Handbook of Microbiological Culture Media

Dehydrated Culture Media ■ Culture Media Ingredients ■ Additives ■ Supplements ■ Reagents

■ Edition No. 10 ■
 TABLE OF CONTENTS

Scharlau Microbiology Introduction I

Scharlau Microbiology Quality Policy II

Safety (Regulations, Hazard Categories, Risk and Safety Phrases, Disposal) V

Guidelines for correct manipulation in Microbiology XIII

Dehydrated Culture Media 1-176

Culture Media Ingredients 177-204

Additives 205-213

Supplements 215-229

Reagents 231-244

Index of products in this Manual 245-251

 Scharlau Microbiology is part of the Scharlau Science Group

Introduction If you work in Spain please contact:

It is a pleasure to introduce to you this edition no. 8 of SCHARLAB, S.L.

our Handbook of Microbiological Culture Media, revised Tel.: 902 20 18 98
and extended, in a new and more attractive format. Fax: 900 50 29 35
E-mail: scharlab@scharlab.com
The range of products has been increased, reaffirming the Post: Gato Pérez, 33. Pol. Ind. Mas d’en Cisa
Scharlau Microbiology brand as an attractive alternative 08181 Sentmenat, Barcelona, Spain
to other manufacturers of culture media for Microbiology.
You will find new products, according to the standards Customers outside of Spain please contact:
of the European Pharmacopoeia, USP and other official
organisms. SCHARLAU CHEMIE, S.A.
In this edition, we have separated additives, reagents Tel.: +34 93 715 18 11
and selective supplements into different chapters, mak- Fax: +34 93 715 31 75
ing it easier to locate each product. E-mail: export@scharlau.com
We have also included additional safety information and Post: Ctra. de Polinyà a Sentmenat, Km. 8,2
R+S sentences, following the EU guidelines 2001/59/ 08181 Sentmenat, Barcelona, Spain
CE.
Or ask for the address of our distributors in any of the
following countries:
The company ALBANIA GUINEA BISSAU QATAR
ANGOLA HAITI REUNION ISLAND
ARGENTINA HONG-KONG ROMANIA
In 1979, Dr. José Sancho Valls, Professor at the Uni- AUSTRALIA HUNGARY RUSSIA
versity of Barcelona, decided to start manufacturing AUSTRIA ICELAND RWANDA
BAHRAIN INDIA SERBIA & MONTENEGRO
dehydrated culture media. A partnership was formed BANGLADESH INDONESIA SAUDI ARABIA
with a local importer of research chemicals and the BARBADOS IRAN SENEGAL
BELGIUM IRAQ SINGAPORE
products sold on the local market under ADSA=MICRO BENIN IRELAND SLOVAKIA
brand name. In 1992 the company was integrated into BOLIVIA ISRAEL SLOVENIA
BOSNIA & HERZEGOVINA ITALY SOUTH AFRICA
the Scharlau group. Thus we look back to more than 25 BOTSWANA JORDAN SRI-LANKA
BRUNEI KENYA SUDAN
years of experience in the manufacture of culture media. BULGARIA KOREA SULTANATE OF OMAN
CHAD KUWAIT SWEDEN
CHILE LATVIA SWITZERLAND
At present, a wide rage of products is offered under the CHINA P.R. LEBANON SYRIA
Scharlau brand, from standard laboratory chemicals COLOMBIA LIBYA TAIWAN
COSTA RICA LITHUANIA TANZANIA
(inorganic salts, acids, buffers, volumetric solution, IVORY COAST MADAGASCAR THAILAND
solvents, etc.), to high purity specialized solvents for CROATIA MALAYSIA THE NETHERLANDS
CUBA MALTA TOGO
HPLC and environmental analysis, up to culture media CYPRUS MAURITANIA TONGA
for microbiology. CZECH REPUBLIC MAURITIUS TRINIDAD & TOBAGO
DENMARK MOLDOVA TUNISIA
The technical supervision remains in the hands of Pro- DOMINICAN REPUBLIC MOROCCO TURKEY
fessor Sancho. Through his wide scientific knowledge, ECUADOR MOZAMBIQUE UGANDA
EGYPT MYANMAR UKRAINE
we can offer good technical support. EL SALVADOR NEW CALEDONIA UNITED ARAB EMIRATES
ERITREA NEW ZEALAND VENEZUELA
ESTONIA NIGERIA VIETNAM
ETHIOPIA NORWAY YEMEN
FINLAND PAKISTAN ZAMBIA
Contact Scharlau FRANCE PANAMA ZIMBAWE
FRENCH POLYNESIA PERU
GERMANY PHILIPPINES
Don’t hesitate. Contact Scharlau for any requirement of GHANA POLAND
GREECE PORTUGAL
culture media for microbiology.
The international presence of Scharlau Microbiology
grows day by day. A wider network of distributors and a
larger portfolio help to better penetrate the markets.
Scharlau Chemie, S.A., which was born 53 years ago to
manufacture exclusively organic chemicals for research
laboratories, has been increasing its products and proc-
esses to become a global manufacturer of laboratory
chemicals and culture media for microbiology.

We stock over 700 different microbiological references in

our new 4.000 m2 warehouse in Sentmenat (Barcelona).

I
 Scharlau Microbiology Quality Policy

www.scharlau.com Environmental care

Please use our website for the latest news and MSDS:
www.scharlau.com.
Our website has been redesigned recently to offer you a
much more modern and intuitive interface.
COA come with each bottle, which is why we don´t pub-
lish them on the web.
One of Scharlau Chemie’s target regarding environmen-
tal issues was achieved in 2003, when we received ISO
14001 approval. Scharlau Chemie has always designed
its processes to minimize environmental impacts on the
rural area where it is located for about 30 years.

As a contribution to the packaging residue minimization

The factory
The microbiological division of Scharlau Chemie, Schar-
lau Microbiology, manufactures a wide range of culture
media, reagents, stains, supplements and additives.
targets, Scharlau has replaced all its packing materials
to avoid the use of expanded polystyrene, which is dif-
ficult to recycle and expensive to dispose of. All internal
and external elements of our packaging are made from
recycled cardboard and are fully biodegradable and
recyclable.

25 years experience in the production of dehydrated

culture media allows us to develop fast new formulations
to keep up with the latest developments in microbiology.

New production facilities have been inaugurated in the

year 2002, thus enabling us to further improve our proc-
esses and expand production capacity. The new factory
has been designed following the most latest directives to
assure the maximum quality of the final product.

More than 700 references are available from Scharlau

Microbiology, ready to be delivered from our new 4000
m warehouse in Sentmenat, Barcelona. The strategic
2

situation, within less than 1000 km from most European

capitals, makes it possible to quickly restock our distribu-
tors. Barcelona, being one of the busiest ports in Europe,
boasts excellent world wide shipping connections.

II
 Scharlau Microbiology Quality Policy

Quality as standard
Our commitment to quality has always been very
important to keep our customers trust.

Scharlau Microbiology purchases raw materials and

transforms them into microbiological culture media,
using various processes and strict analytical controls.
Our target is the production of products of the highest
possible quality.
To achieve this, we have always worked under internal
quality standards, even before ISO regulations were
adopted. This fact not only allows us to manufacture
products of the highest quality, but also to maintain our
quality at a constant level.

The quality concept has evolved and nowadays, qual-

ity is not only understood as an attribute of the product,
but a feature which has to be present in everything
done in the company. It is useless to have an excellent
product, if delivery times or technical assistance are not
adequate.
In Scharlau Microbiology we work under these premises,
injecting quality into all our services, from the develop-
ment of a product designed to cover real customer
requirements, to our after sales assistance.

Since 1997 Scharlau Microbiology is ISO 9002 accred-

ited through a German TUV.

Certificate of Analysis
Every pack is sent together with its Certificate of Analysis,
which guarantees the quality of our products.
Together with the Certificate of Analysis we supply a sec-
ond certificate to assure absence of Bovine Spongiform
Encephalopathy and Foot-and-Mouth Disease in our raw
materials of animal origin.

Each certificate includes the following information:

Physical-chemical specifications:
1. Of the medium before reconstitution:
Colour
Appearance
Particle size / homogenization
2. Of the reconstituted medium ready to use:
Clarity
Precipitates
Gelifying or solidifying strength

Growth control
Results obtained with control strains under optimal
conditions.

III
 Scharlau Microbiology Quality Policy
Quality in our labels
We have changed the design of our label to make it
more useful and modern.
Product name and directions appear now in several
languages with medium specification and formula in
English. Our labels are printed according to CE regula-
tions for dangerous goods.
IV
Expiry date and batch number
Good Laboratory Practices (GLP) which are followed
by many of our customers, indicate that all products in
the laboratory must be labelled showing batch number,
expiry date and other additional data (date of reception,
opening date, opened by...)
Our labels contain batch number and expiry date and
also a specific area is reserved for the user to write
down additional data.
 Safety Regulations

Material Safety Data Sheets

MSDS include physical and chemical data, handling rec-
ommendations, toxicological information and considera-
tions relative to the environment, disposal, storage and
transport of each product.
In some cases, MSDS are delivered with the products but
it is better to get them in advance. We edit MSDS of our
products on CD-ROM and they are also available from
our web:
www.scharlau.com > MSDS

Storage and Transport

Storage and transportation of dangerous goods is sub-
jected to special regulations due to the specific risks they
involve. All parts handling dangerous goods must adopt
the appropriate safety measures to avoid damages.

Safe storage
Dangerous goods must be stored under special safety
conditions to avoid health or environmental injuries. It is
always important to keep in mind chemical in compatibili-
ties and to separate the products to avoid its mixture in
case of accident.
Every substance can be classified into a group of sub-
stances having similar hazards, that can be stored
together. The same security measures will be applied to
all the substances belonging to the same classification.
If a substance has several hazard degrees, it should be classified as per its highest one:
Explosive > Oxidising > Flammable > Toxic > Corrosive > Harmful

There are three basic criteria that should be kept in mind to guarantee a safe storage of chemicals in the laboratories:
• Stock of dangerous products must be limited to the minimum quantity needed.
As a general rule, purchases should never exceed the quantity needed for one year, and preferably should cover a
period of between 3 and 6 months.
• Products must be stored in groups depending on its chemical compatibility, as shown below:

EXPLOSIVES OXIDIZING FLAMMABLE TOXIC CORROSIVES HARMFUL

EXPLOSIVES YES NO NO NO NO NO
OXIDIZING NO YES NO NO NO NO
FLAMMABLE NO NO YES NO YES* YES
TOXIC NO NO NO YES YES YES
CORROSIVES NO NO YES* YES YES YES
HARMFUL NO NO YES YES YES YES
*COULD BE STORED TOGETHER IF CORROSIVE PRODUCTS ARE CONTAINED IN NON FRAGILE BOTTLES

• Some products, that are specially dangerous, should be isolated from the rest.
Carcinogenic or high toxicity substances should be stored in specific cabinets conveniently marked and closed by
key.

Special attention must be paid to the safe storage of flammable liquids, due to the big amounts that are usually
needed in the laboratories. They should be stored in security cabinets and security drums.

Storage regulations of dangerous goods indicate minimum distances that should be kept between different classes
and special storage conditions for each class. Our new warehouse was built following this and other EC regulations
regarding safety and risk prevention.

V
 Safety. Regulations
Safe transportation
Dangerous goods can be transported outside manufac-
turing sites only if they are packed, labelled and deliv-
ered in safe conditions.
• Packaging must be officially approved to contain dan-
gerous goods. Strict tests are performed to evaluate
physical resistance to break.
• Packaging must be labelled according to specific
regulations for transport of dangerous goods.
• Goods must be accompanied by several documents
describing its hazard and how to proceed in case of
damage during loading, transporting or downloading
operations.
Dangerous goods cannot be transported by unauthor-
ized transport means. There are different international
regulations that are applied to the transport by road,
air or sea. The following classes apply to all means of
transport:
CLASS DESCRIPTION
1 Explosive substances
2 Gases
3 Flammable liquids
4.1 Flammable solids
4.2 Spontaneously flammable solids
4.3 Substances that develop flammable gases in contact with water
5.1 Substances that promote combustion (oxidants)
5.2 Organic peroxides
6.1 Toxic substances
6.2 Substances that induce vomiting or infection
7 Radioactive substances
8 Corrosive substances
9 Various hazardous substances
Transport regulations
By road / railway
ADR (Accord européen rélatif au transport international
des merchandises Dangereuses par Route)
This European agreement on the international trans-
port of dangerous goods by road changed last July 1st
2001 and all data in this catalogue have been updated
according to this new version.
The former ADR 1999 can be used until the end of
2002, but, from January 1st 2003, only ADR 2001 will
be applied.
RID (Reglement International concernant le transport
des marchandises Dangereuses par chemin de fer)
Regulates the international transport of dangerous
goods by railway. The new ADR2001 includes all
requirements described in RID and could be used
instead.
VI
By air
IATA (International Air Transport Association) / ICAO
(International Civil Aviation Organization)
IATA regulations for transport of dangerous goods in-
clude all requirements from ICAO as well as additional
technical instructions. All data in this catalogue refer to
the 43rd edition of “Dangerous goods regulation” from
IATA.
You will also find in this catalogue the packing instruc-
tions for passenger aircraft (PAX) and cargo aircraft
(CAO). The “Packing Instruction” indicates the condi-
tions for the packaging, as well as the quantity author-
ized on board the aircraft. If transport of the substance
is forbidden, an “F” is printed instead of the PAX or
CAO data.
The packages that comply PAX conditions, can be
always transported on cargo aircraft.
IATA issues an ID number that is an identification
number for hazardous goods. It is only used in cases
where there is no UN number.
By sea
IMDG (International Maritime
Code for Dangerous Goods)
has been drawn by the IMO (In-
ternational Maritime Organiza-
tion of the UN) and is the regu-
lation applied to any transport of
dangerous goods by see.
Other transport / security data
included in this catalogue:
UN number
The Committee of Experts of
the United Nations (UN) issues
recommendations for all freight
forwarders on the assessment
of hazardous goods for transport purposes. They are
numbered consecutively. These UN numbers are given
to each product so that everyone knows how to handle
it, no matter if it is going to be sent by road, air or sea.
In case there is no UN number, IATA gives an ID number
for air transport of hazardous goods.
EC index number (EC = European Community)
This number is issued by the EC commission for the
classification, packaging and marking of dangerous sub-
stances. The marking with hazard symbols, risk warn-
ings (R phrases) and safety precautions (S phrases), as
used in this catalogue, is linked with this number.
Safety. Hazard categories
E: EXPLOSIVE
Are considered all products and preparatio-
ns, if they can explode through ignition or if
they are more sensitive than dinitrobenzene
towards blows
and friction.
Precautions: Avoid impact, knocks, friction,
sparks, fire and heat.
O: OXIDISING
Are considered all products and preparatio-
ns, which not being flammable, can produce
and enhance fires in contact with flammable
products.
Precautions: Avoid all contact with flamma-
ble substances. Risk of ignition! The substan-
ce promotes fires once started and impedes
fire fighting.
F+: EXTREMELY FLAMMABLE
Are considered all products and preparatio-
ns, with a flash point below 0°C and a boiling
point of 35°C or below.
Precautions: Keep away from naked flames,
sparks and sources of heat.
F: FLAMMABLE
Are considered all products and preparatio-
ns, if:
a) They heat up and finally start burning
in contact with air at normal temperature
without any external energy supply
b) They can start burning in solid condition
ater short contact with a source of ignition
and continue burning after the source has
been taken away,
c) They have a flash point below 21°C in
liquid condition,
d) They form in gaseous condition an explosi-
ve mixture with air under normal pressure,
e) They create in contact with water or wet air
highly flammable gases.
Precautions: Keep away from naked flames,
sparks and sources of heat.
T+: VERY TOXIC
are considered all products and preparations,
if they lead to death or to acute or cronical
health
injuries when inhaled, swallowed or absorbed
through the skin in very small quantities.
Precautions: All contact with the human
body must be avoided, as severe or even
lethal damage to health cannot be excluded.
Particular attention is drawn to the carcinoge-
nic, teratogenic or mutagenic risks associated
with certain substances.
T: TOXIC
Are considered all products and preparations,
if they lead to death or to acute or cronical
health injuries when inhaled, swallowed or
absorbed through the skin in small quantities.
Precautions: All contact with the human
body must be avoided, as severe or even
lethal damage to health cannot be excluded.
Particular attention is drawn to the carcino-
genic, teratogenic or mutagenic risks associ-
ated with certain substances.
Xn: HARMFUL
Are considered all products and prepara-
tions, that if inhaled, swallowed or absorbed
through the skin can cause death or serious
acute or chronic effects.
Precautions: Avoid contact with the human
body, including the inhalation of vapours.
Injury to health is possible with improper use.
With some substances, carcinogenic, tera-
togenic or mutagenic action cannot be fully
excluded, as well as possible sensitization.
C: CORROSIVE
Are considered all products and preparations,
if they can destroy skin by contact.
Precautions: Take special measures to
protect the eyes, skin and clothes. Do not
inhale vapours.
Xi: IRRITANT
Are considered all products and prepara-
tions, if they can produce irritation in a short,
prolonged or repetitive contact with the skin
or the respiratory tract.
Precautions: Avoid contact with eyes and
skin, do not inhale vapours.
N: DANGEROUS FOR THE
ENVIRONMENT
for the Environment can be considered those
products and substances, which can have
detrimental effects upon the ecosystem
of water, soil or air, climate, fauna, flora or
microorganisms in such a way, that they con-
stitute an immediate or future danger for the
environment.
Precautions: According to the kind of haz-
ard do not dispose to wastewater, soil or
environment. Observe special disposal regu-
lations!
VII
 Safety. Risk and Safety Phrases

R: Risk Phrases 58 May cause long-term adverse eflects in the

environment.
1 Explosive when dry. 59 Dangerous for the ozone layer.
2 Risk of explosion by shock, friction, fire or other 60 May impair fertility.
sources of ignition. 61 May cause harm to the unborn child.
3 Extreme risk of explosion by shock, friction, fire or 62 Possible risk of impaired fertility.
other sources of ignition. 63 Possible risks of harm to the unborn child.
4 Forms very sensitive explosive metallic 64 May cause harm to breastfed babies
compounds. 65 Harmful: may cause lung damage if swallowed.
5 Heating may cause an explosion. 67 Vapours may cause drowsiness and dizziness.
6 Explosive with or without contact with air. 66 Repeated esposure may cause skin dryness or
7 May cause fire. cracking.
8 Contact with combustible material may cause fire. 68 Possible risk of irreversible effects.
9 Explosive when mixed with combustible material.
10 Flammable. Combination of particulars risks
11 Highly flammable.
12 Extremely flammable. 14/15 Reacts violently with water, liberating extremely
13 Extremely flammable liquefied gas. flammable gases.
14 Reacts violently with water. 15/29 Contact with water liberates toxic, extremely
15 Contact with water liberates extremely flammable flammable gas.
gases. 20/21 Harmful by inhalation and in contact with skin.
16 Explosive when mixed with oxidising substances. 20/21/22 Harmful by inhalation, in contact with skin and if
17 Spontaneously flammable in air. swallowed.
18 In use, may form flammable/explosive vapour-air 20/22 Harmful by inhalation and if swallowed.
mixture. 21/22 Harmful in contact with skin and if swallowed.
19 May form explosive peroxides. 23/24 Toxic by inhalation and in contact with skin.
20 Harmful by inhalation. 23/24/25 Toxic by inhalation, in contact with skin and if
21 Harmful in contact with skin. swallowed.
22 Harmful if swallowed. 23/25 Toxic by inhalation and if swallowed.
23 Toxic by inhalation. 24/25 Toxic in contact with skin and if swallowed.
24 Toxic in contact with skin. 26/27 Very toxic by inhalation and in contact with skin.
25 Toxic if swallowed. 26/27/28 Very toxic by inhalation, in contact with skin and if
26 Very toxic by inhalation. swallowed.
27 Very toxic in contact with skin. 26/28 Very toxic by inhalation and if swallowed.
28 Very toxic if swallowed. 27/28 Very toxic in contact with skin and if swallowed
29 Contact with water liberates toxic gas. 36/37 Irritating to eyes and respiratory system.
30 Can become highly flammable in use. 36/37/38 Irritating to eyes, respiratory system and skin.
31 Contact with acids liberates toxic gas. 36/38 Irritating to eyes and skin
32 Contact with acids liberates very toxic gas. 37/38 Irritating to respiratory system and skin
33 Danger of cumulative effects. 39/23 Toxic: danger of very serious irreversible effects
34 Causes burns. through inhalation.
35 Causes severe burns. 39/23/24 Toxic: danger of very serious irreversible effects
36 Irritating to eyes. through inhalation and in
37 Irritating to respiratory system. contact with skin.
38 Irritating to skin. 39/23/24/25 Toxic: danger of very serious irreversible
39 Danger of very serious irreversible effects effects through inhalation, in contact with
40 Limited evidence of a carcinogenic effect. skin and if swallowed.
41 Risk of serious damage to eyes. 39/23/25 Toxic danger of very serious irreversible effects
42 May cause sensitisation by inhalation. through inhalation and if swallowed.
43 May cause sensitisation by skin contact. 39/24 Toxic: danger of very serious irreversible effects in
44 Risk of explosion if heated under confinement. contact with skin.
45 May cause cancer. 39/24/25 Toxic: danger of very serious irreversible effects in
46 May cause heritable genetic damage. contact with skin and if swallowed.
47 May cause birth defects. 39/25 Toxic: danger of very serious irreversible effects if
48 Danger of serious damage to health by prolonged swallowed.
exposure. 39/26 Very toxic: danger of very serious irreversible
49 May cause cancer by inhalation. effects through inhalation.
50 Very toxic to aquatic organisms. 39/26/27 Very toxic: danger of very serious irreversible
51 Toxic to aquatic organisms. effects through inhalation and in contact with skin.
52 Harmful to aquatic organisms. 39/26/27/28 Very toxic: danger ol very serious
53 May cause long-term adverse effects in the irreversible effects through inhalation,
aquatic environment. in contact with skin and if swallowed.
54 Toxic to flora. 39/26/28 Very toxic: danger of very serious irreversible
55 Toxic to fauna. effects through inhalation and if swallowed.
56 Toxic to soil organisms. 39/27 Very toxic: danger of very serious irreversible
57 Toxic to bees. effects in contact with skin.

VIII
 Safety. Risk and Safety Phrases

39/27/28 Very toxic: danger of very serious irreversible
effects in contact with skin and if swallowed.
39/28 Very toxic: danger of very serious irreversible
effects if swallowed.
40/20 Harmful: possible risk of irreversible effects
through inhalation.
40/20/21 Harmful: possible risk of irreversible effects
through inhalation and in contact with skin.
40/20/21/22 Harmful: possible risk of irreversible
effects through inhalation, in contact
with skin and if swallowed.
40/20/22 Harmful: possible risk of irreversible effects
through inhalation and if swallowed.
40/21 Harmful: possible risk of irreversible effects in
contact with skin.
40/21/22 Harmful: possible risk of irreversible effects in
contact with skin and if swallowed.
40/22 Harmful possible risk of irreversible effects if
swallowed.
42/43 May cause sensitisation by inhalation and skin
contact.
48/20 Harmful: danger of serious damage to health by
prolonged exposure.
48/20/21 Harmful: danger of serious damage to health by
prolonged exposure
through inhalation and in contact with skin.
48/20/21/22 Harmful: danger of serious damage to
health by prolonged exposure
through inhalation, in contact with skin
and if swallowed.
48/20/22 Harmful: danger of serious damage to health by
prolonged exposure through inhalation and if
swallowed.
48/21 Harmful: danger of serious damage to health by
prolonged exposure in contact with skin.
48/21/22 Harmful: danger of serious damage to health by
prolonged exposure in contact with skin and if
swallowed.
48/22 Harmful: danger of serious damage to health by
prolonged exposure if swallowed.
48/23 Toxic: danger of serious damage to health by
prolonged exposure through inhalation.
48/23/24 Toxic: danger of serious damage to health by
prolonged exposure through
inhalation and in contact with skin.
48/23/24/25 Toxic: danger of serious damage to
health by prolonged exposure through
inhalation, in contact with skin and if
swallowed.
48/23/25 Toxic: danger of serious damage to health by
prolonged exposure through
inhalation and if swallowed.
48/24 Toxic: danger of serious damage to health by
prolonged exposure in contact with skin.
48/24/25 Toxic: danger of serious damage to health by
prolonged exposure in
contact with skin and if swallowed.
48/25 Toxic: danger of serious damage to health by
prolonged exposure if swallowed.
50/53 Very toxic to aquatic organisms, may cause long-
term adverse effects in
the aquatic environment.
52/53 Harmful to acquatic organisms, may cause long-
term adverse effects in the acquatic environment.
51/53 Toxic to acquatic organisms, may cause long-term
effects in the aquatic environment.
68/20 Harmful: possible risk of irreversible effects
through inhalation.
68/21 Harmful: possible risk of irreversible effects in
contact with skin.
68/22 Harmful: possible risk of irreversible effects if
swallowed.
68/20/21 Harmful: possible risk of irreversible effects
through inhalation and in contact with skin.
68/20/22 Harmful: possible risk of irreversible effects
through inhalation and if swallowed.
68/21/22 Harmful: possible risk of irreversible effects in
contact with skin and if swallowed.
68/20/21/22 Harmful: possible risk of irreversible
effects through inhalation, in contact
with skin and if swallowed.
S: Safety phrases
1 Keep locked up.
2 Keep out of reach of children.
3 Keep in a cool place.
4 Keep away from living quarters.
5 Keep contents under _ (appropriate liquid to be
specified by the manufacturer)
5.3 Keep contents under paraffin oil.
6 Keep under (inert gas to be specified by the
manufacturer)
7 Keep container tightly closed.
8 Keep container dry.
9 Keep container in a well ventilated place.
12 Do not keep the container sealed.
13 Keep away from food, drink and animal feeding stuffs.
14 Keep away from _ (incompatible materials to be
indicated by the manufacturer)
14.1 Keep away from alkalis.
14.2 Keep away from oxidizing and acidic substances
as well as heavy metal compounds.
14.9 Keep away from flammable organic substances.
15 Keep away from heat.
16 Keep away from sources of ignition - No Smoking.
17 Keep away from combustible material.
18 Handle and open container with care.
20 When using do not eat or drink.
21 When using do not smoke.
22 Do not breathe dust.
23 Do not breathe gas/fumes/vapour/sray
(appropiate wording to be
specified by the manufacturer).
23.2 Do not breathe vapour.
24 Avoid contact with skin.
25 Avoid contact with eyes.
26 In case of contact with eyes, rinse immediately
with plenty of water and seek medical advice.
27 Take off immediately all contaminated clothing.
28 After contact with skin, wash immediately with
plenty of _ (to be specified by the manufacturer).
28.1 After contact with skin, wash immediately with
plenty of water.
28.2 After contact with skin, wash immediately with
soap and water.
28.3 After contact with skin, wash immediately with
soap and water, if possible
also with polyethylene glycol 400.
28.6 After contact with skin, wash immediately with
polyethylene glycol 400
(then rinse with plenty of water).

IX
 Safety. Risk and Safety Phrases

29 Do not empty into drains. 61 Avoid release to the environment.Refer to special

30 Never add water to this product. instructions/Safety data sheets.
33 Take precautionary measures against static 62 If swallowed, do not induce vomiting: seek
discharges. medical advice immediately
34 Avoid shock and friction. and show this container or label.
35 This material and its container must be disposed 63 In case of accident by inhalation: remove casualty
of in a safe way. to fresh air and keep at rest.
36 Wear suitable protective clothing. 64 If swallowed, rinse mouth with water (only if the
37 Wear suitable gloves. person is conscious).
38 In case of insufficient ventilation, wear suitable
respiratory equipment. Combination of safety precautions
39 Wear eye/face protection.
40 To clean the floor and all objects contaminated by 1/2 Keep locked up and out of reach of children.
this material use _ (to 3/7 Keep container tightly closed in a cool place.
be specified by the manufacturer). 3/7/9 Keep container tightly closed, in a cool well
41 In case of fire and/or explosion do not breathe ventilated place.
fumes. 3/9 Keep in a cool well ventilated place
42 During fumigation/spraying wear suitable 3/9/14 Keep in a cool, well ventilated place away from _
respiratory equipment (incompatible materials
(appropriate wording to be specified) to be indicated by the manufacturer)
43 In case of fire, use _ (indicate in the space the 3/9/14.1 Keep in a cool, well ventilated place away from
precise type of fire-lighting alkalis.
equipment. If water increases the risk add - Never 3/9/14/49 Keep only in the original container in a cool, well-
use water) ventilated place away from ... (incompatible
43.1 In case of fire, use water. materials to be indicated by the manufacturer).
43.3 In case of fire, use powder extinguisher. Never 3/9/49 Keep only in the original container in a cool, well-
use water. ventilated place.
43.6 In case of fire, use sand. Never use water. 3/14 Keep in a cool place away from ... (incompatible
43.7 In case of fire, use metal-fire powder. Never use materials to be indicated by the manufacturer).
water. 7/8 Keep container tightly closed and dry.
43.8 In case of fire, use sand, carbon dioxide or 7/9 Keep container tightly closed and in a well-
powder extinguisher. Never use water. ventilated place.
44 If you feel unwell, seek medical advice (show the 20/21 When using do not eat, drink or smoke.
label where possible) 24/25 Avoid contact with skin and eyes.
45 In case of accident or if you feel unwell, seek 27/28 After contact with skin, take off immediately all
medical advice immediately contaminated clothing, and wash immediately with
(show the label where possible) plenty of ... (to be specified by the manufacturer).
46 If swallowed seek medical advice immediately 29/35 Do not empty into drains; dispose of this material
and show this container or label and its container in a safe way.
47 Keep at temperature not exceeding _ ºC (to be 36/37 Wear suitable protective clothing and gloves.
specified by the manufacturer) 36/37/39 Wear suitable protective clothing, gloves and eye/
48 Keep wetted with _ (appropriate material to be face protection.
specified by the manufacturer) 36/39 Wear suitable protective clothing and eye/face
49 Keep only in the original container. protection.
50 Do not mix with _ (to be specified by the 37/39 Wear suitable gloves and eye/face protection.
manufacturer) 47/49 Keep only in the original container at temperature
50.1 Do not mix with acids. not exceeding _ ºC (to be specified by the
51 Use only in well ventilated areas. manufacturer).
52 Not recommended for interior use on large
surface areas.
53 Avoid exposure - obtain special instruction before
use.
54 Obtain the consent of pollution control authorities
before discharging to wastewater treatment plants.
55 Treat using the best available techniques before
discharge into drains or
the aquatic environment.
56 Dispose of this material and its containes at
hazardous or special waste collection point.
57 Use appropriate containment to avoid
environmental contamination.
58 To be disposed of as hazardous waste.
59 Refer to manufacturer/supplier for information on
recovery/recycling
60 This material and its container must be disposed
of as hazardous waste.

X
 Safety. Disposal
Disposal of Laboratory Waste
Avoid contamination of the water
Elimination of residue through the drain is strictly forbid-
den. You have to bear in mind, that many chemicals
shall not be reduced by the waste water plants and shall
contaminate the environment.
Residue in the laboratory
We highly recommend naming a person in charge of the
residue of your company. This person should be in-
formed of the laws and regulations regulating the waste
of residue in your own country.
In order to hand over the residue to a treatment plant,
it is necessary to organize the collection and storage
previously.
Collection of residue in the laboratory
The material of the residue containers has to comply
with the following conditions:
1. One has to be able to close the containers hermeti-
cally and the material has to be resistant to the con-
tents. Plastic containers should not form toxic fumes
when disposed of through burning.
2. Generally, plastic containers are being used.
3. Corrosive products should be collected in metal
drums with inner plastic lining, like our Combi drum
(Ref. 055-0C0025).
4. Inflammable or oxidizing products should be collect-
ed in metal or plastic drums resistant to the solvent.
5. For products that produce gases or vapours, special
containers with a security valve are required, in order
to avoid the danger of explosions.
If the collection containers are handed over to a trans-
port agency, they have to carry the UN number that
approves them for transport by road.
Hazardous chemicals
In this handbook risk phrases (R-phrases), safety
phrases (S- phrases) and hazard symbols are added in
accordance with the EU Directive (67/548) and (83/467).
The absence of R- and/or S-phrases or hazard symbols
does not mean that those substances are harmless.
The normal safety precautions for handling chemicals
should always be observed.
General Waste -
Disposal procedures
The disposal methods outlined below are intended as a
guideline for the technician. We do not assume respon-
sibility of their use. Careful consideration must be given
to the chemical and physical properties of the substance.
In addition, local laws and regulations may preclude the
use of these methods which were primarily designed
for the quantities we list. Observe all Federal, State and
local laws concerning health and pollution.
All procedures require the use of goggles or face shield,
gloves and apron. Gas masks or self- contained breath-
ing units may also be required.
1. Neutralize the bases with diluted sulphuric acid, then
flush neutral solution down the drain with an abun-
dance of water.
2. Treat the aldehydes or peracids with an excess of
sodium bisulfite solution. Use starch iodide paper to
ensure complete reduction of any peracid. Flush the
resulting solution down the drain with an abundance
of water.
3. The compound should be burned. Ideally, all hydro-
carbons and related solvents should be burned in an
approved presence of sodium carbonate and cal-
cium hydroxide (slaked lime). The solid or the liquid
absorbed on vermiculite should be wrapped in paper
and burned in an approved incinerator.
4. Bury in an approved chemical and hazardous-waste
sand fill site, well away from streams, rivers or wells.
5. Azides and azo-compounds should be treated with
dilute ceric ammonium nitrate solution with cool-
ing. Solutions may be discarded upon completion of
the reaction. Mixtures should be separated and the
organic portion burned.
6. Treat with an alcohol such as ethanol or butanol in
an appropriate solvent. The chemical reaction may be
vigorous and/or exotermic. Provisions must be made
for venting of large volumes of highly flammable
hydrogen and/or hydrocarbon gases. Neutralize the
solution with aqueous acid. Filter off any solid residue
for disposal as hazardous waste. Burn residual
organic materials in an incinerator equipped with an
afterburner and scrubber.
XI
 Safety. Disposal

7. Neutralize the base with dry sodium bisulfate add

water slowly and flush the neutral solution down the
drain with an excess of water.
8. Carefully mix the acidic compound with dry sodium
bicarbonate. Dilute slowly and flush down the drain
with an excess of water.
9. Oxidize the mercaptan or nitrite with a weak aqueous
solution (up to 15%) of sodium or calcium hypoclorite.
Vigorous stirring may be required to ensure comple-
tion of the reaction. Neutralize the resulting mixture,
then discard down the drain with an excess of water.
10. Flush down the drain with an excess of water.
11. Bury in a site approved for disposal of poisonous or
hazardous wastes.
12. Refer to our Material Safety Data Sheet before han-
dling or disposing.

Caution regarding hazardous

components
Some media contain components that are toxic or carci-
nogenic. Appropriate safety precautions must be taken
when using media with such components.

Basic fuchsin and acid fuchsin are carcinogens and

caution must be used in handling media with these com-
pounds to avoid dangerous exposure that could lead to
the development of malignancies . Thalium salts, sodium
azide, sodium biselenite and cyanide are among the
toxic components found in some media. These com-
pounds are poisonous and steps must be taken to avoid
ingestion, inhalation, and skin contact. Azides also react
with many metals, especially copper, to form explosive
metal-azides. The disposal of azides must avoid con-
tact with copper or achieve sufficient dilution to avoid
the formation of such hazardous explosive compounds.
Media with sulphur containing compounds may result in
the formation of hydrogen sulphide which is a toxic gas.
Care must be used to ensure proper ventilation. Media
with human blood or human blood components must be
handled with great caution to avoid exposure to hu-
man immunodeficiency virus and other pathogens that
contaminate some blood supplies. Proper handling and
disposal procedures must be followed with blood-con-
taining as well as other media that are used to cultivate
microorganisms.

XII
 Guidelines for correct manipulation in Microbiology
Following instructions provide the correct guidelines for
the manipulations in microbiology. Every microbiological
laboratory is responsible for their proper implementation.
A. Personal techniques
A.1. Hygiene is the basic element and need for the safe
manipulation.
A.2. All manipulated microorganisms must be presumed
potentially pathogenic, regardless of their nature.
The following procedures or techniques must
therefore be avoided : oral pipetting, application
of cosmetics ,eating, drinking or smoking in the
laboratory. Wrappings and labels in the labortory
should be self-adhesive.
A.3. Manipulation of the microorganisms should be
conducted in such a way that will not provoke
any environmental contamination or hazard. For
example, sprays should always be avoided and
pipettes must be submerged immediately in the
disinfectant after use.
A.4. Manipulators (technicians) must be aware of the
risks and should know how to prevent them.
A.5. Manipulators must make sure that both, the user
and the equipment itself are perfectly safe from
any potential risk.
B. Equipment in general
B.1. A standard equipment for the sterilization of any
potentially hazardous material must be available
(autoclave, jars with disinfectant for the pipettes,
dry heat ovens etc).
B.2. Protective clothes must be used exclusively in the
working area, and should never be worn while in
public areas.
B.3.Toilets and other facilities must be available close
to the working areas, and separate from those of
use to the general public.
B.4. Any device which may probably produce the sprays
must be always handled in microbiological secu-
rity cabins like Laminar Air Flow .
B.5. In case of any accident, all necessary substances
and equipments , either to minimize or neutralize
the risks must be readily available.
C. Basic characteristics of the
laboratory
C.1. The laboratory or plant should not be located in a
passage or corridor so that any outside person
would always remain away from it.
C.2. It must be equiped with an appropiate ventillation
system, mechanically regulated if possible, so as
to create a drift of flowing air from the areas of
low risk to the contaminated areas, and then go-
ing outside, through the HEPA filters.
C.3. The laboratory must be built of easily washable,
waterproof and disinfectant-resistant surfaces.
D. Administrative procedures
D.1. Systems to confirm and assure the effectiveness of
the biological security code carried out by quali-
fied personnel.
D.2. Training programmes regarding the security during
or at work, adapted to the level required by every
maninpulator / technician.
D.3. Adequate medical surveillance and facilities .
D.4. Procedures for the verification and maintenance
of the ventilation system and the equipment in
general.
D.5. Emergency procedures and systems for use in case
of any accident.
D.6. Strategies to restrain the freak entries and exits, to
maintain the security standards of laboratory and
the plant.
D.7. Procedures for the transportation, postage and
reception of the biological material (postage
regulations and related things).
D.8. Training of personnel to act as security officers.
D.9. Systems to eliminate any potentially hazardous
residues.
References
Report of Committee of Enquiry into Smallpox (1974)
Outbreak in March and April 1973. Cmnd.5626, Her
Majesty’s Stationery Office.
Report of the Working Party on the Laboratory Use of
Dangerous Pathogens. (The Godber Report) (1975).
Cmnd. 6054, Her Majesty’s Stationery Office.
Report of the Working Party on the Experimental Manip-
ulation of the Genetic Composition of Micro-organisms.
(1975) Cmnd. 5880, Her Majesty’s Stationery Office.
PUBLIC HEALTH LABORATORY SERVICE (1980)
Safety Precautions-Notes for Guidance. Colindale,
MILLER, B.M. (1987) Laboratory Safety: Principles and
Practices. ASM. Whasington, D.C.
HARTREE, E., U. BROTH (1977) Safety in Biological
Laboratories. Biochem. Soc. Spec. Pub. nº5. The Bio-
chemical Society. London
COLLINS, C.H., E.G. HARTLEY, R. PILSWORT (1976)
The Prevention of Laboratory Adquired Infection. PHLS
Monograph nº6. Her Magesty Stationery Office.
SMITH, J.A. (1996) Laboratory Safety in Clinical Microbi-
ology. Cumitech nº29. ASM. Whasington, D.C.
WHO (1983) Laboratory Biosafety Manual. Geneva.
The appearance and quality of the prepared medium
usually depends on the method used for rehydration
and storage. It is therefore very important to follow the
recommendations stated below.
XIII
 Guidelines for correct manipulation in Microbiology
Rehydration
Directions for the individual reconstitution are detailed
on the container of every media and must be strictly
followed.
The water used for reconstitution purposes must be
distilled or deionized, of «pharmaceutical grade». The
solution or suspension must be completely homogene-
ous and the exposure to heat if necessary, should be
minimal.
In any case, it is always helpful to heat the water previ-
ously to 50-60°C, to help dissolve the media.
For the preparation of the culture media the most recom-
mended technique is the following:
Add necessary amount of powder to half the volume
of water needed. The water should have already been
warmed to 50-60° C. Stir the mixture thoroughly until the
powder is dissolved. Use rest of the water to dissolve the
powder sticking to the walls or sides of the container and
continue stirring and heating if necessary. Heat must
be applied directly and smoothly. Constant agitation will
prevent the components from sticking to the walls of the
XIV
container. Use a container with a capacity of approxi-
mately 2 to 3 times more the volume of the medium, to
allow plenty of space for manipulation.
Sometimes, It is necessary to boil the medium for more
than 1 minute. In such cases, the volume of evaporated
water must be restored.
Media not containing the agar or any solidifying agents
(broths and Nutrient Solutions) tend to dissolve eas-
ily and rapidly in preheated water, however sometimes
boiling is necessary to attain the complete dissolution.
These media have a tendency to form a concentrated
syrup or suspension which settles at the bottom of the
container. This thick suspension usually remains at the
bottom, risking the destruction of the medium contents
by hydrolysis, caramelization and pH drift when the heat
is applied. Therefore, thorough mixing should be as-
sured before heating.
Media containing solidifying agents (Agars and Fluid
Media) must be preheated before sterilization. Media
containing agar must be brought to the boiling, since
agar is insoluble in water below 98-100° C. It is useful to
let the agar soak in preheated water for 5 to 10 minutes
before heating, in order to al-
low it to swell and to secure its
solubility and uniform distribu-
tion and dissolution.
A highly recommended
procedure can be practised
by removing the rehydrated
medium from the source of
heat when the mixture begins
to boil, letting it stand for a
while and then quickly bring-
ing it to the boiling again. This
may be repeated 2 or 3 times
with constant agitation. The
complete dissolution of the
agar will be indicated by the
absence of granules in the
container.
Dissolution of culture media
directly during sterilization in
the autoclave is a frequent
and totally incorrect practice.
It alters the medium’s quali-
ties and frequently causes
the uneven dissolution of the
agar into layers with different
concentration gradients, pH
drifts and browning.
Nowadays, heating the rehy-
drated medium in a microwave
oven in order to melt the agar
 Guidelines for correct manipulation in Microbiology

or dissolve the medium components is a very common dium is introduced in the autoclave , and to let the tem-
procedure. In this case also, it is recommended to let perature drop to 70-80°C before removing the medium
the agar-medium to soak for some time before using so as to avoid severe temperature fluctuations. Although
the microwave and use the containers suitable for the the use of cold water to cool media is widespread, it is
volume of medium to be prepared. Using a microwave not recommended for media containing agar since it
with 800 W for 4 minutes are enough to achieve the total causes flakes and cloud formation.
dissolution or melting of agar. However, it must be noted
that since this is a static procedure, a concentration gra-
dient will appear which will create a stratification. Thus,
it may be necessary to shake the rehydrated medium
vigorously to homogenize the solidifying agent before
using or sterilizing it in the autoclave, in the same way
as if it were an autosterile medium. Microwave heating
can never be adopted as a substitute for autoclave
sterilization.

Dehydrated culture media usually maintain their features

if reconstituted properly. Nevertheless, verification is
encouraged since some of them (colour, pH, etc) may
slightly change subject to the conditions of reconstitution
and the type of water used.

These recommendations are particularly important when

referring to fluid media, since such media contain small
amounts of solidifying agent and are therefore very deli-
cate. Cooling becomes as important as heating, as any
violent process may induce the formation of flakes and
clouds. A slow and smooth cooling process is recom-
mended.

Media containing phosphate buffers together with glu-

cose or other carbohydrates may darken if overheated
as a concentrated solution. Precipitates may also appear
when using poor quality water.

Special attention must be paid to all media containing

agar at a pH under 5, since it hydrolyzes and looses the
ability to form gels. In those cases, unnecessary heating
must be avoided.

Sterilization
The indications given on every container must be fol-
lowed, while bearing in mind that they refer to quantities
of up to 1 litre. As for bigger volumes, autoclaving and
heat penetration conditions of the medium will have to
be taken into consideration.

In all the cases autoclaving must be monitored regu-

larly by the manometers and thermometers, as well
as the even distribution of heat. Nowadays this can be
done mechanically (thermocouples), chemically (ther-
mal indicators) or biologically (thermoresistent spores).
Considering that overheating is one of the main causes
of culture media alteration, it is only natural to prevent,
whenever possible, the treatment of large quantities of
media and the prolonged exposure to heat. For large
autoclaves it is recommended to preheat before the me-

XV
 Guidelines for correct manipulation in Microbiology
Containers used for sterilization purposes must have a
large head space for air to allow foaming. If screw-cap
containers are used,the closures must be a half turned
or screwed to allow inner and outer pressure balancing.
All containers should be chemically inert to prevent al-
teration of the pH of contents. Borosilicate glass contain-
ers are highly recommended.
Media containing carbohydrates darken a little after heat
sterilization, therefore, sterilization should be carried out
at a temperature below 120°C if possible.
Should autoclave not be available, sterilization can be
done in a pressure vessel (at 100°C for 30 minutes). In
such case it is useful to resterilize the medium during
three days, if the medium allows the treatment.
Some media containing selective substances do not
need sterilization. The preparation process is quicker
since they are ready for use after dissolution. Since
these media are usually not very stable and preparation
is brief and easy, the exact quantities required should be
prepared.
Additions to the medium (if any) after sterilization must
be done aseptically, that is, additives should have been
previously sterilized. Additives are generally thermolabi-
les if not they would be part of the dehydrated medium’s
composition. It is therefore necessary to allow the medi-
um to cool to 50-60°C before their addition, so as not to
harm the additive while permitting its correct distribution.
Once the addition is over, reheating must be avoided.
Distribution into final containers must be completed
before sterilizing, to avoid any manipulation. Since it is
rarely possible to do so, manipulation should be done
in safety cabinets instead. The sterile cabinets or rooms
are not be exposed to any radiations and so, no any
kind of highly active reagent could affect the medium’s
components. Excessive steam condensation can be
eluded by distributing the medium at temperatures close
to solidification, ranging from 45 to 50°C.
Storage of dehydrated media
Dehydrated culture media should be stored safely, pro-
tected from humidity or moisture, light and heat, which
are the most frequent causes of their alteration.
SCHARLAU media are supplied in opaque, waterproof,
screw-cap plastic bottles, with an internal relief which
eliminates the need for an intermediate seal. Never-
theless a hermetic seal is assured by accurately fitting
the cap and keeping both its sides and the border of its
mouth clean.
It is important to open the bottles in dry atmosphere
and close them immediately after their use. Refrigera-
tion is not necessary even if low temperatures prolong
the medium’s effectivity. The nature of the containers
of SCHARLAU make culture media are suitable for
prolonged storage in cool, dry places. If the media are
moist they become stiff and hard, lose their properties
XVI
and can even allow direct growth of microorganisms in
them. Under such circumstances they must not be used.
Although they usually have a dry and powdery appear-
ance, the media may occasionally appear moist, oily but
never stiffened, without there being any alteration in their
composition (as e.g.in MRS, Tributyrin Agar, etc).
In any case, storage of dehydrated media is not indefi-
nite. It is not recommended to store large quantities
but to keep small amounts in stock, so as to maintain a
rotation of ready prepared dehydrated media. Register-
ing the reception dates on the containers will avoid the
accumulation of older media.
Storage of ready
prepared culture media
Although it is best to prepare the media as you required,
it is common to store them ready prepared and sterilized
in order to spare preparation time.
Under these circumstances the biggest drawback is the
dehydration and every precaution taken to avoid it will
prolong the medium’s useful life, which is usually be-
tween 4 and 6 weeks. We therefore recommend the use
of hermetically sealed screw-capped containers (either
bottles or tubes) instead of those plugged with cotton
wool. A moderate refrigeration (4°C) usually prolongs
the life of the medium but it should also be noted that a
refrigerated environment is very dry and consequently
renders dehydration. In some cases, as regards in
thioglycolate media and almost all media recommended
for anaerobes, they are best stored at room temperature
for the less penetration of air. Direct light must always
be avoided, particulary when it is specifically indicated
on the label of the medium.
Solid media for the plates are best stored in their original
containers than in the poured plates. Nevertheless,
poured plates may also be prepared and stored if the
indications below are followed:
a) Refrigerate the media immediately after solidification
while incubating «controls» to check the sterility of
the batch.
b) If the media are to be stored for a longer period,
plates should be individually sealed with a waterproof
seal and, if possible, stored in single containers.
c) If storage is to last more than a few days, the plates
should be wrapped in plastic bags to avoid dehydra-
tion of the media.
Since condensation water will inevitably appear, we sug-
gest to store the poured plates inverted. Contamination
with condensation water can be avoided by pouring the
molten medium on the plates as cold as possible.
Media which have been stored in refrigeration should
be allowed to warm up gradually. It is recommended to
keep them at room temperature for a few hours before
inoculation, allowing misted containers to clear and
avoiding the delayed initiation of growth (or eventually its
total failure).
 Guidelines for correct manipulation in Microbiology
Stored ready prepared media which may involve a high
level of dehydration should never be used. Dehydra-
tion is indicated by excess condensation of water or
by a decrease in volume of liquid media; an increase of
concentration or precipitation of fluid and liquid media;
or retraction, cracking and precipitation of solid media.
Plates with very dry surfaces or those which appear to
have changed colour should not be used either.
Remelting solid media
Although most solid culture media can be stored ready
prepared and sterilized, and can be remelted and poured
into plates whenever necessary, this procedure should
not be applied to media which do not need sterilization
or those with a pH 5 or less, since it would alter their
properties.
Remelting is done in a boiling water bath or by fluent
steam autoclaving for 30 minutes. Do not apply direct
heat. However, it must be emphasized that remelted
media are prone to precipitation and darkening if held in
a molten state for more than 1 hour and at temperatures
ranging from 45 to 65°C. Such media may also undergo
nutritional impairment to the desired microorganisms.
It is therefore recommended to avoid remelting of solid
media whenever possible.
One of the quickest and most recommended remelt-
ing methods for culture media is the use of microwave
ovens. In this case all the precautions exposed must be
carefully observed and practised in the media which are
labile and consequently radiation and minimum intensity
should be dosed for the shortest time intervals.
A usual and erroneous practice consists of imposing
strong radiation intensities for short periods, which re-
sults in partial remelting, sudden boiling and overflowing
of the medium and an overall alteration of its properties.
Differentiation of culture media
SCHARLAU names for culture media are based on their
final consistency or appearance. Thus, a medium is
named as:
Agar: Solid media with an agar content of 1% or more.
Fluid Medium (FM): Semisolid media with an agar con-
tent of less than 1%.
Broth: Liquid culture media with undefined organic com-
ponents (peptones, organ and tissue extracts,
etc...)
Nutrient Solution: Culture media with defined chemical
composition.
Selective Medium: Medium that allows the growth of a
biotype or a few biotypes between those that are
present in the inocule.
Differential Medium: Medium that allows to distinguish
or differentiate between similar culture types.
Enrichment Medium: Medium that aids the growth of a
defined biotype over the rest in the inocule.
General Medium: Medium that supports the growth of
a very wide range of microorganisms without
special nutrient needs.
Membrane Filter (MF): Media used when the Mem-
brane Filter Technique is employed.
Membrane Filter Media
Utilization of the Membrane filter technquies has offered
some undoubted advantages to the Microbiology which
has certain specific problems. Membrane filtration allows
to examine big samples (in volume of liquid) with very
low microorganisms concentration or load, to separate
the microorganisms from the culture medium and even
the exchange of the microorganisms between two media
without affecting their growth.
Moreover, the MPN technique is more precise in these
media and also, the membrane with the colonial growth
may be stored and filed.
The membrane filtration system is accepted by most
pharmacopeias as an alternative or the single method to
examine antimicrobial or with strong inhibitors sub-
stances, since a proper wash of the filter remove all the
substances that interfere with the microbial growth.
On other hand, the wide range of filters and qualities
available allows the examination of any product, since
you may find membranes resistant to almost all the dis-
solvents.
Technique
Essentially, the technique consists of filtering the sample
through a filter with a suitable porosity (0,22 microns
for bacteria and 0,45 microns for fungi) with pressure
or suction, in such a way that the microorganisms are
retained on the membrane.
If the fluid that has been filtered contains inhibitors, wash
the membrane several times with rinse liquid to remove
them. Membrane is removed aseptically and it is taken
and placed on the culture medium.
For sterility controls, the membrane is incubated directly
in the classical media. For these tests refer: Thioglyco-
late Broth (Ref. 2-186), Thioglicolate USP Fluid Medium
(Ref. 3-187), Tryptone Soy Broth (Ref. 2-200), Sabour-
aud USP Broth (Ref. 2-165).
Should an enumeration of colonies be desired, incubate
the filter on a solid medium or on a pad soaked in liquid
medium, but beware that the lower surface touches the
culture medium and that there are no air bubbles or air
gap in between.
Usually, general media may be used for these tech-
niques. However, there are several culture media spe-
cifically developed for these techniques, specially in the
water Microbiology.
XVII
Dehydrated Culture Media
 Culture media manufacture process flow chart

2
 Acetamide Medium
Ref. 03-428
Specification
Liquid medium for the enrichment and confirmative test
of presence of Pseudomonas aeruginosa in water acc.
EN 12780:2002 and ISO 16266 standard.
Formula (in g/L)
Acetamide ............................................ 2,0000
Magnesium sulfate, anhydrous ............ 0,2000
Sodium chloride .................................... 0,2000
Ferrous sulfate ..................................... 0,0005
Monopotassium phosphate .................. 1,0000
Sodium molybdate ................................ 0,0050
Final pH 7,0 ± 0,5
Directions
Dissolve 3,4 g of powder in 1 L of distilled water. Heat
only if necessary. Sterilize in the autoclave at 121°C for
15 minutes. Prepared medium may be opalescent and
with precipitate. The prepared medium remain active for
3 months if it is stored in the dark in a cool place.
Description
This nutrient solution has acetamide as the unique
carbon and nitrogen source, and it allows the growth of
only those microorganisms that are able to use aceta-
mide. In water and in almost all the food materials, these
microorganisms are the non fermenting gramnegatives,
thus Pseudomonas aeruginosa is the only one that can
liberate ammonia by deaminating acetamide.
Ref. 03-428 Acetamide Medium. Left: control; center:
Pseudomonas aeruginosa ATCC 27853; right: Pseu-
domonas aeruginosa ATCC 9027.
Xn Some authors suggest the use of this nutrient solution as
a previous enrichment medium before the isolation me-
R-40
S-36/37
dium, especially if you are studing very polluted samples
with companion flora.
Technique
Medium is inoculated with a couple of loops from the
culture or inoculum to be assayed and is incubated at
32-35°C for 24-48 hours before proceeding for the isola-
tion medium.
To confirm Pseudomonas aeruginosa, inoculate a loop
of culture inoculum in Asparagine Broth (Ref. 02-271)
and incubate at 35-37°C for 24 hours. After this period,
pour 1 or 2 drops of Nessler’s Reagent (Ref. 06-084) on
to the culture and observe for ammonia production: a
change in colour to yellow indicates ammonia production
and thus the presence of Pseudomonas aeruginosa .
References
EN 12780 Standard (2002) Water Quality-Detection and
enumeration of Ps.aeruginosa by membrane filtration.
CEN. Brussels.
DIN Standard 3841. Deutsche Einheitsverfahren zür
Wasser, Abwasser und Schlammuntersuchung Mikro-
biologische Verfahren: Nachweiss von Pseudomonas
aeruginosa (K8).
KELLY, N.M., C.T. KEANZ (1983) Acetamide Broth for
Isolation of Pseudomonas aeruginosa from patients with
cystic fibrosis. J. Clin. Microbiol. 17:159-163.
ISO 16266 (2006). Water Quality. Detection and enu-
meration of Pseudomonas aeruginosa. Method by
membrane filtration.
3
 Actinomycete Media
Actinomycete Agar
Ref. 01-003
Specification
Solid culture medium for actinomycete maintenance and
propagation according to Ajello et al. formulation.
Formula (in g/L)
Meat peptone ......................................... 10,00
Casein peptone ........................................ 4,00
Heart extract ........................................... 10,00
Yeast extract ............................................. 5,00
Dextrose ................................................... 5,00
Potassium phosphate ............................. 15,00
Sodium chloride ........................................ 5,00
Starch ....................................................... 1,00
Ammonium sulfate .................................... 1,00
Cysteine ................................................... 1,00
Magnesium sulfate ................................... 0,20
Calcium chloride ....................................... 0,01
Agar ........................................................ 20,00
Final pH 7,0 ± 0,2
Directions
Suspend 77 g of powder into 1 L distilled water and let it
soak. Bring to boiling with constant stirring and distribute
into suitable containers. Sterilize by autoclaving at 121°C
for 15 minutes.
Actinomycete Broth
Ref. 02-003
Specification
Liquid culture medium, able to form a fluid medium, for
the pathogenic actinomycete maintenance and propaga-
tion, according to Ajello et al. formulation.
Formula (in g/L)
Meat peptone ......................................... 10,00
Casein peptone ........................................ 4,00
Heart extract ........................................... 10,00
Yeast extract ............................................. 5,00
Dextrose ................................................... 5,00
Potassium phosphate ............................. 15,00
Sodium chloride ........................................ 5,00
Starch ....................................................... 1,00
Ammonium sulfate .................................... 1,00
Cysteine ................................................... 1,00
Magnesium sulfate ................................... 0,20
Calcium chloride ....................................... 0,01
Final pH 7,0 ± 0,2
4
Directions
Dissolve 57 g of powder into1 L of distilled water. Heat if
necessary. Distribute into suitable containers and steri-
lize by autoclaving at 121°C for 15 minutes.
Should a fluid medium be required, incorporate 7 g of
Agar and follow the directions for the solid medium. After
the sterilization, cool it quickly but gently to avoid agar
flocculation.
Description
Basically Pine and Watson formulated this medium for
the maintenance of pathogenic actinomycytes which was
then further modified by Ajello et al. Those media do not
possess any inhibitory agents, and so are not recom-
mended for the isolation, but their slight reducing power
and their high nutritive capacity make them suitable for
facultative anaerobes also.
Technique
If growth conditions require strict anaerobiosis, the incu-
bation must be done in special flasks, but if the condi-
tions are only moderate or not strict anaerobic, a good
anaerobiosis can be obtained by sealing the flasks with
Sealing Anaerobic Agar (Ref. 01-174) or with the classic
alkaline pyrogallate caps.
Best results are obtained using an Anaerobic Jar with a
CO2 mixture generator.
The use of a liquid medium allows freezing of the cul-
tures after their growth and the use also as a massive
production system for cellular preparations. In the fluid
format, the Actinomycete Broth reaches a high reduction
potential allowing an easy growth to strict anaerobics.
There is only one difference between solid and broth
formulation which is the addition of agar 20,0 g/L to the
solid medium as a solidifying agent, providing the suf-
ficient strength to support the surface growth.
References
AJELLO, GEORG, KAPLAN & KAUFFMAN (1963) CDC
Lab Manual for Medical Mycology, Publication nº994 US
Govt. Prmtmg. Office. Washington.
LENNETTE, SPAULDING & TROULANT (1974) Manual
of Clinical Microbiology. ASM 2d. Ed. Washington.
PINE, L., A.HOWELL, S.J.WATSON (1960) Studies on
the morphological characters of Actynomyces bovis. J.
Gen. Microbiol. 23:403-424.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press. London.
 AFP Agar (Aspergillus flavus/parasiticus Agar)

Ref. 01-405 T Technique

Specification
Selective solid medium for the isolation and enumeration
of Aspergillus flavus and Aspergillus parasiticus.
Formula (in g/L)
Peptone ................................................ 10,000
Yeast extract ......................................... 20,000
Ammoniun Iron Citrate .......................... 0,500
Dichloran ................................................ 0,002
Chloramphenicol .................................... 0,100
Agar ...................................................... 15,000
Final pH 6,3 ± 0,2
R-45
The sample is inoculate on the surface of the AFP Agar
S-45-53 ad the plates are incubated at 30ºC for 42-48 hours.
Count the fungal colonies that shows the typical yellow-
orange pigmentation on the reverse. Express the results
as “Aspergillus flavus/parasiticus colonies per g or mL of
sample”.
In this conditions Aspergillus orizae can produce a very
similar pigmentation than induce error in the counting.
Also, Aspergillus niger can be present but its pigment is
pale yellow and never turns to orange and after 48 hours
of incubation begin to produce the typical black conidi-
ophora.

Directions
Suspend 45,6 g of powder in 1 L of distilled water and
bring to the boil. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes.
Description
AFP Agar is a modification of the original medium devel-
oped by King, Hocking & Pitt for the enumeration and
isolation of moulds in foods. The inclusion of ammonium
iron citrate and the high temperature (30ºC) of incubation
enhances the quickly typical pigmentation in Aspergillus
flavus and A. parasiticus.
References
KING D.A. A.D. HOCKING & J.I. PITT (1979) Dichloran-
rose bengal médium fot enumeration and isolation of
moulds from foods. Appl. Environm. Microbiol 37:959-
964
VANDERZANT, C. & D.F. SPLITTSTOESSER (1992)
Compendium of methods for the microbiological exami-
nation of foods. APHA. Washington.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press. London

The elimination of glucose from the formula, with the

low value of pH and the presence of chloramphenicol
inhibits the bacterial growth and the dichloran repress
the spreading of the fungal colonies.

Ref. 01-405 AFP Agar. Left: control; right: Aspergillus ssp.

5
 Algae Media
Algae Agar
Ref. 01-007
Specification
Solid culture medium for the isolation and cultivation of
algae from soil, water and waste water.
Formula (in g/L)
Sodium nitrate ........................................ 1,000
Dipotassium phosphate .......................... 0,250
Magnesium sulfate ................................. 0,513
Ammonium chloride ................................ 0,050
Calcium chloride ..................................... 0,058
Ferrous chloride ..................................... 0,003
Agar ...................................................... 15,000
Final pH 7,0 ± 0,2
Directions
Suspend 17 g of powder into 1 L of distilled water and let
it soak . Bring to boiling with constant stirring. Distribute
into appropriate containers and sterilize in the autoclave
at 121°C for 15 minutes.
Description
The balanced nutrient medium composition provide all
necessary nutrients for the good growth of the algae
, but it does not support the good growth of fungi and
bacteria which can not grow well or have difficulties in
the medium.
It is also suitable for algicide testing, however it is essen-
tially recommended for the algae-pattern maintenance
and cultivation or for the isolation of water contaminants.
Technique
For the maintenance of algal strains it is recommended
to incubate at room temperature, under a suitable
light source (natural, fluorescent tube or incandescent
lamp) until a good growth is obtained (within one to two
weeks).
In these conditions, and without gel dehydration, cultures
can be maintained up to two months.
Algae Broth
Ref. 02-007
Specification
Nutritive solution for algae and cyanobacteria, suitable
for water algicide biotesting.
6
Formula (in g/L)
Sodium nitrate ........................................ 1,000
Dipotassium phosphate .......................... 0,250
Magnesium sulfate ................................. 0,513
Ammonium chloride ................................ 0,050
Calcium chloride ..................................... 0,058
Ferrous chloride ..................................... 0,003
Final pH 7,0 ± 0,2
Directions
Dissolve 1,87 g of powder in 1 L of distilled water and
distribute into suitable containers. Sterilize in the auto-
clave at 121°C for 15 minutes.
Description
This liquid medium is suitable for algae and cyanobac-
teria cultivation, and it is especially adapted for inocule
preparation and algicide biotesting, as per Fitzgerald’s
technique.
Due to the less content of energy source, fungi and
bacteria do not grow or grow badly unlike algae, which
obtain all the necessary nutrients, except the energy
source.
Technique
Fitzgerald’s procedure for the algicide ability verification
of chemical products is:
a) Inoculum preparation
Prepare the Algae Broth and distribute 20 mL. each in
the 50 mL capacity conical flasks. Sterilize and keep
them cool until usage.
Regularly, one of the conical flasks is inoculated with a
couple of loops from Chlorella emersonii culture from
slanted Agar (Ref 1-007) and it is incubated at room
temperature until good growth is observed.
This culture can be used as biotest inoculum, but for
only up to 30 days.
b) Biotest
1- Samples
Prepare one litre of pure distilled water and 1 litre of
distilled water containing the inhibitor. Add 120 mg of
Sodium nitrate and 2,5 g of Di-potassium phosphate to
each sample.
2- Technique of the Test
Prepare a double series of 50 mL capacity conical flasks
and add 5, 12.5 and 25 mL respectively of water algicidal
mixture, and then refill with pure water to get 25 mL in
each conical flask.
Add only 25 mL of pure water in one or two conical
flasks to use them for control purposes.
All the conical flasks are inoculated with the same vol-
ume of inoculum, the necessary amount to get an algae
concentration about 300.000 cells/mL in each flask. As
a practice (not exactly) this concentration produces a
slight greenish tinge. If necessary, adjust the inoculum
by counting or photocolorimetry.
 Algae Media

Incubate the inoculated flasks at room temperature

under an homogeneous and standardized light (i.e. 20 W
fluorescent light).
Countings of all the flasks is carried out daily with a glob-
ule-cell count (Thoma, Neubauer type or similar).
The test is said to be over when the control conical
flasks have an average concentration greater than 5x106
cells/mL, comparing the other flasks to them.
3-Interpretation
Inhibitor concentration in the flasks with equal growth
to the control is considered non toxic or ineffective. If
algal concentration is maintained or remains same as
at the starting of experiment, it is considered algistatic.
Concentrations that have reduced starting population are
considered as algicidic, with the different effectivity ratio
depending on the amount of loss.

References
CLESCERI, L., A.E. GREENBERG, A.D. EATON (1998)
Standard Methods for Examination of Water and Waste-
water. APHA-AWWA-WEF. Washington, D.C. Ref. 01-007 Algae Agar. Chlorella sp.
ALLEN, (1952) Arch. Microbiol. 17:34
FITZGERALD (1962) Water and Sewage Works.
109:361.

Antibiotic Media

While performing the antibiotic assays, the only meth-
odology accepted universally is the microbiological
methodology. National pharmacopeia provides different
directives to adapt to new compounds. Nowadays, the
European Pharmacopeia (1996), following a path to
standardize all the countries in the EU, provides several
rules and recommendations but it does not impose strict
criteria, since they could interfere with the local legisla-
tion. Instead of this, the USP 25/NF 20 (2002) incorpo-
rates more concrete guidelines about the antibiotics that
are accepted for human consumption and their assay
methodology. However, many times the USP refers
to the US/FDA, which is the more detailed publication
about the antibiotics assay.
Kirshbaun and Arret, in 1959, published a report with all
the variations of this methodology. The success of their
first attempt, and the discovery of new antibiotics made
the same authors revise their work and , in 1967, they
published a new report detailing 57 official methods, and
in 1971 last edition they detailed 83 official methods and
10 non official methods. The XIX edition of the USP con-
sider this last 1971 edition as its information source.
The present article simply pretends to resume the most
important characteristics of this methodology, following
the guidelines of the current pharmacopeia. There are
no detailed directions to perform the assay because it
is considered that this is going to be read by microbiol-
ogists who are familiar with the basic techniques.If you
are interested in this subject, we would ask you to read
the bibliography mentioned at the end of this publication,
especially 21CFR for the details about the preparation of
samples and the extraction of antibiotics for each phar-
maceutical preparation.
The reference substances used in the assays are sub-
stances whose activity has been precisely determined
with references to the corresponding international stand-
ard or international reference preparation.
Microorganisms
Strains used in the antibiotics assay as well as the
preparation methods and inocula are resumed in the
Table I. Strains may be obtained from the following and
other collections:
ATCC: American Type Culture Collection, 12301 Park-
lawn Drive, Rockville, Maryland 20852-176. USA.
Fax +1 301-231-5826
CIP/CNCM: Collection de l’Institut Pasteur / Collection
Nationale de Cultures de Microorganismes,
Institut Pasteur, 25 rue du Docteur Roux, 75724
PARIS CEDEX 15. France. Fax +33 143 06 98
35
CECT: Colección Española de Cultivos Tipo, Dpto Micro-
biologia, Fac. Ciencias Biológicas, Dr. Moliner
50, 46100 Burjasot (Valencia) Spain. Fax +34
63 86 43 72

7
 Antibiotic Media

NCTC:National Collection of Type Cultures, PHLS Method #2

Central Public Health Laboratory, 61 Colindale
Avenue, LONDON NW9 5HT England.
NCIB: The National Collections of Industrial and Marine
Bacteria Ltd., 23 St Machar Drove, ABERDEEN
AB2 1RY, Scotland. U.K. Fax +4402 24 487
658
NCYC: National Collection of Yeast Cultures. Institute
of Food Research. Norwich Laboratory. Colney
Lane. NORWICH NR4 7UA. U.K.
Preparation methods
Method #1
Strains to be assayed are mantained in slanted tubes
of Antibiotic Medium 1 (Ref. 01-009), subculturing
to fresh tubes every week. The growth on a slanted
tube, incubated at 32-37° for 24 hours, is washed
with 3 mL of Ringer Solution (Ref. 06-073), and the
resulting suspension is used to inoculate a Roux
flask containing 250 mL of sterile and controlled
Antibiotic Medium 1 (Ref. 01-009).
For the maintenance of antibiotic resistent strains
prepare Antibiotic Medium 1 (Ref. 01-009) with the
addition of proper amounts of antibiotic, as described
in the Table II. The growth obtained in the Roux flask
after a 24 hours incubation at 32-37°C is washed and
collected with 50 mL of Ringer Solution (Ref. 06-073)
and it is stored in refrigeration (4-6°C)
Process is the same as the Method #1 but the
standardization is performed in the following way:
Centrifuge and collect the pellet. Sediment is sus-
pended with 50-70 mL of Ringer 1/4 Solution (Ref.
06-073) and heat up to 70°C for 30 minutes. Refrig-
erate the spore suspension obtained in this way.
Method #3
Proceed as per Method #2 performing the thermal
shock before the centrifugation. Wash the spore
suspension 3 consecutive times with distilled water
(25-50 mL each time) and centrifuge. Reconstitute
the final suspension with 50-70 mL of sterile distilled
water and store it in refrigeration.
Method #4
Let the fungi grow for 6-8 weeks at room temperature
(20-25°C) in several 3 L Erlenmeyer flasks, contain-
ing each one about 200 mL of Antibiotic Medium 22.
After six weeks, test the sporulation regularly and,
when it reaches 80%, collect the spores of the aerial
micelium with a sterile spatula or any other suitable
instrument. The collected spores are suspended in
20 mL of Ringer 1/4 Solution (Ref. 06-073) and store
refrigerated.

Table I. Strains (Test organisms) for the Antibiotic Assays

# Strain Genus & species Strain and Type culture collection # Method
American Collection European Collections

A Staphylococcus aureus ATCC 6538P CIP 53.156 NCTC 7447 1

B Staphylococcus aureus ATCC 29737 1
C Staphylococcus aureus ATCC 9144 CIP 53.134 NCIB 6751 1
D Staphylococcus epidermidis ATCC 12228 CIP 68.21 NCIB 8853 1
E Micrococcus luteus ATCC 9341 CIP 53.45 NCTC 8340 1
F Micrococcus luteus (flavus) ATCC 10240 CIP 53.160 NCTC 7743 1
G Enterococcus hirae ATCC 10541 CIP 58.55 NCIB 8192 5
H Bacillus pumilus CIP 76.18 NCTC 8241 2
I Bacillus cereus ATCC 11778 CECT 193 NCTC 10320 3
J Bacillus subtilis ATCC 6633 CIP 52.62 NCTC 10400 2
K Bacillus subtilis CIP 1.83 NCTC 8236 2
L Klebsiella pneumoniae ATCC 10031 CIP 53.153 NCTC 7427 1
M Escherichia coli ATCC 10536 CIP 54.127 NCIB 8879 1
N Escherichia coli ATCC 9637 CIP 2.83 NCIB 8666 1
O Bordetella bronchiseptica ATCC 4617 CIP 53.157 NCTC 8344 1
P Saccharomyces cerevisiae ATCC 9763 CIP 1432.83 NCYC 87 61
Q Saccharomyces cerevisiae ATCC 2601 CECT 1324 NCYC 853 6
R Pseudomonas aeruginosa ATCC 25619 NCIB 10817 1
S Mycobacterium smegmatis ATCC 607 NCTC 7017 7
T Candida tropicalis CIP 1433.83 NCYC 1393 6
1
For candicidine, use method #1

8
 Antibiotic Media
Method #5
With the help of a loop inoculate a flask containing
100 mL of sterile and controlled Antibiotic Medium 3
(Ref. 02-011). from a slant culture. Incubate for 16-18
hours.
Method #6
Follow the directions stated in the Method #1 but in-
cubate the slanted subcultures at 30°C for 24 hours,
and the Roux flasks at 30°C for 48 hours.
Method #7
Organisms are maintained on Antibiotic Medium 36
in slanted tubes, subculturing them each week with
an incubation at 37°C for 48 hours.
Collect the growth of a recent subculture with 3 mL
of Ringer 1/4 Solution (Ref. 06-073) and use it to
inoculate a 500 mL Erlenmeyer flask containing 100
mL of Antibiotic Medium 34. Add asseptically to the
flask 50 g of sterile glass balls and incubate at 27°C
for 5 days with constant stirring (120 rpm and 3,5 cm
radius). Keep the suspension in refrigeration.
Inoculum Determination
For many years the official US/FDA methods provided
specific instructions for the inoculum standardization. Af-
ter 1985 the USA Federal Regulations and the USP XIX
adopted the criterion suggested by Kirshbaum, Kramer
and Gorth that recommends to dilute the microorgan-
isms suspension until it provides a 25% transmitance at
580 nm in a 13 mm tube. However, even with this stand-
Table II. Buffer solutions and diluents
# Buffer1
1 Phosphate buffer 1% at pH 6,0±0,05
3 Phosphate buffer 0,1M at pH 7,9±0,1
4 Phosphate buffer 0,1M at pH 4,5±0,05
6 Phosphate buffer 10% at pH 6,0±0,05
10 Phosphate buffer 0,2M at pH 10,5±0,1
16 Phosphate buffer 0,1N at pH 7,0
17 Phosphate-methanol buffer at pH 6
18 Sodium carbonate solution 1%
1
May require adjustment with phosphoric acid 18 N or KOH 10N before or after sterilization.
ardization it is necessary to test every batch of inoculum
to determine the exact volume that must be added to
each assay medium.
If the inoculum suspensions are prepared as described
in the above sections, they will be homogeneous enough
to determine the volumes that are going to be added,
with tests, without any other previous standardization
step.
Diffusion agar Assay
Prepare a series of flasks with 100 mL of sterilized,
melted and cooled Seed Layer Medium (Ref. 01-009)
and add to each flask a different volume of micro-
organisms suspension, as per the volume pattern
suggested in Table IV.
Prepare Petri plates with the inoculated medium
according to the specificacions of each antibiotic and
put a steel or glass cylinder or a disc with the refer-
ence concentration of antibiotic. Incubate the plates
and measure the inhibition zones. The volume of
suspension that produces optimum zones, in defini-
tion as well as in diameter, is the one that is adopted
for the inoculum. In Table IV the optimum inhibition
zones are shown that are supposed to appear in the
different assays.
Turbidimetric Assay
Proceed in the same way as in the previous case but
instead of inoculating over the melted medium, inoc-
ulate in the flasks containing 100 mL of the suitable
liquid medium, using the volumes pattern specified in
Formula (in g/L of distilled water)
K2HPO4 ................................. 2,000 g
KH2PO4 ................................. 8,000 g
K2HPO4 ............................... 16,730 g
KH2PO4 ................................. 0,523 g
KH2PO4 ............................... 13,600 g
K2HPO4 ............................... 20,000 g
KH2PO4 ............................... 80,000 g
K2HPO4 ............................... 35,000 g
KOH 10N ............................... 2,000 mL
K2HPO4 ............................... 13,600 g
KH2PO4 ................................. 4,000 g
Methanol .............................. 50,000 mL
Buffer #1 ........................................ q.s.
Sodium carbonate ............... 10,000 g
9
 Antibiotic Media

Table III . With these volumes of inoculated medium
continue as if you were performing an assay, using
just the highest and lowest concentrations in relation
to the pattern. Incubate for 3-4 hours and read the
absorbances. With all these data you will be able to
establish the right inoculum that provides a better
answer between the high and low point, and so adopt
it for the assay.
Strain maintenance
Assay strains are maintained by vegetative propaga-
tion in slants and in duplicate. One of the duplicates is
used only for the next subculture, and the other one is
used for all the other operations. Medium, if there is no
other directive, is Antibiotic Medium 1 (Ref. 01-009) with
antibiotics or other additive if necessary. Details may be
obtained in Table II. Nonetheless, other media may be
used if considered necessary.
Buffer Solutions and diluents
The components of the culture media accomplishes the
specifications of the several pharmacopeia, however the
technician is responsible for the end use of dehydrated
culture media or any other variation in it, as long as they
still have the same characteristics described for the
above media. pH of the medium must be checked when
it is completely reconstituted and at 25°C.
Antibiotic Medium 1
(Eur. Phar. Antibiotic Medium A
at pH 6,6)
Ref. 01-009
Specification
Antibiotic Medium 1 or Seed Layer is used for the anti-
biotic assays by the diffusion method in agar, that may
be performed in several ways (cylinder, punched-hole or
paper disc method).

Composition of the most common buffer solutions and
diluents is given in Table II. Dilutions of pure chemical
products as well as organic diluents have been om-
mited. In anycase, the compounds must be according
to the purity standards of the corresponding pharmaco-
peia. Water must always be distilled and be of reagent
grade. All buffer solutions and diluents have to be sterile.
Sometimes a little pH readjustment is necessary after
the sterilization.
Culture Media
The composition of the several SCHARLAU Microbiol-
ogy Antibiotic Media is given below. Media still have the
nomenclature according to the US/FDA, which uses the
one described by Grove and Randall (media 1 to 13)
and Kirshbaum and Arret (media 18 to 21). The Euro-
pean Pharmacopeia names the media with alphabetical
letters, and the corresponding Scharlau media are as
follows:
Formula (in g/L)
Peptone ..................................................... 6,0
Casein Peptone .......................................... 4,0
Yeast extract ............................................... 3,0
Beef extract ................................................ 1,5
Dextrose ..................................................... 1,0
Agar .......................................................... 15,0
Final pH 6,6 ± 0,2
Directions
Add 30,5 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Steri-
lize in the autoclave at 121°C for 15 minutes.

Medium A..............Antibiotic Medium 1 and 11

Medium B .......................Antibiotic Medium 10
Medium C ........................ Antibiotic Medium C
Medium D ........................ Antibiotic Medium D
Medium E ........................ Antibiotic Medium E
Medium F........................Antibiotic Medium 19
Medium G .......................Antibiotic Medium 35

Note: Medium D is very similar to Antibiotic Medium 3

but with the addition of 2 g/L of potassium nitrate
in it.

SCHARLAU media described following are prepared

with the ingredients specified in the USP 23/NF 18, Eur.
Pharm. 3 and 21 CFR for these components, providing
an absolute reproducubility and reliability.

10
 Antibiotic Media
Antibiotic Medium 2
Ref. 01-010
Specification
Antibiotic Medium 2 or Basal Layer is used for the anti-
biotic assays by the diffusion method in agar, that may
be performed in several ways (cylinder, punched-hole or
paper disc method).
Formula (in g/L)
Peptone ..................................................... 6,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Agar .......................................................... 15,0
Final pH 6,5 ± 0,2
Directions
Add 25,5 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Steri-
lize in the autoclave at 121°C for 15 minutes.
Antibiotic Medium 3
Ref. 02-011
Specification
Antibiotic medium 3 or Antibiotic Assay Broth may be
used in the inoculum preparation, serial dilutions or turbi-
dimetric antibiotic assays.
Formula (in g/L)
Peptone ................................................... 5,00
Yeast extract ............................................. 1,50
Meat extract .............................................. 1,50
Sodium chloride ........................................ 3,50
Dextrose ................................................... 1,00
Monopotassium phosphate ...................... 1,32
Dipotassium phosphate ............................ 3,68
Final pH 7 ± 0,05
Directions
Add 17,5 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Steri-
lize in the autoclave at 121°C for 15 minutes.
Antibiotic Medium 4
Ref. 01-012
Specification
Antibiotic Medium 4 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method)
Formula (in g/L)
Peptone ..................................................... 6,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Dextrose ..................................................... 1,0
Agar .......................................................... 15,0
Final pH 6,5 ± 0,1
Directions
Add 26,5 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Steri-
lize in the autoclave at 121°C for 15 minutes.
Antibiotic Medium 5
Ref. 01-013
Specification
Antibiotic Medium 5 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method).
Formula (in g/L)
Peptone ..................................................... 6,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Agar .......................................................... 15,0
Final pH 7,9 ± 0,1
Directions
Add 25,5 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Steri-
lize in the autoclave at 121°C for 15 minutes.
Antibiotic Medium 8
Ref. 01-014
Specification
Antibiotic Medium 8 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method).
Formula (in g/L)
Peptone ..................................................... 6,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Agar .......................................................... 15,0
Final pH 5,9 ± 0,1
Directions
Add 25,5 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Steri-
lize in the autoclave at 121°C for 15 minutes.
11
 Antibiotic Media

Antibiotic Medium 9 Antibiotic Medium 11

Ref. 01-015
Specification
Antibiotic Medium 9 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method).
Formula (in g/L)
Casein peptone ........................................ 17,0
Soy peptone ............................................... 3,0
Sodium chloride .......................................... 5,0
Dipotassium phosphate .............................. 2,5
Dextrose ..................................................... 2,5
Agar .......................................................... 20,0
Final pH 7,2 ± 0,2
Directions
Add 50 g of medium to 1 L of distilled water. Heat to the
boiling and dispense into suitable containers. Sterilize in
the autoclave at 121°C for 15 minutes.
Antibiotic Medium 10
(Eur. Phar. Antibiotic Medium B)
Ref. 01-016
Specification
Antibiotic Medium 10 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method).
Formula (in g/L)
Casein peptone ........................................ 17,0
Soy peptone ............................................... 3,0
Sodium chloride ......................................... 5,0
Dipotassium phosphate .............................. 2,5
Dextrose ..................................................... 2,5
Agar .......................................................... 12,0
Final pH 7,2 ± 0,2
(Eur. Phar. Antibiotic Medium A
at pH 8,0)
Ref. 01-017
Specification
Antibiotic Medium 11 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method)
Formula (in g/L)
Peptone ..................................................... 6,0
Casein peptone .......................................... 4,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Dextrose ..................................................... 1,0
Agar .......................................................... 15,0
Final pH 8,0± 0,2
Directions
Add 30,5 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Steri-
lize in the autoclave at 121°C for 15 minutes.
Antibiotic Medium 13
Ref. 02-544
Specification
Antibiotic Medium 13 is used in the turbidimetric antibi-
otic assays.
Formula (in g/L)
Peptone .................................................... 10,0
Dextrose ................................................... 20,0
Final pH 5,5 ± 0,2
Directions
Add 30 g of medium to 1 L of distilled water. Heat to the
boiling and dispense into suitable containers. Sterilize in
the autoclave at 121°C for 15 minutes.

Directions
Add 42 g of medium to 1 L of distilled water. After disso-
Antibiotic Medium 19
lution, add 10 mL of Polysorbate 80. Heat to the boiling (Eur. Phar. Antibiotic Medium F)
and dispense into suitable containers. Sterilize in the
autoclave at 121°C for 15 minutes. Ref. 01-434

Specification
Antibiotic Medium 19 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method)

12
 Antibiotic Media
Formula (in g/L)
Peptone ..................................................... 9,4
Yeast extract ............................................... 4,7
Beef extract ................................................ 2,4
Sodium chloride ........................................ 10,0
Dextrose ................................................... 10,0
Agar .......................................................... 23,5
Final pH 6,0 ± 0,1
Directions
Add 60 g of medium to 1 L of distilled water. Heat to the
boiling and dispense into suitable containers. Sterilize in
the autoclave at 121°C for 15 minutes.
Antibiotic Medium 32
Ref. 01-069
This medium is sold by Scharlay Microbiology under the
name Sporulating AK Agar.
Specification
Antibiotic Medium 32 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method).
Formula (in g/L)
Peptone ................................................. 6,000
Casein peptone ...................................... 4,000
Yeast extract ........................................... 3,000
Meat extract ............................................ 1,500
Dextrose ................................................. 1,000
MnSO4.H20 ............................................. 0,003
Agar ...................................................... 15,000
Final pH 6,5 ± 0,2
Directions
Add 30,503 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Steri-
lize in the autoclave at 121°C for 15 minutes.
Antibiotic Medium 34 Base
Ref. 02-545
Specification
Antibiotic Medium 34 is used in the turbidimetric antibi-
otic assays.
Formula (in g/L)
Peptone .................................................... 10,0
Meat extract .............................................. 10,0
Sodium chloride .......................................... 3,0
Final pH 7,0 ± 0,1
Directions
Add 33 g of medium to 1 L of distilled water.Add 10 mL
of glycerol. Heat to the boiling and dispense into suitable
containers. Sterilize in the autoclave at 121°C for 15
minutes.
Antibiotic Medium 35 Base
(Eur. Phar. Antibiotic Medium G)
Ref. 01-545
Specification
Antibiotic Medium 35 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method).
Formula (in g/L)
Peptone .................................................... 10,0
Meat extract .............................................. 10,0
Sodium chloride .......................................... 3,0
Agar .......................................................... 15,0
Final pH 7,0 ± 0,1
Directions
Add 38 g of medium to 1 L of distilled water containing
10 mL glycerol. Heat to the boiling and dispense into
suitable containers. Sterilize in the autoclave at 121°C
for 15 minutes.
Antibiotic Medium 36
Ref. 01-200
Sharlau Microbiology sold this medium as Tryptic Soy
Agar (TSA).
Specification
Antibiotic Medium 36 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method)
Formula (in g/L)
Casein peptone ........................................ 15,0
Soy peptone ............................................... 5,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,3 ± 0,1
Directions
Add 40 g of medium to 1 L of distilled water.Heat to the
boiling and dispense into suitable containers. Sterilize in
the autoclave at 121°C for 15 minutes.
13
 Antibiotic Media
Antibiotic Medium 39
Ref. 02-547
Specification
Antibiotic Medium 39 may be used in the inoculum
preparation, serial dilutions or turbidimetric antibiotic
assays.
Formula (in g/L)
Peptone ................................................... 5,00
Yeast extract ............................................. 1,50
Meat extract .............................................. 1,50
Sodium chloride ........................................ 3,50
Dextrose ................................................... 1,00
Monopotassium phosphate ...................... 1,32
Dipotassium phosphate ............................ 3,68
Final pH 7,9 ± 0,1
Directions
Add 17,5 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Steri-
lize in the autoclave at 121°C for 15 minutes.
Antibiotic Medium 40
Ref. 01-546
Specification
Antibiotic Medium 40 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
metho
Formula (in g/L)
Polypeptone ............................................... 5,0
Dextrose ................................................... 10,0
Yeast extract ............................................. 20,0
Polysorbate 80 ........................................... 0,1
Monopotassium phosphate ........................ 2,0
Agar .......................................................... 12,0
Final pH 6,8 ± 0,1
Directions
Add 49 g of medium to 1 L of distilled water. Heat to the
boiling and dispense into suitable containers. Sterilize in
the autoclave at 121°C for 15 minutes.
Antibiotic Medium 41
Ref. 02-548
Specification
Antibiotic Medium 41 is used in the antibiotic assays by
the turbidimetric method.
14
Formula (in g/L)
Casein Peptone ....................................... 9,00
Yeast extract ............................................. 5,00
Sodium citrate ........................................ 10,00
Dextrose ................................................. 20,00
Monopotassium phosphate ...................... 1,00
Dipotassium phosphate ............................ 1,00
Final pH 6,8 ± 0,1
Directions
Add 46g of medium to 1 L of distilled water. Heat to the
boiling and dispense into suitable containers. Sterilize in
the autoclave at 121°C for 15 minutes.
Antibiotic Medium C
(Eur. Phar. Antibiotic Medium C)
Ref. 02-601
Specification
Antibiotic Medium C may be used for the inoculum prepa-
ration, serial dilutions or turbidimetric antibiotic assays.
Formula (in g/L)
Peptone ................................................... 6,00
Yeast extract ............................................. 3,00
Beef extract .............................................. 1,50
Sodium chloride ........................................ 3,50
Dextrose ................................................... 1,00
Monopotassium phosphate ...................... 1,32
Dipotassium phosphate ............................ 3,68
Final pH 7,0 ± 0,2
Directions
Add 20 g of medium to 1 L of distilled water. Heat to the
boiling and dispense into suitable containers. Sterilize
in the autoclave at 121°C for 15 minutes. If pH 8,0 is
desired, adjust with NaOH1N.
Antibiotic Medium D
(Eur. Phar. Antibiotic Medium D)
Ref. 02-549
Specification
Antibiotic Medium D may be used for the inoculum prepa-
ration, serial dilutions or turbidimetric antibiotic assays.
Formula (in g/L)
Heart extract ............................................. 1,50
Yeast extract ............................................. 1,50
Casein peptone ........................................ 5,00
Sodium chloride ........................................ 3,50
Dextrose ................................................... 1,00
Monopotassium phosphate ...................... 1,32
Dipotassium phosphate ............................ 3,68
Potassium nitrate ..................................... 2,00
Final pH 7,0 ± 0,2
 Antibiotic Media

Directions Formula (in g/L)

Add 19,5 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Steri-
lize in the autoclave at 121°C for 15 minutes.
Antibiotic Medium E
(Eur. Phar. Antibiotic Medium E)
Ref. 01-430
Specification
Antibiotic Medium E is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method).
Peptone ................................................... 5,00
Meat extract .............................................. 3,00
Disodium phosphate ............................... 26,90
Agar ........................................................ 12,00
Final pH 7,9 ± 0,2
Directions
Add 46,9 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into containers. Sterilize in the
autoclave at 121°C for 15 minutes. Precipitates ap-
pearing after sterilization do not interfere in its normal
operation.

Table III. Specifications for the antibiotic master standard solutions

Drying cond. Initial solvent Master soln Days (cool) Final solvent

Amikacin 6 Distilled water 1,0 mg 15 Distilled water

Amphotericin B1 1 Dimethylsulfoxide 1,0 mg 1 10
Bacitracin 1 1 100,0 ui 7 1
Bleomycin 7 16 2,0 ui 15 16
Carbenicillin None 1 1,0 mg 15 1
Cephalothin 1 1 1,0 mg 5 1
Cycloserine 1 Distilled water 1,0 mg 30 1
Chloramphenicol None Ethanol 1,0 mg 30 1
Chlortetracycline None HCl 0,01N 1,0 mg 4 4
Cloxacillin None 1 1,0 mg 7 1
Colistimethate Na 1 Distilled water 1,0 mg 1 6
Colistin 1 Distilled water 1,0 mg 15 6
Demeclocycline None HCl 0,01N 1,0 mg 5 4
Dihydrostreptomycin 5 3 1,0 mg 30 3
Doxycycline None HCl 0,01N 1,0 mg 5 Distilled water
Erythromycin 1 Methanol 1,0 mg 14 3
Framicetine 3 Distilled water 1,0 mg 7 3
Gentamicin 3 3 1,0 mg 30 3
Gramicidin 1 Ethanol 1,0 mg 30 Ethanol
Kanamycin SO4 None 3 1,0 mg 30 3
Methacycline None HCl 0,01 N 1,0 mg 15 4
Methylmicin None 3 1,0 mg 7 3
Nafcillin None 1 1,0 mg 2 1
Natamycin 4 Dimethylsulfoxide 1,0 mg 1 10
Neomycin 1 3 1,0 mg 15 3
Novobiocin 5 Absolute Ethanol 1,0 mg 5 6
Nystatin 4 Dimethylformamide 1000,0 ui 1 6
Oxytetracycline None HCl 0,1N 1,0 mg 4 4
Paromomycin 1 3 1,0 mg 21 3
Penicillin G None 1 1000,0 ui 2 1
Polymyxin B 1 Distilled water 10000,0 ui 15 6
Rifampin None Methanol 1,0 mg 1 1
Rolitetracycline None Distilled water 1,0 mg 1 Distilled water
Sisomycin None 3 1,0 mg 15 3
Spectinomycin 6 Distilled water 1,0 mg 30 Distilled water
Spiramycin 1 Methanol 1,0 mg 7 3
Streptomycin 1 3 1,0 mg 30 3
Tetracycline None HCl 0,1N 1,0 mg 1 4
Ticarcillin None 1 1,0 mg 1 1
Thiostreptone None Dimethylsulfoxide 1,0 ui 1 Dimethylsulfoxide
Tobramycin 3 Distilled water 1,0 mg 15 16
Troleandomycin None Isopropanol 1,0 mg 1 Distilled water
Vancomycin 1 Distilled water 0,4 mg 7 4

1
Use amber glass or work under red light

15
 Antibiotic Media
Reference Standard
Each antibiotic must be assayed with a proper reference
standard or master standard pattern. Patterns may
be obtained from the USP NF Convention Inc. or other
organisms, such as:
European Pharmacopeia Secretariat, B.P. 907, F-67029
Strasbourg. CEDEX 1. Fax +33 388 41 20 36. e-mail:10
0536.3157@compuserve.com
USP-NF Reference Standards, 12601 Twinbrook Park-
way, Rockville. Md. 20852, USA
British Pharmacopoeia Comission Laboratory, Govern-
ment Buildings, Block 2, Honeypot Lane, Stan-
more, Middlesex, HA7 1A4. England.
However, you may use domestic or working standards
if they have been compared to a reference pattern. In
Table III you may find details about the reference stand-
ard: dessication conditions, initial diluent to obtain the
mother solution, activity period,etc...
Following are described the dessication methods for the
standards. US/FDA numeration have been maintained.
US/FDA Method #1
In vacuum oven, dry the pattern or master reference
standard at 60°C for 3 hours, at pressure 5 mm Hg.
US/FDA Method #3
In vacuum oven, dry the pattern or master reference
standard at 40°C for 3 hours, at pressure 5 mm Hg.
US/FDA Method #4
In vacuum oven, dry the pattern or master reference
standard at 40°C for 2 hours, at pressure 5 mm Hg.
US/FDA Method #5
In vacuum oven, dry the pattern or master reference
standard at 100°C for 4 hours, at pressure 5 mm Hg.
US/FDA Method #6
In vacuum oven, dry the pattern or master reference
standard at 45°C for 3 hours, at pressure 5 mm Hg .
US/FDA Method #7
In vacuum oven, dry the pattern or master reference
standard at 35°C for 4 hours, at pressure 5 mm Hg .
16
Diffusion Method in Agar
(Plate assay)
Once we decide the assay conditions (tabulated in
Table IV), we can prepare the plates as per the following
method .
Plate preparation
..Pour basal medium, melted and cooled to 50°C, into
each plate, and let it solidify horizontally. Using 10 cm.
diameter plates, suggested volumes are 21 mL for each
basal layer and 4 mL for the seed layer. Nonetheless, in
the case of amphotericin and nystatin a single seed
layer of 8 mL is used, without any basal layer. Another
exception to this rule is bleomycin, for which a 10 mL of
basal layer and a 6 mL of seed layer is used. In practise,
when the standard 9 cm diameter plastic plates are used
it is advisable to use the volumes of about 15 mL for the
basal layer and 4 mL for the seed layer, in order to avoid
the contact between the cylinders and the plate cover.
While the basal layer solidifies, adjust the temperature
for the medium of seed layer to 45-48°C, and inocu-
late with the suggested (or tested) volume of inoculum
suspension. Homogenize by gentle stirring and carefully
pour the medium over the basal layer in 4 mL volumes,
except for the cases mentioned above. Be very careful
in this operation, since the final results may be affected
if the layer is not very homogeneous, and forms bubbles
or clots. Once the agar solidifies, place the cylinders or
make the holes. Put or make 6 cylinders or holes per
plate, centered in the plate at 60° angle from each other.
Reference pattern preparation and
standard curve
Master pattern is prepared according to the specifica-
tions in Table IV, starting from the stock solution and
using the adequate diluent until the desired concentra-
tion is obtained. Reference level is always the medium
concentration among the 5 concentrations used to obtain
the standard curve (see Table IV).
Assay technique
To obtain the curve, 12 plates are used, three for each
level, except for the reference standard, which is in-
cluded in all. In each batch of three plates, 3 alternate
holes or cylinders are filled with the reference concentra-
tion and the other three are filled with the corresponding
concentration. By proceeding in this way we can obtain
30 inhibition halos or inhibition zones corresponding to
the reference concentration and 9 halos for each one of
the other assay standards.
Three plates, with 6 holes or cylinders, are used in
each test. Holes or cylinders are filled alternatively with
the reference level and the diluted sample. Plates are
incubated at appropriate temperature (see Table IV) for
16-18 hours. After this period, inhibition halo readings
are performed using the proper measuring instrument.
 Antibiotic Media

Table IV. Agar diffusion test specifications

Medium Final concentrations: assay levels for the standard curve in

Assay Maintenance Inoculum Incubation Reference
IU/mL or in mcg/mL
Antibiotic strain Medium (mL/100 mL Basal Seed Temperat. halo
(Table I) (A.M. nº) medium) strain strain (°C) Ref. (mm)

Amphotericin B P 19 1,00 - 19 30 0,640 0,800 1,000 1,250 1,560 16-18

Bacitracin F 1 0,30 9 10 32-35 0,640 0,800 1,000 1,250 1,560 16-18
Bleomycin S 36 1,00 35 35 32-35 0,010 0,020 0,040 0,080 0,160 18-20
Carbenicillin R 1 0,50 9 10 3712,800 16,000 20,000 25,000 31,200 16-18
Cephalothin A 1 0,10 2 1 32-35 0,640 0,800 1,000 1,250 1,560 14-16
Cloxacillin A 1 0,10 2 1 32-35 3,200 4,000 5,000 6,250 7,810 18-20
Colistimethate sodium O 1 0,10 9 10 37 0,640 0,800 1,000 1,250 1,560 13-15
Colistin O 1 0,10 9 10 37 0,640 0,800 1,000 1,250 1,560 15-17
Dihydrostreptomycin J 1 0,05 5 5 37 0,640 0,800 1,000 1,250 1,560 14-16
Doxycyclin I 1 0,40 8 8 30 0,640 0,800 1,000 1,250 1,560 18-20
Erythromycin E 1 1,50 11 11 32-35 0,640 0,800 1,000 1,250 1,560 17-19
Framicetin J,H 1 0,40 1 1 35-37 0,640 0,800 1,000 1,250 1,560 14-16
Gentamicin D 1 0,03 11 11 37 0,640 0,800 1,000 1,250 1,560 15-17
Nafcillin A 1 0,30 2 1 32-35 1,280 1,600 2,000 2,500 3,120 14-16
Natamycin P 19 1,00 19 19 28-30 3,200 4,000 5,000 6,250 7,810 17-19
Neomycin A,H,J 1 0,40 11 11 32-35 6,400 8,000 10,000 12,500 15,600 16-18
Netilmycin D 1 0,25 11 11 32-35 0,064 0,080 0,100 0,125 0,156 14-16
Novobiocin D 1 4,00 2 1 35 0,320 0,400 0,500 0,625 0,781 16-18
Nystatin Q 19 1,00 - 19 3012,800 16,000 20,000 25,000 31,200 16-18
Paromomycin D 1 2,00 11 11 37 0,640 0,800 1,000 1,250 1,560 19-21
Penicillin G A 1 1,00 2 1 32-35 0,640 0,800 1,000 1,250 1,560 17-19
Polymyxin O 1 0,10 9 10 37 6,400 8,000 10,000 12,500 15,600 15-17
Rifamycin J 1 0,10 2 2 30 3,200 4,000 5,000 6,250 7,810 15-17
Sisomycin D 1 0,03 11 11 32-35 0,064 0,080 0,100 0,125 0,156 15-17
Spiramycin J 1 0,20 1 1 35-37 0,640 0,800 1,000 1,250 1,560 15-17
Ticarcillin B 1 0,40 8 8 32-35 3,200 4,000 5,000 6,250 7,810 17-19
Vancomycin J 1 0,20 8 8 37 6,400 8,000 10,000 12,500 15,600 16-18

Potency calculation
To extrapolate from the standard curve, use the average
of the 36 diameters of the reference level and the sev-
eral batches of 9 halos obtained in each assay level. The
average of the 36 halos of reference is the correction
value of the standard curve, and thus we can achieve
each point as the function of the average value of the
reference values of the corresponding standard.
Values obtained in this way are plotted over a semiloga-
rithmic graph paper, drawing the antibiotic concentration
(in mcg/mL) on X axis, and the halo or zone diameters
on the Y axis. Standard curve is drawn by linking the
five points or by tracing a straight line between the high
and low point, which are calculated using the following
statistical formulae:
3a+2b+c-e 3e+2d+c-a
A= B=
5 5
where:
A: Halo diameter, calculated for the highest standard
concentration.
B: Halo diameter, calculated for the lowest pattern con-
centration.
c: Reference halo diameter, average of the 36 halos
of the pattern plates.
a,b,d,e: Corrected average values of the other pattern
assay levels,from the highest to the lowest order.
To estimate the potency of the test sample, calculate
average of the inhibition halo diameters and the refer-
ence and sample levels of the three plates, and perform
the corrections related to the correction point. Corrected
value of the problem is projected on the standard curve
and thus the theoritical concentration is obtained. Theo-
ritical concentraion multiplied by the dilution factor gives
the real concentration of antibiotic in the sample.
Turbidimetric assay
Reference pattern preparation and
standard curve
Once the assay conditions are determined as per Table
III, prepare the pattern according to the specifications in
Table V. At the time of assay experiment, take an aliq-
uote of mother solution and prepare appropriate dilutions
to obtain the desired concentrations. Reference level is
the medium concentration between the five proposed for
the standard curve.

17
 Antibiotic Media

Table V. Turbidimetric assay specifications

Final concentrations: assay levels for the pattern curve in IU/mL or
Assay strain Inoculum Assay Incubation mcg/mL
Antibiotic (Table I) (mL/100 mL medium tempera-
medium) ture Ref.
(°C)

Amikacin A 0,50 3 32 6,000 8,000 10,000 12,500 15,600

Candicidin P 0,20 13 29 0,030 0,042 0,060 0,084 0,120
Capreomycin L 0,05 3 37 64,000 80,000 100,000 125,000 156,000
Chloramphenicol M 0,70 3 32 2,000 2,240 2,500 2,800 3,120
Chlortetracycline A 0,10 3 32 0,038 0,048 0,060 0,075 0,094
Cycloserine A 0,40 3 32 32,000 40,000 50,000 62,500 78,100
Demeclocycline A 0,10 3 32 0,064 0,080 0,100 0,125 0,156
Dihydrostreptomycin L 0,10 3 37 24,000 26,800 30,000 33,500 37,500
Doxycyclin A 0,10 3 32 0,064 0,080 0,100 0,125 0,156
Gramicidin G 1,00 3 37 0,028 0,034 0,040 0,048 0,057
Kanamycin B 0,20 3 32 8,000 8,900 10,000 11,200 12,500
Methacycline A 0,10 3 32 0,038 0,048 0,060 0,075 0,094
Neomycin L 2,00 39 37 0,640 0,800 1,000 1,250 1,560
Oxytetracycline A 0,10 3 32 0,160 0,200 0,250 0,312 0,390
Rolitetracycline A 0,10 3 32 0,160 0,200 0,250 0,312 0,390
Spectinomycin M 0,10 3 32 24,000 26,800 30,000 33,500 37,500
Streptomycin L 0,10 3 37 24,000 26,800 30,000 33,500 37,500
Tetracycline A 0,10 3 32 0,160 0,200 0,250 0,312 0,390
Thiostreptone G 0,20 3 37 0,400 0,600 0,800 1,000 1,250
Tobramycin A 0,15 41 32 2,000 2,236 2,500 2,795 3,125
Troleandomycin L 0,10 3 32 16,000 20,000 25,000 31,200 39,000

Assay Technique where:

The assay is performed in the following way:
Prepare 3 tubes and put 1 mL (or 0,1 mL if the antibiotic
is gramicidin or tyrothricin) of every assay level of stand-
ard and sample. Thus, 15 tubes will be employed for the
pattern and 3 tubes for each problem.
Add asseptically 9 mL of inoculated assay broth to
each tube, and put them into a boiling water bath for
2-4 hours. The exact incubation time is determined by
observing the growth inside the tube, taking the standard
tube as the reference.
Tubes are removed from the boiling water bath and
0,5 mL of 12% formaldehyde is added in each tube in
order to stop the growth. Growth in the form of turbidity
is measured by a Spectrophotometer at the wavelength
530 nm. The zero is fixed by the device and 100% trans-
mission is provided by a blank containing only the sterile
medium and formaldehyde at the same proportion or
concentration as that of the assay.
Potency calculation
Statistical values corresponding to the highest and
lowest concentrations are determined according to the
following formulae:
3a+2b+c-e 3e+2d+c-a
A= B=
5 5
A: Absorbance value, calculated for the highest
value of the pattern curve.
B: Absorbance value, calculated for the lowest value of
the pattern curve.
a,b,c,d,e: Average values for each value in the triplicate
standard curve, from the highest to the lowest
concentration.
Potency estimation is performed by taking the average
value of each assay level in the pattern curve over a
semilogarithmic paper. Draw the absorbance over the
logarithmic scale and concentration over the abscissa.
If the calculated values are used, the result is a straight
line, but a curve may be drawn by linking the five points.
The absorbance average value of the test sample is pro-
jected over the standard curve, obtaining in this way the
theoritical concentration which, multiplied by the dilution
factor, gives the real potency of the test sample.
Cross Contamination
with Penicillin
To perform this assay all the instrumentation and even
the environment must be free of penicillin. Plates are
prepared following the general method, using 10 mL of
Antibiotic Medium 1 (Ref. 01-009) in the basal layer and
4 mL of Antibiotic Medium 4 in seed layer and inoculating
the C strain of microorganism (see Table I).

18
 Antibiotic Media

Reference standard is prepared according to the stand- with the pattern reference level. Fill another 2 holes or
ard for penicillin, proposed in Table II, but taking the cylinders in each plate with the untreated sample, and
mother solution of 100 units and the final concentrations the last 2 holes or cylinders in each plate with the treated
at 0,0005;0,0125; 0,0050; 0,100 and 0,200 penicillin sample.
units. Reference is the one with final concentration 0,05 Plates are incubated at 30°C for overnight. Presence
units (see Table III). of inhibition halos in the untreated sample, and the
absence of holes in the treated sample are a sign of
Sample is prepared by dissolving 1 g in 18 mL of distilled penicillin contamination. Should a qualitative estimation
water. From this initial solution, take 9 mL into a separat- of penicillin be desired, measure the halos and proceed
ing funnel, and add 20 mL of amyl acetate. Add then 1 as in the normal penicillin assay.
mL of Solution #11 (see Table II) and stir it vigorously.
Let it rest and once the two layers are separated, take Special lab requirements
the aqueous layer to another separating funnel. Check
the pH of the solution and if it is higher than 3, readjust The instruments used in all these assays must be
to 2,5 with HCl. Extract again with amyl acetate and washed carefully before and after each test, in order to
discard the aqueous layer. remove all the traces of antibiotic.
Mix the two parts of amyl acetate, and wash them with To sterilize lab commodities, cover them well and use a
10 mL of Solution #2 (see Table II). Discard the aqueous hot air oven at 200-220°C for 2 hours. Volumetric flasks ,
layer. Extract penicillin from the amyl acetate with 10 mL pipets, ... must be washed carefully.
of phosphate buffer pH 6 (Solution #1, Table II). Penicillin
presence is determined in this solution.
Use 15 plates to perform the assay: three for each
standard curve assay, except for the reference stand-
ard, which is present in all of them. Place 6 holes or
cylinders in each plate and fill them alternatively with the
reference solution and the corresponding assay level. In
this way, you will obtain 45 inhibition halos for the refer-
ence level and 9 halos more for each one of the other
levels.
A part of sample (2-5 mL) is treated with 0,1 mL of peni-
cillinase and incubated at 37°C for 1 hour. Use 3 plates
for each sample, and fill 2 holes or cylinders in each one

Plate external Ø 9 mm
Cylinder ext. Ø 6 mm
Cylinder int. Ø 8 mm

Figure 1. Holes, discs or cylinders at 90° Figure 2. Holes, discs or cylinders at 60°

19
 Antibiotic Media
Scharlau Chemie may supply all the necessary instru-
ments to perform antibiotic assays, which is detailed
below:
Volumetric material
If it is possible, use guaranteed, checked and class
A glass.
Cylinders
Cylinders must be made of stainless steel. Exterior
diameter: 8 mm, interior diameter: 6 mm, height: 8-10
mm.
Holes
Holes should be made with a suitable punching
instrument of the cylinder dimension.
Discs
Use normalized paper filter discs, 6 mm diameter.
Plates
Use plastic or glass plates. If glass plates are em-
ployed, they must be washed and sterilized.
Tubes
All the tubes used in one assay must be equal and
uniform in their dimensions.
20
Colourimeter / Spectrophotometer
It must be able to read at 530 nm, have its own zero
adjustment and 100% transmission can be deter-
mined with a sterile medium solution at the same
conditions of assay.
References
ARRET, B. DIANA, P. JOHNSON y A. KIRSHBAUM
(1971) Outline of Details for Microbiological Assays
of Antibiotics: Second Revision. J. PHARM, Sci.
60,11,1689-1694.
EUROPEAN PHARMACOPEIA (1997) 3rd Ed. 2.7 Bio-
logical Assays. 2.7.2 Microbiological Assay of Antibiotics.
Council of Europe. Strasbourg.
SANCHO, GUINEA, PARES. (1980) Microbiología
Analítica Básica. Ed. JIMS. Barcelona,
U.S. PHARMACOPEIA XIX (1975) Antibiotic Assays.
U.S./F.D.A.: 21 CFR (1976 and following) 436.100 and
following.
U.S. PHARMACOPEIA XX/National Formulary XV
(1980) Antibiotic Assays.
U.S. PHARMACOPEIA 23/National Formulary 18 (1995)
Biological Tests and Assays. {81} Antibiotics Microbial
Assays.
U.S. PHARMACOPEIA 25/National Formulary 20 (2002)
Biological Tests and Assays. {81} Antibiotics Microbial
Assays.
EUROPEAN PHARMACOPEIA, Supplement (2002), 4th
Ed.,Council of Europe, Strasbourg,
 APT Media
APT Agar
Ref. 01-026
Specification
Solid medium for general purposes, especially designed
for the cultivation of the heterofermentative lactic acid
bacteria that causes meat greening.
Formula (in g/L)
Casein Peptone .................................... 12,500
Yeast Extract .......................................... 7,500
Sodium Chloride ..................................... 5,000
Dipotassium Phosphate ......................... 5,000
Trisodium Citrate .................................... 5,000
Dextrose ............................................... 10,000
Magnesium Sulfate ................................. 0,800
Manganous Chloride .............................. 0,140
Iron Sulfate ............................................. 0,040
Thiamine HCl .......................................... 0,001
Polysorbate 80 ....................................... 0,200
Agar ...................................................... 15,000
Final pH 6,7 ± 0,2
Directions
Suspend 61,2 g of powder into 1 L of distilled water and
let it soak . Heat to boiling with constant stirring. Distrib-
ute in suitable containers and sterilize in the autoclave at
121°C for 15 minutes.
APT Broth
Ref. 02-026
Specification
Liquid version of the medium with the same name. It
is especially designed for the cultivation of lactic acid
bacteria.
Formula (in g/L)
Casein Peptone .................................... 12,500
Yeast Extract .......................................... 7,500
Sodium Chloride ..................................... 5,000
Dipotassium Phosphate ......................... 5,000
Trisodium Citrate .................................... 5,000
Dextrose ............................................... 10,000
Magnesium Sulfate ................................. 0,800
Manganous Chloride .............................. 0,140
Iron Sulfate ............................................. 0,040
Thiamine HCl .......................................... 0,001
Polysorbate 80 ....................................... 0,200
Final pH 6,7 ± 0,2
Directions
Dissolve 46,2 g of powder into 1 L of distilled water,
heating up slightly if necessary. Distribute into suitable
containers and sterilize in the autoclave at 121°C for 15
minutes.
Description
These two general purpose media (APT= All Purpose
with Tween), originally formulated by Evans and Niven,
have been used successfully for the isolation and culti-
vation of lactic acid bacteria that alter the food quality
and composition (especially meat) and require a high
thiamine level. For this reason, ignoring what peptone
and yeast extract could provide, the medium has been
complemented with an extra amount of thiamine.
Both versions, solid and liquid, have demonstrated their
efficacy for detection of lactobacilli that produce meat
greening. Moreover, if media are supplemented with
5% fruit juices, as APHA states, they are converted into
irreplaceable prospection media for any kind of food
biomodifier.
However, without the inclusion of any inhibitory agent in
the formulation signifies that the media have no selective
ability, and thus they can support the growth of almost all
the microbial types.
Technique
Usual detection technique for the bacteria those causes
the greening is as mentioned below:
Products to be examined are crushed carefully in Tryp-
tone Water (Ref. 03-156), and with the same diluent a
dilution bank is prepared. From each dilution, APT Agar
plates are inoculated in mass and in triplicate, and they
are incubated at 32°C for 48 hours. After the incubation
period, colonies are counted using usual technique, and
different types are selected. Every type is inoculated
in APT Broth, and is incubated at 32°C for 24 hours or
more. From these pure cultures, streak on Frankfurt
sausages slices, and incubate those slices and also the
one without the culture as a Control, in a humid room
or atmosphere to verify its greening capacity. Final
identification is done by morphological and biochemical
characteristics.
References
EVANS, J.B. & C.F. NIVEN (1951) Nutrition of the het-
erofermentative lactobacilli that cause greening of cured
meat products J.Bact. 62:599
DEIBEL, R.H, J.B. EVANS & C.F. NIVEN (1957) Micro-
biological assay for the thiamin using Lactobacillus
viridescens. J. Bact. 74:818-821
DOWNES, F.P., K. ITO (2002) Compendium of methods
for the microbiological examination of food. 4rd. ed.
APHA. Washington.
21
 Asparagine Broth
Ref. 02-271
Specification
Liquid medium for the presumptive assay and enumera-
tion of Pseudomonas aeruginosa in bottled water by
MPN method.
Formula (in g/L)
Asparagine ................................................. 2,0
Dipotassium phosphate .............................. 1,0
Monopotassium phosphate ...................... 10,0
Magnesium sulfate ..................................... 0,5
Final pH 7,0 ± 0,2
Directions
Dissolve 13,5 g of powder in 1 L of distilled water con-
taining 8 mL of glycerol. Sterilize by filtration and dis-
tribute in tubes (10 mL/tube). To obtain broth of double
strength, dissolve 27 g of powder in 1 L of distilled water
containing 16 mL of glycerol.
22
Description
Asparagine medium is recommended for the microbio-
logical analysis of bottled water. This is an excellent
enrichment medium for Pseudomonas aeruginosa, since
it is composed of a mineral base and the only carbon
source is asparagine. It may also be used in the multiple
tube technique in microbiological analysis of recreational
waters and as a presumptive test medium for the differ-
entiation of non fermentative gramnegative bacteria.
Technique
Some standards suggests the viable enumeration by
MPN method with 5 tubes per series, inoculating 10 mL,
1 mL and 0,1 mL. All the tubes are incubated at 37°C
for 48 hours. Growth, with or without pigmentation, is
estimated as a presumptive evidence of presence of
Pseudomonas aeruginosa. Enumeration is carried out
with MPN tables for 5 tubes (See MPN chapter in this
book).
Confirmation is performed subculturing a loop of each
tube in Acetamide Medium (Ref. 03-428).
References
PASCUAL ANDERSON, M.R. (1992) Microbiología
Alimentaria. Diaz de Santos. Madrid.
 Azide Dextrose Media
Azide Dextrose Broth
Xn
Ref. 02-343
R-22-32-52/53
S-7-46-61
Specification
Liquid and selective medium for the detection of strepto-
cocci in water, waste water and milk.
Formula (in g/L)
Meat extract ................................................ 4,5
Tryptose .................................................... 15,0
Dextrose ..................................................... 7,5
Sodium chloride .......................................... 7,5
Sodium azide .............................................. 0,2
Final pH 7,2 ± 0,2
Directions
Dissolve 35 g of powder in 1 L of distilled water, heat if
necessary. Distribute into tubes and sterilize in the auto-
clave at 121°C for 15 minutes. To make double strength
broth, dissolve 70 g of powder in 1 L of distilled water.
Description
Azide Dextrose Broth is formulated according to the
“Standard Methods for the Examination of Water and
Wastewater”.
Azide concentration in the medium restrains the growth
of gramnegative bacteria and lets a good growth of en-
terococci. This medium composition is very similar to the
Azide Dextrose Broth acc. to Rothe (Ref. 02-027), but
without phosphate buffering. Therefore, it is advisable to
use this medium with samples with a high salts concen-
tration, to avoid any precipitation in the medium.
Azide Dextrose Broth acc. to
Rothe (Rothe Azide Broth)
Xn
Ref. 02-027
R-22-32-52/53
S-7-46-61
Specification
Medium for the detection and enumeration of entero-
cocci in water.
Formula (in g/L)
Meat peptone ........................................... 10,0
Casein peptone ........................................ 10,0
Dextrose ..................................................... 5,0
Sodium chloride .......................................... 5,0
Dipotassium hydrogen phosphate .............. 2,7
Potassium dihydrogen phosphate .............. 2,7
Sodium azide .............................................. 0,2
Final pH 7.0 ± 0,2
Directions
Dissolve 35,6 g in 1 L of distilled water. Heat if neces-
sary to help dissolution. Divide into 10 mL portions and
pour into tubes. Sterilize by autoclaving at 121°C for 15
minutes. As for the double strength medium, dissolve
71,2 g/L and proceed as indicated above.
Description
The Azide Dextrose Broth acc. to Rothe has been widely
used since 1948 for the detection of fecal streptococci. It
usually provides higher positive results than other similar
media. Its efficiency is due to the Sodium azide, which
is both selective for enterococci and inhibitor of the ac-
companying flora through interference of the electron
transport chain.
This medium is also used for the primary enrichment of
food samples, particularly frozen vegetables.
Technique
Streptometry in Water
Add 10 mL of water to be examined to each one of three
tubes containing 10 mL of double strength medium.
Add 1 mL of sample to another three tubes containing
10 mL of medium each, of single strength. Then add 0.1
mL of water to each one of three tubes containing 10
mL of medium of single strength. Incubate at 37° C and
examine after 24 hours and 48 hours. All tubes which
show turbidity due to growth will be considered as PRE-
SUMPTIVLY POSITIVE and will have to be confirmed on
EVA Broth (Ref.2-028). All tubes which result positive on
this second testing should be considered for the count
of the Most Probable Number (MPN).
When considering other type of samples, dilute them in
Ringer solution 1/4 or peptone water and then inoculate
the tubes as done before.
In case of the highly contaminated samples, dilutions
should be done before inoculation.
References
APHA-AWWA-WPCF (1980) Standard Methods for the
Examination of Water and Wastewater. 15th. Ed. APHA
Inc., Washington, D.C.
CLESCERI, L., A.E. GREENBERG, A.E. EATON (1998)
Standard Methods for the Examination of Water and
Wastewater. APHA-AWWA-WEA. Washington.
GUINEA, SANCHO y PARÉS. (1979) Análisis
Microbiológico de Aguas: Aspectos Aplicados. Ed.
Omega,Barcelona,.
ROTHE (1948) Illinois State Health Department.
DOWNES, F.C. & K.ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 4th ed.
APHA. Washington.
23
 Bacillus cereus Media
Bacillus cereus Agar (MYP Agar)
Ref. 01-262
Specification
Selective solid medium, according to Mossel, for the
isolation and identification of Bacillus cereus from food
samples, acc. ISO 7932 (2003) and ISO 21871:2006.
Formula (in g/L)
Peptone ................................................ 10,000
Mannitol ................................................ 10,000
Sodium chloride .................................... 10,000
Meat extract ............................................ 1,000
Phenol red .............................................. 0,025
Agar ...................................................... 15,000
Final pH 7,2 ± 0,2
Directions
Suspend 46 g of powder in 900 mL of distilled water.
Let it soak and bring to boil. Sterilize in the autoclave at
121°C for 15 minutes. Let it cool to 50°C and then add
100 mL of Egg’s Yolk Sterile Emulsion (Ref. 06-016) and
100 mg/L of Polymyxin (Ref. 06-021CASE). Homogenize
well and distribute into plates. Do not reheat or remelt
the complete medium.
Description
Mossel’s formulation is developed to detect and enu-
merate B.cereus in any kind of food, since it lets a good
differentiation and selection of these microorganisms.
Polymyxin addition inhibits most of accompanying
bacteria, but it does not affect the growth of B.cereus.
This bacteria do not ferment mannitol and thus there is
no change in the indicator around the colonies. On other
hand, due to lecithinase activity of B.cereus it produces
a halo or zone of white precipitate around the colonies.
A count of B.cereus over 100.000 cells/g of the food
sample is considered to be hazardous, since the ac-
cumulated phosphoril-choline may cause intoxication
symptoms in children. For this reason, besides common
isolation and identification, a viable enumeration must be
performed to evaluate the real population of cells.
Technique
According to the authors, dehydrated or dry samples
must follow a recovery process in the following way: 20
g of sample is mixed with 90 mL of Tryptone Water (Ref.
03-156) for a minimum period of 1 hour, at room tem-
perature. Afterwards, add 90 mL more of Tryptone Water
and homogenize it. A dilution of 1:10 should be obtained.
If necessary, a decimal dilution bank using Tryptone wa-
ter as the diluent can be prepared. With a Drigalsky loop
(Ref. 5-010), spread aliquotes of 0,1 mL over the surface
of the agar plates and let the agar medium absorb those
aliquots. Incubate the plates at 30°C for 18-24 hours to
allow spore germination before giving definite results,
which are referred as B. cereus colonies per gram of
sample.
Suspected colonies show the following appearance:
irregular borders, pink colour and even purple in the
center, and with a halo of white precipitate. Colonies
with yellow halos must be discarded surely.
24
Sometimes,the confusion with other colonies of gram-
positive bacilli is possible, and hence to identity this,
confirmation has to be performed verifying the glucose
fermentation, gelatin degradation and nitrate reduction,
which are positive tests for Bacillus cereus.
References
ATLAS, R.M. & L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press. Londres.
CORRY, J.E.L., G.D.W. CURTIS & R.M. BAIRD. (2003)
Handbook of Culture Media for Food Microbiology. Elsevier
Sci. B.V. Amsterdam. The Netherlands.
DOWNES, F.P. & K. ITO (2001) Compendium of methods
for the microbiological examination of foods. 4th ed. APHA.
Washington DC. USA.
FIL-IDF 181:1998 Provisional Int. Standard. Dried Milk
Products. Enumeration of Bacillus cereus.- Most probable
number technique
ISO 7932 Standard (2004) 3rd ed. Microbiology of food
and animal feeding stuffs. Horizontal method for the
enumeration of presumptive Bacillus cereus. Colony count
technique at 30ºC.
ISO 21871 Standard (2006) Microbiology of food
and animal feeding stuffs.- Horizontal method for the
determination of low numbers of presumptive Bacillus
cereus.- Most probable number technique and detection
method.
MOSSEL, D.A.A., KOOPMAN. M.J. y JONGERIUS, E.
(1967) Enumeration of Bacillus cereus in foods. Appl.
Microbiol. 15:650-653.
PASCUAL ANDERSON, Mª.Rª (1992) Microbiología
Alimentaria. Díaz de Santos, S.A. Madrid.
Bacillus cereus Selective Agar (PEMBA)
Ref. 01-487
Specification
Selective solid medium for the enumeration of Bacillus
cereus in food, according to ISO 21871.
Formula (in g/L)
Peptone .................................................... 1,00
Mannitol .................................................. 10,00
Sodium chloride ........................................ 2,00
Magnesium sulfate ................................... 0,20
Disodium phosphate ................................. 2,50
Potassium phosphate ............................... 0,25
Brom thymol blue ..................................... 0,12
Sodium pyruvate .................................... 10,00
Agar ........................................................ 14,00
Final pH 7,2 ± 0,2
Directions
Suspend 40 g of powder in 950 mL of distilled water. Let
it soak and bring to the boiling. Distribute into suitable
containers and sterilize by autoclaving at 121ºC for 15
minutes. Let it cool to 50ºC and then add 50 mL/L of
Egg’s Yolk Sterile Emulsion (Ref. 06-016) and Polymyxin
B Sulfate (Ref. 06-021CASE) to reach a 100 U/mL con-
centration. Homogenize and pour into plates.
 Bacillus cereus Media
Description
Bacillus Cereus Selective Agar is formulated according
to the Food Analysis Nordic Committee (NMLK) stand-
ards. This standard uses this medium and Blood Agar
Base (Ref. 01-352) simultaneously for the detection and
enumeration of B. cereus in any type of food. This me-
dium can also be used to confirm doubtful colonies, and
in this last case Polymyxin addition may be ommitted.
Technique
NMLK proposes the simultaneous use of Bacillus cereus
Selective Agar and Blood Agar Base (Ref. 01-352). Both
media are inoculated by surface streaking with 0,1 mL
aliquotes which are spread with a Drigalsky loop. Both
series of plates are incubated at 30ºC for 24 hours.
Typical B.cereus colonies over Blood Agar are big, irreg-
ular, dirty white or grey-like colour with a hemolysis halo
surrounding them. In B.cereus Selective Agar, colonies
are blue, surrounded by a clear zone of egg yolk diges-
tion (lecithinase positive).
If there is an equal amount of typical or similar colonies
in both the media, later confirmation is not necessary.
References
ISO 21871 Standard (2006) Microbiology of food and
animal feeding stuffs.- Horizontal method for the
Baird Parker Agar Base (Eur. Phar. Medium O)
Ref. 01-030
Specification
Solid and selective culture medium for the screening of
staphylococci from a variety of samples, acc. to pharma-
copeias and ISO standard.
Formula (in g/L)
Tryptone ................................................... 10,0
Sodium pyruvate ...................................... 10,0
Glycine ..................................................... 12,0
Meat extract ................................................ 5,0
Lithium chloride .......................................... 5,0
Yeast extract ............................................... 1,0
Agar ......................................................... 17,0
Final pH 7,0 ± 0,2
Directions
Suspend 60 g in 950 mL of distilled water. Allow it to
soak and then bring to the boil with constant stirring.
Sterilize by autoclaving at 121° C for 15 minutes. Cool to
50° C and add 50 mL of Egg’s Yolk Tellurite Sterile Emul-
sion (Ref. 06-026). Homogenize
and distribute into plates. Once prepared, the medium
must not be reheated nor sterilized again.
Description
The Baird Parker Agar Base is specially recommended
for the detection and enumeration of staphylococci in
food and other material, since it allows a good differen-
tiation of coagulase-positive strains. The growth of the
determination of low numbers of presumptive Bacillus
cereus.- Most probable number technique and detection
method.
Ref. 01-262 Bacillus cereus Agar
Control
Bacillus cereus ATCC 10876
accompanying bacteria is usually suppressed by the high
concentration in lithium, glycine and pyruvate. Lithium
and glycine enhances the growth of staphylococci. Even
if its high selectivity does not affect staphylococci it may
occasionally permit the growth of some Bacillus species,
yeast and very rarely, Proteus. The growth of Proteus
species can be suppressed by adding 50 mg/l of sul-
phamethazine.
The presence of tellurite and egg’s yolk, which must
always be added to the medium after sterilization, allows
the differentiation of presumptly pathogenic staphylococ-
cal colonies. A high correlation has been found between
the coagulase test and the presence of clearing zones
of lypolysis in this medium, which is due to the staphylo-
coccal lecithinase. On the other hand, studies show that
almost 100% of coagulase-positive staphylococci are
capable of reducing tellurite, which produces black colo-
nies, whereas other staphylococci can not always do so.
When using sterile reagents other than SCHARLAU
MICROBIOLOGY, the complete medium may be ob-
tained by adding 50 mL sterile egg’s yolk and 10 mL of
1% potassium tellurite solution. Plates should be used
on the same day of preparation or within 48 hours, to
avoid the loss of definition in the precipitated zones . The
basal medium, without the yolk or the tellurite, is perfect-
ly stable and therefore can be repeatedly melted.
Technique
The inoculation is done by spreading 0,5 mL of sample
over each plate with a Drigalski loop (Ref. 5-010). After
18-24 hours of incubation at 35° C, select the colonies
25
 Baird Parker Agar Base (Eur. Phar. Medium O)
which are black, shiny and convex with regular margins
surrounded by a zone of clearing. These can be pre-
sumptly identified as coagulase-positive Staphylococcus
aureus.
Colonial appearance after 24 h. at 35°C:
Staphylococcus aureus: Black, shiny, convex, regular
margins, 1.0-1.5 mm diameter, surrounded by a
clearing zone of lipolysis 2-5 mm in width. May
produce wide opaque precipitate zones extending
into the cleared medium after 48 hours.
Other species of Staphylococcus: Black, usually dully,
with regular margins. Sometimes they are brown
with zones of clearing but these present wide
opaque zones.
Micrococcus spp: Brown, very small and without clear-
ing.
Bacillus spp: Various shades of brown, big. May produce
clearing after 48 hours.
Yeasts: White, big and smooth.
The egg’s yolk emulsion can be prepared by mixing
a fresh egg’s yolk with an equivalent quantity (w/v) of
saline solution. Sterilize by filtration and aseptically add
to the medium. This reagents´s reference, already steri-
lized, is SCHARLAU 6-016.
The potassium tellurite solution is prepared by dissolv-
ing 3,5 g potassium tellurite in 100 mL distilled water.
Sterilize by filtration. This sterile reagent’s SCHARLAU
MICROBIOLOGY reference is 6-011.
Although these solutions can be mixed to be added
to the Baird Parker Agar Base forming the commonly
known Egg Yolk Tellurite Sterile Emulsion (Ref. 06-026),
they are also stable as the separate supplement and can
be used in many other culture media.
Bile Esculin Media
Bile Esculin Agar
Ref. 01-265
Specification
Solid culture medium for the identification of probiotic
streptococci in food samples.
Formula (in g/L)
Meat extract ................................................ 3,0
Peptone ...................................................... 5,0
Bile .......................................................... 40,0
Ferric citrate................................................ 0,5
Esculin ........................................................ 1,0
Agar .......................................................... 15,0
Final pH 7,0 ± 0,2
Directions
Suspend 64,5 g of powder in 1 L of distilled water and
let it soak. Heat to boiling and distribute into containers.
Sterilize in the autoclave at 121°C for 15 minutes.
26
References
ATLAS R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press. Londres
BAIRD-PARKER, A.C. (1962) An improved diagnostic
and selective médium for isolating coagulase-positive
staphylococci. J. Appl. Bact. 25:12.
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Foods. 4rd
ed. APHA. Washington. USA
EUROPEAN PHARMACOPOEIA (2007) 5ª ed. Suppl.
5.6 § 2.6.13 Microbiological examination of non-sterile
products. EDQM Council of Europe. Strasbourg.
ISO 5944:2001 Standard. Milk and Milk based products
– Detection of coagulase positive staphylococci – MPN
Technique. Geneva.
ISO 6888-1:1999 Standard.Microbiology of food and
animal feeding stuffs – Horizontal method for the enu-
meration of coagulase-positive staphylococci – Part 1
Technique using Baird-Parker Agar médium. Geneva.
ISO 22718:2006 Standard. Cosmetics – Detection of
Staphylococcus aureus.
FIL-IDF 60:2001 Standard. Latí et produits à base de
lait – Détection des staphylocoques à coagulase positive
– Technique du nombre le plus probable. Brussels.
USP 29 – NF 25 (2006) <61> Microbial Limit Tests. US
Phamacopoeial Conv. Inc. Rockville. Md, USA
ZANGERL, P. & H. ASPERGER. (2003) Media used
in the detection and enumeration of Staphylococcus
aureus in Handbook of Culture Media for Food Microbiol-
ogy Corry et als. Eds. Elsevier Sci. BV Ámsterdam
Description
This medium formulation is based on the modification by
Facklam and Moody of the original formulation by Swan
to verify the esculin hydrolysing capacity of streptococci
and their resistance to bile salts which inhibit gram posi-
tive bacteria.
In fact, this medium can substitute KAA Confirmative
Agar (Ref. 01-263), but it has not the same selectivity.
Hence, it is used just as a substrate to verify the two as-
says simultaneously in the biochemical tests that identify
enterococci.
Technique
Assay is performed by inoculating the surface of a slant
with the pure culture that is going to be verified. After the
24 hours incubation at 35°C, it might produce translucid
colonies, surrounded by black halos or zones, due to
esculin hydrolysis. Resistance to bile salts is indicated
by the growth.
References
LEUCHNER, R.G.K., J.BEW, K.J.DOMIG, &
W.KNEIFEL. (2002) A collaborative study of a method
for enumeration of probiotic enterococci in animal feed J.
Appl. Microbiol. 93:781-786
 Bile Esculin Media
DEIBEL, R.H., HARTMAN, P.A. (1976) The enterococci,
Compendium of Methods for the Microbiological Exami-
nation of Food. APHA.
FACKLAM, R.R., MOODY, M.D. (1970) Presumptive
identification of group D streptococci: The bile-esculin
test. Appl. Microbiol. 20:245.
PASCUAL ANDERSON, MªRª. (1992) Microbiología
Alimentaria. Diaz de Santos, S.A.,Madrid,SPAIN.
SWAN,A. (1954), The use of bile-esculin medium and
Maxted’s technique of Lancefield grouping in the identifi-
cation of enterococci. J.Clin.Pathol. 7:160.
FDA (1998) Bacterilogical Analitical Manual. 8th ed. Rev.
A. APHA International. Gaitherburg, VA
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Prss. London.
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological examination of Foods. APHA.
Washington
Bile Esculin Azide Agar
Xn
Ref. 01-592
R-22-32-52/53
S-7-46-61
Specification
Solid medium for the enterococci confirmative test in
water by the membrane filtration method according to
ISO 7899-2.
Formula (in g/L)
Tryptone ................................................. 17,00
Peptone .................................................... 3,00
Yeast extract ............................................. 5,00
Bile ......................................................... 10,00
Sodium chloride ....................................... 5,00
Esculin ...................................................... 1,00
Ammonium-ferric citrate ........................... 0,50
Sodium azide ............................................ 0,15
Agar ........................................................ 15,00
Final pH 7,2 ± 0,2
Directions
Suspend 56.6 g of powder in 1 L distilled water and bring
to the boil. Distribute in suitable containers and sterilize
in autoclave at 121ºC for 15 minutes. Cool to 50-60ºC
and pour plates to 3-5 mm thickness. These plates can
be stocked at 2-8ºC until two weeks.
Ref. 01-592 Bile Esculin Azide Agar. Entero-
coccus faecalis ATCC 29212
Description
The Bile Esculin Azide Medium is a modification of the
classical Bile Esculin proposed by Isenberg, Goldberg
and Sampson in 1970, with a reduction in the amount of
bile and the addition of the sodium azide. Brodsky and
Schieman shown that this medium, also know as Pfizer
Enterococci Selective gave best results with the filtration
technique.
The actual formulation is according the ISO Standard
7899-2:2000 for the second step in the confirmation and
enumeration of enterococci in water by the membrane
filtration method. The colonies previously selected in
the Slanetz-Bartley Agar (Ref. 01-579 + 6-023) must
be confirmed by a short incubation on the Bile Esculin
Azide Medium that permits the verification of the esculin
hydrolysis in a selective environment.
Technique
After an incubation of 24-48 hours on Slanetz Bartley
Agar (Ref. 01-579 + 6-023), the membrane filter that
show typical colonies is transferred, with sterile forceps
and upright position, to a pre-warmed plate of Bile
Esculin Azide Agar. After two hours of incubation at 44 ±
0,5ºC the membrane filter is inspected. All the typical col-
onies that show a brown to black colour in the surround-
ing medium are considered as positives and enumerate
as intestinal enterococci.
A heterogeneous distribution of the colonies or the pres-
ence of abundant and different micro organisms can
interfere with the differentiation of positive colonies.
References
ISO Standard 7899-2 (2000) Water Quality. Detection
and enumeration of intestinal enterococci. Part 2: Mem-
brane filtration method.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press Boca Raton. Fla.
ISENBERG, H.D., D. GOLDBERG & J. SAMPSON
(1970) Laboratory studies with a selective enterococcus
medium. Appl. Microbiol. 20:433
BRODSKY M.H. & D.A. SCHIEMANN (1976) Evalua-
tion of Pfizer Selective Enterococcus and KF media for
recovery of fecal streptococci from water by membrane
filtration. Appl. Environ. Microbiol. 31:695-699
27
 Blood Media
Blood Agar Base
Ref. 01-352
Specification
General purpose medium for the isolation and cultivation
of microorganisms.
Formula (in g/L)
Meat extract .............................................. 10,0
Tryptone ................................................... 10,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,3 ± 0,2
Directions
Suspend 40 g of powder in 950 mL. of distilled water and
let it soak. Heat to the boiling and distribute into contain-
ers. Sterilize by autoclaving at 121°C for 15 minutes. Let
it cool to 45-50°C and then add defibrinated blood in a
proportion about 7% or the desired enrichment.
Description
Blood Agar Base may be used for the cultivation of non
fastidious microorganisms, since it has a balanced nutri-
ent base .
For the fastidious microorganisms, it is advisable to add
special enrichments, such as ascitic liquid, egg yolk,
etc..
This medium, with the addition of blood, is very suitable
for studies in hemolytic activity, but for the isolation of
pathogens Blood Agar Base Columbia type (Ref. 01-
034) is more suitable.
References
ATLAS, R.M. and L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press, London.
Blood Agar Base No. 2
Ref. 01-505
Specification
Improved base with high nutrient properties especially
adapted for the growth of very fastidious
microorganisms, acc. ISO 7932 (2003).
Formula (in g/L)
Proteose peptone ..................................... 15,0
Liver extract ................................................ 2,5
Yeast extract ............................................... 5,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,0 ± 0,2
28
Directions
Suspend 42,5 g in 950 mL of distilled water and bring to
boil. Distribute into flasks and sterilize by autoclaving at
121ºC for 15 minutes. Cool to 45-50ºC and aseptically
add 7% of sterile defibrinated blood. Mix gently and pour
into plates.
Note: Blood and medium should be mixed in a big flask
to assure proper blood oxidation and mixing.
Description
Blood Agar Base No. 2 allows a maximum recovery of
weak organisms without altering or interfering in their
hemolytic reactions. Compared to other Blood Agar
bases, this one shows an equal or higher stimulatory
growth ability, however it specially helps the formation of
pigment in the chromogenic bacteria.
References
ATLAS, R.M. and L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press, London.
CASMAN, E. (1947) A noninfusion blood agar base for
neisseriae, pneumococci and streptococci. Am. J. Clin
Path. 17:281-289
FDA (1998) Bacterilogical Analitical Manual. 8th ed. Rev.
A. APHA International. Gaitherburg, VA
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological examination of Foods. APHA.
Washington
ISO 7932 Standard (2003). Microbiology of food and
animal feeding stuffs. Horizontal Methods for the enu-
meration of presumptive Bacillus cereus. Colony count
tecnique at 30°C.
Blood Agar Base (Columbia)
Ref. 01-034
Specification
Medium especially rich in peptone, appropriate for blood
addition or to prepare Chocolate Agar.
Formula (in g/L)
Casein Peptone ........................................ 12,0
Meat peptone ........................................... 11,0
Starch ......................................................... 1,5
Sodium Chloride ......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,3 ± 0,2
Directions
Add 44,5 g of powder to 950 mL of distilled water and
bring it to the boil. Distribute into suitable containers and
sterilize at 121°C for 15 minutes. To obtain Blood Agar
cool it to 45-50°C and aseptically add sterile defibrinated
blood at 5% proportion.
Description
Blood Agar Base contains an equilibrated mixture of
meat and casein peptones, being suitable for preparing
selective and as diagnostic media with the addition of
 Blood Media
blood or inhibitors. As it is presented, without additions, it
is also an excellent general culture medium.
Generally, Blood Agar base contains a casein peptone,
that aids big size colonies formation, or a meat petone,
that provides a well defined hemolysis halos or zones.
Blood Agar Base is prepared according to the Columbia
University formulation, and meets the two conditions
mentioned above.
Technique
Some applications for this base are:
Base Agar without either enrichment and inhibitors:
This medium supports growth of normal micro-
organisms as enterobacteriaand othyers more
strengh as Pasteurella, Brucella and Clostridium
perfringens.
Clostridium Selective Base Agar: Should a selective
clostridium medium be desired, add 240 mg/L
Sodium Azide and 180 mg/l Neomycin before the
sterilization, or alternatively the contents of SC
Inhibitor container (Ref. 06-012CASE).
Blood Agar: Aseptically add to the sterile medium 5%
sterile defibrinated sheep blood and cool it to
45°C. This way, medium is enriched and allows
the determination of typical hemolytic reactions
necessary for the identification of enterococci,
staphylococci and other microorganisms.
Selective Gram-positive cocci Blood Agar: Simulta-
neously at the time blood addition, also add 10
mg/L of colistine and 15 mg/L of Nalidíxic Acid,
or, the contents of a CP Inhibitor container (Ref.
06-013CASE), to obtain an excellent selective
medium for gram-positive cocci.
Note: Some authors recommend Blood Agar Base as
the maintenance medium for Campylobacter.
References
CASMAN, E. (1947) A non infusion blood agar base for
neiseriae, pneumococci and streptococci. Am. J. Clin.
Path. 17:281-289.
ATLAS, R.M. and L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press, London.
Blood Azide Agar Base
Xn
Ref. 01-242
R-22-32-52/53
S-7-46-61
Specification
Selective basal medium for staphylococci and strepto-
cocci isolation. With blood added can be used for the
determination of haemolytic reactions.
Formula (in g/L)
Tryptose .................................................. 10,00
Meat extract .............................................. 3,00
Sodium chloride ........................................ 5,00
Sodium azide ............................................ 0,20
Agar ........................................................ 15,00
Final pH 7,2 ± 0,2
Directions
Suspend 33 g of powder in 1 L of distilled water and
bring to the boil. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes. Cool to 45-
50ºC and aseptically add 5% of defibrinated blood. Pour
in sterile plates.
Description
This medium is a nutrient base (Tryptose and Meat
extract) with a suitable osmotic value (Sodium chlo-
ride) that includes a selective agent (Sodium azide) to
suppressing the growth of gram-negative bacteria. The
addition of 5% defibrinated blood supplies growth factors
for the fastidious microorganisms and is used for the
determination of the haemolytic patterns.
Technique
The plates are inoculated by the surface striking and
stabbing the agar several times to deposit inoculum be-
neath the agar surface, to show the hemolytic reaction of
both oxygen-stable and oxygen-labile streptolysins. After
an incubation of 18-24 and 48 ours in a suitable environ-
ment the hemolytic reactions are displayed:
Alpha-(α)-haemolysis is the reduction of haemo-
globin to met-haemoglobin that sows a greenish
decolourisation of the medium surrounding the
colony.
Beta-(β)-haemolysis is the total lysis of erythro-
cytes that produce a clear zone surrounding the
colony.
Gamma-(γ)-haemolysis means no haemolysis
and there is no change in the medium
Alpha-prime-(α’)-haemolysis is a small zone of
complete haemolysis that is surrounded by area
of partial lysis around the colony.
In the haemolysis studies must be in mind that the
haemolytic reactions are affected by several conditions:
atmosphere (aerobic, anaerobic or CO2 enriched) of in-
cubation, the composition of the culture media (presence
of sugars or growth factors) and the source of the blood
(horse rabbit, sheep, human…)
References
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press. London
ISENBERG, H.D. (1992) Clinical microbiology proce-
dures handbook. Vol. I ASM. Washington DC.
PACKER, R.A (1943) The use of sodium azide as an
inhibition substance of gram-negative bacteria. J. Infect.
Dis. 67:113
29
 Blood Media

RUOFF, K.L. (1995) Streptococcus p. 299-305, in P.R.
Murray et al. (ed.) Manual of Clinical Microbiology. 6th
ed. ASM Washington DC.
Blood Tryptose Agar Base
Ref. 01-551
Specification
Solid highly nutrient medium developed as Base for
Blood Agar for the isolation and cultivation of fastidious
microorganisms.
Formula (in g/L)
Tryptose ................................................. 10,00
Meat extract .............................................. 3,00
Sodium chloride ........................................ 5,00
Agar ........................................................ 15,00
Final pH 7,3 ± 0,2
Directions
Suspend 33 g of powder in 1 L of distilled water and let
it soak. Bring to the boiling and distribute into suitable
containers. Sterilize autoclaving at 121°C for 15 minutes.
Let it cool to 45-50ºC and then add 5-10% defibrinated
blood or the suitable enrichment. Homogenize and pour
plates.
Gamma- (γ)-haemolysis means no haemolysis
and there is no change in the medium
Alpha-prime- (α’)-haemolysis is a small zone of
complete haemolysis that is surrounded by area
of partial lysis around the colony.
In the haemolysis studies must be in mind that the
haemolytic reactions are affected by the environmental
conditions (aerobic, anaerobic or CO2 enriched) of incu-
bation, the composition of the culture media (presence
of sugars or growth factors) and the source of the blood
(horse rabbit, sheep, human…)
References
ATLAS, R.M. and L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press. London.
BALLOWS, A. & W.J. HAUSLER (1981) Diagnostic Pro-
cedures for Bacterial, Mycotic and Parasitic Infections 6th
Ed. APHA Washington D.C.
CASMAN, E.P. (1947) A non-infusion blood agar base for
neisseriae, pneumococci and streptococci. Am. J. Clin.
Pathol. 17:281-289
HARMON, S.M. et al. (1998) FDA Bacteriological
Analytical Manual. 8th ed. AOAC International. Gaithers-
burg.
ISENBERG, H.D. (1992) Clinical Microbiology Proce-
dures Handbook. Vol. I ASM. Washington DC.

Description
Casman proposed Blood Tryptose Agar Base in 1947 as
alternative medium without tissue infusion components.
Their original formulations include dextrose that inter-
feres with haemolytic reactions and it is omitted in the
present formulation.
This medium, with the addition of blood, is very suitable
for studies in haemolytic activity, but to isolate pathogens
Blood Agar Base Columbia type (Ref. 1-034) is more
suitable. This medium support the growth of a wide vari-
ety of fastidious microorganisms but several species of
Streptococcus and Neisseria require the addition of 1 g/L
of Yeast Extract for the optimal growth.

Technique
The plates are inoculate by the surface striking and stab-
bing the agar several times to deposit inoculum beneath
the agar surface, to show the hemolytic reaction of both
oxygen-stable and oxygen-labile streptolysins. After an
incubation of 18-24 and 48 ours in a suitable environ-
ment the hemolytic reactions are displayed:
Alpha- (α)-haemolysis is the reduction of haemo-
globin to met-haemoglobin that sows a greenish
decolourisation of the medium surrounding the
colony.
Beta- (β)-haemolysis is the total lysis of erythro-
cytes that produce a clear zone surrounding the
colony.

30
 BPRM Broth Base

Ref. 02-409 Description

BPRM (Bacteroides Phage Recovery Medium) was
Specification formulated according the specifications by the Microbi-
Base medium to cultivate Bacteroides fragilis and phage ology Department of the Barcelona University. Tartera
recovery from the environmental samples according to and cols. verified its efficiency in the Bacteroides fragilis
ISO Standard 10705-4. phage recovery used as human fecal pollution indica-
tors in environmental samples. Initially Kanamycin (100
mg/L) and Vancomycin (7,5 mg/L) were added to the
Formula (in g/L)
medium to prevent the growth of unwanted accompany-
Peptone .................................................. 10,00
ing bacteria, but later the Vancomycin was replaced by
Tryptone ................................................. 10,00
Nalidixic acid (100 mg/L).
Yeast extract ............................................. 2,00
BPRM Basal Broth can be used in double strengh and,
Sodium chloride ........................................ 5,00
with the addition of adequate amounts of agar, in the
Glucose .................................................... 1,80
preparation of solid or semisolid media.
Magnesium sulfate ................................... 0,12
L-Cysteine HCl ......................................... 0,50
Final pH 6,8 ± 0,2 References
TARTERA, C., R. ARAUJO, T. MICHEL and JOFRE
(1992) Culture and decontamination methods affecting
Directions
enumeration of phages infecting Bacteroides fragilis in
Dissolve 29,42 g of powder in 1 L of distilled water. Add
sewage. Appl. Environm. Microbiol. 58:8:2670-2673.
1 mL of a CaCl2 5% solution. Distribute in flasks and
ISO Standard 10705-4 (2001) Water Quality-Detection
sterilize in the autoclave at 121°C for 15 minutes. Cool
and enumeration of bacterigophages. Part 4: Enumera-
to 40°C and add 1 mL/L of 1% sterile soution of hemin
tion of bacteriophages infecting Bacteroides fragilis.
prepared in 0,02% NaOH. Mix well. Just before the
utilization add 25mL/L of a sterile solution of 10,6% (w/v)
NaCO3. Adjust the pH to 7,0 with HCl.

31
 Brain Heart Infusion Media (BHI Media)
Brain Heart Infusion Agar
(BHI Agar)
Ref. 01-599
Specification
General purpose solid medium for various fastidious
pathogenic microorganisms.
Formula (in g/L)
Brain Extract ............................................. 12,5
Heart Extract .............................................. 5,0
Proteose peptone ..................................... 10,0
Sodium chloride .......................................... 5,0
Di-sodium phosphate ................................. 2,5
Dextrose ..................................................... 2,0
Agar .......................................................... 15,0
Final pH 7,4 ± 0,2
Directions
Suspend 52 g of powder in 1 L of distilled water and
bring to the boil . Distribute in tubes or flasks and steri-
lize by autoclaving at 121°C for 15 minutes.
Brain Heart Infusion Broth
(BHI Broth)
Ref. 02-599
Specification
Liquid culture medium for general purpose according to
ISO 5944.
Formula (in g/L)
Brain extract ............................................. 12,5
Heart extract ............................................... 5,0
Proteose peptone ..................................... 10,0
Dextrose ..................................................... 2,0
Sodium chloride .......................................... 5,0
Di-sodium phosphate ................................. 2,5
Final pH 7,4 ± 0,2
Directions
Dissolve 37 g of powder in 1 L of distilled water, heating
up if necessary. Distribute into containers and sterilize in
the autoclave at 121°C for 15 minutes.
32
Description
Brain Heart Infusion is used for the cultivation of fastidi-
ous bacteria (streptococci, pnemococci, meningococci,
etc.) and also is recommended for the cultivation of
pathogenic fungi.
Growth of the accompanying bacterial flora can be
almost completely suppresed by adding 20 I.U. penicillin
and 40 µg streptomycin per mL culture medium.
If this medium is to be used for the selective isolation of
fastidious fungi (especially of Histoplasma capsulatum
and Blastomyces) add 10% sterile defibrinated blood
and from mixinfected samples add also 0,05 µg cy-
cloheximide/mL and 0,5 µg choramphenicol/mL.
This medium is not suitable for obtaining the character-
istic hemolytic reactions even after addition of the blood
because of its glucose contents.
References
ROSENOW, E.C. (1919) Studies on elective localization.
J. Dent. Research. 1:205-209
HAYDEN R.L. (1923) Elective localization in the eye of
bacteria from infected teeth. Arch. Int. Med. 32:828-849
HOWELL(1948) The efficiency of methods for the isola-
tion of Histoplasma capsulatum Public. Health. Reports
63, 173, 178.
CONANT (1950) Diagnostic Procedures and Reagents
3rd Ed., APHA, Inc., New York, 452.
DIN Standard 10163. Bestimmung koagulase positiver
Staphylokokken.
APHA-AWWA-AWPC (1998) Standard methods for the
examination of Water and Wastewater. 20th ed. Wash-
ington.
FDA (1998) Bacterilogical Analitical Manual. 8th ed. Rev.
A. APHA International. Gaitherburg, VA
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Prss. London.
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological examination of Foods. APHA.
Washington
ISO 5944 Standard (2001) Milk and milk based products:
Detection of coagulase-positive staphylococci - MPN
Technique.
 Brilliant Green Media
Brilliant Green Agar (BGA)
(Eur. Phar. Agar Medium L)
Ref. 01-203
Specification
Medium for Salmonella isolation, according to the Euro-
pean Pharmacopoeia
Formula (in g/L)
Meat peptone ....................................... 5,0000
Casein peptone .................................... 5,0000
Sodium chloride .................................... 5,0000
Yeast extract ......................................... 3,0000
Lactose ............................................... 10,0000
Sucrose .............................................. 10,0000
Phenol Red ........................................... 0,0800
Brilliant Green ....................................... 0,0125
Agar .................................................... 15,0000
Final pH 7,0 ± 0,2
Directions
Suspend 53 g of powder in 1 L of distilled water and heat
to boiling with constant stirring. Dispense into containers
and sterilize at 121°C for 15 minutes.
Description
BGA is a differential selective medium, able to detect
the presence of enteropathogenic bacteria in different
samples. This medium is a modification to Kauffman’s
original formulation, and it complies with the ISO, HMO,
Eur. Phar., USP and APHA
specifications.
Since it has a high brilliant green concentration, it inhibits
notably the growth of most bacteria, except Salmonella.
However, S. typhi and S. paratyphi are also inhibited.
Therefore, when their presence or Shigella is suspected,
it is recommended to use other media in parallel, as
Deoxycholate Lactose Agar (Ref. 01-057), MacConkey
Agar (Ref. 01-118), Salmonella Shigella Agar (Ref. 01-
171), Xylose Lysine Deoxycholate Agar (Ref. 01-211 or
1-552) or Endo Agar Base (Ref. 01-589) which are less
inhibitory.
Presence of lactose and sucrose allows a good dif-
ferentiation between Salmonella, which produce pink
or colourless colonies with a red halo or zone, and the
companion flora, which produce smaller and green yel-
lowish colonies with yellow halo, due to acid created by
lactose and/or sucrose fermentation.
Osborn and Stokes suggested the addition of 0,08 g/L of
sulfadiacine or 1 g/L of sulfapyridin in order to make this
medium more selective for Salmonella and provide suit-
able qualities to this medium to perform the examination
of food and eggs and their derivatives.
References
U.S.PHARMACOPOEIA (2002) 25 ed. Chap. <61> “Mi-
crobial Limit Tests”. USP/NF Conv.Inc. Rockville.MD
KAUFFMAN, F. (1935) Weitere Erfahrungen mit der
kombinierten Anreicherungsverfahren für Salmonellaba-
zillen Z. Hyg. Infekt. Krhn, 117; 26-32
EUROPEAN PHARMACOPOEIA,Supplement 4.2
(2002), 4th Ed., Council of Europe,Strasbourg, France
Brilliant Green Bile 2% Broth
Ref. 02-041
Specification
Liquid medium for the detection of coliforms in water,
as recommended by APHA and ISO 4831 and 9308-1
Standards.
Formula (in g/L)
Bile ....................................................... 20,000
Lactose ................................................. 10,000
Peptone ................................................ 10,000
Brilliant Green ......................................... 0,013
Final pH 7,2 ± 0,2
Directions
Dissolve 40 g of powder in 1 L of distilled water and
bring to the boil. Distribute into containers containing
Durham tubes and sterilize by autoclaving at 121°C for
15 minutes.
Description
Brilliant Green Bile 2% Broth has been widely used as
a medium for the assay of presumptive colimetries in
food, milk and water, through the Most Probable Number
Technique. This broth offers some advantages over
other similar broths as its balanced composition of bile
and Brilliant Green effectively suppresses the growth of
gram-positive bacteria, even that of the more fastidious
Clostridium perfringens.
It is recommended by the APHA for colimetries in water,
milk and food.
British and Australian methodology use the broth as an
intermediate stage between presumptive and confirma-
tive colimetry, as it was an enrichment at 32°C. Other
authors suggest it as an optimal base for the Eijkman
testing of gas production at 44°C, for the identification
of E. coli.
This medium can be used as Presumptive broth for
E.coli (by fluorescent reaction) if before sterilization
MUG (Ref. 06-102CASE) is added.
References
APHA (1985) Standard Methods for the Examination of
Water and Wastewater, 16th ed. Washington.
VANDERZANT, C., SPLITTSTOESSER, D.F.(1992)
Compendium of Methods for the Microbiological Exami-
nation of Food. 3rd. Ed. APHA. Washington.
33
 Brilliant Green Media

DOWNES F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 4rd. Ed.
APHA. Washington.
FDA (1998). Bacteriological Analitical Manual.8th ed.
Rev. A AOAC International, Gaithersburg MD..
PASCUAL ANDERSON, MªRª (1992) Microbiologia
Alimentaria. Diaz de Santos, S.A.,Madrid.
ISO 4831 Standard (1991) General guidance for the
enumeration of coliforms. MPN technique.
ISO 9308-1. Standard (1990) Water Quality-Detection
and enumeration of coliforms, thermotolerants coliforms
and presumptive E.coli. MPN Method.
Brilliant Green Modified Agar
Ref. 01-309
Specification
Solid culture medium for selective isolation of Salmonel-
lae in food (except S. typhi) according ISO 6579, 6340,
6785 and IDF 93 Standards.
Formula (in g/L)
Peptone ................................................ 10,000
Meat extract ............................................ 5,000
Yeast extract ........................................... 3,000
Lactose ................................................. 10,000
Sucrose ................................................ 10,000
Disodium phosphate ............................... 1,000
Sodium phosphate ................................. 0,600
Phenol red .............................................. 0,090
Brilliant green ......................................... 0,005
Agar ...................................................... 15,000
Final pH 6,9 ± 0,2
Directions
Suspend 54,5 g of powder in 1 L of distilled water. Let it
soak and heat up to boiling with constant stirring. Distrib-
ute in plates. Do not autoclave.
This formulation was subsequently adopted by the ISO
and DIN official method for detecting Salmonellae in
meat.
Technique
A previous enrichment in Tetrathionate Base Broth
(Ref.2-033) is recommended. Inoculate on the surface
of this plate medium in order to get separate colonies.
Incubate at 35-37°C for a 18-24 hours period.
Salmonella colonies (except S.typhi) are red, pinkish or
white, but they are always surrounded by a red halo or
zone, which shows the non lactose or sucrose fermen-
tation. Colonies of lactose and/or sucrose fermenting
bacteria produce yellow-green colonies surrounded by a
yellow halo. Sometimes, Proteus or Pseudomonas may
appear, and they produce red pointed colonies.
In very polluted samples, it is recommended to include
1 g/L of sodium sulfacetamide and 250 mg/L of sodium
mandelate.
References
DIN. 10181 Mikrobiologische Milchuntersuchung. Nach-
weis von Salmonellen. Referenzverfahren.
DIN 10160. Untersuchung von fleisch und fleischerzeng-
nissen. Nachweis von Salmonellen. Referenzverfahren
ISO Standard 6579 Meat and meat Product. Detection
of Salmonellae. Reference Method. (1993)
PASCUAL ANDERSON MªRª (1992) Microbiología Ali-
mentaria. Diaz de Santos, S.A.Madrid,.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc.,Boca Raton, Fla.
ISO 6785 Standard (2001) Milk and milk products - De-
tection of Salmonella spp.
FIL-IDF Standard 93 (2001) Milk and milk based prod-
ucts - Detection of Salmonella spp.
ISO 6340 Standard (1995) Water Quality - Detection of
Salmonella spp.

Description
In this modification of the classical medium for Salmo-
nellae, the concentration of brilliant green has been
reduced to obtain a less inhibitory medium. At the same
time, the nutrient basis has been enriched to enhance
the recovery of those microorganisms that are weakened
during the food production process.

Ref. 01-203 Brilliant Green Agar.

Salmonella
typhimurium E. coli
ATCC 14028 ATCC 25922

Ref. 02-041 Brilliant Green Bile 2% Broth. Left: control;

center: E.coli ATCC 25922; right: Salmonella typhimu-
rium ATCC 14028
Control
34
 Bromcresol Purple Base Broth
Ref. 02-031
Specification
Liquid culture medium with indicator to carry out sugar
fermentation studies.
Formula (in g/L)
Gelatin peptone ...................................... 10,00
Sodium chloride ........................................ 5,00
Bromocresol purple .................................. 0,02
Final pH 6,8 ± 0,2
Directions
Dissolve 15 g of powder in 1 litre of distilled water. Add
substrate to assay in the desired concentration and
distribute into containers provided with Durham’s tubes.
Sterilize in the autoclave at 121°C for 10 minutes. Heat
up the autoclave before putting in the tubes to avoid
sugar caramelization.
Addition of some kind of sugars may require a pH adjust-
ment.
To study the fermentation of some sugars like Glucose
(Ref. 06-048), Lactose (Ref. 06-051), Maltose (Ref. 06-
052), Mannitol (Ref. 06-050) and Sucrose (Ref. 06-049)
it is advisable to add 10 g/L of each one.
Brucella Media
Brucella Agar
Ref. 01-042
Specification
Solid medium for the cultivation of Brucella and other
fastidious microorganisms
Formula (in g/L)
Peptone of casein ..................................... 10,0
Meat Peptone ........................................... 10,0
Yeast Extract .............................................. 2,0
D(+) Glucose ............................................. 1,0
Sodium chloride .......................................... 5,0
Sodium bisulphite ...................................... 0,1
Agar .......................................................... 15,0
Final pH 7,0 ± 0,2
Directions
Suspend 43 g of powder in 1 L of distilled water and
bring to the boil. Distribute into suitable containers and
sterilize in autoclave at 121ºC for 15 minutes. If a high
selectivity medium is wanted, add 2 flasks of Brucella
Selective Supplement (Ref. 06-025CASE). When Ethyl
Violet 1,25 mg/L is added to the culture medium, the
growth of biotype 2 of Brucella abortus is inhibited.
Some microbiologists promote this medium as suitable
for Campylobacter maintenance.
Description
Bromcresol Purple Base Broth is the liquid version suit-
able to determine gas production (by Durham´s tubes)
by enterobacteria which are sensitive to phenol red.
In the SCHARLAU formulation, meat extract has been
omitted as it was found unnecessary and it also provid-
ed low concentrations of fermentable sugars that could
change or give erroneous results.
The bacteria when ferment the carbohydrates, changes
the medium colour to yellow due to Bromcresol purple
pH indicator, and if they produce gas, it is retained in the
Durham’s tube.
References
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc.,Boca Raton, Fla.
Brucella Broth
Ref. 02-042
Specification
Liquid culture medium for Brucella and other fastidious
microorganisms
Formula (in g/L)
Peptone of casein ..................................... 10,0
Meat Peptone ........................................... 10,0
Yeast Extract .............................................. 2,0
D(+) Glucose ............................................. 1,0
Sodium chloride .......................................... 5,0
Sodium bisulphite ...................................... 0,1
Final pH 7,0 ± 0,2
Directions
Dissolve 28 g of powder in 1 L of distilled water, heat-
ing if necessary. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes. If a highly
selectivity is wanted, add aseptically after autoclave,
Brucella Selective Supplement (Ref. 06-025CASE).
When Ethyl Violet 1,25 mg/L is added to the culture
medium, the growth of biotype 2 of Brucella abortus is
inhibited.
35
 Brucella Media
Description
The Brucella Media are prepared from composition of
the APHA’s Albimi Broth used for isolation of Brucella
species and the only difference between Broth and Agar
is the solidifying agent. Both media are suitable for the
isolation and cultivation of a lot of fastidious microorgan-
isms including Streptococcus, Neisseria and Campylo-
bacter, but they became selective with the addition of
antibiotics like polymyxin or bacitracin or chemical inhibi-
tors like cycloheximide and ethyl violet. With some dyes
(fuchsin and thionin) the media became differential. See
the suitable reference for the technique in every case.
Caution
Brucella species are classified as Biosafety Level
3 pathogens. All manipulations with live cultures and
antigens must be confined to a Class II Biological Safety
Cabinet. Follow proper established laboratory proce-
dures in handling and disposing of infectious materials.
Bryant & Burkey Media
Bryant & Burkey Lactate Broth
(BB-Lactate Broth)
Ref. 02-421
Specification
Liquid medium for the enumeration of spores of lactate-
fermenting clostridia in milk and dairy products
Formula (in g/L)
Peptone .............................................. 15,0000
Meat extract .......................................... 7,5000
Yeast extract ......................................... 5,0000
L-Cysteine HCl ..................................... 0,5000
Resarzurine .......................................... 0,0025
Sodium acetate .................................... 5,0000
Sodium lactate ...................................... 5,0000
Final pH 6,2 ± 0,2
Directions
Dissolve 38 g of powder in 1 L of distilled water, heating
up only if necessary. Distribute in suitable containers for
every procedure and sterilize in the autoclave at 121°C
for 15 minutes.
36
References
ALTON, G.G, L.M. JONES & D.E. PIETZ (1976) Las
técnicas de laboratorio en la Brucelosis, 21 ed. Mono-
graph nº.55 FAO/WHO. Geneve.
CRUICKSHANK.(1965) Medical Microbiology. 11th ed.
E.S. Livingstone. Edimburgo.
ISENBERG H.D. (1992) Clinical Microbiology Proce-
dures Handbook. ASM. Washington D.C.
MacFADDIN J.D. (1985) Media for Isolation-cultivation-
identification-maintenance of medical bacteria. William &
Wilkins Baltimore MD.
VANDERZANT, C & D.F. SPLITTSTOESSER (1992)
Compendium of methods for the microbiological exami-
nation of food 3rd Ed. APHA. Washington D.C.
Description
BB medium is used to enumerate, by MPN technique,
the spores of gasogenic clostridia that are the produc-
ers of the swelling and rancidness of cheese in the dairy
industry (late blowing or butyric swelling). In normal
conditions of use,the medium allows the growth of other
microorganims also which are not directly related to the
cheese alteration, e.g. Cl. butyricum or Cl. sporogenes,
besides the main responsible organism Cl. tyrobutyri-
cum, in presence of enough acetate, Clostridium tyrobu-
tyricum ferments lactate, producing acetic and butyric
acids, CO2 and hydrogen.
Technique
Recommended technique is to enumerate the spores by
the MPN technique with fresh made medium, covered
with a vaspar layer of 2 mL that acts as a cap that as-
sures a low redox potential and at the same time retains
gas produced in the reaction.
Sample must be previously decontaminated by heat-
ing up for 10 minutes at 75°C in order to destroy all the
vegetative forms and only leaving the alive spores.
Incubation is performed for 7 days at 37°C. Tubes will
be declared positive if they show a clear gas produc-
tion. Despite the fact that results are often expressed as
butyric spores/mL, the limitations of the method suggest
that they should be expressed as lactate fermenting
clostridial spores.
 Bryant & Burkey Media

Bryant & Burkey Modified
Fluid Medium
Ref. 03-557
Specification
Fluid medium for the enumeration of spores of lactate-
fermenting clostridia in dairy products
Formula (in g/L)
Tryptose .............................................. 15,0000
Meat Extract ......................................... 7,5000
Yeast Extract ........................................ 5,0000
Cystine HCl........................................... 0,6000
Resazurine ........................................... 0,0025
Sodium lactate ..................................... 3,0000
Sodium acetate .................................... 5,0000
Sodium thioglycollate ........................... 0,2000
Agar ...................................................... 0,7500
Final pH 5,9 ± 0,2
maintaining its anaerobic condition by the thioglycollate
added. Also the amount of lactate is reduced because
the density of the medium retains easily the gas bubbles
produced.
Technique
Samples are processed by established procedures in
every product.
References
BERGÈRE, J. L. & S. SIVELA (1989) Detection and enu-
meration of clostridial spores related to cheese quality.
Classical and new method. FIL-IDF Bull. 51:18-23
BRYANT M.P. & L.A. BURKEY (1956) The characteris-
tics of lactate-fermenting spore-forming anaerobes from
silage. J. Bacteriol. 71:43-46
ROSENBERGER, K.F. (1951) The development of meth-
ods for the study of obligate anaerobes in silage. Proc.
Soc. Appl. Bacteriol. 14:161-164

Directions
Suspend 37 g of powder in 1 L of distilled water and
bring to the boil. Distribute in suitable containers for
every procedure and sterilize in autoclave at 121ºC for
15 minutes.

Description
This modification of the Bryant & Burkey Lactate Broth
(Ref. 02-421) add a little amount of agar that makes
the medium more thick to be used in greater volumes

Buffered Peptone Water Eur Phar (Buffered

Sodium Chloride-Peptone Solution pH 7.0)
Ref. 02-494
Specification
Diluent for the homogenization of samples according to
the European Pharmacopeia and ISO 21149.
Formula (in g/L)
Peptone .................................................... 1,00
Sodium chloride ........................................ 4,30
Disodium phosphate ................................. 7,23
Potassium phosphate ............................... 3,56
Final pH 7,0 ± 0,2
Directions
Dissolve 16 g of powder in 1 L of distilled water, heating
up if necessary. Add 1 to 10 mL of Polysorbate 80 (Ref.
06-088) or Polysorbate 20 depending on the type of food
to be diluted. Homogenize and distribute into containers.
Sterilize by autoclaving at 121ºC for 15 minutes.
Description
This solution is recommended by European Pharmaco-
poeia to dilute the sample for microbiological examina-
tion. Depending on the amount of fat in the sample to
examine it is the technician’s decision regarding the kind
and quantity of emulsifying agent to be used.
References
EUROPEAN PHARMACOPOEIA (2007) 5ª ed. Suppl.
5.6 § 2.6.13 Microbiological examination of non-sterile
products. EDQM Council of Europe. Strasbourg.
ISO 21149:2006 Cosmetics – Enumeration and detec-
tion of aerobic mesophilic bacteria.

37
 Casein Lecithin Polysorbate Broth Base

Ref. 02-539 Description

Specification
Liquid medium to dilute and neutralize samples of phar-
maceutical, cosmetic, raw material or end-products for
the purpose of enumeration
Formula (in g/L)
Casein peptone ..................................... 20,00
Soya lecithin ............................................ 5,00
Final pH 7,3 ± 0,2
Directions
Dissolve 25 g of powder in 960 mL of distilled water pre-
warmed at 50ºC . Add 40 mL of polysorbate 20, homog-
enize and distribute in suitable containers. Sterilize in
autoclave at 121ºC for 15 minutes.
This medium is produced according the formulation of
the U.S. Pharmacopoeia. In the Section <61> “Microbial
Limit Tests” is proposed as alternative system to neutral-
ize preservatives and didsinfectants before to proceed
with the enumeration process, specially by the mem-
brane filtration method.
References
US PHARMACOPOEIA (2002) <61> Microbial Limits
Tests. 25th ed. US Pharmacopoeial Conv. Inc. Rockville.
MD

Caseinate Agar

Ref. 01-569 Technique

Pour the caseinate agar into plates so that the medium
Specification is 2 mm thick (e.g. 12 mL for a 9-cm plate). After the me-
Solid medium, acc. Standard Methods, for the detection dium is hardened, plate 0,1 mL quantities of the sample
and enumeration of proteolytic organisms in dairy prod- or diluted sample on the agar surface and spread evenly
ucts with a sterile Drigalski rod. To ensure absorption of the
sample allow inoculated plates to dry for 15 minutes,
and incubate them for 48 to 72 hours at 32±1ºC or for 72
Formula (in g/L)
hours at 21±1ºC. Colonies surrounded by a white or off-
Tryptone ................................................... 5,00
white zone of casein precipitate are proteolytic. Highly
Yeast extract ............................................. 2,50
proteolytic bacteria will also produce a clear inner zone.
Dextrose ................................................... 1,00
Report results as “proteolytic count per gram or mL”.
tri-Sodium citrate ...................................... 4,41
Designate the medium used and the incubation condi-
Sodium caseinate ................................... 10,00
tions.
Calcium chloride ....................................... 4,40
Agar ........................................................ 15,00
Final pH 7,0 ± 0,2 References
MARTLEY, F.G., S.R. JAYASHANKAR & R.C. LAW-
RENCE. (1070) An improved agar medium for the detec-
Directions
tion of proteolytic organisms in total bacterial counts. J.
Suspend 42,3 g of powder in 1 L of distilled water and
Appl. Bacteriol. 33:363-370
bring to the boil. Distribute in suitable containers and
FRANK, J.F. G.L CHRISTEN & L.B. BULLERMAN
sterilize in autoclave at 121ºC for 15 minutes.
(1992) Test for groups of Microorganisms. In R. T.
Marshall (ed) Standard Methods for the examination of
Description Dairy Products. APHA. Washington D.C.
Caseinate Agar is preferred to other media like Skimmed
Milk Agar for the detection of proteolytic organisms
because its better recovery of stressed cells and the
absence of false positives.

38
 Cetrimide Agar (Pseudomonas Selective Agar)
(Eur. Phar. Agar Medium N)
Ref. 01-160
R-52/53
Specification
Solid culture medium for selective isolation of Pseu-
domonas aeruginosa acc. to ISO 22717.
Formula (in g/L)
Gelatin peptone ........................................ 20,0
Magnesium chloride ................................... 1,4
Potassium sulfate ..................................... 10,0
Cetyltrimethyl-Ammonium Bromide .......... 0,3
Agar .......................................................... 15,0
Final pH 7,2 ± 0,2
Directions
Suspend 46,7 g of powder in 1 L of distilled water and
add 10 mL of Glycerol. Bring to the boil and distribute
into suitable containers. Sterilize at 121°C for 15 min-
utes.
N References
LOWBURY,E.J.L. & A.G. COLLINS (1955) The use of a
S-61 new cetrimide product in a selective medium for Pseu-
domonas aeruginosa J. Clin. Path. 8.47
BROWN, V.I. & J.L. LOWBURY (1965) Use of an im-
proved Cetrimide Agar Medium and of culture methods
for Pseudomonas aeruginosa. J. Clin. Path. 18.752
FDA (1998) Bacteriological Analytical Manual. 8th ed.
Rev. A. AOAC International. Gaitherburg. VA.
ATLAS, R.M. and L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press Inc.,Boca Raton,Fla.
EUROPEAN PHARMACOPOEIA,(2002) Supplement4.2
4th ed., 2.6.13 Microbiological examination of non-sterile
products. Council of Europe,Strasbourg,.
U.S. PHARMACOPOEIA (2002) 25 ed. <61> Microbial
limit tests. Us Pharmacopoeial Conv. Inc. Rockville, MD
ISO 22717:2006 Cosmetics – Detection of Pseu-
domonas aeruginosa.

Description control
The Cetrimide Agar is based on the enormous resistance
of Ps. aeruginosa strains to the Quaternary Ammonium
Compounds (QAC’s). With regard to the Cetyltrimethyl-
Ammonium Bromide there has been growth at 1 g/L
concentrations, but in such cases it has been very poor
and slow.
An inhibitor concentration of 0,3-0,5 g/L does not seem
to affect the viability of the pyogenic species. Neverthe-
less, it does inhibit the rest of the fastidious accompa-
nying bacteria, both gram-positive and gram-negative,
as well as other species of Pseudomonas which may Pseudomonas aeruginosa ATCC
develop at lower inhibitory concentrations. 27853

Although Ps. aeruginosa prevails over any other fastidi-

ous bacteria after a 48 hour incubation at 35°C, it is
recommended to first isolate at 42°C with an incubation
of 48 hours. By this method, almost complete inhibition
of other microorganisms is obtained.

39
 Chapman-Stone Agar
Ref. 01-052
Specification
Solid and differential medium with a high selective ability,
for the isolation of staphylococci from food.
Formula (in g/L)
Peptone .................................................... 10,0
Yeast extract ............................................... 2,0
Gelatin ...................................................... 30,0
D-Mannitol ................................................ 10,0
Sodium chloride ........................................ 55,0
Ammonium sulfate .................................... 75,0
Dipotassium phosphate .............................. 5,0
Agar .......................................................... 15,0
Final pH 7,0 ± 0,2
Directions
Suspend 202 g of powder in 1 L of distilled water and
heat up in boiling water bath until the total dissolution of
gelatin. Bring to the boiling. Distribute in tubes or flasks
and sterilize at 121°C for 15 minutes. Pour into plates
immediately. Avoid overheating.
Description
Chapman-Stone medium is according to a modification
of the classical 110 medium for Staphylococcus. The
main modification consists the inclusion of ammonium
sulfate, that allows the direct reading or observation of
gelatin hidrolysis, instead of adding reagents to the plate
medium. Another modification is the reduction in the
amount of sodium chloride. The saline content is selec-
tive itself, but it is reinforced by the ammonium sulfate,
which is also selective. Finally, lactose has been omitted
, making the difference between the colonies that fer-
Ref. 01-053 Chapman TTC Agar.
control
E. coli ATCC 25922
(MF technique)
40
ment mannitol (biqger size) and the colonies that do not
ferment mannitol (smaller size) greater, since the latter
must grow solely depending on peptone as the nutrient
source.
Technique
Material under test is inoculated on the surface to pro-
duce separated colonies, and is incubated at 30°C for
a 48 hours period. After this time, examine and select
colonies on the basis of these criteria:
White or non pigmented colonies are discarded, even if
they show a gelatin liquefaction halo (coagulase nega-
tive).
Golden yellow pigmented colonies, surrounded by a
clear zone of gelatin hydrolysis (positive Stone’s reac-
tion) are selected to verify mannitol fermentation and
further, coagulase and hemolysis.
Mannitol fermentation is checked by adding a few drops
of Bromcresol purple indicator (aq. sol. 0,04%) over the
colonies. Any colour change indicates positive mannitol
fermentation.
Coagulase production and hemolysis tests are per-
formed in later subcultures on suitable media.
Chapman-Stone medium composition is very selective,
and so, medium can be employed without real steriliza-
tion. However, it is always advisable to sterilize it and
keep one control before use.
References
CHAPMAN (1948).An improved Stone Medium for the
isolation and testing of food-poisoning staphylococci
Food Research 13:100-105
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc.London
E. coli ATCC 25922 Salmonella typhimurium ATCC 14028
Salmonella typhimurium
ATCC 14028
(MF technique)
 Chapman TTC Agar (Tergitol® 7 Agar)
Ref. 01-053
Specification
Medium for the detection of coliforms by membrane fil-
tration technique in water analysis acc. ISO 9308-1:2000
standard.
Formula (in g/L)
Meat peptone ......................................... 10,00
Meat extract .............................................. 5,00
Lactose .................................................. 20,00
Yeast extract ............................................. 6,00
Bromothymol blue ................................... 0,05
Sodium heptadecyl sulfate ....................... 0,10
Agar ........................................................ 15,00
Final pH 7,2 ± 0,2
Directions
Suspend 56,2 g in 1 L of distilled water and bring to the
boil. Distribute into suitable containers and sterilize by
autoclaving at 121°C for 15 minutes. Cool to 45-50°C.
Add 2-3 mL/L of filter sterile 1% aqueous 2,3,5-triphe-
nyltetrazolium chloride (TTC) (Ref. 06-023) and pour
plates. Do not reheat.
Description
This medium is formulated for the presumptive iden-
tification of coliforms in drinking water, by membrane
filtration technique.
Due to the instability the triphenyltetrazolium is provided
in a separate container, sterilized and ready to use.
Poured plates can be stored refrigerated for up to 8 days
without losing their effectiveness. They should not be
used if any dehydration or drying signs appear.
Technique
While using the membrane filter technique for the pre-
sumptive identification of coliforms in water, it should be
kept in mind that to every type of water corresponds a
minimum volume to be filtered and depends on the type
of water. Dilute with sterile phosphate buffer if necessary
to obtain the number of colonies on the membrane which
are easy to count.
For every water sample two volumes or double quantity
must be filtered over different membranes and incubated
on Tergitol 7 Agar at 35°C and 44°C respectively.
After 48 hours the typical colonies have the appearance
shown in the table.
Most coliform can not grow on this medium when incu-
bated at 44°C, except E. coli which forms a characteris-
tic appearance.
Results are always expressed per 100 mL sample in-
cluding any applied dilutions. Estimation is done by tak-
ing typical colonies which have grown at 35°C as fecal
coliform, together with those grown at 44°C as E.coli.
Nevertheless, according to the legislation and despite
the medium’s selectivity, results can only be considered
as presumptive and all coliform colonies have to be con-
firmed by following the criteria stated below:
Typical appearance in EMB Agar (Ref.1-068) or Endo
Agar Base (Ref.1-589); characteristic reactions in Kligler
Iron Agar (Ref.1-103). For the confirmation of faecal E.
coli, verification of it is: a motile,gram-negative bacillus
and lactose fermentator with acid and gas production,
which gives negative results on the citrate test and
indole production positive.
References
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc. London.
CHAPMAN G.H. (1951). A culture medium for detect-
ing and confirming E. coli in ten hours. Am. J. Publ. Hlth
41:1381-1386.
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Foods. 3rd
ed. APHA.Washington.
GUINEA, SANCHO,PARES (1979). Análisis Microbiológ-
ico de Aguas. Ed. Omega. Barcelona.
ISO 9309-1:2000 Standard. Water Quality. Detection and
enumeration of Escherichia coli and coliform bacteria.-
Part 1: Membrane filtration method.
SPECK, M (Ed.) (1982) Compendium of Methods for the
Microbiological Examination of Foods. 2nd. Ed. APHA.
Washington.
41
 Chloramphenicol Glucose Media
Chloramphenicol Glucose Agar
(CGA) (Yeast Extract Dextrose
Chloramphenicol Agar
acc. FIL-IDF)
T T
Ref. 01-366
R-45
S-53-45
Specification
Solid and selective medium for the isolation and enu-
meration of fungi in milk and dairy products according
ISO 7954.
Formula (in g/L)
Dextrose ................................................... 20,0
Yeast extract ............................................... 5,0
Chloramphenicol ........................................ 0,1
Agar .......................................................... 15,0
Final pH 6,6 ± 0,2
Directions
Suspend 40 g of powder in 1 L of distilled water and let
it soak. Bring to the boil and distribute into containers.
Sterilize in the autoclave at 121°C for 15 minutes.
Description
This is the medium recommended by the Federation In-
ternational Laitrere - International Dairy Federation (FIL-
IDF) for the isolation and enumeration of fungi (moulds
and yeast) in milk and dairy products. This medium has
also been adopted by the DIN and ISO standard.
Medium relies its selectivity on the bactericidal action of
Chloramphenicol which, due to its thermostabity, may
be sterilized with the complete medium in the autoclave.
Moreover, pH may be adjusted near to neutrality, and
this fact allows the medium to be remelted several times
without affecting its stability, selectivity and efficacy.
Remeltings and overheatings may make the medium
darker.
Technique
Generally the mass seed method or poured plate
method is used to inoculate the medium, and an incuba-
tion at 22-25°C for 4 to 5 days is carried out.
42
Chloramphenicol Glucose Broth
(CGB) (Yeast Extract Dextrose
Chloramphenicol Broth
acc. FIL-IDF)
Ref. 02-366
R-45
S-53-45
Specification
Liquid and selective medium for the enumeration of fungi
in milk and dairy products using the MPN technique.
Formula (in g/L)
Dextrose ................................................... 20,0
Yeast extract ............................................... 5,0
Chloramphenicol ........................................ 0,1
Final pH 6,6 ± 0,2
Directions
Dissolve 25 g of powder in 1 L of distilled water. Distrib-
ute in tubes and sterilize in the autoclave at 121°C for 15
minutes.
Description
This is the liquid version of the medium with the same
name (Ref. 01-366) recommended by the Federation
International Laitrere - International Dairy Federation
(FIL-IDF) for the enumeration of fungi (moulds and
yeasts in liquid products).
Medium is specially adapted to enumerate yeast and,
eventually, moulds by the Most Probable Number tech-
nique (MPN).
References
FIL-IDF 94B Standard (1991). Enumeration of yeast and
moulds. Colony Count Technique at 25 °C.
ISO 7954 Standard (1987) General guidance for enu-
meration of yeast and moulds - Colony count at 25ºC.
DIN Standard 10186. Mikrobiologische Milch Untersu-
chung. Bestimmung der Anzahl von Hefen und Schim-
melpilzen. Referenzverfahren.
 Citrate Azide Agar
Ref. 01-558
R-45-22-32-52/53
Specification
Selective differential solid medium for the detection and
enumeration of streptococci in dairy products.
Formula in g/L
Tryptone ................................................. 10,00
Yeast extract ........................................... 10,00
Sodium citrate ........................................ 20,00
Sodium azide ............................................ 0,40
Tetrazolium blue ....................................... 0,10
Agar ........................................................ 15,00
Final pH 7,0 ± 0,2
Directions
Suspend 55,5 g of powder in 1 L of distilled water and
bring to the boil. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes.
Description
Citrate Azide Agar is the streptococci detection medium
preferred in dairy microbiology over the other media like
KF-Streptococcal Agar (Ref. 01-294) used in food micro-
biology because its high selectivity and security of use.
T Technique
Pour the melted medium cooled to 45-50ºC over the
S-53-45-7-61 sample in a petri dish. Let it solidify and dose another
5-7 sterile volume of medium on the surface to facilitate
the micro-aerofilic environment. The plates are incubated
at 37ºC for 48-72 hours and then the blue colonies are
counted. Express the results as “streptococci per g or
mL of sample”
References
MARSHALL, R.T. (1992) Standard Methods for the
Examination of Dairy Products. 16th Ed. APHA. Washing-
ton. DC
HARTMANN P.A, R.H. DEIBEL, & L.M. SIEVERDING
(1992) Enterococci. en Vanderzant & Splittstoesser:
Compendium of methods for the microbiological exami-
nation of foods. 3rd Ed. APHA. Washington DC.
EFTHYMIOU C.J., P. BACCASH, V.J. LABOMBARDI, &
D.S. EPSTEIN (1974) Improved isolation and differentia-
tion of enterococci in cheese. Appl. Microbiol. 28:417-
422
SARASWAT, D.S. G.W. REINBOLD & W.S. CLARK
(1963) Selection of a medium for the isolation and enu-
meration of enterococci in dairy products. J. Milk Food
Technol. 26:114-118
43
 CLED Agar (Brolacin Agar)

Ref. 01-047 Staphylococci coagulase(-ve): White or light yellow

Specification
Cystine,lactose,electrolyte deficient medium, recom-
mended for the isolation and identification of urinary
pathogenic bacteria.
Formula (in g/L)
Peptone ................................................. 4,000
Trypsic Peptone ..................................... 4,000
Meat extract ........................................... 3,000
Lactose ................................................. 10,000
L-Cystine ............................................... 0,128
Brom Thymol Blue ................................. 0,020
Agar ...................................................... 15,000
Final pH 7,4 ± 0,2
colonies, with halo and the same size as of
Enterococci.
Proteus sp.: Blue, translucent and smaller than E.coli.
Pseudomonas aeruginosa: Plain, matt and wrinkled
colonies. Green colour and irregular boundary.
Corynebacteria: Pointed and grey colonies
Lactobacilli: Matt colonies, like Corynebacteria.
References
SANDYS, G.H. (1960) A new method of preventive
swarming of Proteus sp. J.Med.Lab.Tech. 17:224
MACKEY J.P. and G.H.SANDYS (1966) Diagnosis of
urinary tract infections. Brit. Med. J. 1.173

Directions
Add 36 g of powder to 1 L of distilled water and heat to
boiling. Sterilize by autoclaving at 121°C for 15 minutes.

Description
This general purpose medium has been recommended
for bacteriological urine analysis. Current formulation is
a modification of the original one reported by Sandys,
that achieves an excellent colony differentiation without
inhibitors. This fact, and also the careful selection of nu-
tritive components, makes this medium a substrate able
to support growth of most urinary
pathogenic bacteria.
Presence of lactose as a fermentable sugar allows clas-
sic differentiation and, at the same time, lack of electro-
lytes suppresses swarming waves on the members of
the Proteus species and sometimes growth of Shigella
sp. also.
The characteristics of colonies that grow on C.L.E.D.
Agar (after 18 hours of incubation):
Escherichia coli: Yellowish colonies, opaque, with core,
1,25 mm diameter. Non fermentative strains give control
blue colonies.
Klebsiella sp.: Very mucuous colonies of variable colour,
from yellow to blue-white.
Salmonella sp.: Plain and blue colonies.
Enterococcus faecalis: Yellow colonies. 0,5 mm diam-
eter.
Staphylococcus aureus: Convex yellow colonies. 0,75
mm diameter.

Proteus mirabilis ATCC 10975

44
 CN Selective Agar Base for Pseudomonas
Ref. 01-609
R-52/53
Specifications
Selective solid medium for the detection of Pseu-
domonas aeruginosa according the EN 12780-2002 and
ISO 16266 Standard.
Formula (in g/L)
Gelatine Peptone .................................... 16,00
Casein Peptone ...................................... 10,00
Potassium sulphate ................................ 10,00
Magnesium chloride ................................. 1,40
Cetrimide® ................................................ 0,20
Agar ........................................................ 15,00
Final pH 7,1 ± 0,2
Directions
Add 52,6 g of powder to 1 L of distilled water with 10
mL of glycerol. Heat to complete solution and sterilize in
autoclave at 121ºC for 15 min. Cool to 45-50ºC and add
to 500 mL of medium a flask of the Nalidixic Acid Selec-
tive Supplement (Ref. 06-124CASE). Homogenise and
pour plates.
Do not maintain the complete medium melted for more
than 4 hours. Do not re-melt. The finished plates can be
used without lose its efficiency for one month if they are
refrigerated and in a dark place.
Description
The CN Selective Medium for Pseudomonas was pro-
gressively developed from the basic media of King, Ward
and Raney for the production of pigments. Browne and
Lowbury add the cetrimide as selective agent and Goto
and Enomoto improves efficiency by adding nalidixic
acid. The presence of both inhibitors eliminates the con-
taminant microbiota from heavily polluted specimens and
was adopted by the CEN (Centre Europeen de Normali-
sation) in its EN Standard 12780 for the detection of Ps.
aeruginosa by filtering membrane in water.
control
N Technique
A volume of the sample is filtered thorough a filtering
S-61 membrane of 0,45 µm pore and the membrane is depos-
ited on the surface of the CN medium. The plates are
incubated at 36±2ºC for a period of 44±4 h with a partial
examination at 22±2 h.
All the colonies producing a green or blue (pyocianin)
pigmentation in this period must be considered as Pseu-
domonas aeruginosa without any other confirmation.
All the colonies that produces fluorescence under the
Wood’s light (without pyocianin production) era consid-
ered presumptive Ps. aeruginosa and must be confir-
mate on Acetamide Medium (Ref. 03-428).
All the colonies producing a brown-reddish pigment and
no produces fluorescence nor pyocianine are considered
presumptive Ps. aeruginosa and must be confirmed by
the oxidase test and typical growth on Acetamide Me-
dium (Ref. 03-428) and King B Agar (Ref. 01-029)
References
KING, E.O., M.K. WARD & E.E. RANEY (1954) Two
simple media for the demonstration of pyocianin and
fluorescein. J. Lab. Clin. Med. 44:301
BROWN, V.L. & E.J.L. LOWBURY (1965) Use of an im-
proved Cetrimide Agar Medium and of culture methods
for Ps. aeruginosa. J., Clin. Pathol. 18:752
GOTO S. & S. ENOMOTO (1970) Nalidixic acid cetrim-
ide agar. A new selective plating medium for the selec-
tive isolation of Ps. aeruginosa. Jpn. J. Microbiol. 14:65
ROBIN, T. & J.M. JANDA (1984) Enhanced recovery of
Ps. aruginosa from diverse clinical specimens on a new
selective agar. Diag. Microbiol. Infect Dis. 2:207
EN STANDARD 12780 (2002) Water Quality. Detection
and enumeration of Ps. aeruginosa by membrane filtra-
tion. Brussels.
ISO 16266:2006 Standard. Water Quality – Detection
and enumeration of Pseudomonas aeruginosa. Method
by membrane filtration.
Pseudomonas aeruginosa ATCC 27853
45
 Cosmetic Diluent of Beerens
Ref. 02-257
Specification
Diluent to neutralize preservative systems in common
examination of cosmetic products.
Formula (in g/L)
Lecithine ................................................... 3,00
Sodium thiosulfate .................................... 5,00
L-Histidine HCl ......................................... 1,00
Peptone .................................................... 1,00
Sodium chloride ........................................ 8,50
Dipotassium phosphate ............................ 1,00
Final pH 7,0 ± 0,2
Directions
Dissolve 19,5 g of powder in 1 L of distilled water con-
taining 30 mL of polysorbate 80.(Ref. 06-080). Distribute
into suitable containers and sterilize by autoclaving at
121°C for 15 minutes.
Let it cool to 50°C and shake gently to redissolve polys-
orbate.
m-CP Agar Base
Ref. 01-513
Specification
Solid medium for the enumeration and isolation of
Clostridium perfringens in water acc. the European
Directive 12767/97
Formula (in g/L)
Tryptose .................................................. 30,00
Yeast extract ........................................... 20,00
Sucrose .................................................... 5,00
L-Cisteine HCl .......................................... 1,00
Magnesium sulfate.7H2O .......................... 0,10
Bromocresol purple .................................. 0,04
Agar ........................................................ 15,00
Final pH 7,6 ± 0,2
Directions
Suspend 71 g of powder in 1 L of distilled water and
bring to the boil. Distributein suitable containers and
sterilize in autoclave at 121ºC for 15 minutes. Cool to
45-50ºC and aseptically add 2 flasks of Selective Sup-
plement m-CP (Ref. 06-125CASE) with the following
composition:
D-Cicloserine ............................................. 400 mg/L
Polymyxin B sulphate .................................. 25 mg/L
Indoxil-b-D-Glucoside .................................. 60 mg/L
Fenolftalein diphosphate ........................... 100 mg/L
Iron (III) Chloride ......................................... 90 mg/L
46
Description
Cosmetic diluent of Beerens has all the necessary
compounds to neutralize most of the chemical agents
included in cosmetic products to maintain and preserve it
free of microorganisms.
It complies with the EU recommendation that states that
before any microbiological examination, a treatment to
remove all the growth inhibitor systems in the cosmetics
must be performed.
However, this standard also declares that later dilutions
have to be performed in less aggressive media, that
may be considered as an enrichment and revitalization
system, and it suggests Letheen Broth (Ref. 02-236) or
Letheen Modified Broth (Ref. 02-237).
References
BEERENS, H., RAMONS, C., LEMAIRE, D. (1976). Rev.
Inst. Past. Lyon. 9:127.
BRIGIDI, P., MATTEUZZI, D. (1982) II Farmaco Ed. Pr.
37:8:260. Commission del Communautes Europeennes,
Groupe Special. Methodes de Controle Microbiologique
des Produits Cosmetiques: Limites Numeriques Appli-
cables au controle Officiel de la Qualité Microbiologique
des Produits Cosmetiques. XI/405A, ISPRA, 1976.
Homogenize avoiding bubble formation and pour in
plates.
Description
The m-CP Agar Base is a solid medium for counting
and isolating vegetative cells and spores of Clostridium
perfringens by the membrane filtration method. Its use is
compulsory in determining the quality of water for human
consumption in European Union by Directive 12767 (12-
07-1997) of the European Council.
Technique
A suitable volume of water is filtered through a mem-
brane filter of 47 mm diameter and 0,45 mm pore. Put
the membrane on the surface of a plate of m-CP medium
freshly prepared and incubate in an anaerobic atmos-
phere at 44±1ºC for 21±3 hours. Expose the growth
obtained to amonium hydroxide vapours for 20-30 sec-
onds. Count as Clostridium perfringens all the opaque
yellow colonies that turn to pink or red after the ammo-
nium hydroxide exposure. Express the results as cfu/mL.
References
European Council (1998) Directive 98/83/CE on the
quality of the water destined to the human consuption.
EC Bull. 11-03-1998.
 m-CP Agar Base

control Clostridium perfringens Clostridium perfringens

before the exposure after the exposure
to Ammonia vapours (AM0257) to Ammonia vapours (AM0257)

Cystine-Tryptone Fluid Medium (CTA Medium)

Ref. 03-045 Technique

On this medium, we can maintain by deep stab a lot of
Specification fastidious microorganisms like Neisseria, Enterococcus,
Basic medium for strain maintenance or sugar fermenta- Pasteurella, Shigella and in most cases without needing
tion assays. Its fluidity makes it suitable to observe the any additives or CO2. Even some light-sensitive anaero-
motility. bic microorganisms can grow without special conditions
though in reduced atmopheres they give ideal growth on
this medium.
Formula (in g/L)
In the few cases as per the nutritive requirements of the
L-Cystine HCl ......................................... 0,500
microorganisms like serum or ascitic liquid, which can
Casein peptone .................................... 20,000
be added in an aseptic way to the molten medium, when
Sodium chloride ...................................... 5,000
cooled to 45-50°C.
Sodium sulfite ......................................... 0,500
Microorganism motility can be easily detected by growth
Phenol red .............................................. 0,017
in the stab, and helped by the medium fluidity.
Agar ........................................................ 2,500
Medium, with added sugars, may be used to study sugar
Final pH 7,3 ± 0,2
fermentation with microorganisms that do not grow on
phenol red classical media because of the additional
Directions nutritional requirements. Acidification can be easily ob-
Suspend 28,5 g of powder in 1 L of distilled water and served with the change in colour of phenol red indicator.
heat to boiling. Dispense and sterilize at 121°C for 15
minutes. Addition of certain sugars may require a pH
readjustment.
References
VERA, H.D. (1948) A simple medium for identification
and maintenance of the gonococcus and other bacteria.
Description J.Bact. 55:531
Cystine and Tryptone fluid medium is appropiate for the ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
propagation and maintenance of bacterial strains, even biological Media. CRC Press, Inc.
the fastidious ones, without additives.
The lack of fermentative sugars allows the long survival
of microorganims, and sometimes it has been used for
studies on severe microaerials fermentation, due to the
fact that these microorganisms do not grow on current
basal media. Medium has its maximum efficacy when
freshly prepared, but it can be stored for long periods
of time, taking care to avoid its dehydration. To achieve
this, screw caps or hermetic sealing are strongly recom-
mended. In any case, if medium has been stored for a
long time, it is better to put it in a boiling water bath for a
few minutes before using it.

47
 Czapek-Dox Media

Czapek-Dox Agar lactic acid 10% solution, producing a pH drop to 3,5. In

Ref. 01-051
Specification
Semisynthetic solid medium for the cultivation of fungi,
with sodium nitrate as the only nitrogen source.
Formula (in g/L)
Sucrose .................................................. 30,00
Sodium nitrate .......................................... 2,00
Magnesium glycerophosphate ................. 0,50
Potassium sulfate ..................................... 0,35
Potassium chloride ................................... 0,50
Ferrous sulfate ......................................... 0,01
Agar ........................................................ 15,00
Final pH 6,8 ± 0,2
Directions
Suspend 48,5 g of powder in 1 L of distilled water and
heat to boiling. Dispense and sterilize by autoclaving at
121°C for 15 minutes. Should a lower pH be desired,
acidify the medium with sterile lactic acid after cooling to
45°C.
Czapek-Dox Broth
Ref. 02-051
Specification
Semisynthetic liquid culture medium with sodium nitrate
as the only nitrogen source.
this medium, Warcup adds 5 g/L of Yeast extract (Ref.
07-079) and an antibiotic mixture (Streptomycin 30 mg/L
and Tetracycline 2 mg/L) to achieve total efficacy and
selectivity. In any case, bacterial growth is very poor.
Czapek-Dox medium has been adopted for the morpho-
logical studies of soil fungi, and it aids chlamydospora
formation by Candida albicans in shorter periods (Daw-
son, 1962). Temperatures and times of incubation al-
ways varies, and they can go from 1 to 5 weeks at room
temperature. Usually, it is 8-15 days at 15°C. In case of
Candida, 28°C for 48 hours seems to be optimum ; for
Penicillium, 22°C and nonetheless, Aspergillus grows
better at 30-32°C.
References
CZAPEK, F. (1903) Untersuchung uber die sticstoff-
gewinnungund einweissbildung der Pflanze.Beitr. Chem.
Physiol. Pathol. 1:540
DOX, A.W. (1910) The intracellular enzymes of Penicil-
liumand Aspergillus with special references to those of P.
camemberti. US Dept Agr. Bur. Animal Ind. Bull. 120:70
APHA-AWWA-WPCF (1992) Standard Methods for the
examination of Water and Wastewater. 18th ed. APHA.
Washington.
RAPER, K.B. & D.J.FENELL (1965) The genus Aspergil-
lus. William & Wilkins Co. Baltimore.
THOM,C & M.B. CHURCH (1926 The aspergilli. William
& Wilkins Co. Baltimore.
WARCUP, J.H. (1950) The soil-plate method for isolation
of fungi from soil. Natur 166:117-118

Formula (in g/L)

Sucrose .................................................. 30,00
Sodium nitrate .......................................... 2,00
Potassium chloride ................................... 0,50
Magnesium glycerophosphate ................. 0,50
Ferrous Sulfate ........................................ 0,01
Potassium sulfate ..................................... 0,35
Final pH 6,8 ± 0,2

Directions
Dissolve 33,5 g of powder in 1 L of distilled water. Dis-
pense in suitable containers and sterilize in the auto-
clave at 121°C for 15 minutes. Do not overheat

Description
Czapek-Dox medium, both liquid and solid version, is a
general cultivation medium of defined chemical composi-
tion, where the sole nitrogen source is sodium nitrate,
and the carbon source is sucrose. It has been employed
successfully in the isolation and cultivation of soil micro-
organisms especially fungi.

Its original version had a pH near neutrality, but it can be Fusarium sp.
rendered selective medium for the fungi by adding, (after
sterilization and before solidification), 10 mL of sterile

48
 D/E Neutralizing Agar
Xi
Ref. 01-610
R-43
S-24-37
Specification
Solid culture medium for the neutralization and testing of
antiseptics and disinfectants acc. ISO 22717 and 22718
standards.
Formula (in g/L)
Tryptone ................................................... 5,00
Yeast extract ............................................. 2,50
Dextrose ................................................. 10,00
Lecithin ..................................................... 7,00
Sodium thiosulphate ................................. 6,00
Sodium sulphite ........................................ 2,50
Sodium thioglycollate ............................... 1,00
Polysorbate 80 ......................................... 5,00
Bromcresol purple .................................... 0,02
Agar ........................................................ 15,00
Final pH 7,6 ± 0,2
Directions
Suspend 54 g of powder in 1 L of distilled water and
bring to the boil. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes. The ap-
pearance of precipitates is normal and do not interferes
the results.
Description
Dey & Engley developed this medium in 1983 to recov-
ery chemically damaged staphylococci, At the present
its use is generalized for testing by the contact method
(RODAC Plates) the efficiency of antiseptics and disin-
fectants on impervious surfaces. The present formulation
incorporate neutralizing substances for almost all the
active products used as antiseptics and disinfectants.
Lecithin neutralizes quaternary ammonium compounds
(QAC’s); Polysorbate acts on phenolics and formalin;
thioglycollate neutralizes the organic-mercurial com-
pounds; thiosulfate-sulfite inactive halogen-compounds
and lecithin + polysorbate neutralizes ethanol and other
alcoholic compounds.
Technique
When the RODAC (Replicate Organisms Detection and
Counting) plates are filled in the laboratory be careful
with the meniscus of the agar: It should rise above the
rim of the plate to give a slightly convex surface to make
a proper contact with the surface to be sampled.
For sampling remove the cover of the RODAC plate and
carefully press the agar surface to the surface being
sampled. Make certain that the entire agar meniscus
contacts the surface. Replace the cover and incubate
in an inverted position under the time and temperature
conditions for the microorganisms in question. Express
the results as “colonies per RODAC plate” or “Colonies
per cm2”
References
DEY, B.P. & F.B. ENGLEY (1983) Methodology for
recovery of chemically treated Staphilococcus aureus
with neutralizing medium. Appl. Environm. Microbiol.
453:1533-1537
HICKEY, P.J., C.E. BECKELHEIMER, & T. PARROW
(1992) Microbiological tests for equipment, containers,
water and air. In R.T. Marshall (Ed.) Standard Methods
for the examination of Dairy Products 16th ed. APHA
Washington.
EVANCHO, G.M., W.H. SVEUM, LL. J. MOBERG & J.F.
FRANK (2001) Microbiological Monitoring of the Food
Processing Environment. In DOWNES & ITO (Eds)
Compendium of Methods for the Microbiological Exami-
nation of Foods. 4th ed. APHA. Washington DC.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Culture Media. CRC Press. Boca Ratón, Fla.
ISO 22717:2006 Standard. Cosmetics – Detection of
Pseudomonas aeruginosa.
ISO 22718:2006 Standard. Cosmetics – Detection of
Staphylococcus aureus.
49
 Decarboxylase Lysine Broth acc. to Taylor
Ref. 02-336
Specification
Liquid medium to differentiate enteric bacteria in the
L-Lysine decarboxylation assays according to ISO 6785,
21567 and IDF 93 Standards
Formula (in g/L)
Yeast extract ........................................... 3,000
D(+)Glucose ........................................... 1,000
Bromcresol purple .................................. 0,016
L-Lysine .................................................. 5,000
Final pH 6,8 ± 0,2
Directions
Dissolve 9 g of powder in 1 L of distilled water. Distribute
in thin tubes in an amount of 2 or 5 mL per tube. Steri-
lize in the autoclave at 121°C for 10 minutes.
Description
The capacity to decarboxylate some aminoacids has
been widely employed to classify Enterobacteriaceae.
Taylor’s formulation, including lysine, has been recently
included in several standards for the identification of Sal-
monella. This modification shows an improved perform-
ance, in comparison to Falkow’s formulation.
Ref. 01-057 Deoxycholate Lactose Agar
control
Salmonella typhimurium ATCC 14028
50
Technique
It is advisable to use a vaseline seal to avoid spontane-
ous oxidation. The use of glucose in anaerobic condi-
tions produces an acidification of the medium, the indica-
tor will then turn to yellow if there is growth. The use of
the aminoacid will acidify the medium again, and then
it turns to grey and finally to violet, thereby showing the
positive reaction. The observations of these biochemi-
cal tests are performed after an incubation period of 24
hours at 37°C.
References
DOWNES, F.P. & K. ITO (2001) Compendium of meth-
ods for the microbiological examination of foods. APHA.
Washington. DC.
TAYLOR, W.I. (1961) Isolation of Salmonellae from Food
Supplies. V Determination of the Method Choice for
Enumeration of Salmonella. App. Microbiol. 9, 487-490.
ISO 6785 Standard (2001) Milk and milk products - De-
tection of Salmonella spp.
FIL-IDF 93 Standard (2001) Detection of Salmonella
spp.
ISO 21567:2004 Food and feeding stuffs – Horizontal
method for the detection of Shigella ssp.
Escherichia coli ATCC 25922
 Deoxycholate Media
Deoxycholate Citrate Agar
(Eur. Phar. Medium J)
Ref. 01-056
Specification
Differential and moderately selective plating medium for
enteric pathogenic bacteria, according the European
Pharmacopoeia.
Formula (in g/L)
Peptone ................................................. 10,00
Meat extract ............................................ 10,00
Lactose ................................................... 10,00
Ferric Citrate ............................................. 1,00
Sodium Citrate ........................................ 20,00
Sodium deoxycholate .............................. 5,00
Neutral red ............................................... 0,02
Agar ........................................................ 15,00
Final pH 7,3 ± 0,2
Directions
Suspend 71 g of powder in 1 L of distilled water and
bring to the boil. Immediatelly pour into plates. Plates
may be used at once or refrigerated for a few days. Do
not autoclave or overheat.
Description
The European Pharmacopoeia formulation is one of
these modifications to rhr original medium developed by
Leifson in 1935. The inhibition of gram-positive microor-
ganisms is due primarily to its content of sodium deoxy-
cholate, although the two citrate compounds also are
active inhibitors. The lactose achieves differentiation of
enteric bacilli. Organisms that ferment lactose produce
acid that, in presence of neutral red indicator, results in
the formation of red colonies. Lactose non-fermenting
produces colourless colonies. The black centres due to
the ferric sulphide depot detect the production of SH2.
Technique
Inoculate the specimen as soon as possible directly onto
surface of medium. Incubate the plates at 35 ± 2ºC for
18-24 hours. Plates can be incubated for an additional
24 hours if no lactose-fermenting are observed.
Typical colonial morphology on Deoxycholate Citrate
Agar is as follows:
Escherichia coli: Large, flat, rose-red
Enterobacter / Klesiella: Large, mucoid, pale with pink
centre.
Proteus: Large, colourless to tan.
Salmonella: Large, colourless to tan.
Shigella: Colourless to pink
Pseudomonas: Irregular, colourless to brown
Gram-positive bacteria: No growth to slight growth
References
LEIFSON, E. (1935) New culture media based on so-
dium deoxycholate for the isolation of intestinal patògens
and for the enumeration of colon bacilli in milk and water.
J. Path.. Bact. 40:581-599.
HYNES, M. (1942) The isolation of intestinal pathogens
by selective media. J. Path. Bact. 54:193-207
EUROPEAN PHARMACOPOEIA (2002) 4th ed. Suppl.
4.2 . § 2.6.13 Test for specified micro-organisms. Council
of Europe. Strasbourg.
MAC FADDIN, J.F. (1985) Media for isolation-cultivation-
identification-maintenance of medical bacteria. William &
Wilkins, Baltimore, MD.
ATLAS, R.M. & L.C. PARKS (1991) Handbook of Micro-
biological Media. CRC Press London.
Deoxycholate Lactose Agar
Ref. 01-057
Specificacion
Differential solid medium for the isolation of enterobacte-
ria according to APHA.
Formula (in g/L)
Peptone .................................................. 10,00
Lactose ................................................... 10,00
Sodium chloride ........................................ 5,00
Sodium citrate .......................................... 2,00
Sodium deoxycholate ............................... 0,50
Neutral red ................................................ 0,03
Agar ........................................................ 15,00
Final pH 7,1 ± 0,2
Directions
Suspend 42,5 g of powder in 1 L of distilled water and
heat to the boil. Do not autoclave and pour into sterile
petri plates. The medium loses its efficiency if overheat-
ed and so avoid autoclaving and remelting.
Description
The Deoxycholate-Lactose Agar is very close to the
Deoxycholate Agar, differing only in the deoxycholate
amount and in its reduced inhibitory power. The present
formulation is made according to the recommendation of
APHA and AOAC.
References
GREENBERG, A.E., L.S. CLESCERI & A.D. EATON
(1995) Standard Methods for the examination of Water
and Wastwater. 19th ed. APHA-AWWA-WEF. Washing-
ton D.C.
SPECK, M.L (1984) Compemdium of methods for the
microbiological examination of food.2nd ed. APHA.
Washington D.C.
ATLAS, R.M. and L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press, Boca Raton, Fla.
51
 Dextrose Media
Dextrose Agar
Ref. 01-089
Specification
General purpose solid culture medium.
Formula (in g/L)
Meat peptone ............................................. 5,0
Casein peptone .......................................... 5,0
Meat extract ................................................ 3,0
D(+)Glucose ............................................. 10,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 6,9 ± 0,2
Directions
Suspend 43 g of powder in 1 L of distilled water and
heat to boiling . Sterilize by autoclaving at 121°C for
15 minutes. Should the acid pH is required, add sterile
tartaric acid solution when the medium is at 45°C. Do
not reheat.
Description
This solid culture medium is suitable for many objectives
and it supports growth of most non fastidious microor-
ganisms. Adding 5% sterile defibrinated blood to this me-
dium, makes it an excellent culture medium which satisfy
nearly all kinds of nutritive needs, even for meningococci
and pneumococci, however due to its high glucose con-
tent it is not suitable for hemolytic reaction studies.
This formulation is also in accordance with the one
suggested for the study of frozen food, and also for
fruit juices. In this latter case, it is suggested to use it in
duplicate, and one of the samples must be acidified in
order to facilitate the growth of moulds and yeast in such
a selective way.
Acidification can be easily achievevd by adding 7-8 mL
of 10% sterile tartaric acid to the sterile, and cooled me-
dium at 45°C,producing a pH drop to 3,5 ± 0,2. In these
conditions, do not remelt the medium, as then agar
tends to get hydrolysed and do not solidify again.
References
APHA (1958) Recommended Methods for the Microbio-
logical Examination of Food. APHA. Inc, New York.
SPECK, M. L. (1984) Compendium of Methods for the
Microbiological Examination of Food. 2nd ed. APHA.
Washington.
VANDERZANT & SPLITTSTOESSER (1992) Compen-
dium of Methods for the Microbiological Examination of
Food. 3rd ed. APHA. Washington.
52
Dextrose Broth
Ref. 02-089
Specification
Liquid culture medium for general purposes.
Formula (in g/L)
Casein peptone ........................................ 10,0
D(+)Glucose ............................................... 5,0
Sodium chloride .......................................... 5,0
Final pH 7,2 ± 0,2
Directions
Dissolve 20 g of powder in 1 L of distilled water, heating
up if necessary. Dispense into containers and sterilize by
autoclaving at 121°C for 10 minutes.
Description
This version of Dextrose Broth is formulated without
meat extract in order to make dextrose the single carbo-
hydrate in the medium. It is a liquid culture medium for
the faster growth, since most microorganisms can use
glucose as their energy source, but it has one draw-
back that it is not buffered, and the strong acidification
produced by fermentation may damage its maintenance.
Hence, though it is used many times in blood cultures, it
has been replaced by other, more buffered media.
Due to the relatively high proportion of glucose, it is
advisable to use it freshly prepared, and with short
sterilization period, as otherwise toxic furfurales may be
developed.
On the other hand, its simple formulation makes it the
best medium for checking gas production from glucose
if Durham’s tubes are used, as it has no indicator that
could interfere with it.
References
APHA (1958) Recommended Methods for the Microbio-
logical Examination of Food. APHA. Inc, New York.
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food.4th ed.
APHA. Washington.
MacFADDIN, J.D. (1985) Media for isolation-cultivation-
identification-maintenance of medical bacteria. William &
Wilkins Co. Baltimore
Dextrose Purple Broth
Ref. 02-416
Specification
Liquid medium for E.coli detection in water, according to
German legislation (DEV).
 Dextrose Media
Formula (in g/L)
Peptone .................................................. 10,00
Meat extract .............................................. 3,00
Sodium chloride ........................................ 5,00
Dextrose ................................................. 10,00
Bromocresol purple .................................. 0,02
Final pH 7,1 ± 0,2
Directions
Dissolve 28 g of powder in 1 L of distilled water. Dis-
tribute into tubes with Durham’s tubes and sterilize by
autoclaving at 121°C for 15 minutes.
Description
Medium can be used for any assay of degradation of
sucrose. It has been adopted by the German Federal
Government for detecting E.coli in water, according to
Eijkman’s test, which is based on the production of acid
and gas (yellow change of indicator) from sucrose after
an incubation of 20 hours at 44°C (±0,5).
References
DIN Normative (Standards) 38411 (1991) Teil 6 (Juni
1991): Mikrobiologische Verfahren (Gruppe K): Nach-
weis von Escherichia coli und coliformen keimen (K6).
DEUTSCHE EINHEITSVERFAHREN zur Wasser-,
Abwasser- Und Schlammuntersuchung. VCH Verlags-
gesellschaft D-6940 Weinheim.
VERORDNUNG über Trinkwasser und über wasser fur
Lebensmittelbetreibe vom 12/12/1990. Bundesgesetzbl.
Teil I 2613-2629 (1990)
Dextrose Tryptone PB Agar
Ref. 01-556
Specification
Solid medium for the cultivating the “flat-sour” food spoil-
ing microorganisms
Formula (in g/L)
Tryptone ................................................. 10,00
Dextrose ................................................... 5,00
Bromocresol Purple .................................. 0,04
Agar ........................................................ 15,00
Final pH 6,9 ± 0,2
Directions
Suspend 30 g of powder in 1 L of distilled water and
bring to the boil. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes.
Description
This medium was adopted in 1930 by the National Can-
ners Association for the detection of the microorganisms
causing the “flat-sour” spoilage in the canned foods.
Latter it was used for the detection and enumeration
of all the micro-organisms related with acid spoilage
of foods, like Bacillus coagulans (causing the typical
“flat-sour”) other Bacillus and Sporolactobacillus and the
thermophilic Bacillus stearothermophilus.
Technique
The samples or its dilutions are inoculated in the me-
dium, melted and cooled to 50ºC. Then are poured in
petri dishes and incubated for 72 h at 32ºC (mesophiles)
or for 48 hours at 55ºC (thermophiles). After incubation
the acid-producing colonies can be easily enumerated
because they show a yellow zone that contrast with the
purple medium.
References
NATIONAL CANNERS ASSOCIATION (1968) Labora-
tory Manual for food canners and Processors. Vol. 1.
NCA. Washington
DOWNES, F.P. & K. ITO (2001) Compendium of meth-
ods for the Microbiological Examination of Foods. 4th ed.
APHA. Washington.
HORWITZ, W. (2000) Official Methods of Analysis.
AOAC International, Gaithersburg. MD.
Dextrose Tryptone PB Broth
Ref. 02-556
Specification
Liquid medium for the microbiological examination of
canned foods
Formula (in g/L)
Tryptone ................................................. 10,00
Dextrose ................................................... 5,00
Bromocresol Purple .................................. 0,04
Final pH 6,9 ± 0,2
Directions
Dissolve 15 g of powder in 1 L of distilled water, heating
if necessary. Distribute in suitable containers and steri-
lize in autoclave at 121ºC for 15 minutes.
Description
Baumgartner and Hersom proposed this medium, in
1956 for the microbial examination of canned foods and
at the present it is recommended for all the medium and
low acidity (pH 4,5) canned or heat-processed foods.
The microbial growth is supported by the peptone and
the pH indicator that turns from purple to yellow detects
the acid-producers from glucose.
Technique
Volumes of 10-20 mL of medium in duplicate are inocu-
lated with 1-2 g or mL of sample to detect the aerobic
microorganisms. For the anaerobic ones it is convenient
inoculate other series of tubes with a more reducer me-
dium like Liver Broth (Ref. 02-098). The incubation must
be at 35 and 55ºC for both series.
53
 Dextrose Media

References
BAUMGARTNER, J.G. & A.C. HERSOM (1956) Canned
Foods. 4th ed. Churchill Ltd. London
DOWNES F.P. & K. Ito (2001) Compendium of methods
for the Microbiological Examination of Foods. 4th ed.
APHA. Washington. D.C.

Dichloran Glycerin Selective Agar (DG18 Agar)

Ref. 01-485 T The inclusion of 18% (w/v) of Glycerine makes the

Specification
Solid differential and low water activity medium for the
determination of xerophilic fungi from low moisture food.
Formula (in g/L)
Peptone ................................................. 5,000
Dextrose ............................................... 10,000
Monopotassium phosphate .................... 1,000
Magnesium sulfate ................................. 0,500
Dichloran ................................................ 0,002
Chloramphenicol .................................... 0,100
Agar ...................................................... 15,000
Final pH 5,6 ± 0,2
Directions
Suspend 31,7 g of powder in 820 mL of distilled water
and heat to the boiling. Add 180 mL of glycerin. Distrib-
ute into suitable containers and sterilize by autoclaving
at 121ºC for 15 minutes.
Description
Among the culture media for xerophilic fungi, those that
have played a more successful role are the ones which
include any agent that restrains the continuous growth
of zygomicete fungal colonies. Dichloran (Dichloreben-
zalkonium cloride) and Rose bengal are two of those
inhibitors.
DG18 Agar medium is according to the formulation pro-
posed by Hocking & Pitt in 1980, and it includes Dichlo-
ran which limits the size of fungal colonies more ef-
ficiently than Rose bengal. Chloramphenicol inhibits the
bacterial growth very well and its thermostability allows it
to be included in the medium before sterilization.
R-45
medium with a water activity (aw) of 0,995 without caus-
S-53-45 ing any problems that generally appear when this water
activity is reached with sodium chloride or sugar.
Technique
It is advisable to inoculate in mass and thus a surface
inoculation is recommended. This seed may be by
surface streaking as well as swab spreading or by Dri-
gaslky loop. Never use inoculum more than 0,1 mL.
According to the standardized technique, plates must be
incubated at 22-25ºC, with partial readings after 3 and
5 days, and a definitive readings after 7-8 days. Results
are expressed in cfu xerophiles/g or mL of food sample.
References
HOCKING, A.D., J.I. PITT (1980) Dichloran-glycerol me-
dium for enumeration of xerophilic funghi from low-mois-
ture food. Appl. Environm. Microbiol. 39:488-492
PITT, J.I., A.D. HOCKING, D.R. GLENN (1983) An
improved medium for the detection of Aspergillus flavus
and A. parasiticus. J. Appl. Bacteriol. 54:109-114.
PITT, J.I., A.D. HOCKING (1985) Fungi and Food Spoil-
age. Academic Press: Sydney
SAMSON, R.A., E.S. HOEKSTRA (2001) Introduction
to the Food Borne fungi. 6th. Ed. Centralbureau voor
Schimmelcultures: Baarn.

54
 Differential Reinforced Clostridial Medium (DRCM)
Ref. 02-410
Specification
Liquid medium for the enumeration of clostridia in food
samples and other products by MPN technique.
Formula (in g/L)
Peptone ................................................ 10,000
Meat extract ............................................ 8,000
Yeast extract ........................................... 1,000
Starch ..................................................... 1,000
Glucose .................................................. 1,000
L-Cysteine HCl ....................................... 0,500
Sodium acetate ...................................... 5,000
Sodium bisulfite ...................................... 0,500
Ferric-ammonium citrate......................... 0,500
Resazurine ............................................. 0,002
Final pH 7,0 ± 0,2
Directions
Dissolve 27,5 g of powder in 1 L of distilled water. Bring
to the boil, distribute in tubes and sterilize in the auto-
clave at 121°C for 15 minutes.
Description
This medium is a modification by Freame and Fitzpatrick
of the Gibb’s classic medium, to easily detect the pres-
ence of sulfite reducing clostridia. The modification is,
mainly, an addition of sodium bisulfite and ferric citrate,
that make colonies black and thus more conspicu-
ously visible. The current version of this medium has no
agar in order to facilitate the blackness of the medium.
Resazurine,the redox indicator allows the verification
of anaerobiosis in the medium in the same assay. L-
Cysteine acts as reducing agent in this medium.
Technique
Sample to be examined is distributed in tubes as per the
MPN technique, and is covered with paraffin or vaseline
oil to help the anaerobiosis. The bank of tubes is kept in
a boiling water bath at 75°C for 30 minutes to remove
all the dissolved oxygen and vegetative cells. Then,
incubate at 30°C up to 7 days before concluding any
negative results.
Generally, the spores of sulfate reducing clostridia
germinate between the second and fourth day, and the
medium turns black, in which case the test is positive.
The medium can be rendered selective by the addition of
70 IU/mL of Polymyxin sulftate.
Prepared tubes without the inoculation may be stored
up to 2 weeks provided the resazurine band does not
show an excessive oxidation (more than a 1/3 part of the
column).
References
GIBBS, B.M., FREAME, B. (1965) Methods for the re-
covery Clostridia from food. J.Appl. Bact. 36:23-33
FREAME, B., FITZPATRICK, B.W.F. (1972) The use of
DRCM for the isolation and enumeration of Clostridia
from Food. In Isolation of Anaerobics. Ed. Shapton AND
Board. Academic Press. London.
DIN Standard 38411 (1991) Teil 6 (Juni 1991): Mikro-
biologische Verfahren (Gruppe K): Nachweis von Es-
cherichia coli und coliformen keimen (K6).
55
 DNAse Agar

Ref. 01-346 Mannitol fermentation may be simultaneously deter-

mined if 10 g of mannitol and 0,025 g of phenol red are
Specification added to 1 L of DNAse Agar, before sterilization. Positive
Solid culture medium for the determination of the deox- results in both tests will determine with more certainty
yribonuclease activity of microorganisms, especially of that the microorganism is a pathogenic Staphylococcus
staphylococci and Serratia sp. aureus.
This medium is also useful to identify Serratia marces-
cens in clinical specimens, since it is a DNAse producer.
Formula (in g/L) Davis (1967) modified the medium by adding toluidine
Tryptose .................................................. 20,00
blue and crystal violet, and stated that gramnegative
DNA ......................................................... 2,00
DNAse producing bacilli that grew on this medium may
Sodium chloride ........................................ 5,00
be accepted as Serratia species.
Agar ........................................................ 15,00
Final pH 7,3 ± 0,2
Tehnique
DNAse Agar plates are inoculated with the microorgan-
Directions ism to be studied by thick streak or inoculating at the
Suspend 42 g of powder in 1 L of distilled water and heat
bottom and are incubated at 35-37°C for a 18-24 hours
to the boiling with constant stirring. Distribute into suit-
period.
able flasks and sterilize in the autoclave at 121°C for 15
To read, flood the plates with 1N chlorhydric acid and
minutes. Cool it to 50°C and pour it into the plates.
observe if there are any clear or transparent zones sur-
rounding the streak. If the plate becomes totally turbid
Description without any clear zone then the test is Negative however
Jeffries, Holtman and Guse (1957) incorporated DNA if any clear zone developes around the growth, then the
into a general medium with agar to study bacterial and test is accepted as Positive.
fungal DNAse production. Microorganisms that are able
to produce DNAse, break DNA, reducing it to nucleotide
References
fragments.
JEFFRIES, C.D., D.F. HOLTMAN y D.G. GUSE (1957).
This reaction is observed by the appearance of a clear
Rapid Method for Determining the Activity of Microorgan-
zone surrounding the growth, the rest of the plate re-
isms on NucleicAcids. J. Bacteriol. 73:590-591
maining turbid. The process is as follows: Hydrochloric
DISALVO, J.W. (1958). Desoxyrribonuclease and
acid reacts with DNA producing white precipitates that
Coagulase Activity of Micrococci. Med. Tech. Bull. U.S.
make the medium turbid, although it does not react with
Armed Forces. Med. J. 9:191
nucleotide fragments.
SMITH, P.B., G.A. HANCOCK & D.L. RHODEN (1969)
The same authors also observed that there is a correla-
Improved médium for detecting deoxyrribonuclease-pro-
tion between coagulase production and DNase activity,
ducing bacteria. Appl. Microbiol. 18:991-993
thus DNAse Medium may be used as a laboratory test to
diagnose pathogenic staphylococci.

control

Serratia marcescens ATCC 13880 Staphylococcus aureus ATCC 25923

56
 EC Broth
Ref. 02-060
Specification
Selective medium for the detection of enterobacteria, in
water and foodstuff according to ISO 9308-2 and 7251
standards.
Formula (in g/L)
Peptone .................................................... 20,0
Bile Salt n.3 ................................................ 1,5
Lactose ....................................................... 5,0
Dipotassium phosphate .............................. 4,0
Potassium dihydrogen phosphate .............. 1,5
Sodium chloride .......................................... 5,0
Final pH 6,9 ± 0,2
Directions
Dissolve 37 g of powder in 1 L of distilled water. Distrib-
ute into tubes or containers with inverted Durham tubes
(for gas production). Sterilize at 121°C for 15 minutes.
Description
EC Broth is a buffered medium containing lactose. It is in
the range of selective broths for Enterobacteriaceae. Its
efficiency or selectivity is based on bile salts’ inhibitory
effect on other microorganisms.
This broth may be used for routine testing of water and
food, either alone or by using the Most Probable Number
method of enumeration.
The type of sample will determine how precise the
results are. If the incubation is at 35-37°C for 48 hours,
gas formation may be interpreted as presumptive evi-
E. coli Direct Agar (ECD Agar)
Ref. 01-484
Specification
Solid culture medium for the detection of coliforms and
E.coli in water and food.
Formula (in g/L)
Tryptone ................................................. 20,00
Yeast extract ............................................. 5,00
Bile salts ................................................... 1,50
Disodium phosphate ................................. 5,00
Monopotassium phosphate ...................... 1,50
Sodium chloride ........................................ 5,00
Agar ........................................................ 15,00
Final pH 7,2 ± 0,2
Directions
Suspend 53 g of powder in 1 L of distilled water and add
5 g/L of carbohydrate. Heat to the boiling. Distribute into
suitable containers and sterilize by autoclaving at 121ºC
for 15 minutes. Cool to 50ºC and add 2 flasks/L of MUG
Supplement (Ref. 06-102CASE)
dence of coliform bacteria. Later confirmation will have
to be done using any of the classical methods.
Should the incubation take place at 44,5°C, gas forma-
tion could be interpreted as a confirmation of the pres-
ence of Escherichia coli. Nevertheless, it must be taken
into account that the validity of this test is highly limited
by technical variations. A maximum incubation time of
24 hours in a water bath with a very precise temperature
regulation, is therefore recommended.
When using the samples more than 10 mL, the medium
must be reconstituted at a concentration equivalent
to that specified on the directions, once the sample is
added.
References
APHA-AWWA-WEF (1998). Standard Methods for the
Examination of Water and Wastewater, 20th ed. APHA,
Inc. Washington D.C., U.S.A.
PERRY and HAJNA, (1943). Am. J. Pub. Hlth. 33:550.
DOWNES, F.P. & K. ITO (2001) Compendium of meth-
ods for the microbiological examination of foods. 4th ed.
APHA. Washigton DC
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc. London.
ISO Standard 9308-2 (1990) Water Quality Detection
and enumeration of coliform organisms, thermotoler-
ant coliform organisms and pressumptive E. coli - MPN
method.
ISO Standard 7251 (1993). Microbiology General
Guidance for enumeration of presumptive E. coli. M.P.N.
Technique.
Description
This medium is formulated according to the Swiss Stand-
ards for the detection of coliforms in the food. Although
the medium is complete by itself, it is recommended
to add 5 g/L of glucose or lactose in order to make the
colonial growth more evident.
Direct detection is achieved by adding to the preparation
the fluorescent agent (MUG, Ref. 06-102). After incuba-
tion of the sample dilution or filter membrane, E.coli
colonies show a light blue fluorescence when examined
under UV light.
References
ANONYMOUS. Schweizerisches Lebensmittelbuch. 5th.
Ed. Chap. 56A.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press. Boca Raton. Fla.
57
 EE Broth (Eur. Phar. Enrichment Broth Medium E)
Ref. 02-064
Specification
Liquid culture medium for the enrichment of enterobacte-
ria from food samples according to ISO 8523 standard.
Formula (in g/L)
Gelatin peptone .................................... 10,000
Dextrose ................................................. 5,000
Ox bile .................................................. 20,000
Di-sodium phosphate ............................. 6,450
Monopotassium phosphate .................... 2,000
Brilliant green ......................................... 0,015
Final pH 7,2 ± 0,2
Directions
Suspend 43,5 g of powder in 1 L of distilled water and
heat at 100°C for 30 min. and cool immediately.
Description
As the name suggests, this medium is for the enrichment
of enterobacteria, and is a modification by Mossel(1963)
of the classic Brilliant Green Bile 2% Broth (Ref. 02-041).
Substitution of lactose by glucose makes it more suitable
for enteric bacteria detection, whether gas or non-gas-
producer, in food and different samples.
Elliker Broth
Ref. 02-288
Specification
Liquid culture medium for the enrichment of lactobacilli
and streptococci in the dairy industry.
Formula (in g/L)
Casein peptone ........................................ 20,0
Yeast extract ............................................... 5,0
Gelatin ........................................................ 2,5
Dextrose ..................................................... 5,0
Lactose ....................................................... 5,0
Sucrose ...................................................... 5,0
Sodium chloride .......................................... 4,0
Sodium acetate .......................................... 1,5
Ascorbic acid .............................................. 0,5
Final pH 6,8 ± 0,2
Directions
Dissolve 48,5 g of powder in 1 L of distilled water, heat-
ing up to 50°C to totally dissolve gelatin. Distribute into
suitable containers, and sterilize by autoclaving at 121°C
for 15 minutes.
58
Usual recommended technique is as follows: sample
to be studied is added to sterile broth at a proportion of
10%. After strong homogenization, the mixture is incu-
bated for a period of18-20 hours at 35-37°C. Afterwards,
subcultures are performed on a solid media appropiate
for the selective enterobacteria isolation. For this step,
Violet Red Bile Agar (Ref. 01-164) is specially recom-
mended, though there are also the MacConkey (Ref.
01-118), Deoxycholate or Brilliant green based media.
From the suspected colonies on this media, identification
can be performed following the common methodology.
References
MOSSEL, VISSER and CORNELISSEN (1963) The ex-
amination of foods for Enterobacteriaceae using a test of
the type generally adopted for the detection of salmonel-
lae J. Appl.Bact. 26:444-452
PASCUAL ANDERSON, MªRª. (1992) Microbiología
Alimentaria. Díaz de Santos, S.A. Madrid,.
EUROPEAN PHARMACOPOEIA,Supplement 4.2
(2002),2.6.13 Test for specified micro-organisms 4th
ed.,Council of Europe, Strasbourg.
ISO 8523 Standard (1991) General guidance for the
detection of Enterobacteriaceae with pre-enrichment.
Description
Medium was formulated by Elliker, Anderson and Han-
nesson to cultivate lactobacilli and streptococci from milk
and other dairy products. Sodium acetate, in this me-
dium at this concentration, gives two advantages: on one
hand it restrains gramnegative bacteria growth, and on
the other hand, it enhances lactic bacteria growth. The
latter produce massive growth due to the high amount of
sugars.
Technique
Readings are performed after 3-5 days of incubation at
37°C. Should a solid medium be desired, add 15 g/L of
Agar Bacteriological (Ref. 07-004).
References
ELLIKER, P.R., ANDERSON, A.W., HANNESSON, G.
(1956) An Agar Culture Medium for Lactic Acid Strepto-
cocci and Lactobacilli. J. Dairy Sci. 39:1611.
HAUSLER, W.J. (1976) Standard Methods for the
Examination of Dairy Products, 14 Ed. (APHA). Wash-
ington, D.C.
MARSHALL, R.T. (1992) Standard Methods for the
Examination of Dairy Products, 16 Ed. (APHA). Wash-
ington, D.C
 Endo Media
Endo Agar Base
Ref. 01-589
Specification
Solid selective medium for the detection of coliform and
other enteric organisms, in milk and water, according to
the APHA specifications.
Formula (in g/L)
Peptone .................................................... 10,0
Lactose ..................................................... 10,0
Sodium sulfite ............................................. 2,5
Di-potassium hydrogen phosphate ............. 3,5
Agar .......................................................... 15,0
Final pH 7,2 ± 0,2
Directions
Suspend 41 g of powder in 1 L of distilled water. Bring
to the boil and add 2 vials of Basic Fuchsin 250 Supple-
ment (Ref. 06-607CASE). Homogenize and distribute
into suitable containers. Sterilize at 121°C for 15 min-
utes.Cool to 45-50°C, homogenize and pour into plates.
Medium must appear slightly pinkish. If colour is very in-
tense red, it can be decolourised by adding a few drops
of a sterile solution of sodium sulfite 10% before pouring
it into the plates. Medium must be freshly prepared for
the use, and must not be used when it is red.
Description
Endo Agar is used to confirm the detection of and to
count coliform bacteria following presumptive test of
drinking water, as well as for the detection and isolation
of coliforms and fecal coliforms from milk, dairy products
and other food.
Inoculate the plates by the streak-plate method and
incubate for 24 hours at 37°C.
Colonies of coliform bacilli, which ferment lactose, are
pink to rose red, with or without green metallic sheen:
marked reddening of the medium may occur. Colonies
of other enteric bacilli, including Salmonella and of non
lactose fermentors are about the same colour as the
medium, being almost colourless to faint pink.
On exposure to oxygen, the plated medium gradually
becomes red due to the oxidation of sulfite and can thus
no longer be used. It can only be kept for a few days
even if it is stored in the dark and at refrigerator tem-
perature.
References
APHA /AWWA/WEF, (1985). Standard Methods for the
Examination of Water and Wastewater, 15th ed., Inc.
Washington D.C., U.S.A.
APHA (1967). Standard Methods for the Examination of
Dairy Products, 12th ed. , APHA Inc. Washington D.C.,
U.S.A.
ENDO, S (1904), Über ein Verfahren Zum Nachweis von
typhusbazillen. Zbl BaKt. Hyg. Abt. I, Orig, 35:109
MARSHALL, R.T. (1992) Standard Methods for the
Examination of Dairy Products, 16th ed. , APHA Inc.
Washington D.C., U.S.A.
Endo Dev Agar Base
Ref. 01-606
Specification
Selective agar for the isolation and differentiation of
E.coli in water, according to the german legislation.
Formula (in g/L)
Meat extract ............................................ 10,00
Peptone .................................................. 10,00
Lactose ................................................... 10,00
Sodium chloride ........................................ 5,00
Sodium sulfite ........................................... 2,50
Agar ........................................................ 20,00
Final pH 7,3 ± 0,2
Directions
Suspend 57,5 g of powder in 1 L of distilled water and
bring to the boil. Add 2 vials of Basic Fuchsin 250 Sup-
plement (Ref. 06-607CASE). Homogenize and distribute
into suitable containers. Sterilize in the autoclave at
121°C for 15 minutes. Cool it to 45-50°C, homogenize
and pour into plates.
In these conditions, medium must appear slightly pink-
ish. If colour is very intense red, it can be decolourised
by adding a few drops of a sterile solution of sodium
sulfite 10% before pouring it into the plates. Medium
must be freshly prepared for the use, and must not be
used when it is red.
Description
Medium is a modification over the classical ENDO (Ref.
01-589), according to the German legislation, to obtain a
better detection of damaged coliforms. Since the buffer
system is removed in this medium, this formulation
includes a more rich nutrient base and sodium chloride
to restore the osmotic balance. Agar concentration has
been increased to keep the strengh of gel after the water
sample is incorporated.
On this medium, E.coli colonies appear red, with metallic
sheen, meanwhile Klebsiella and Enterobacter only take
on the red colour. Colonies of other enteric bacteria are
colourless.
Technique
DEV standards recommends this medium to incubate
membrane filters used in the coliform detection and
in the re-seed on the surface of suspicious colonies
for their confirmation and isolation. However, the agar
strength allows the incorporation of the sample to be as-
sayed in the medium mass, without any loss of consist-
ency.
For times and temperatures of incubation, it is recom-
mended to follow the standard for every purpose, or fol-
low the technician’s criteria. In any case, an incubation at
25-30°C for 24-48 hours usually provides good results.
59
 Endo Media
References
DEUTSCHE EINHEITSVERFAHREN zur Wasser-,
Abwasser- Und Schlammuntersuchung. VCH Verlags-
gesellschaft D-6940 Weinheim.
BUNDESGESUNDHEITSAMT: Amtliche Sammlung von
Untersuchungverfahren nach. 35 LMBG Beuth Verlag
Berlin Köln.
DIN 38411: Teil 6 (Juni 1991): Mikrobiologische Ver-
fahren (Gruppe K): Nachweis von Escherichia coli und
coliformen keimen (K6).
Endo LES Agar Base
Ref. 01-604
Specification
Medium for detecting and enumerating coliforms in water
and food, by the membrane filtration method.
Formula (in g/L)
Peptone .................................................. 15,00
Yeast extract ............................................. 1,20
Lactose ..................................................... 9,40
Sodium chloride ........................................ 3,70
Dipotassium phosphate ............................ 3,30
Potassium phosphate ............................... 1,00
Sodium deoxycholate ............................... 0,10
Sodium laurylsulfate ................................. 0,05
Sodium sulfite ........................................... 1,60
Agar ........................................................ 15,00
Final pH 7,2 ± 0,2
Directions
Suspend 50,3 g of powder in 1 L of distilled water and
bring to the boil. Add 4 vials of Basic Fuchsin 200 Sup-
plement (Ref. 06-617CASE). Homogenize and distribute
in sterile plates. Do not autoclave. Final medium is
intense red in colour.
Description
Medium is a modification of the classical Endo’s me-
dium, reformulated to use it with the membrane filtration
technique and a previous enrichment in Tryptose Lauryl
Sulfate Broth (Ref. 02-108) is always helpful and recom-
mended for achieving in more growths and more brilliant
colonies.
Technique
Membrane(s) that have filtered the sample are incu-
bated at 35°C for 2-3 hours over a pad with Tryptose
Lauryl Sulfate Broth (Ref. 02-108), and after this they are
transferred to a plate with Endo LES Agar. Incubate at
35°C for 24 hours. Coliform colonies appear red with a
characteristic metallic sheen.
60
References
APHA-AWWA-WEF (1985) Standard Methods for the
Examination of Water and Wastewater. 16th. Ed. Wash-
ington DC.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press Inc.,London
Endo MF Broth Base
Ref. 02-605
Specification
Liquid medium especially formulated for the incubation
of membrane filters over the absorbent pads.
Formula (in g/L)
Casein peptone ...................................... 10,00
Meat peptone ......................................... 10,00
Yeast extract ............................................. 1,50
Lactose ................................................... 12,50
Sodium chloride ....................................... 5,00
Potassium phosphate .............................. 5,75
Sodium lauryl-sulfate ............................... 0,05
Sodium deoxycholate .............................. 0,10
Sodium sulfite .......................................... 2,10
Final pH 7,2 ± 0,2
Directions
Suspend 47g of powder in 1 L of distilled water. Heat
to boiling. Add 4 vials of Basic Fuchsin 250 Supplement
(Ref. 06-607CASE) and homogenize. Optimum results
are obtained using the media on the same day it is pre-
pared. Do not sterilize in the autoclave.
Description
Endo Broth for membrane filtration has the suggested
composition according to “Standard Methods” for detec-
tion and enumeration of coliforms by the membrane
filtration method.
Basically, it is the classical Endo medium, but selectivity
has been improved by adding lauryl-sulfate and sodium
deoxycholate and also the sodium sulfite and basic
fuchsin. The mixture of peptones and yeast extract pro-
vide the medium with a nutritive substrate by which most
of the enteric bacteria can have the better recovery and
the colonies of coliforms take on a characteristic metal-
lic sheen. So,the members of Enterobacter, Citrobacter
and Klebsiella types, after 20-22 hours of incubation,
take a metallic green-blue sheen, but not so intense as
Escherichia coli.
Technique
Endo Broth for membrane filtration may be used directly
or soaked in a pad as just by solidifying it with agar. In
some cases, it seems that a better results are obtained
if double-step method is used. That is, a short incuba-
tion of 2-3 hours on Tryptose Lauryl Sulfate Broth (Ref.
 Endo Media
02-108) and a later incubation of 20-22 hours over Endo
Broth at 35°C (see Endo LES Agar Base, Ref. 01-604).
The procedure suggested by APHA is filtration of mini-
mal amounts of water (50 mL), but in any case, mem-
branes with more than 400 colonies must be rejected for
enumeration. In the direct method, as in the double-step
method, lactose fermenting colonies show a characteris-
tic sheen, and they have to be considered as presump-
tive coliforms, meanwhile the lactose non fermenting
colonies are colouress and transparent.
Salmonella typhimurium ATCC 14028
control
Eosin Methylene Blue Agar (EMB Agar)
Ref. 01-068
Specification
Selective differential medium for the isolation of coliforms
from water acc. ISO 21150 Standard.
Formula (in g/L)
Peptone ................................................ 10,000
Lactose ................................................. 10,000
Dipotassium Hydrogen phosphate ......... 2,000
Yellowish Eosin ...................................... 0,400
Methylene Blue ....................................... 0,065
Agar ...................................................... 15,000
Final pH 7,1 ± 0,2
Directions
Add 37,5 g to 1 L of distilled water. Bring to the boil and
distribute in suitable containers. Sterilize in the autoclave
at 121°C for 15 minutes.
Description
A very versatile medium originally developed for the
differentiation of E. coli and Enterobacter aerogenes. It
has also proved very effective in the rapid identification
of Candida albicans and presents a high correlation with
the coagulase test for staphylococci.
References
APHA-AWWA-WEF (1995) Standard Methods for the
Examination of Water and Wastewater, 19th ed. APHA
Washington. D.C.
FIFIELD, C.W. & C.P. SCHAUFUS (1958) Improved
membrane filter medium for the detection of coliform
organisms.J. Amer.Water Works Assoc. 50:193
Escherichia coli ATCC 25922
It has been repeatedly recommended for the detec-
tion, enumeration and differentiation of members of the
coliform bacteria.
Technique
The Weld method for the identification of Candida albi-
cans uses this medium with Chlortetracycline (100 mg/l)
in a 10% CO2 environment. The method’s effectivity has
been tested with a variety of samples, such as sputum,
oral secretions, faeces, nails and vaginal secretions, all
of which provide definite results within 24-48 hours. sta-
phylococci are also easily identified, particularly coagu-
lase-positive strains. These have a very characteristic
appearance: small colourless colonies with a central red
nucleus.
Nevertheless, the medium’s prevailing application is in
the differentiation of E. coli and E. aerogenes.
The medium should be sterilized once distributed into
tubes containing 20 mL of product each, and then be
refrigerated. Melt in a boiling water bath before use and
stir until it acquires a dark purple colour. Pour a tube into
each sterile plate and allow to solidify. It is advisable to
dry the medium’s surface before use, leaving the plate
open but inverted on a heater.
For each doubtful lactose broth tube,inoculate one plate
by streaking , and incubate for 24 to 48 hours at 37°C.
Examine afterwards.
61
 Eosin Methylene Blue Agar (EMB Agar)
Escherichia coli and Citrobacter form flat colonies of 2-3
mm in diameter and are dark violet in colour with
a black centre which produces a distinctive green
metallic glow when light is reflected on it.
Enterobacter and Klebsiella form convex colonies
which are twice as big as the very smooth E. coli
, have no metallic glow and are pink in colour with
a dark blue centre. Non-lactose fermenting organ-
isms produce colourless colonies.
Candida albicans colonies incubated in a CO2 atmos-
phere have a very peculiar cotton-like appear-
ance which distinguishes them from other
Candida species that produce classical yeast like
colonies.
Ethyl Violet Azide Broth (EVA Broth)
Ref. 02-028
R-22-32-52/53
Specification
Medium for the confirmation of enterococci in water.
Formula (in g/L)
Meat Peptone ..................................... 10,0000
Casein Peptone .................................. 10,0000
Dextrose ............................................... 5,0000
Sodium chloride .................................... 5,0000
Monopotasium phosphate .................... 2,7000
Dipotassium phosphate ........................ 2,7000
Sodium Azide ....................................... 0,3000
Ethyl Violet............................................ 0,0005
Final pH 6,8 ± 0,2
Directions
Dissolve 35,6 g of powder in 1L of distilled water, heating
up slightly if necessary. Distribute in tubes or flasks and
sterilize in the autoclave at 121°C for 15 minutes.
Description
EVA Broth is a highly selective medium for some kinds
of enterococci, and it has been adopted by many Official
Organisations, National and International.
Medium´s high selectivity is due to the presence of
sodium azide and ethyl violet, as they inhibit other ac-
companying bacteria, blocking their respiratory chains,
leaving enterococci unaffected. In general, this medium
is always used as a confirmation medium in the sec-
ond stage, recommending an inoculum from a suitable
medium such as Rothe Azide Broth (Ref. 02-027) to be
inoculated in this medium.
62
References
CLESCERI, L.S., A.E. GREENBERG & A.D. EATON.
(1998) Standard Methods for the Examination of Water
and Wastewater. 20th edition. APHA-AWWA-WEF.
Washington D. C.
HOLT-HARRIS, J. E. y TEAGUE O.A. (1916) A New Cul-
ture Medium for the Isolation of Bacillus typhosus from
Stools J. Infect. Dis. 18:596-600.
ISO 21150:2006 Cosmetics – Detection of Escherichia
coli.
LEVINE, M (1918) Diferentation of E. coli and A. aero-
genes on simplified Eosin-ethylene Blue Agar. J. Infect.
Dis. 23:43-47.
MENOLASINO, N.I., GRIEVES B. Y PAYNE P. (1960)
Isolation and Identification of Coagulase Positive Sta-
phylococci on Levine’s Eosin-Methylene Blue Agar. J.
Lab. Clin. Med. 56(6) 908-910.
WELD, J. (1953) Candida albicans: Rapid Identification
in Cultures made directly from Human materials Arch.
Dermat. Syph. 67(5):473-478.
WINDLE TAYLOR, E. (1958)The Examination of Water
and Water Supplies. Churchill Ltd. 7th ed. Londres.
USP 29 – NF 24 (2006) 2nd Suppl. <61> Microbial Tests.
USP Con. Inc. Rockville, MD, USA
Xn
Technique
S-7-46-61 Each of the EVA Broth tubes is inoculated with one or
two loops from a presumed positive Rothe Azide Broth
(Ref. 02-027) flask, and are incubated for a 24-48 hours
period at 37°C. Enterococcus presence is noted by the
turbidity in the medium.
Occasionally a slight turbidity may appear accompanied
by ample violet sediment at the bottom of the tube.
Commonly, growth confirmation in this medium is
considered enough to state Enterococcus presence.
However, confirmative identification must be carried out
by isolation in solid media and classification in one of the
four faecal enterococci species: Enterococcus faecalis,
Enterococcus faecium, Enterococcus bovis and Entero-
coccus equinum.
References
LITSKY, MALLMAN and FIFIELD (1953) Amer.J.Publ.
Hlth 43:873
APHA-AWWA-WPCF (1995) Standard Methods for the
Examination of Water and Wastewater. 19th. ed. APHA
Washington.
SPECK, APHA/Intersociety (1976). Compendium of
methods for the microbiological examination of food.
Washington.
GUINEA, SANCHO and PARES (1979). Análisis micro-
biologicos de aguas: aspectos aplicados. Ed. Omega,
Barcelona.
 FDA Broth (AATCC Bacteriostasis Broth)
Ref. 02-532
Specification
Liquid medium used in routine antibacterial testing of
antiseptics and disinfectants.
Formula (in g/L)
Meat peptone ......................................... 10,00
Meat Extract ............................................. 5,00
Sodium chloride ........................................ 5,00
Final pH 6,9 ± 0,2
Directions
Dissolve 20 g of powder in 1 L distilled water, heating if
necessary. Distribute in suitable containers and sterilize
in autoclave at 121ºC for 15 minutes.
Description
This medium is produced according the formulation
specified in U.S. Food ad Drug Administration (FDA),
Association of Official Analytical Chemists (AOAC) and
American Association of Textile Chemists and Colour-
ists (AATCC) procedures for the testing of antiseptics
Fecal Coliforms Media (FC Media)
Fecal Coliforms Agar (FC Agar)
Ref. 01-287
Specification
Solid, selective and differential medium for coliform enu-
meration by membrane filter technique.
Formula (in g/L)
Tryptose .................................................... 10,0
Yeast extract ............................................... 3,0
Proteose peptone ....................................... 5,0
Bile salts # 3 ............................................... 1,5
Sodium chloride .......................................... 5,0
Lactose ..................................................... 12,5
Aniline blue ................................................. 0,1
Agar .......................................................... 15,0
Final pH 7,4 ± 0,2
Directions
Suspend 52,1 g of powder in 1 L of distilled water and
heat to boiling. Add two vials of Rosolic Acid Selective
Supplement (Ref. 06-085CASE). Mix and pour into ster-
ile Petri plates. Do not autoclave nor overheat. Use
freshly prepared medium.
and disinfectants for antibacterial activities. It has the
formula required by the AOAC for “Nutrient Broth” used
in Phenol Coefficient and others determinations. The
AATCC uses this medium in the procedures for the de-
tection of antibacterial activity of fabrics and in the pro-
duction of the solid medium for this type of evaluations.
References
AATCC (1985) 1986 Technical Manual of the AATCC
Vol. 61. Research Triangle Park. N.C.
MACFADDIN J.F. (1985) Media for Isolation Cultivation-
Identification-Maintenance of Medical Bacteria. William &
Wilkins. Baltimore
ATLAS R.M, & LC PARKS (1993) Handbook of Microbio-
logical Media. CRC Press. Boca Raton Fla.
HORWITZ, W. (2000) Official Methods of Analysis. 17th.
Ed. AOAC International. Gaithersbourg, M.D.
Fecal Coliforms Broth
(FC Broth)
Ref. 02-287
Specification
Liquid, selective and differential medium for coliform
enumeration by membrane filter technique.
Formula (in g/L)
Tryptose .................................................... 10,0
Yeast extract ............................................... 3,0
Proteose peptone ....................................... 5,0
Bile salts # 3 ............................................... 1,5
Sodium chloride .......................................... 5,0
Lactose ..................................................... 12,5
Aniline blue ................................................. 0,1
Final pH 7,4 ± 0,2
Directions
Suspend 37,1 g of powder in 1 L of distilled water and
heat to boiling. Add two vials of Rosolic Acid Selective
Supplement (Ref. 06-085CASE). Do not autoclave nor
overheat. Use freshly prepared medium.
63
 Fecal Coliforms Media (FC Media)
Description
FC Agar and Broth are formulated according to Geldre-
ich et al., to detect the faecal coliforms in polluted water.
The bile salts included in these media make these
media selective for enterobacteria, and also selective
for coliforms due to the high temperature of incubation:
44,5°C±0,5°C.
Freshly prepared medium has a red-garnet colour. Fae-
cal coliform colonies are greenish-blue, and the medium
also turns to this colour. In case of other bacteria, when
they grow, show pinkish colonies, and then the medium
turns to fluorescent red.
Technique
Essentially, the technique consists of filtering the test
sample to be examined through a membrane filter of
suitable porocity (0,22-0,45 µm), assisting the filtration
by pressure or suction, so that the microorganisms are
retained on the membrane. Remove the membrane
carefully and aseptically and take it to the culture me-
dium.
Put the membrane over the agar,if using the solid me-
dium, or over the impregnated pad if using the liquid ver-
sion. Cover the Petri plates and incubate them at 37°C
for 24 hours. After incubation, proceed with the counting
GE Motility Medium
Ref. 03-593
Specification
Semi-solid culture media for the motility test perform-
ance.
Formula (in g/L)
Gelatin .................................................... 52,00
Tryptose .................................................. 10,00
Heart extract ........................................... 10,00
Sodium chloride ........................................ 5,00
Agar .......................................................... 5,00
Final pH 7,2 ± 0,2
Directions
Suspend 82 g of powder in 1 L distilled water, heating
until total solution. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes.
Description
In this medium, the presence of diffuse growth away
from the line or spot of inoculation evidences motil-
ity. Non-motile organisms growth only along the line of
inoculation. In the medium all the components supplies
nutrients and the little amount of agar introduces enough
strength to maintain solidity even at incubation tempera-
tures, therefore, it is adaptable to use in both tubes and
64
of coliforms. Should a total E.coli selectivity be desired,
incubate at 44,5°C.
References
GELDREICH, E.E, H.F.CLARK, C.B. HUFF and
L.C.BEST (1965) Fecal-Coliform-organism medium for
the membrane filter technique. J. Am. Water Works As-
soc, 57:208-214
APHA-AWWA-WPCF (1995) Standard Method for the
examination of water and wastewater. 19th ed. APHA
Washington DC.
Without
Rosolic Acid
With
Rosolic Acid
Escherichia coli ATCC Salmonella typhimurium
25922 ATCC 14028
plates for motility studies, improving the original formula-
tion by Jordan et al.
Technique
After sterilization, cool the tubes by placing in cool
water bath up to the depth of the medium (in a 16x160
mm tube 8 cm depth or 15 mL of medium). For plates,
cool flasks of medium to 50ºC and pour into sterile petri
dishes to a dept of 5 mm or more and allow solidifying.
From an overnight culture spot the inoculum on the
surface or stab just below the medium surface. If tubes
are used, inoculate by depth stab inoculation. Incubation
must be suitable in time and temperature to the suspect-
ed organism being tested. Periodically examine tubes or
plates for growth and signs of motility at last for 7 days.
References
ATLAS, R.M. & L.C. PARKS. (1993) Handbook of
Microbiological Media. CRC Press. London
MACFADDIN, J.F. (1985) Media for isolation-cultivation-
identification-maintenance of medical bacteria. William &
Wilkins, Baltimore MD
D’AMATO, R.F. & K.M. TOMFOHRDE (1981) Influence
of media on temperature-dependant motility test for
Yersinia enterocolitica. J. Clin Microbiol. 14:347-348
JORDAN, E.O. M.E CALDWELL & D. REITER (1934)
Bacterial motility. J. Bacteriol. 27:165-173.
 Giolitti-Cantoni Broth
Ref. 02-230
Specification
Liquid medium for the recovery and enumeration of low
numbers of coagulase-positive staphylococci in foods
acc. to the ISO 5944:2001, IDF 60:2001 and EN-ISO
6888-3:2003 Standards
Formula (in g/L)
Tryptone ................................................... 10,0
Meat extract ................................................ 5,0
Yeast extract ............................................... 5,0
Lithium chloride .......................................... 5,0
D-Mannitol ................................................ 20,0
Sodium chloride .......................................... 5,0
Glycine ....................................................... 1,2
Sodium pyruvate ........................................ 3,0
Polysorbate 80 ........................................... 1,0
Final pH 6,9 ± 0,2
Directions
Dissolve 55,2 g of powder in 1 L of distilled water. The
medium can be prepared at single strength or double
strength using double quantity of powder Distribute into
tubes dispensing 10 mL/tube (Single strength) or 20
mL/tube (double strength). Sterilize by autoclaving at
121°C for 15 minutes. Cool and add 1% Potassium Tel-
lurite Sterile Solution (Ref. 06-089) using 0,1 mL/tube for
single strength and 0,2 mL/tube for double strength.
Description
This medium for the selective enrichment of staphyloco-
cci was formulated by Giolitti and Cantoni in 1966.
The growth of staphylococci is promoted by pyruvate,
glycine and above all by a high concentration of man-
nitol. Addition of Polysorbate 80 is necessary for the suc-
cessful recovery of Staphylococcus aureus (Chopin et
al., 1985). Competitive microbiota is inhibited by lithium
chloride and potassium tellurite. Anaerobic growth condi-
tions increase the selectivity of the medium. Generally,
growth of staphylococci can be recognized by a blacken-
ing or black precipitates in the culture medium due to
reduction of tellurite to metallic tellurium.
The prepared basal culture medium can be stored for
about 1-2 weeks in the refrigerator. The ready-to-use
medium must be used the same day of preparation. It is
advisable that the stored basal medium be degasified at
the moment of use by heating for 15 minutes at 100°C,
cool rapidly and add sterile potassium tellurite solution.
Technique
Refer to the standard protocol for specific products
(Food and animal feeding stuffs EN-ISO 6888-3:2003;
Milk and milk based products ISO 5944:2001 and FIL-
IDF 60:2001). As a general technique the following is
suggested:
Use food macerates or decimal dilutions and inoculate
1 mL to single strength medium. To lower the detection
limit 10 mL of the test sample (liquid products) or of the
Left: control; right: Staphylococcus
aureus ATCC 25923
first dilution (other products) may be inoculated in double
strength medium. MPN procedures need at least three
tubes for at least three dilution steps. If no anaerobic jar
is available, overlay with a layer of sterilized vaseline
(Ref. 6-077) or vaspar. Incubate anaerobically for 24-48
h at 37°C.
After 24 hours, subculture any tubes showing blackening
or black precipitate by streaking onto Baird-Parker Agar
(Ref. 01-030). Incubate the remainder of the tubes for
a further 24 h and subculture all tubes showing growth
(irrespective of blackening) to Baird-Parker Agar.
When determining the bacterial count by the MPN
method, all tubes showing growth are considered as
presumptive positive for staphylococci and they are
confirmed only if they produce a positive result in the
coagulase test.
References
CHOPIN, A. et altri (1985) ICMSF Methods Studies XV.
Comparison of four media and methods for enumerat-
ing Staphylococcus aureus in powdered milk. J. Food
Protect. 48:21-27
EN-ISO 6888-3 Standard (2003) Microbiology of food
and animal feeding stuffs. Horizontal method for the enu-
meration of coagulase positive staphylococci (Staphylo-
coccus aureus and other species). Part 3: Detection and
MPN technique for low numbers.
FIL-IDF (2001) Milk and milk based Products. Detection
of coagulase-positive staphylococci. MPN technique.
Standard 60:2001. Brussels.
GIOLITTI, G. A. CANTONI, C (1966) A medium for the
isolation of staphylococci from foodtuffs. J.Appl. Bact.
29, 395-398.
HARRIGAN, WF. a. McCANCE, M.E. (1976) Labora-
tory Methods in Food and Dairy Microbiology. Academic
Press. London.
ISO 5944 Standard (2001) Milk and milk based Prod-
ucts. Detection of coagulase-positive staphylococci.
MPN technique.
65
 Glucose Bromcresol Purple Agar
Ref. 01-502
Specification
Solid medium for the confirmation of enterobacteria
in diverse samples according to ISO 4702 and 8523
standards.
Formula (in g/L)
Tryptone ............................................... 10,000
Yeast extract ........................................... 1,500
Dextrose ............................................... 10,000
Sodium chloride ...................................... 5,000
Bromcresol purple .................................. 0,015
Agar ...................................................... 15,000
Final pH 7,0 ± 0,2
Directions
Suspend 41,5 g of powder in 1 L of distilled water and
bring to the boil. Distribute into containers and sterilize
by autoclaving at 121ºC for 15 minutes.
Glutamate Starch Pseudomonas Agar (GSP Agar)
Ref. 01-092
Specification
Solid, semiselective and differential solid medium for the
isolation of Pseudomonas and Aeromonas from very
contaminated samples.
Formula (in g/L)
Sodium L(+) glutamate ........................... 10,00
Soluble starch ......................................... 20,00
Monopotassium phosphate ...................... 2,00
Magnesium sulfate ................................... 0,50
Phenol red ................................................ 0,36
Agar ........................................................ 15,00
Final pH 7,2 ± 0,2
Directions
Suspend 48 g of powder in 1 L of distilled water and
bring to the boil. Dispense in tubes or flasks and sterilize
by autoclaving at 121°C for 15 minutes. Cool to 50°C
and add 100.000 I.U. of sodium G penicillin and 0,01g of
pimaricine per litre. Pour into sterile plates.
Description
This formulation is according to Kielwein’s modification
to Korth’s medium, improving the latter because it sup-
ports growth of almost all types of Pseudomonas and
Aeromonas.
Selectivity is achieved due to the antibiotics (penicillin
and pimaricine) and glutamate, which is hard to metabo-
lize by gram-negative bacteria. Differentiation between
Pseudomonas and Aeromonas is based on the utiliza-
66
Description
This medium is used to confirm enterobacteria by their
ability to ferment glucose. Glucose fermentation can be
detected by the production of acid that change the colour
of the medium to yellow.
References
ISO 4702 Standard (1993) Microbiology General Guid-
ance for the enumeration of entrobacteriaceae without
ressucitation. MPN technique and colony count tech-
nique.
ISO 8523 Standard (1991) Microbiology General Guid-
ance for the detection of Enterobacteriaceae with pre-
enrichment.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press. Boca Raton. Fla.
tion of starch, which is degraded by Aeromonas with
acid production which causes change in colour of phenol
red to yellow. Pseudomonas also grows on this medium
but do not degrade starch and there is no acid produc-
tion and thus their colonies remain blue-violet.
Technique
Antibiotics are added to this medium after sterilization
and cooling to 50°C, in such a way that final concentra-
tions in the medium are 100 u/mL of penicillin and 10
mcg/mL of piramicine (it may be replaced by amphoter-
icin or nystatin).
After solidification in plates, medium may be used by
surface inoculation or by leaving for the membrane
filters. Aeromonas colonies turn to yellow, and Pseu-
domonas ones do not.
The incubation is performed at room temperature (20-
25°C) for 3 days. Sometimes, enterobacteria may also
grow but very slowly with pinpoint colonies.
References
KORTH, H. (1963) Ein Nährboden zur Züchtung von
Pseudomonaden. Zbl.Backt.Parasit. Hyg. Abt. 190:225
STANIER, R., N. PALLERONI, M. DOUDOROFF. (1966)
The aerobic pseudomonads: A taxonomic study. J. Gen.
Microbiol. 42:159-271
KIELWEIN, G. (1971) Die Isolierung und Differenzierung
von Pseudomonaden aus Lebensmitteln. Arch G. Leb-
ensmillelhyg. 22:29-37.
 Gram Negative Broth (GN Broth)
Ref. 02-093
Specification
Liquid culture medium for enteric bacteria according
Hajna’s formulation.
Formula (in g/L)
Peptone .................................................... 20,0
Dextrose ..................................................... 1,0
D-Mannitol .................................................. 2,0
Sodium citrate ............................................ 5,0
Sodium deoxycholate ................................. 0,5
Di-potassium phosphate ............................. 4,0
Monopotassium phosphate ........................ 1,5
Sodium chloride .......................................... 5,0
Final pH 7,0 ± 0,2
Directions
Dissolve 39 g of powder in 1 L of distilled water. Dis-
pense in tubes or flasks and sterilize in the autoclave at
121°C for 15 minutes.
Description
GN Broth (Gram Negative Broth) is an enrichment and
selective medium for enterobacteria, with a strong inhibi-
tory action against gram-positive because of its high
HC Agar Base
T
Ref. 01-298
R-45
S-53-45
Specification
Selective solid medium for the enumeration of molds in
cosmetic products.
Formula (in g/L)
Tryptone ................................................... 2,50
Proteose peptone ..................................... 2,50
Yeast Extract ............................................ 5,00
Dextrose ................................................. 20,00
Disodium phosphate ................................. 3,50
Monopotasium phosphate ........................ 3,40
Ammonium chloride .................................. 1,40
Magnesium sulphate ................................ 0,06
Sodium carbonate .................................... 1,00
Chloramphenicol ...................................... 0,10
Agar ........................................................ 15,00
Final pH 7,0 ± 0,2
Directions
Suspend 54,6 g of powder in 1 L of distilled water and
bring to boil. Add 20 mL of polysorbate 80 and homog-
enize. Distribute in suitable containers and sterilize in
autoclave at 121ºC for 15 minutes.
content of citrate and deoxycholate. On the other hand,
mannitol restrains the growth of Proteus and facilitates
the proliferation of Salmonella and Shigella.
The medium is strongly recommended for primary
enrichment, 14-16 first hours, before going to selective
media such as EMB (Ref. 01-068) or MacConkey (Ref.
01-118). Its author, Hajna, declares an extraordinary
selectivity of the medium, whatever may the origin of the
sample, if everything is kept in a transport medium upto
the inoculation.
References
HAJNA, A.A. (1955) A new enrichment medium for gram-
negative organisms of the intestinal group Pub.Hlth.Lab
13:83
EDWARDS and EWING (1973). Identification of Entero-
bactericeae. Burgess Pub.Co. Minneapolis.
VANDERZANT & SPLITTSTOESSER (1992). Compen-
dium of Methods for the Microbiological Examination of
Food. 3rd. Ed. APHA. Washington.
DOWNES, F.P. & K.ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 4th ed.
APHA. Washington.
Description
The HC Agar was developed by Mead & O’Neill in 1986
to attain reliable enumeration of moulds in cosmetic
products in short time. The nutrient basis of the medium
s the dextrose with the peptones and yeast extract that
supplies the energy, nitrogen and vitamins and growth
factors. The inorganic ions are given by ammonium chlo-
ride and magnesium sulphate and both phosphates acts
buffering the medium. Sodium carbonate and polysorb-
ate are detoxifiers and neutralising preservatives and
others toxic substances. The selectivity against bacteria
is due to the chloramphenicol.
Technique
Suitable sample is inoculated into surface of me-
dium plates per duplicate and incubate aerobically at
27,5±0,5ºC for 72 hours. Count colonies of moulds from
duplicate plates and record average count of mould
count per g or mL of sample.
References
MEAD, C. & J. O’NEILL (1986) A three-day mould as-
say for cosmetics and toiletries. J. Soc. Cosmet. Chem.
37:49-57
67
 Heart Extract Broth
Ref. 02-564
Specification
Liquid medium for fastidious microorganisms cultivation
Formula (in g/L)
Heart Extract .......................................... 10,00
Tryptose .................................................. 10,00
Sodium Chloride ....................................... 5,00
Final pH 7,4 ± 0,2
Directions
Dissolve 25 g of powder in 1 L of distilled water, heating
if necessary. Distribute in suitable containers and steri-
lize in autoclave at 121ºC for 15 minutes.
Hektoen Enteric Agar
Ref. 01-216
Specification
Solid, selective and differential culture medium for isola-
tion of pathogenic enterobacteria from very contami-
nated samples acc. ISO 21567.
Formula (in g/L)
Peptone .................................................. 12,00
Yeast extract ............................................. 3,00
Bile salts ................................................... 9,00
Lactose ................................................... 12,00
Sucrose .................................................. 12,00
Salicin ....................................................... 2,00
Sodium chloride ........................................ 5,00
Sodium thiosulfate .................................... 5,00
Ammonium ferric citrate............................ 1,50
Acid fuchsin .............................................. 0,10
Bromothymol blue .................................... 0,06
Agar ........................................................ 15,00
Final pH 7,7 ± 0,2
Directions
Suspend 77 g of powder in 1 litre of distilled water and
let it soak. Heat up by constant stirring until boiling.
Cool to 55-60°C and pour into sterile plates. Do not
autoclave. This medium is very thermolabile and thus
overheating should be avoided.
Description
This culture medium, originally developed by King and
Metzger, has a high nutrient content like peptones,
fermentable sugars and combination of indicators which
makes this medium less toxic. All these characteristics
and the bile salts makes it a very selective and effective
medium.
68
Description
The Heart Extract Broth is a very old and classical gen-
eral purpose medium that can be used for the cultivation
of fastidious microorganisms. Its components supply a
very rich nutritive base which supplemented with suitable
additives can be used in all laboratory purposes, from
Blood Agar Base until Base for carbohydrate fermenta-
tion studies.
References
HUNTOON, F.M. (1918) “Hormone” Medium. A simple
medium employable as a substitute for serum medium.
J. of infect. Dis. 23:169-72
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press Boca Ratón Fl.
MAC FADDIN, J.F. (1985) Media for Isolation-Cultiva-
tion-Identification-Maintenance of Medical Bacteria.
William & Wilkins. Baltimore.
FDA (1998) Bacteriological Analytical Manual. 8th ed.
Rev.A. AOAC International. Gaithersburg. MD.
Technique
In order to avoid the spreading of Proteus, it is neces-
sary that the agar surface be perfectly dry at the moment
of inoculation. Inoculation must be carried out by surface
streaking, directly from rectal swabs or faeces dilutions.
If colonies are well separated after 18 hours of incuba-
tion, first characteristic appearances or colony charac-
ters may be observed, as then those colonies become
more prominent after a longer period:
Shigella spp., Proteus inconstans: Raised colonies,
humid, green colour.
Salmonella sp.: Green-blue colonies, with or without
black core.
Pseudomonas spp.: Irregular colonies, plain, green or
brown.
Companion and non pathogenic bacteria: Salmon colour
colonies.
References
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media CRC Press. BocaRaton. Fla. USA
HORWITZ, W. (2000). Official Methods of Analysis of the
AOAC Internacional 17th ed. Gaithersburg Md. USA
DOWNES, F.P. & K. ITO (2001) Compendium of
Methods for the Microbiological Examination of
Foods.4th ed. APHA. Washington DC. USA.
FORBES, B.A., D.F SAHM & A.S. WEISSFELD (Eds)
(1998) Bailey & Scott’s Diagnostic Microbiology 10th ed.
Mosby. St Louis, Mo. USA
MURRAY, P.R., E.J. BARON, J.H. JORGENSEN, M.A.
PFALLER & R.H. YOLKEN (Eds) (2003) Manual of Clini-
cal Mcrobiology 8th ed. ASM Press. Washington DC, USA
KING S.y METZGER W. Y. (1968). A new plating method
for the isolation of the enteric pathogens. Appl. Microbiol.
16:577.
US FDA (1998) Bacteriological Analytical Manual 8th
ed. AOAC Internacional. Gaithersburg, Md. USA.
ISO 21567. Standard (2004) . Horizontal method for the
detection of Shigella ssp.
 m-HPC Agar
Ref. 01-593
Specification
Solid medium for the enumeration of heterotrophic mi-
cro-organisms from water by filter membrane technique
Formula (in g/L)
Peptone ................................................. 20,00
Gelatine .................................................. 25,00
Agar ........................................................ 15,00
Final pH 7,1 ± 0,2
Directions
Suspend 60 g of powder in 1 L of distilled water and
bring to the boil. Add 10 mL of glycerol and homogenize.
Distribute in suitable containers and sterilize in autoclave
at 121ºC for 15 minutes.
Description
The membrane-Heterotrophic Plate Count Agar was
developed in 1979 by Taylor and Geldreich as an adap-
tation to the membrane filtration method of the Standard
Plate Count Medium and it is also know as m-SPC.
In this medium peptone supplies all he nutrients and
gelatine overcomes the trouble of liquefaction and over-
growth of the colonies. The m-HCP is recommended by
Standard Methods for Examination of Water and Waste-
water in the filtration technique applied to the greats
volumes of water with a low microbial population.
Indole Nitrite Fluid Medium
Ref. 03-101
Specification
General purpose medium for indole production tests and
nitrite detection.
Formula (in g/L)
Casein peptone ........................................ 20,0
Di-sodium phosphate ................................. 2,0
Dextrose ..................................................... 1,0
Potassium nitrate ........................................ 1,0
Agar ............................................................ 1,0
Final pH 7,2 ± 0,2
Directions
Suspend 25 g of powder in 1 L of distilled water. Heat to
boiling and dispense in tubes. Sterilize at 121°C for 15
minutes. If tubes are kept in refrigerator, heat them up in
boiling water bath for 2 minutes before using them.
Description
The extraordinary richness of this medium, due to high
quality and quantity of peptone, allows its usage as a
general medium. Even the fastidious microorganisms
Technique
Sampling
Refer to the Section 9060 of the Standard Methods or
the ISO 5667 Standard. In any case the time between
the sampling and analysis must exceed 8 hours (6 for
transfer and 2 for analysis). The samples can be refriger-
ated but never chilled. After 24 hours refrigeration the
samples must be rejected.
Procedure
A suitable volume to obtain 20-200 colonies on the 47
mm filter must be filtered. Grilled filter of 0,45 µm pore
are preferred. The funnel and the filter are washed three
times with 20-30 mL of sterile water. The filter is placed
on the surface of a m-HPC Agar plate and incubated
for 48 h. at 35±2ºC. Express the results as “Cfu/unity of
volume filtered”
References
TAYLOR, R.H & E.E. GELDREICH (1979) A new mem-
brane filter procedure for bacterial counts in potable wa-
ter and swimming pool samples. J. Amer. Water Works
Assoc. 71:402-405
EATON, A.D., .S. CLESCERI & A.E. GREENBERG (Eds)
(1995) Standard Methods for the Examination of Water and
Wastewater. 19th Ed. APHA Washington DC.
such as Lactobacillus or Clostridium grow well on this
medium, and indole and nitrite tests can be carried out.
Adding 2 g/L of Bacteriological Agar to this medium can
make it suitable for motility test. Indole production can
be observed with Kovacs’ Reagent (Ref. 06-018) with or
without previous extraction with chloroform, and in an-
other tube, nitrate assay can be carried out with Nitrate
reagents (Ref. 06-003 and Ref. 06-004).
This medium is not the most suitable one for indole
production detection in enterobacteria, because some
times there are some interferences. For that assay, SIM
Medium (Ref.3-176) or Nitrate Broth (Ref. 02-138) are
more suitable.
Nonetheless, Indole Nitrite Broth is the medium of choice
for nitrate reduction assays or denitrification assays with
any other bacterial biotype especially the grampositive
ones .
References
VANDERZANT & SPLITTSTOESSER (1992). Compen-
dium of Methods for the Microbiological Examination of
Food.3rd. Ed. APHA. Washington.
69
 Inhibitory Substances Test Media
Specification
Solid media for the verification of antimicrobial inhibi-
tor substances in food and packaging material.
Assay Agar at pH 6,0
Ref. 01-437
Formula (in g/L)
Casein peptone ........................................ 3,45
Meat peptone ........................................... 3,45
Sodium chloride ........................................ 5,10
Agar ........................................................ 15,00
Final pH 6,0 ± 0,1
Directions
Suspend 27 g of powder in 1 L of distilled water and heat
to the boil. Distribute in the suitable containers and steri-
lize in the autoclave at 121°C for 15 minutes. Check the
pH and adjust it if necessary. Cool to 50°C and inoculate
with the spore suspension of Bacillus subtilis to obtain a
confluent culture.
Homogenize well and pour the medium into sterile
plates, (15 mL per plate). Once the agar is solidified, put
the plates into the refrigerator until their use.
Assay Agar at pH 8,0
Ref. 01-438
Formula (in g/L)
Casein peptone ........................................ 3,45
Meat peptone ........................................... 3,45
Sodium chloride ........................................ 5,10
Trisodium phosphate ................................ 2,40
Agar ........................................................ 15,00
Final pH 8,0 ± 0,1
Directions
Suspend 29,4 g of powder in 1 L of distilled water and
heat to the boiling. Distribute into suitable containers and
sterilize in the autoclave at 121°C for 15 minutes. Check
the pH and adjust it if necessary. Cool to 50°C. Inoculate
a part of medium with the spore suspension of Bacillus
subtilis to obtain a confluent culture. Inoculate the other
part of medium with a cell suspension of Micrococcus
luteus ATCC 9341.
Homogenize well and pour the medium into sterile
plates, (15 mL per plate). Once the agar is solidified, put
all the plates into the refrigerator until their use.
70
Assay Agar at pH 7,2
Ref. 01-439
Formula (in g/L)
Casein peptone ........................................ 3,60
Meat peptone ........................................... 3,60
Sodium chloride ........................................ 5,00
Trisodium phosphate ................................ 0,80
Agar ........................................................ 15,00
Final pH 7,2 ± 0,1
Directions
Suspend 27 g of powder in 1 L of distilled water and
heat to the boiling. Distribute into suitable containers and
sterilize in the autoclave at 121°C for 15 minutes. Check
the pH and adjust it if necessary. Cool to 50°C and add
50 mcg/L of Trimetoprim. Inoculate with the spore sus-
pension of Bacillus subtilis to obtain a confluent culture.
Homogenize well and pour the medium into sterile
plates, 15 mL per plate. Once the agar is solidified, put
the plates into the refrigerator until their use.
All the plates must be sealed with an adhesive and
water-proof tape, named, and packed in plastic bags
before storing them into the refrigerator until their use. If
they are stored at 3-6°C, plates may be thus stored up
to 3 weeks. Do not freeze the plates, and never put them
back in the refrigerator once they have reached 10°C.
Technique
Assay may be performed with 2 or 4 plates. If two plates
are used, one must be at pH 6.0 and the other one at pH
8.0, and both must be inoculated with Bacillus subtilis.
If four plates are used, include another plate at pH 7,2,
inoculated with Bacillus subtilis too, and another plate at
pH 8,0 inoculated by Micrococcus luteus.
If the sample is solid, use a punch to extract 8 mm x 2
mm diameter cylinders. If the sample is liquid, use anti-
biogram assay discs. Sample pieces or discs are placed
over the plates surface, 2 per plate, on all the plates.
As a control, put discs alternatively with the samples
in each plate, impregnated with the following standard
substances:
Assay Medium at pH 6,0: Penicillin 0,01 IU/disc
Assay Medium at pH 8,0: Streptomycin 0,5 mcg/disc
Assay Medium at pH 7,2: Sulfamidin 0,5 mcg/disc
Plates are incubated at 30°C for 18-24 hours, except
those inoculated with M. luteus, which are incubated at
37°C.
After the incubation period, read the inhibition halos
(zones of inhibition), but do not consider the diameter of
the discs or cylinders.
Assay is considered reliable when control discs provide
ø12 mm halos, and then samples with average diameter
greater than ø4 mm are considered positive (with inhibi-
tor presence), and samples with ø1-4 mm halos must be
considered doubtful.
 Inhibitory Substances Test Media

References
BAUR, E. (1975) Untersuchungen von Fleisch und
Wurstwaren mit dem Hemmstoff im Rahmen das Tierärt-
zlichen Lebensmittelüberwachung. Fleischwirtschaft
55:843-845.
BOGAERTS, R., F. WOLF (1980) Eine standardisierte
Methode sum Nachweis von Rückständen antibakterial
wirksauer substanzen in fischen Fleich. Fleischwirtschaft
60: 667-675
DEUTSCHES FLEICHBESCHANGESETZ: Aus-
führungsbestimmungen A über die Untersuchung und
Gesundheitspolizeiliche Behandlung der Schlachtiere
und des Fleisches bei Schlachtungen im Inland. Anlage
1 zu § 20 Abs. 4: Vorschriften über die Bakteriologische
Fleischuntersuchung.

Iron Sulfite Agar

Ref. 01-328 suitable for clostridia. The actual formulation is according

Specification
Medium for detection and identification of sulfite reduc-
ing clostridia.
Formula (in g/L)
Tryptone ................................................... 10,0
Sodium sulfite ............................................. 0,5
Ferric citrate................................................ 0,5
Agar .......................................................... 15,0
Final pH 7,1 ± 0,2
Directions
Dissolve 26 g of powder in 1 L of distilled water, heating
up to boiling with constantl stirring. Distribute in suitable
containers and sterilize in the autoclave at 121°C for 15
minutes.
Description
Although the medium was originally described by Wilson
Blair, it remained unused because it was not safe. There
have been many modifications, and the one by Tanner
in 1944 for the National Canners Association of America
was the more lasting. However, it was also modified
because it was demonstrated that clostridia were highly
inhibited at concentrations of sulfite over 0,1%, this be-
ing the reason why the Wilson Blair formulation was un-
to the one widely experimented by several authors, and
provides a relatively low false negative results.
Most of clostridia have sulfite reductases in their cito-
plasm, but they are unable to expel them to the exte-
rior. So, when H2S is produced from sulfite, the colony
becomes dark due to the formation of precipitates of iron
sulfide from citrate, which is less toxica than the alum
described by Wilson and Blair.
Note that there are many gram negative bacteria able to
reduce sulfite with iron sulfide production in this medium,
but in these cases the enzymes are extracellular and all
the medium becomes dark, rendering their enumeration
impossible.
Technique
To enumerate sulfite reducing clostridia, vegetative
cells have to be eliminated by heating up the sample to
80°C for 10 minutes. Sample is cooled under water and
mixed aseptically with an equal volume of sterile, double
concentrated, melted and cooled to 60°C medium. Let it
solidify and incubate at 37°C for 48 hours, with visual ex-
amination after 24 hours. Enumerate the black colonies,
which clearly contrast with the medium, and express
the result as spores of sulfite reducing clostridium per
volume of sample.
Tubes with blackish medium, without discrete colonies,
must be rejected and the assay has to be restarted heat-

71
 Iron Sulfite Agar

ing up the sample at the same temperature but for ad-

ditional 5 minutes more than the previous heating time.
Should thermophilic species are desired to be enumer-
ated, incubate at 55°C.
If the assay is performed by membrane filtration, it is
advisable to put the membrane over a layer of medium
solidified in the plate, and then cover it with another layer
of medium (20 mL) melted and cooled to 60°C.

References
TANNER, F.W. (1944) The Microbiology of Food. 2 Ed.
Garrad Press U.S.A.
BUTTON, A.W.J. (1959) A note on the enumeration of
thermophilic sulfate-reducing bacteria. J. Appl. Bact.,
22(2) 278-280.
SCARR, M.P. (1959) Selective Media Used in the Micro-
biological Examination of Sugar Products. J. Sci. Food
Agri., 678-681.

Left and center: Clostridium perfringens

ATCC 13124; right: control.

Kanamycin Esculin Azide Media (KAA Media)

Kanamycin Esculin Azide Agar Kanamycin Esculin Azide Broth

(KAA Agar) (KAA Broth)
Xn Xn
Ref. 01-263 Ref. 02-263
R-22-32-52/53 R-22-32-52/53
S-7-46-61 S-7-46-61
Specification Specification
Solid medium for confirmative detection and isolation of Liquid medium for the presumptive detection of Lance-
Lancefield’s group D streptococci in food samples, ac- field’s group D streptococci in food samples, according
cording to Mossel et al. to Mossel et al.

Formula (in g/L) Formula (in g/L)

Tryptone ................................................. 20,00
Yeast extract ............................................. 5,00
Sodium chloride ........................................ 5,00
Disodium citrate ........................................ 1,00
Esculin ...................................................... 1,00
Ferric-Ammonium citrate .......................... 0,50
Sodium azide ............................................ 0,15
Kanamycin sulfate .................................... 0,02
Agar ........................................................ 15,00
Final pH 7,0 ± 0,2
Directions
Suspend 48 g of powder in 1 L of distilled water and let it
soak. Heat to boiling and distribute into suitable contain-
ers. Sterilize in the autoclave at 121°C for 15 minutes.
Tryptone ................................................. 20,00
Yeast extract ............................................. 5,00
Sodium chloride ........................................ 5,00
Disodium citrate ........................................ 1,00
Esculin ...................................................... 1,00
Ferric-Ammonium citrate .......................... 0,50
Sodium azide ............................................ 0,15
Kanamycin sulfate .................................... 0,02
Final pH 7,0 ± 0,2
Directions
Dissolve 33 g of powder in 1 L of distilled water. Distrib-
ute in suitable containers and sterilize in the autoclave at
121°C for 15 minutes.

72
 Kanamycin Esculin Azide Media (KAA Media)
Description
KAA Presumptive Broth and Confirmative Agar are the
two media that the Spanish National Center for Food
and Nutrition (CeNAN) recommend to detect, enumerate
and isolate Lancefield’s group D streptococci in samples
of food and beverages like: bottled water, fresh, refriger-
ated, frozen or mashed meat, fish and molluscs, soft
drinks, pastries, spices and semiconserves. Kanamycin
and sodium azide are the selective inhibitory com-
pounds.
Technique
Prepare tubes with 9 mL of broth, and Petri plates with
the agar. Make a decimal dilution bank from the sample
in duplicate, and inoculate 1 mL fractions in the tubes.
Incubate at 37°C for 24 hours.
Presumptive presence of streptococci is indicated by the
development of a blackish-brown colour and the loss of
fluorescence behind Wood’s light. These tubes are con-
sidered as positive, and then inoculate 0,1 mL aliquotes
from them over the surface of plates with Confirmative
Agar, spreading with a Drigalsky loop (Ref. 5-010).
Incubate those plates, in inverted position, at 37°C for 24
hours. Colonies that appear surrounded by a black halo
are considered as group D streptoccoci, and are isolated
to confirm them biochemically and morphologically with
the following tests: microscopical examination, catalase
assay (that should be negative) in an azideless medium,
growth at 45°C and resistance to a high saline concen-
tration (6,5% of NaCl in BHI Broth (Ref. 02-102). Finally,
they have to grow in Bile Esculin Agar (Ref. 01-265) with
a similar appearance of the colonies in the Confirmative
Agar. Nonetheless, there are some exceptions to this
rule, i.e. Streptococcus equinus and S.bovis do not grow
in the hypersaline broth, and therefore, definitive identifi-
cation has to be performed by serological methods.
This methodology does not allow the enumeration of
cells from the original sample, and as this is a neces-
sary data, it is recommended to use the Most Probable
Number (MPN) technique with the presumptive broth,
doing it at double strength if necessary. For bottled
water, soft drinks and molluscs, CeNAN suggest the fol-
lowing technique:
Prepare broth tubes at normal concentration and at
double strength. Using a sterile pipet, inoculate five broth
tubes of double strength with 10 mL of sample. Inoculate
five tubes of normal concentration with 1 mL of sample
and five tubes of normal concentration with 0,1 mL of
sample. Homogenize them well and incubate at 37°C
for 48 hours. Tubes that show a blackish-brown colour
after the incubation period, are considered positive. Note
down the results and carry out the counting using the
MPN tables.
References
GUINEA, J., SANCHO, J., PARES, R. (1979). Análisis
Microbiológico de Aguas. Ed. Omega, Barcelona,.
MOSSEL, D.A.A., P.G.M. BUKER, J. ELDERING (1978)
Streptokokken der Lancefield Gruppe D in Lebensmitteln
und Trinkwasser. Arch F. Lebensmittelhyg. 29:121-127
PASCUAL ANDERSON, MªRª (1992) Microbiología
Alimentaria. Diaz de Santos, S.A. Madrid.
VANDERZANT & SPLITTSTOESSER (1992). Compen-
dium of Methods for the Microbiological Examination of
Food.3rd. Ed. APHA. Washington.
73
 Kenner Fecal Media (KF Media)
Kenner Fecal Agar (KF Agar)
Xn
Ref. 01-294
R-22-32-52/53
S-7-46-61
Specification
Solid and selective medium for enterococci enumeration
and detection.
Formula (in g/L)
Proteose peptone ................................. 10,000
Yeast extract ......................................... 10,000
Sodium chloride ...................................... 5,000
Sodium glycerophosphate .................... 10,000
Maltose ................................................. 20,000
Lactose ................................................... 1,000
Sodium azide .......................................... 0,400
Bromocresol purple ................................ 0,015
Agar ...................................................... 20,000
Final pH 7,2 ± 0,2
Directions
Suspend 76,4 g of powder in 1 L of distilled water and
heat to boiling by constant stirring. If it has to be used
immediately, it may not to be sterilized. Otherwise,
sterilize in the autoclave in small volumes, at 121°C and
for 10 minutes maximum. In both the cases, let it cool to
50°C and add 10 mL/L of TTC Sterile Solution 1% (Ref.
06-023). Homogenize well and distribute in sterile plates.
Note: Non homogeneous appearance is normal, and it
does not affect the medium´s quality and efficacy.
Kenner Fecal Broth (KF Broth)
Xn
Ref. 02-294
R-22-32-52/53
S-7-46-61
Specification
Liquid and selective medium for enterococci enumera-
tion by the MPN or membrane filter methods.
Formula (in g/L)
Proteose peptone ................................. 10,000
Yeast extract ......................................... 10,000
Sodium chloride ...................................... 5,000
Sodium glycerophosphate .................... 10,000
Maltose ................................................. 20,000
Lactose ................................................... 1,000
Sodium azide .......................................... 0,400
Bromocresol purple ................................ 0,015
Final pH 7,2 ± 0,2
74
Directions
Suspend 56,4 g of powder in 1 L of distilled water. If it
has to be used immediately, it may not to be sterilized,
just heat to boiling for one or two minutes, with constant
stirring. Otherwise, sterilize in the autoclave in small
volumes, at 121°C and 10 minutes maximum. In both
cases, let it cool to 50°C and add 10 mL/L of TTC Sterile
Solution 1% (Ref. 06-023). Homogenize well and distrib-
ute in sterile tubes.
Note: Non homogeneous appearance of medium is nor-
mal, and it does not affect the medium´s quality
and efficacy.
Description
Kenner, Clark and Kabler (1960,1961) discovered that
KF media were excellent for detecting enterococci in pol-
luted water. Carbohydrates in this medium viz. lactose
and maltose, are utilised by most of enterococci, produc-
ing a big amount of acid and making the indicator turn
from violet to yellow. streptococci that do not belong to
D group may also grow in the medium, but they do not
produce enough acid to change the indicator colour.
Other microorganisms, are strongly inhibited by sodium
azide. enterococci reduce TTC to formazan and so their
colonies are red coloured.
Technique
Media can be used by following several techniques
depending on the sample. When a marginal contami-
nation is thought, then often it is performed using the
membrane filter or MPN method. On the contrary, if a
high population of enterococci is suspected, it is more
advisable to make a plate count.
To use the membrane filter technique, incubate at 37°C
for 48 hours on the surface of KF Agar or on an absorb-
ent pad impregnated in KF Broth.
To use MPN technique, directly inoculate samples, of up
to 1 mL to tubes with 10 mL of KF Broth in normal con-
centration, and for larger samples, to tubes with 10 mL of
KF Broth in double strength.
If the sample is suspected of being highly contaminated,
prepare a decimal dilution bank and inoculate on the
surface 0,1 mL with a Drigalsky Loop (Ref. 5-010) or, if
desired, inoculate by stabbing 1 mL. Anyway, incubation
should be carried out at 37°C and for a 48 hours period.
After the incubation, readings are performed by observ-
ing the indicator colour turning from violet to yellow, and
on the solid medium, colonies will be also pink or red
coloured.
It is very important to maintain the pH of the medium
over 7,0, or otherwise, false results may appear. Steri-
lization longer than the specified period could result in
caramelization and thereby a decrease in the pH.
 Kenner Fecal Media (KF Media)
References
KENNER, B.A., CLARK, H.F., KABLER, P.W. (1961)
Fecal streptococcci I. Cultivation and Enumeration of
streptococci in Surface Waters. Appl. Microbiol. 9:15.
KENNER, B.A., CLARK, H.F., KABLER, P.W. (1960)
Fecal streptococci. II. Quantification of streptococci in
faeces. Am. I. Publ. Health, 50:1553.
VANDERZANT & SPLITTSTOESSER (1992). Compen-
dium of Methods for the Microbiological Examination of
Food.3rd. Ed. APHA. Washington.
APHA-AWWA-WEF (1998) Standard Methods for the
Examination of Water an WasteWater. 20th. Ed. Wash-
ington.
King Media
King A Agar
Ref. 01-001
Specification
Solid Medium to enhance the pyocyanine production
by Pseudomonas aeruginosa acc. ISO 16266, 22717
standards.
Formula (in g/L)
Peptone .................................................... 20,0
Magnesium Chloride .................................. 1,4
Potassium Sulfate .................................... 10,0
Agar .......................................................... 15,0
Final pH 7,2 ± 0,2
Directions
Suspend 46,4 g of powder in 1 L of distilled water with
glycerol 10 mL and let it soak . Heat with constant stirring
until it boils. Distribute into suitable containers and steri-
lize by autoclaving at 121°C for 15 minutes. If tubes are
used, let them solidify with short slant and good butt.
Description
This A medium was formulated by King, Ward and
Raney in 1954 to enhance the pyocyanine production by
Pseudomonas aeruginosa. The blue pigment Pyocyanine
diffuses into the culture medium and its production varies
depending on the strains of Pseudomonas aeruginosa
and on the growth conditions.
Sometimes, although this medium enhances especially
blue pigment production, it is possible that green (piover-
dine) or brown (piomelanine) pigments also appear and
mask the pyocyanine. Anyway, fluorescence and other
pseudomonas pigments can be noticed on other more
suitable media, like King B Agar (1-029).
Technique
Slanted tubes or Petri dishes are inoculated by surface
inoculation by streaking and are then incubated at 30-
32°C for 4-5 days. Petri plates usage has the disadvan-
tage of the dehydration of the medium during incubation.
Therefore, it is better to use slanted tubes being careful
for the aeration by loosening the screw caps or replacing
them with cotton or aluminium caps.
In the recently isolated pathogenic strains from the
pathological material, pigment production is often shown
early i.e. after 24-48 hours of incubation, however if
the material is non pathogenic or if they come from
water, food or soil,then the pigmentation can be later or
delayed.
When pigment has not got the usual blue colour, it is due
to the production of two or more coloured substances.
At this time, and if it is not confirmed on other culture
medium, it is recommended to confirm by extraction:
on the culture slant, 0,5-1 mL chloroform is added,
and it is shaken for a few minutes until the pyocyanine
is diffused, which makes the solvent blue. After that,
chloroform is acidified with a few drops of HCl, obtaining
a rapid change in colour from blue to red,the fact that
confirms the presence of pyocyanine.
References
KING E.O., M. WARD y D.E. RANEY (1954) Two simple
media for the demonstration of pyocyanin and fluores-
cein. J.Lab.Clin.Med. 44:301-307
US Pharmacopeia (2002) 25th ed. <61> Microbial Limit
Test. US Pharmacopoeial Conv. Inc. Rockville MD.
ISO 16266:2006 Standard. Water Quality.– Detection
and enumeration of Pseudomonas aeruginosa. Method
by membrane filtration
ISO 22717:2006 Standard. Cosmetics – Detection of
Pseudomonas aeruginosa.
75
 King Media
King B Agar (F Agar)
Ref. 01-029
Specification
Culture media for enhancing the fluorescein production
by Pseudomonas species.acc. EN 12780:2002 and ISO
16266, 22717 standards.
Formula (in g/L)
Meat peptone ........................................... 10,0
Casein peptone ........................................ 10,0
Dipotassium phosphate .............................. 1,5
Magnesium sulfate ..................................... 1,5
Agar .......................................................... 15,0
Final pH 7,2 ± 0,2
Directions
Suspend 38 g of powder in 1L of distilled water with
10 mL of glycerol and let it soak . Heat to boiling and
distribute in suitable containers. Sterilize in the autoclave
at 121°C for 15 minutes. Cool by solidifying in slanted
position with a long slant.
Description
F medium was formulated by King, Ward and Raney
in 1954 to enhance green fluorescent pigment (pio-
verdine) production by Pseudomonas fluorescens
and Ps.aeruginosa, in which pyocyanine production is
restricted.
Green-yellowish pigments, soluble and from fluorescent
type, define the Pseudomonas group I according to the
9th. edition of Bergey’s Manual of Systematic Bacteriol-
ogy, and therefore, detection of its productive capacity is
critical.
76
Technique
Slanted tubes are inoculated with Pseudomonas strains
and incubated at 30-31°C for a 2-4 days period. If after
this time a green-yellowish colour does not appear on
the medium, the tubes should be kept under observa-
tion at room temperature for a period of 6-20 days more
before the culture can be rejected as negative. It can
be observed that Pseudomonas aeruginosa and Pseu-
domonas putida strains coming from water, soil or food,
produce pigments in a very slow way.
Pioverdine is not soluble in chloroform, so the confirma-
tion of presence is usually done by characteristic fluo-
rescence verification under Wood’s light, comparing the
dubious tube to another uninoculated F medium , which
is considered as the control.
References
KING, E.O., M.WARD and D.E. RANEY (1954) Two
simple media for the demonstration of pyocyanin and
fluorescein J.Lab.Clin.Med. 44:30-307
LENNETTE, E.H., E.H. SPAULDING and J.P. TROUANT
(1974) Manual of Clinical Microbiology. 2nd. Ed. ASM.
Washington.
US PHARMACOPOEIA (2002) <61> Microbial Limit
Tests. 25th ed. US Phamacopoeial Conv. Inc. Rockville.
MD.
DIN Standard 38411Teil 6 (Juni 1991): Mikrobiologische
Verfahren (Gruppe K): Nachweis von Escherichia coli
und coliformen keimen (K6).
PALLERONI, N. (1984) The genus Pseudomonas, in
Bergey’s Manual of Systematic Bacteriology.
EUROPEAN STANDARD EN 12780:2002. Water quality.
Detection and enumeration of Pseudomonas aeruginosa
by membrane filtration. CEN. Brussels.
ISO 16266:2006 Standard. Water Quality.– Detection
and enumeration of Pseudomonas aeruginosa. Method
by membrane filtration
ISO 22717:2006 Standard. Cosmetics – Detection of
Pseudomonas aeruginosa.
 Kligler Iron Agar (KIA)
Ref. 01-103
Specification
Solid and differential medium for primary identification of
enterobacteria based on the fermentation of two sugars
and the hydrogen sulphide production according ISO
Standard 6340.
Formula (in g/L)
Meat extract .............................................. 3,00
Yeast extract ............................................. 3,00
Peptone .................................................. 20,00
Lactose ................................................... 10,00
Sodium chloride ........................................ 5,00
Dextrose ................................................... 1,00
Ammonium iron citrate ............................. 0,50
Sodium tiosulfate ...................................... 0,50
Phenol red ................................................ 0,03
Agar ........................................................ 15,00
Final pH 7,4 ± 0,2
Directions
Add 58 g of powder to 1 L of distilled water and heat to
the boiling. Distribute in tubes and sterilize in the auto-
clave at 121°C for 15 minutes. Let it solidify with short
slant and plenty of butt.
Description
Kligler Agar is a differential medium that has all the
characteristics of the 2-Sugar Russell Agar and the
Lead Acetate Medium for H2S detection. In this medium,
lactose fermentation and hydrogen sulphide production
can be detected, so it allows a presumptive diagnostic
of most enterobacteria. Glucose fermentation is shown
by acid production, which makes the indicator turn from
red to yellow, but since there is little sugar (dextrose),
acid production is very limited and then a reoxidation of
the indicator is produced on the surface of the medium,
and the indicator remains red. Otherwise, when lactose
is fermented, the large amount of acid produced avoids
reoxidation and then all the medium turns to yellow.
Hydrogen sulphide production is indicated by the me-
dium turning black, due to the reaction of H2S (liberated
from tiosulfate) with the Fe ions from ammonium iron
citrate.
Technique
Kligler Iron agar is used in slanted tubes with short slant
and plenty of butt, that are inoculated on the surface as
much as in stab. Inoculum must be copious, it has to
come from a solid medium, because otherwise, readings
may be delayed (up to 2-3 days more). Normal incubation
is 18 hours at 37°C.
It is recommended to use tubes with caps that allow
ventilation, like cotton caps, cellullose caps or cap-o-
test. Should screw caps be used, do not tighten them
because otherwise they can hinder the reoxidation of the
indicator.
Kligler medium provides excellent results if it is used
freshly prepared, however if it has been prepared before
a few days then, it is advisable to remelt it and solidify it
again to obtain more precision.
When H2 S production is more, it may make the read-
ings difficult, and hence the early readings are strongly
recommended. Anyway, one can obtain more precise
readings if Three Sugar Iron Agar (Ref. 01-192) is used,
since this one has the sucrose that allows a greater dif-
ferentiation between members of Proteus, Salmonella
and Shigella types.
References
KLIGLER (1918) Modification of Culture Media used in
the Isolation and Differentiation of Typhoid, Dyesentery
and allied Bacilli. J. Exper. Med. 28:319-332
KLIGLER (1917) A simple medium for the differentiation
of members of typhoid-paratyphoid groups. Am. J. Pub.
Hlth 7:1042-1044
VANDERZANT & SPLITTSTOESSER (1992) Compen-
dium of Methods for the Microbiological Examination of
Food. 3rd. Ed. APHA. Washington.
DOWNES, F.P. & K.ITO (2001) Compendium of Methods
for the Microbiological Examination of Food. 4th. ed.
APHA. Washington.
Left: control; center: Escherichia coli
ATCC 25922; right: Salmonella typhimu-
rium ATCC 14028.
77
 Kligler Iron Agar (KIA)

Typical reactions of enterobacteria on Kligler Iron Agar

78
 Lactose Media
Lactose Broth
(Eur. Phar. Broth Medium D)
Ref. 02-105
Specification
Medium for the pre-enrichment and the detection of
enterobacteria and coliforms in milk and water according
ISO 9308-2 and 21150 standards.
Formula (in g/L)
Peptone ...................................................... 5,0
Meat extract ................................................ 3,0
Lactose ....................................................... 5,0
Final pH 6.9 ± 0,2
Directions
Add 13 g of powder to 1 L of distilled water, or in the
quantity required for the desired concentration. Dissolve
it and distribute into containers fitted with Durham tubes.
Sterilize by autoclaving at 121°C for 15 minutes. Avoid
any further reheating.
Description
Lactose Broth is a classical medium for use in the
presumptive testing for coliforms and for the enrichment
of Salmonella. This formulation is as per the standards
recommended by APHA, AWWA, USP-NF and Euro-
pean Pharmacopoeia.
It is commonly used with Durham fermentation tubes for
the examination of gas formation. If the volume of sam-
ple to inoculate is important, reconstitute the medium
at a concentration such, as to remain normal once the
sample has been added to it.
Although it is not the original formulation, this broth pro-
vides excellent results in Eijkman assays of gas produc-
tion at 45°C, which is a characteristic of Escherichia coli.
While preparing this medium it is important to avoid
overheating and to distribute it into tubes before sterili-
zation.
References
FDA (1998) Bacteriological Analitical Manual 8th ed.
Rev A. AOAC International. Gaithersburg. Va. USA..
VANDERZANT & SPLITTSTOESSER (1992) Compen-
dium of Methods for the Microbiological Examination of
Foods. 3rd. Ed. APHA. Washington.
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Foods. 4th ed.
APHA. Washington.
EUROPEAN PHARMACOPOEIA (2005) 5th ed. §2.6.13
Test for specified micro-organisms. EDQM. Council of
Europe. Strasbourg.
ISO 9308-2 Standard. (1990) Water Quality – Detection
and enumeration of coliform organisms, thermotolerant
coliform and presumptive E. coli – MPN technique.
ISO 21150:2006 Standard. Cosmetics – Detection of
Escherichia coli
US PHARMACOPOEIA (2005) <61> Microbial limit test.
US Pharmacopoeial Conv. Inc. Rockville. Md USA
APHA-AWWA-WPCF (1998) Standard methods for the
examination of water and wastewater. 20th ed. APHA
Washington
Lactose Peptone Broth
Ref. 02-414
Specification
Liquid medium for the enrichment and enumeration of
coliforms in water.
Formula (in g/L)
Casein peptone ...................................... 17,00
Soy peptone ............................................. 3,00
Lactose ................................................... 10,00
Sodium chloride ........................................ 5,00
Bromocresol purple .................................. 0,02
Final pH 7,2 ± 0,2
Directions
Dissolve 35 g of powder (or 70 g if double concentrated
medium is desired) in 1 L of distilled water. Distribute in
tubes provided with Durham’s tubes and sterilize in the
autoclave at 121°C for 15 minutes.
Description
This medium is according to the German standards for
quality control of water.
Technique
German standards suggests the use of MPN technique
with 0,1, 1 and 10 mL of sample and an incubation at
36±1°C for 44±4 hours. Tubes that change to yellow
and eventual gas production/accumulation in Durham´s
tubes are considered positive.
References
DEUTSCHE EINHEITSVERFAHREN zur Wasser-,
Abwasser- Und Schlammuntersuchung. Mikrobiologie
Verfahren (Gruppe K) Nachweiss vom E. coli und colifor-
men keimen.
VERORDNÜNG über Trinkwasser und Wasser für Leb-
ensmittelbetriebe vom 12- Dezember-1990- Bundesges-
etzblatt; Teil I, 2613-2629 (1990)
MURRAY, PR, EJ BARON, MA PFALLER, FC TENO-
VER & RH YOLKEN (eds) (1995) Manual of Clinical
Microbiology, 6th ed. ASM Washington.
MaCFADDIN J.A. (1985) Media for Isolation-Cultivation-
Identification-Maintenance of Medical Bacteria. William&
Wilkins.Baltimore.
79
 Lactose Media

Lactose Purple Modified Broth Directions

Ref. 02-417
Specification
Liquid medium for coliforms and E. coli identification.
Formula (in g/L)
Meat peptone ......................................... 10,00
Meat extract .............................................. 3,00
Sodium chloride ........................................ 5,00
Lactose ................................................... 10,00
Bromocresol purple .................................. 0,02
Final pH 7,2 ± 0,2
Dissolve completely 10,1 g of powder in 500 mL of
distilled water and sterilize in the autoclave at 121°C
for 15 minutes. Cool and aseptically add a flask sodium
meta-bisulfite sterile solution (Ref. 06-114CASE) and a
flask of sterile solution of ammonium ferric citrate (Ref.
06-113CASE). Mix well and distribute into sterile tubes
provided with Durham´s tubes.
Description
This is a simple medium that selects Cl. perfringens
among other sulfite reducing clostridia by their ability to
produce gas from lactose, at 46°C. It has interferences
only with Cl. paraperfringens, however this microorgan-
ism is very rare in food samples.

Directions
Dissolve 28 g of powder in 1 L of distilled water. Distrib-
ute in tubes provided with Durham’s tubes. Sterilize in
the autoclave at 121°C for 15 minutes.
Description
This medium is according to the German standards for
quality control of water.
Technique
German standards suggests the use of MPN technique
with 0,1, 1 and 10 mL of sample and an incubation at
36±1°C for 44±4 hours. Tubes that change to yellow
and eventual gas production/accumulation in Durham´s
tubes are considered
positive.
References
DEUTSCHE EINHEITSVERFAHREN zur Wasser-,
Abwasser- Und Schlammuntersuchung. VCM Verlags-
gesellschaft, D-6940. Weinhem.
VERORDNÜNG über Trinkwasser und Wasser für Leb-
ensmittelbetriebe vom 12- Dezember-1990- Bundesges-
etzblatt; Teil I, 2613-2629 (1990)
Technique
All of the freshly prepared media tubes (or regenereated)
are inoculated in duplicate with 1 mL of sample dilution.
Sample dilution must be previously kept in boiling water
bath, boiling it for 10 minutes. Tubes are incubated in
anaerobic conditions at 46°C for a period of 18-24 hours.
Cl. perfringens presence is observed by an iron sulfide
precipitate appearing in the tubes. It indicates the sulfite
reducing activity. Accumulation of gas in the Durham’s
tubes is a sign of lactose fermentation.
References
PASCUAL ANDERSON, MªRª (1992) Microbiología
Alimentaria. Diaz de Santos, S.A.,Madrid,.
EUROPEAN PHARMACOPOEIA (2002) Suppl. 4.2
(2001). Chap. 2.6.13 Tests for specified microo-organ-
isms 4th Ed., Council of Europe, Strasbourg.
ISO 7937 Standard (2004) Microbiology of food and
animal feeding stuff. Horizontal method for enumeration
of Cl. perfringens. Colony-count technique.

Lactose Sulfite Broth Base

(Eur. Phar. Medium R)

Ref. 02-519

Specification
Liquid medium for the determination of H2S produc-
tion by Clostridium perfringens according to ISO 7937
standard.

Formula (in g/L)

Peptone .................................................. 5,000
Yeast extract ........................................... 2,500
Sodium chloride ...................................... 2,500
Lactose ................................................. 10,000
L-Cysteine HCl ....................................... 0,300
Final pH 7,1 ± 0,2

80
 LB Media
Under this generic name there are several formulations
included which are derived from the base medium that
was originally described by Luria and lately modified by
different authors.
This nutrient base has become very popular among
culture media for the maintenance and propagation of
Escherichia coli in the assays about molecular genetics.
The nutrient richness and simplicity in their composi-
tion allow easy and quick modifications, where the most
common are the addition of different antibiotics and
inhibitors as well as the variations in the sodium chloride
concentration.
The current SCHARLAU formulation program covers the
following formulations:
LB Agar
Ref. 01-384
Specification
Solid medium for general purposes recommended for
the molecular genetics studies with E. coli.
Formula (in g/L)
Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Agar .......................................................... 15,0
Final pH 7,2 ± 0,2
Directions
Suspend 30 g of powder in 1 L of distilled water and heat
to the boil with constant stirring. Distribute into suitable
containers and sterilize by autoclaving at 121°C for 15
minutes.
LB Broth
Ref. 02-384
Specification
Liquid medium for general purposes, recommended for
molecular genetics studies with E. coli.
Formula (in g/L)
Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Final pH 7,2 ± 0,2
Directions
Dissolve 15 g of powder in 1 L of distilled water. Distrib-
ute into suitable containers and sterilize by autoclaving
at 121°C for 15 minutes.
Description
Formulation of both the media, solid and liquid, are ac-
cording to the Luria and Bartani base, in which sodium
chloride has been omitted to help in the variation of the
saline concentration with other additives.
LB Agar acc. to Lennox
Ref. 01-406
Specification
Solid medium for general purposes recommended for
the molecular genetics studies with E. coli.
Formula (in g/L)
Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,0 ± 0,2
Directions
Suspend 35 g of powder in 1 L of distilled water and heat
to the boil with constant stirring. Distribute into suitable
containers and sterilize by autoclaving at 121°C for 15
minutes.
LB Broth acc. to Lennox
Ref. 02-406
Specification
Liquid medium for general purposes, recommended for
molecular genetics studies with E. coli.
Formula (in g/L)
Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Sodium chloride .......................................... 5,0
Final pH 7,0 ± 0,2
Directions
Dissolve 20 g of powder in 1 L of distilled water. Distrib-
ute into suitable containers and sterilize by autoclaving
at 121°C for 15 minutes.
Description
Formulation of both the media, solid and liquid, are
according to the Luria and Bartani base modified by
Lennox.
81
 LB Media

LB Agar acc. to Miller Directions

Ref. 01-385
Specification
Solid medium for general purposes recommended for
the molecular genetics studies with E. coli.
Formula (in g/L)
Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Sodium chloride ........................................ 10,0
Agar .......................................................... 15,0
Final pH 7,2 ± 0,2
Directions
Suspend 40 g of powder in 1 L of distilled water and heat
to the boil with constant stirring. Distribute into suitable
containers and sterilize by autoclaving at 121°C for 15
minutes.
LB Broth acc. to Miller
Ref. 02-385
Specification
Liquid medium for general purposes, recommended for
molecular genetics studies with E. coli.
Formula (in g/L)
Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Sodium chloride ........................................ 10,0
Final pH 7,2 ± 0,2
Dissolve 25 g of powder in 1 L of distilled water. Distrib-
ute into suitable containers and sterilize by autoclaving
at 121°C for 15 minutes.
Description
Formulation of both the media, solid and liquid, are ac-
cording to the Luria and Bartani base modified by Miller,
who has increased the sodium chloride concentration.
References
AUSUBEL, F.M., R. BRENT, R.E. KINGSTON, D.D.
MOORE, J.G. SEIDMAN, J.A. SMITH & K. STRUHL
(1994) Current protocols in molecular biology. Greene
Pub. Assoc. Inc. Brooklyn, N.Y.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc. London.
GHERNA, R., P. PIENTA, R. COTE (Eds.) 1992. ATCC
Catalogue of Bacteria and Bacteriophages. Media
#1065, #1226, #1226, #1235, #1236, #1315, #1364.
American Type Culture Collection. Rockville MD. USA.
LURIA, S.E. & J.W. BURROUS (1955) Hybridization
between Escherichia coli and Shigella. J. Bacteriol.
74:461-476
LENNOX, E.S. (1955) Transduction of linked genetic
character of the host bacteriophage P1. Virology 1:190-
206.
MILLER, J.H. (1972) Experiments in Molecular Genetics.
Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.
SAMBROOK, J., E.F. FITSCH & T. MANIATIS (1989)
Molecular cloning: A laboratory manual. 2nd ed. Cold
Spring Harbor Laboratory. Cold Spring Harbor, N.Y.

82
 Letheen Media
Letheen Agar
Ref. 01-236
Specification
Solid medium for assays of antimicrobial action of qua-
ternary ammonium compounds (QAC’s).
Formula (in g/L)
Tryptone ................................................... 5,00
Meat extract .............................................. 3,00
Dextrose ................................................... 1,00
Lecithin ..................................................... 1,00
Agar ....................................................... 15,00
Final pH 7,0 ± 0,2
Directions
Suspend 25 g of powder in 1 L of distilled water with 7
mL of Polysorbate 80 (Ref. 06-088). Let it soak and heat
to boiling with constant stirring. Distribute in suitable
containers and sterilize in the autoclave at 121°C for 15
minutes.
Description
Letheen Agar is formulated according to AOAC direc-
tions, that were taken from the research work of Weber
and Black for the assay of bactericidal action of quater-
nary ammonium compounds (QAC’s).
In fact, the medium is the classical one for the standard-
ized counting with the addition of lecithin and polysorb-
ate, which act as the neutralizers of the QAC’s.
Letheen Broth
Ref. 02-236
Specification
Liquid culture medium for the determination of germicidal
activity coefficients of cationic detergents.
Formula (in g/L)
Peptone .................................................... 10,0
Meat extract ................................................ 5,0
Lecithin ....................................................... 0,7
Sodium chloride .......................................... 5,0
Final pH 7,0 ± 0,2
Directions
Dissolve 20,7g of powder in 1 L of distilled water with 5
mL of Polysorbate 80 (Ref. 06-088). Distribute in suitable
containers and sterilize by autoclaving at 121°C for 15
minutes.
Description
This is the liquid version of Letheen Agar (Ref. 01-236),
recommended by AOAC to verify the germicidal activity
coefficients in cationic soaps, although its formulation is
not the same.
Letheen Modified Agar
Ref. 01-237
Specification
Solid medium for the primary screening of microorgan-
isms in cosmetics according to the FDA
Formula (in g/L)
Casein peptone ........................................ 10,0
Meat peptone ........................................... 10,0
Meat extract ............................................... 3,0
Yeast extract ............................................... 2,0
Dextrose ..................................................... 1,0
Lecithin ....................................................... 1,0
Sodium chloride .......................................... 5,0
Sodium bisulphite ....................................... 0,1
Agar .......................................................... 15,0
Final pH 7.0 ± 0,2
Directions
Suspend 47 g of powder in 1 L of distilled water and add
7 mL of Polysorbate 80 (Ref. 06-088). Allow to soak and
heat to the boil with constant agitation.
Distribute into final containers and sterilize by autoclav-
ing at 121°C for 15 minutes.
Letheen Modified Broth
Ref. 02-237
Specification
FDA recommended liquid medium for the primary recov-
ery of stressed microorganisms in the microbial exami-
nation of cosmetics.
Formula (in g/L)
Casein Peptone ........................................ 15,0
Meat Peptone ........................................... 10,0
Meat extract ................................................ 5,0
Yeast extract ............................................... 2,0
Sodium chloride ........................................ 10,0
Lecithin ....................................................... 0,7
Sodium bisulfite .......................................... 0,1
Final pH 7,0 ± 0,2
Directions
Dissolve 43 g of the dry powder in 1 L of distilled water
with 5 mL of Polysorbate 80 (Ref. 06-088). Distribute
in suitable containers and autoclave at 121°C for 15
minutes.
83
 Letheen Media
Description
In the early 40’s, Weber and Black recommended the
use of lecithin and polysorbates to neutralize the anti-
microbial action of the quaternary amonium compounds
(QAC’s).
In 1965 the methodology was accepted by AOAC for the
antimicrobial assays and extendes their use to all the
cationic tensoactives (detergents).
The TAT (Tryptone Azolactin Tween) medium in the
Newburger Cosmetic Analysis Manual (2nd. Ed., 1977)
is closely similar in formulation and uses to the AOAC
recipe. In 1978 the FDA (Bacteriological Analytical
Manual, 1978) incorporated it as previous presumptive
and enrichment medium for every microbial examination
in cosmetics.
The SCHARLAU formulation of the Letheen Media
covers the old recipe of Lucas (1977) in the Newburger
Manual (Ref.1-236 Letheen Agar and Ref. 02-236
Letheen Broth) and the new recipe (Ref. 01-237 Letheen
Modified Agar and Ref. 02-237 Letheen Modified Broth)
of the FDA (B.A.M., 1979)
84
References
ASTM Standard E 640-78 (1991) Test method for pre-
servatives in water-containing cosmetics. Philadelphia.
PA
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc. London.
FDA (1998) Bacteriogical Analytical Manual. 8th ed.
Revision A. AOAC International. Gaithersburg. MD
HORWITZ, W. (2000) Official Methods of Analysis. 17th
ed. AOAC International. Gaithersburg. MD
LUCAS, J.P. (1977) Microbiological Examination of
Cosmetics. Newburguers’ Manual of Cosmetic Analysis.
AOAC
WEBER, G.R. & L.A. BLACK (1948) Relative efficiency
of quaternary inhibitors. Soap and Sanit Chem. 24:134-
139
ISO 21149:2006 Cosmetics – Enumeration and detec-
tion of aerobic mesophilic bacteria.
ISO 22717:2006 Standard. Cosmetics – Detection of
Pseudomonas aeruginosa.
ISO 22718:2006 Standard. Cosmetics – Detection of
Staphylococcus aureus.
 Liebermeister & Braveny Agar
Ref. 01-446
Specification
Solid medium for the selective isolation of ß-hemolytic
streptococci from throat samples.
Formula (in g/L)
Meat peptone ........................................... 1,00
Meat extract .............................................. 0,60
Yeast extract ............................................. 0,50
L(+)Lysine ................................................. 0,02
Sodium chloride ........................................ 6,00
Disodium phosphate ................................. 2,00
Agar ........................................................ 15,00
Final pH 7,2 ± 0,2
Directions
Suspend 25 g of powder in 930 mL of distilled water and
heat to the boil. Distribute into suitable containers and
sterilize in an autoclave at 121°C for 15 minutes. Cool
to 45°C, then add 70 mL/L of sterile defibrinated lamb’s
blood. Homogenize well and pour into sterile plates.
Description
Despite its simplicity, this medium has a better yield in
the recovery of ß-hemolytic streptococci than the com-
monly used Blood Agar.
Okamoto et al. and, later Bernheimer and Rodbart
demonstrated the strong stimulatory effect of the nucleic
acids on the hemolytic properties of streptococci. Lieber-
meister and Braveny formulated a medium with insuf-
ficient nutrients for the development of normal microor-
ganisms but with an increased amount of nucleic acids
in the yeast extract. Moreover, they also included lysine,
which has an effect on the hemolysis similar to that of
the nucleic acids.
The result is that microorganims fail to grow or form only
small colonies that have hemolysis zones(ß-hemolysis)
of normal or greater than normal size. Viridans strep-
tococci (alpha-hemolytic) virtually do not grow, and if
hemolysis zones form at all, they are minimal.
Technique
Plates are inoculated by surface seeding and incubated
at 37°C for 24-48 hours. After the incubation small colo-
nies form which are surrounded by a large, well defined
hemolytic zone. Staphylococci and enterococci are
almost viridans streptococci.
References
OKAMOTO, H., S. KYODA, R. ITO (1939) Jap. J. Med.
Sci. VI Pharmacol. 12:167.
BERNHEIMER, A.W., M. RODBART (1948) The effect
of nucleic acids and carbohydrates on the formation of
streptolysin. J. Exp. Med. 88:149.
LIEBERMEISTER, K., J. BRAVENY (1971) Ein Nährsub-
strat zur Isolierung von haemolytischen Streptokokken.
Z. Med. Mikrobiol. Inmunol. 156:149-153.
MILATOVIC, D. (1981) Comparison of five selective
media for beta-haemolytic streptococci. J. Clin. Pathol.
34:556-558
85
 Lingby Iron Agar Base
Ref. 01-584
Specification
Solid medium for the isolation and enumeration of
heterotrophic H2S producing bacteria from fish and fish
products.
Formula (in g/L)
Tryptone .................................................. 20,0
Meat extract ................................................ 3,0
Yeast extract ............................................... 3,0
Ferric citrate................................................ 0,3
Sodium thiosulfate ...................................... 0,3
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,4 ± 0,2
Directions
Suspend 46,6 g of powder in 1L of distilled water and
heat to the boil. Distribute into suitable containers and
sterilize by autoclaving at 121ºC for 15 minutes. Cool to
45-50ºC and aseptically add 8 mL/L of a filter sterilized
solution of 5% L-Cysteine hydrochloride. Homogenize
well and pour into sterile plates.
Description
Listeria Enrichment Media
Listeriosis
Listeriosis is the generic name for the whole group of
disorders caused by Listeria monocytogenes, which is
the only species of the Listeria genus that is important
as a human pathogen, howewer occasionally L.seeligeri,
L.welshimeri and L.ivanovii have been related with
human diseases. In anycase, all the species are patho-
genic between the ovin and bovine cattle.
Positive diagnosis of listeriosis can be obtained only by
the isolation and cultivation of the responsible bacte-
ria from blood or cerebrospinnal fluid samples of the
affected organism. Faecal samples have a very debat-
able value, since actually it is estimated that between
1-10% of human population may be an intestinal carrier
of L.monocytogenes. This species of bacteria have been
isolated from 37 species of mammals, domestic or wild,
as well as from 17 species of birds and some species of
fishes and shellfishes.
Listeria monocytogenes bears a notorious tolerance to
heat, cold and desiccation, and it acts as a real psi-
chrotrophic. These facts and the high morbidity when it
infects annimals and humans increase its interest as a
food-borne pathogen.
Its presence has been related with food as raw milk or
supposedly pasteurized milk, soft-ripped cheese, ice
creams, vegetables, raw meat, cured fermented meat,
raw or smoked fish, but it has been isolated also from
soil, silages and other environmental samples.
86
Lingby Iron Agar Base has been formulated accord-
ing to the Food Analysis Nordic Committee standard.
This standard states that cysteine addition must be
done separately in order to achieve better results in the
recovery of the H2S producing bacteria from fish and fish
products that have not been under heat treatment or with
any addition of preservatives.
Technique
For the total count of H2S producing bacteria, the Com-
mittee prescribes the following technique:
Transfer 1 mL of sample dilution to sterile Petri plates.
Add 10-12 mL of molten medium and cool to 45ºC. Ho-
mogenize by shaking it gently. When cooled, add a new
layer of medium and incubate at 20±1ºC for 3 days.
Select the adequate plates and proceed with total count.
H2S producing bacteria form black colonies.
If incubation temperature is too high or the covering
layer is too thin, black colonies loses the colour rapidly.
References
Nordisk Metodikkommité för Livsmedil. (1994) UDC.
637.56:576.8.08-No96. 2ntg- Bacteriological examina-
tion of fresh and frozen seafood.
Detection and confirmation of
Listeria
Food control requires to culture the samples that use
to need 5-7 days before provide definitive results, and
although the commercial production of DNA probes to
help the diagnosis, they are not available yet.
The traditional technique of low temperature (4ºC) en-
richment has been substituted for selective enrichments
at temperatures near to the optimum growth tempera-
ture. Isolation can be also performed over selective
and differential media, that most of the cases allow to
disregard the special Henry lighting technique, but final
identification must be carried out by biochemical and
serological methods.
The type of sample restrains the technique of detection
because the companion flora is very important. If a pop-
ulation more than 100 Listeria cells/g is suspected, the
sample can be inoculated directly over the solid selective
media, but in other cases, an enrichment technique, in
one or two stages, must be adopted.
 Listeria Enrichment Media
Specific identification
All the Listeria species are grampositive, oxidase- and
urease-negative. They produce catalase, do not reduce
nitrate and hydrolyze esculin. Provide +/+ result in the
O/F test for glucose and they also acidify the bottom
and surface in TSI Agar. Provide a positive result in the
Voges-Proskauer and Phenol Red tests. Differential
characteristics are shown in the table above.
References
DONNELLY, C.W., R.E. BRACHETT, S. DOORES, W.H.
LEE, J. LOVETT (1992)
Listeria, in Compendium of method for the microbiologi-
cal examination of foods, Vanderzant & Splittstoesser
(eds) APHA. Washington.
ADAMS, M.R., M. O. MOSS (1995) Food Microbiology .
The Royal Society of Chemistry. Cambridge. U.K.
FDA/ Center for Food Safety & Applied Nutrition. (March
1999) Foodborne Pathogenic Microorganisms and
Natural Toxins Handbook: Listeria monocytogenes http://
vm.cfsan.fda.gov/mow
Following you will find the Scharlau Microbiology produc-
tion program in selective and differential media for the
enrichment and isolation of Listeria.
Listeria Enrichment Broth Base
(UVM)
Ref. 02-472
Specification
Liquid culture medium for the enrichment of Listeria sp.
Formula (in g/L)
Proteose Peptone ..................................... 5,00
Tryptone ................................................... 5,00
Meat extract .............................................. 5,00
Yeast extract ............................................. 5,00
Sodium chloride ...................................... 20,00
Esculin ...................................................... 1,00
Disodium phosphate ............................... 12,00
Dipotassium phosphate ............................ 1,35
Final pH 7,4 ± 0,2
Directions
Dissolve 54,5 g of powder into 1 L of distilled water. Dis-
tribute 500 mL in each container and autoclave at 121ºC
for 15 minutes. Cool to 50ºC and then aseptically add
the suitable selective supplement toeach 500 mL: UVM
I for primary enrichment (Ref. 06-106CASE) and UVM
II/Fraser for secondary enrichment (Ref. 06-111CASE).
Note: Prepared medium (broth+supplement) must be
kept away from light, since it helps the produc-
tion of acriflavine-oxidised photocomplexes that
repress Listeria growth.
Description
This basal broth for the enrichment of listeria is made ac-
cording the AOAC modifications over the medium of the
Vermont University (UVM), since it was demonstrated
that a slight increase of acriflavine concentration in the
secondary enrichment and a strong reduction in the
amount of nalidixic acid in all the stages allowed more
positive isolations.
Technique
Primary enrichment
Add 25 g or 25 mL of sample to 225 mL of primary
enrichment broth (Base Broth, Ref. 02-472, and UVM I,
Ref. 06-106CASE) and homogenize it all in a stomacher
for 2 minutes. Incubate the mixture at 30ºC for 24 hours,
but after the first 4 hours take aliquots of 0,2 mL to plates
with Oxford Selective Agar (Ref. 01-471) in order to
make the isolation.
Secondary enrichment
After 24 hours of primary enrichment, inoculate the sec-
ondary enrichment broth (Base Broth, Ref. 02-472, and
UVM II/Fraser, (Ref. 06-111CASE) at the rate of 1:100.
Incubate at 30ºC. After 4 and 24 hours take aliquots of
0,2 mL to plates with Oxford Selective Agar (Ref. 01-
471) in order to make the isolation.
Isolation
Isolation is carried out on the Oxford Selective Agar (Ref.
01-471) plates prepared during the primary and second-
ary enrichment, after 24-48 of incubation at 30-37ºC.
Sometimes it is advisable to alkalinize the inoculum
before the seeding, by mixing 1 mL of enrichment broth
with 5 mL of 0,5% sterile KOH solution.
87
 Listeria Enrichment Media
Listeria Enrichment Broth Base
acc. to Lovett
Ref. 02-498
Specification
Liquid culture medium for the enrichment of Listeria, ac-
cording Lovett et al.
Formula (in g/L)
Tryptone ................................................. 17,00
Yeast extract ............................................. 6,00
Soy peptone ............................................. 3,00
Sodium chloride ........................................ 5,00
Dextrose ................................................... 2,50
Dipotassium phosphate ............................ 2,50
Final pH 7,3 ± 0,2
Directions
Dissolve 36 g of powder into 1 L of distilled water and
distribute 500 mL per flask. Sterilize by autoclaving at
121ºC for 15 minutes. Cool to 50ºC and aseptically add
to each flask the content of one vial of Listeria Supple-
ment for Selective Enrichment acc. to FDA/IDF (Ref.
06-107CASE). Homogenize and distribute into suitable
containers.
Note: Prepared medium (broth+supplement) must be
kept away from light, since it helps the produc-
tion of acriflavine oxidised photocomplexs that
repress Listeria growth.
Description
This medium according to Lovett et cols. formulation has
been adopted by the FDA for the analysis of food, and it
is recommended by the IDF/FIL for the selective enrich-
ment of Listeria in milk samples, due to its good results
in the recovery of stressed bacteria, with only an enrich-
ment stage.
Technique
Mix the sample (25 mL or 25 g) with 225 mL of complete
enrichment broth and incubate at 30ºC for 7 days. Make
subcultures after 24 hours, 48 hours and 7 days in the
following way:
Inoculate 0,5 mL of enrichment culture in a solid medium
for the Listeria isolation (Oxford Agar Base, Ref. 01-471,
or Palcam Agar Base, Ref. 01-470, with their respective
selective supplements).
Alkalinize 0,5 mL of enrichment culture by mixing with
4,5 mL of 0,5% sterile KOH solution and inoculate on a
solid medium for Listeria isolation.
88
Listeria Enrichment Broth Base
acc. to Fraser
Ref. 02-496
Specification
Liquid culture medium for the enrichment and detection
of Listeria spp. according ISO standards 11290-1 and
11290-2.
Formula (in g/L)
Proteose peptone ..................................... 5,00
Tryptone ................................................... 5,00
Meat extract .............................................. 5,00
Yeast extract ............................................. 5,00
Sodium chloride ...................................... 20,00
Esculin ...................................................... 1,00
Dissodium phosphate ............................. 12,00
Monopotassium phosphate ...................... 1,35
Lithium chloride ........................................ 3,00
Final pH 7,2 ± 0,2
Directions
Dissolve 57,4 g of powder into 1 L of distilled water.
Distribute 500 mL per flask and sterilize in the autoclave
at 121ºC for 15 minutes. Cool to 50ºC. Aseptically add
the Ferric Ammonium Citrate for Bacteriology (Ref.
06-112CASE) and Listeria Supplement for Secondary
Enrichment UVM II/Fraser (Ref. 06-111CASE) to each
flask and homogenize well.
Note: Prepared medium (broth+supplement) must be
kept away from light, since it helps the production
of acriflavine oxidised photocomplex that repress
Listeria growth.
Description
This broth base for Listeria enrichment is made accord-
ing to the modifications of Fraser and Sparber over the
UVM medium, which have been adopted by the USDA-
FSIS. The inclusion of lithium chloride inhibits the devel-
opment of enterococci which also may hydrolyze esculin
in the same way of Listeria. Thus, any darkness in the
medium produced by the reaction of esculetin coming
from esculin hydrolysis with iron present in the medium
can be taken as a presumptive presence of Listeria.
Moreover, it seems the ferric citrate helps L. monocy-
togenes development.
Technique
Although some authors use the Fraser Broth as the only
enrichment, it has been verified than better results are
obtained if it is employed as secondary enrichment, ac-
cording the following methodology:
Inoculate the sample to examine in a primary enrichment
broth (UVM I, Ref. 02-472 or Lovett Broth, Ref. 02-498)
and incubate for 18-24 hours.
 Listeria Enrichment Media
Take aliquots of 0,1 mL, inoculate them in tubes with 10
mL of Fraser Broth and incubate for 24-28 hours.
Tubes that become dark are considered presumptively
positive and must be subcultured over isolation and
confirmation solid media, such as Oxford Agar Base
(Ref. 01-471) or Palcam Agar Base (Ref. 01-470). Tubes
that remain clear are considered negative and can be
discarded or kept in incubation for 24 hours more to
clear the doubts if any.
Palcam Agar Base
Ref. 01-470
Specification
Solid, selective and differential medium for the detection,
enumeration and isolation of Listeria spp. according ISO
standards 11290-1 and 11290-2.
Formula (in g/L)
Tryptone ................................................. 23,00
Lithium chloride ...................................... 15,00
Mannitol .................................................. 10,00
Sodium chloride ........................................ 5,00
Yeast extract ............................................. 3,00
Starch ....................................................... 1,00
Esculin ...................................................... 0,80
Ferric Ammonium citrate........................... 0,50
Dextrose ................................................... 0,50
Phenol red ................................................ 0,08
Agar ........................................................ 13,00
Final pH 7,2 ± 0,2
Directions
Suspend 72 g of powder in 1 L of distilled water and
let it soak. Heat to boil and distribute 500 mL per flask.
Sterilize by autoclaving at 121ºC for 15 minutes. Cool
to 50ºC and aseptically add the Palcam Agar Selective
Supplement (Ref. 06-110CASE) to each flask. Mix well
and pour into sterile plates.
Note: Prepared medium (broth+supplement) must be
kept away from light, since it helps the production
of acriflavine oxidised photocomplex that repress
Listeria growth.
Description
Palcam Agar is based in the formulation described initial-
ly by van Netten et cols., which has a high selectivity and
a good colonial differentiation. Selectivity is achieved by
the inclusion of lithium chloride, acriflavine, polymixin B
and ceftacidine, since they inhibit the growth of almost
all the gramnegative bacteria and most of grampositive
companion bacteria.
Listeria hydrolyze esculin to esculetin, which reacts with
ferric ammonium citrate producing a dark precipitate
colouring the colonies to green-grey with beige halos.
The colonies of enterococci or staphylococci that may
overpass the high selectivity of this medium can be
easily recognized, since they use mannitol and produce
yellow colonies and halos, contrasting with the cherry
red colour of medium.
However, when there are many Listeria colonies, the
entire medium turns dark, and the differentiation can be
interfered. In these cases it is advisable to perform a
more diluted inoculation.
Technique
Seed the Palcam Agar with a growth from a primary
enrichment broth (UVM I, Ref. 02-472 or Lovett, Ref.
02-498) or a secondary enrichment broth (UVM II, Ref.
02-472 or Fraser, Ref. 02-496). Incubate in a microaer-
ophile atmosphere for 48 hours at 37ºC.
In these conditions, Listeria colonies have a size approx.
2 mm diameter, green-grey coloured with black core
and halo. Enterococcus and Staphylococcus colonies
are bigger, grey with green-brown halo if they do not
use mannitol or form yellow colonies with yellow halo if
they do. Anyway, suspicious colonies must be confirmed
biochemically and serologically.
Oxford Agar Base
Xn
Ref. 01-471
R-22-36/38
S-26-37/39-46
Specification
Solid, selective and differential medium for the detection,
enumeration and isolation of Listeria sp. according ISO
standards 11290-1 and 11290-2.
Formula (in g/L)
Tryptone ................................................. 10,00
Lithium chloride ...................................... 15,00
Proteose peptone ................................... 10,00
Sodium chloride ........................................ 5,00
Yeast extract ............................................. 3,00
Starch ....................................................... 1,00
Esculin ...................................................... 1,00
Ferric Ammonium citrate........................... 0,50
Agar ........................................................ 13,00
Final pH 7,2 ± 0,2
Directions
Suspend 58,5 g of powder in 1 L of distilled water and
let it soak. Heat to boil and distribute 500 mL per flask.
Sterilize by autoclaving at 121ºC for 15 minutes. Cool to
50ºC and aseptically add the Oxford Agar Selective Sup-
plement (Ref. 06-109CASE) to each flask . Mix well and
pour into sterile plates.
Note: Prepared medium (broth+supplement) must be
kept away from light, since it helps the production
of acriflavine oxidised photocomplex that repress
Listeria growth.
89
 Listeria Enrichment Media
Description
Oxford Agar is derivated from the original formulation by
Curtis et al, which worked a medium with a high nutritive
ability as the Columbia Agar and added inhibitor agents
to remove all the undesirable companion bacteria.
The current formulation keep the high capacity to sup-
port growth and restrain all the gramnegative flora and
most of grampositive, including yeast. Thanks to the
inhibitors incorporated in the selective supplement:
cycloheximide, acriflavine, colystin, phosphomicyn
and ceftacidine. These inhibitors in addition to lithium
chloride restrain the growth of all other bacteria except
Listeria.
However, selectivity is not total, but Listeria colonies are
easily recognizable since as they hydrolyze esculin. Free
esculetin that reacts with the ferric ions and produce
a dark precipitate around the colonies, which typically
present a grey-blue colour with a very dark core.
Technique
Although the selectivity of the medium is enough to allow
the isolation and differentiation by direct surface inocula-
tion, a previous dilution of the inoculum is advisable, or
even more when the sample is highly polluted.
In anycase, most authors prefer one or two previous
cultures in any of the primary enrichment media (UVM I,
Ref. 02-472 or Lovett, Ref. 02-498) or secondary enrich-
ment media (UVM II, Ref. 02-472 or Fraser, Ref. 02-496)
before inoculating in Oxford Agar.
Incubation is carried out at 37ºC, and after 24 hours
typical colonies of Listeria monocytogenes are visible.
However, it is recommended to extend incubation for
more 20-24 hours in order to evidence the slow grow-
ing strains even though this could allow staphylococci
or streptococci development, since they would grow
weakly.
Ref. 02-496 Listeria Enrichment Broth acc. Fraser.
Left: control; right: positive.
90
References
McCLAIN, D., W.H. LEE (1988) Development of USDA-
FSIS Method for isolation of Listeria monocytogenes
from raw meat and poultry. JAOAC 71:3:660-664.
ATLAS, R.M. (1993) Handbook of Microbiological Media.
CRC Press. Boca Raton, Florida.
VANDERZANT, C., D.F. SPLITTSTOESSER (1992)
Compendium of methods for the microbiological exami-
nation of food. APHA, Washington D.C.
LOVETT, J., D.W. FRANCIS, J.M. HUNT (1988) Listeria
monocytogenes in raw milk; Detection, incidence and
pathogenicity. J. Food Protect. 50;188-192
LOVETT,J., A.D. HITCHINS (1989) Listeria isolation.
FDA Bacteriological Analytical Manual. 6th Ed. Supp.
Sept. 1987 (2nd Print):29.01
FRASER, J.A., W.H. SPERBER (1988) Rapid detection
of Listeria sp. in food and environmental samples by
esculin hydrolysis. J. Food Prot. 51:762-765.
van NETTEN, P., J.PERALES, A. van de MOODSDUCK,
G.D.W. CURTIS, D.A.A. MOSSEL (1989) Liquid and
solid selective differential media for the detection and
enumeration of Listeria monocytogenes. Int. J. Food
Microbiol. 8:299-316.
CURTIS, G.D., R.G. MITCHELL, A.F. KING, E.J. GRIF-
FIN (1989) A selective differential medium for the isola-
tion of Listeria monocytogenes. Letters Appl. Microbiol.
8:95-98
ISO 11290 standard (1996) Microbiology of food and
animal feeding stuff. Horizontal method for the detec-
tion and enumeration of Listeria monocytogenes. Part 1
- Detection method. Part 2 - Enumeration method.
 Lysine Media
Lysine Iron Agar (LIA)
Ref. 01-094
Specification
Differential medium for Enterobacteria, recommended by
Edwars and Ewing for Salmonella identification.
Formula (in g/L)
Gelatin peptone ........................................ 5,00
Yeast extract ............................................. 3,00
Dextrose ................................................... 1,00
L-Lysine .................................................. 10,00
Ammonium Ferric citrate .......................... 0,50
Sodium thiosulfate .................................... 0,04
Bromocresol purple .................................. 0,02
Agar ........................................................ 15,00
Final pH 6,7 ± 0,2
Directions
Suspend 34,5 g of powder in 1 L of distilled water and
heat to boiling . Dispense in tubes and sterilize by
autoclaving at 121°C for 15 minutes. Allow to solidify in
slanted position, with copious depth and short slant.
Description
Lysine and Iron medium has been widely used for the
differentiation among different biotypes of Salmonella,
above all those corresponding to S. arizona, which, on
usual selective isolation in plate media, like MacConkey
or Deoxycholate, may give colour or colourless colonies
due to the fact that their lactose fermentative capacity is
quite variable.
If it is considered that these microorganisms, when
cultured in tubes with Kligler Iron Agar (Ref. 01-103) or
Triple Sugar Iron (Ref. 01-192), produce enough acid to
avoid sulfide formation, it is comprehensible that they
are sometimes not recognized or
overlooked as pathogen, and wrong or negative results
are consequently obtained. On the other hand, Salmo-
nella is the only genus of enterobacteria that normally
decarboxylates lysine and produces important amounts
of hydrogen sulfide.
LIA works perfectly verifying these two characteristics,
and that is the reason why it is used at the same time of
use of Kligler Iron Agar (Ref. 01-103) and/or TSI (Ref.
01-192) in the second phase of isolation of pathogenic
enterobacteria.
Technique
From some suspicious colonies in the isolation media,
and with a needle, inoculate a Kligler’s medium tube,
and without recharging the inoculation loop, pass it to
LIA by surface streaking and depth stab. Incubate them
closed, but at the same time sufficiently ventilated, at
35-37°C for 24 hours.
Microorganisms that decarboxylate the lysine, quickly,
produce a strong alkalinization in all the medium, that
is observed by the indicator turning to purple. Whereas,
those that have no lysine decarboxylase activity, acidify
the medium at the bottom producing a yellow coloura-
tion, meanwhile the surface of the medium remains with
the same original colour or with alkaline reaction.
Proteus type members are distinguished easily, since,
over the yellow bottom, they produce a typical red or
orange colour on the surface, due to oxidative deamina-
tion of lysine. The microorganisms which produces of
hydrogen sulfide blacken the medium because of iron
sulfur precipitates.
Although the gas production may be observed, generally
this medium does not offer optimal conditions for this,
and gives very irregular results, even giving total inhibi-
tion in some cases.
References
EDWARDS, P.R. and M.A. FIFE (1961) Lysine-Iron Agar
in the detection of Arizona cultures. Appl. Microbiol. 99,
478-480.
EWING, J. (1982) Edwards and Ewing’s identification of
Enterobacteriaceae. 4th. Ed. Elsevier Sci. Pub. Co. Inc.
N.Y.
McFADDIN, J.F. (1985) Media for the isolation, cultiva-
tion, identification and maintenance of medical bacteria.
William & Wilkins. Baltimore
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press,London
DOWNES, F.P. & K. ITO (2001)) Compendium of Meth-
ods for the Microbiological Examination of Food. 4th ed
APHA. Washington
HORWITZ, W. (2000) Official Methods of Analysis. 17th
ed. AOAC International. Gaitherburg. MD.
MARSHALL R.T. (1992) Methods for the examination of
dairy products. APHA. Washington.
91
 Lysine Media

92
 M-17 Media
M-17 Agar
Ref. 01-245
Specification
Solid selective medium for Streptococcus thermophilus
in the common examination of yoghurt acc. to ISO 7889:
2003 and IDF 117: 2003.
Formula (in g/L)
Tryptone ................................................... 2,50
Meat peptone ........................................... 2,50
Soya peptone ........................................... 5,00
Yeast extract ............................................. 2,50
Meat extract .............................................. 5,00
Sodium glycerophosphate ...................... 19,00
Magnesium sulfate ................................... 0,25
Ascorbic acid ............................................ 0,50
Lactose ..................................................... 5,00
Agar ....................................................... 15,00
Final pH 7,0 ± 0,2
Directions
Suspend 57 g of powder in 1 L of distilled water and let it
soak. Heat to boiling and dispense into suitable contain-
ers. Sterilize by autoclaving at 121°C for 15 minutes.
Avoid unnecessary overheating and remelting.
M-17 Broth
Ref. 02-245
Specification
Liquid selective medium for Streptococcus thermophilus
in the common examination of yoghurt.
Formula (in g/L)
Tryptone ................................................... 2,50
Meat peptone ........................................... 2,50
Soya peptone ........................................... 5,00
Yeast extract ............................................. 2,50
Meat extract .............................................. 5,00
Sodium glycerophosphate ...................... 19,00
Magnesium sulfate ................................... 0,25
Ascorbic acid ............................................ 0,50
Lactose ..................................................... 5,00
Final pH 7,0 ± 0,2
Directions
Suspend 42 g of powder in 1 L of distilled water and let it
soak. Heat if necessary and dispense into suitable con-
tainers. Sterilize by autoclaving at 121°C for 15 minutes.
M-17 w/o Lactose Broth
Ref. 02-580
Specification
Liquid selective medium for Streptococcus thermophilus
in the examination of yoghurt and other dairy products.
Formula (in g/L)
Tryptone ................................................... 2,50
Meat peptone ........................................... 2,50
Soya peptone ........................................... 5,00
Yeast extract ............................................. 2,50
Meat extract .............................................. 5,00
Sodium glycerophosphate ...................... 19,00
Magnesium sulfate ................................... 0,25
Ascorbic acid ............................................ 0,50
Final pH 7,0 ± 0,2
Directions
Disolve 37 g of powder in 1 L of distilled water, heating if
necessary. Dispense into suitable containers. Sterilize by
autoclaving at 121°C for 15 minutes.
Description
M-17 Agar was developed by Terzaghi and Sandine for
the screening of bacteriophages in streptococci of dairy
industry. Afterward, Shankar and Davies demonstrated
the efficacy of this medium for selective isolation of
Streptococcus thermophilus in yoghurt. Medium com-
bines a strong buffer, which aids development of strep-
tococci, with a high concentration of glycerophosphate,
which inhibits the growth of lactobacilli.
Technique
Suggested technique to enumerate streptococci is to
seed in mass or by stabbing, with agar melted and
cooled to 50-55°C, and then to incubate them at 42°C for
a 24 hours period. With these conditions, all the colo-
nies might be streptococci. Longer incubation periods or
lower temperatures may cause morphological changes
in the colonies which hinders in the the recognition of the
colonies.
FIL-IDF has adopted this medium for yoghurt examina-
tion, and uses it simultaneously with MRS Agar (Ref.
01-135).
Lactobacilli count is performed analogously in MRS Me-
dium, at 30°C and in CO2 enriched atmosphere.
Colonies of lactose positive streptococci are visible after
15 hours, and after 5 days they may reach a diameter of
about 3-4 mm, whereas those lactose negatives are 1
mm of diameter. However, longer incubation period may
hinder the observations, due to the growth of lactobacilli.
Bacteriophages presence is observed by the appear-
ance of characteristic plaques over the bacterial growth.
93
 M-17 Media

References
TERZAGHI, B.E. and SANDINE, W.E. (1975) Improved
medium for lactic streptococcacal phages from cheese
factories. Appl. Environm. Microbiol 29:807-813
SHANKAR, P.A. and DAVIES, F.L. (1977) Selective
Technique for yogurt Bacteria Enumeration. J. Soc. Dairy
Technol. 30:28.
FIL-IDF Standard 146A (1998) Identification of charac-
teristic micro-organisma of yoghurt.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press,London
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 4th
ed.APHA. Washington.
ISO 7889 standard (2003). Yogurt - Enumeration of
characteristic microorganisms - Colony- count technique
at 37°C.

94
 MacConkey Media
In the production program of SCHARLAU you can find
the following formulations of the MacConkey media:
MacConkey Agar ....................................... Ref. 01-118
(Eur. Phar. Medium H)
MacConkey Broth ...................................... Ref. 02-118
MacConkey Modified Broth ...................... Ref. 02-120
MacConkey Sorbitol Agar .......................... Ref. 01-541
MacConkey w/o Salt Agar ......................... Ref. 01-320
MacConkey WHO Broth ........................... Ref. 01-121
Mac Conkey G Broth ................................ Ref. 02-611
(Eur Pharm Medium G)
MacConkey Agar
(Eur. Phar. Medium H)
Ref. 01-118
Specification
A selective and differential medium for the detection,
isolation and enumeration of coliforms from a variety of
samples according European Pharmacopoeia and ISO
standard.
Formula (in g/L)
Peptone ................................................ 20,000
Lactose ................................................. 10,000
Bile Salts #3 ........................................... 1,500
Sodium chloride ...................................... 5,000
Neutral red .............................................. 0,030
Crystal violet ........................................... 0,001
Agar ...................................................... 15,000
Final pH 7,2 ± 0,2
Directions
Suspend 51,5 g of powder to 1 L of distilled water. Bring
to the boil and sterilize by autoclaving at 121°C for 15
minutes.
MacConkey Broth
Ref. 02-118
Specification
Liquid medium for the detection and enumeration of
coliforms by MPN technique.
Formula (in g/L)
Peptone ................................................ 20,000
Lactose ................................................. 10,000
Bile Salts ................................................ 5,000
Sodium chloride ...................................... 5,000
Neutral Red ............................................ 0,075
Final pH 7,4 ± 0,2
Directions
Dissolve 40 g of powder in 1 L of distilled water. Bring to
the boil and distribute into suitable containers fitted with
Durham tubes. Sterilize by autoclaving at 121°C for 15
minutes.
MacConkey G Broth
(Eur. Phar. Medium G)
Ref. 02-611
Specification
Liquid medium for the detection and enumeration of
coliforms by MPN technique. It is a modification of the
classic medium, where neutral red is replaced by a less
aggressive indicator, according European Pharmaco-
poeia.
Formula (in g/L)
Peptone .................................................. 20,00
Lactose ................................................... 10,00
Bile Salts .................................................. 5,00
Bromocresol purple .................................. 0,01
Final pH 7,4 ± 0,2
Directions
Dissolve 35 g of powder in 1 L of distilled water. Heat
only if necessary to help the dissolution. Distribute into
suitable containers fitted with Durham tubes. Sterilize by
autoclaving at 121°C for 15 minutes.
MacConkey Modified Broth
Ref. 02-120
Specification
Liquid medium for the detection and enumeration of
coliforms by MPN technique according ISO standard.
Formula (in g/L)
Peptone .................................................. 20,00
Lactose ................................................... 10,00
Bile Salts .................................................. 5,00
Sodium chloride ........................................ 5,00
Bromocresol purple .................................. 0,01
Final pH 7,4 ± 0,2
Directions
Dissolve 40 g of powder in 1 L of distilled water. Heat
only if necessary to help the dissolution. Distribute into
suitable containers fitted with Durham tubes. Sterilize by
autoclaving at 121°C for 15 minutes.
95
 MacConkey Media
MacConkey Sorbitol Agar
Ref. 01-541
Specification
Selective and differential solid medium for the detection
of Enterohaemorrhagic Escherichia coli (EHEC O157:
H7) according ISO standard.
Formula (in g/L)
Peptone ................................................ 20,000
Sorbitol ................................................. 10,000
Bile salts ................................................. 1,500
Sodium chloride ...................................... 5,000
Neutral Red ............................................ 0,030
Crystal violet ........................................... 0,001
Agar ...................................................... 15,000
Final pH 7,1 ± 0,2
Directions
Suspend 51,5 g of powder into 1 L of distilled water and
heat to boiling. Sterilize in the autoclave at 121ºC for 15
minutes. If the medium is used the same day of prepara-
tion autoclaving is not necessary but the boiling must be
for 3 minutes at least.
Description
The substitution of lactose by sorbitol for the isolation of
the enteropathogenic serotypes O111 y O55 of Es-
cherichia coli was proposed in 1952 by Rappaport and
Hening. The usefulness of the medium was showed by
March and Ratman (1986) and Adas (1991) for the de-
tection, differentiation and isolation of the enterohaemor-
rhagic (EHEC) and the verocytoxin-producing (VTEC)
strains of the serotype O17:H7 of E. coli.
The only modification on the typical MacConkey Media
formulations is the replacement of lactose with sorbitol.
The enterohaemorrhagic strains do not use this sub-
strate and produce colourless colonies, but the other
serotypes can ferment sorbitol and produce red colonies
In all others aspects, MacConkey Agar with Sorbitol
works similarly as the other media in the MacConkey
group. Peptone supply the nitrogen and sodium chloride
the osmotic environment. Crystal violet and bile salts
inhibits the growth of grampositive bacteria. Neutral red
acts as the pH indicator.
Technique
Spread the inoculum on the dry surface of the medium
and incubate at 35±2ºC for 24 hours. Usually, the O157:
H7 serotype forms colourless colonies and the other
strains of E. coli red colonies. The results must be
recorded at 24 hours because an extended incubation
produce a decreasing colouring in the sorbitol-ferment-
96
ing colonies and some times there is some colony of the
o157:H7 serotype that begins to ferment sorbitol.
Some gramnegative bacteria like Pseudomonas, Pro-
teus and Klebsiella can growth on the MacConkey Agar
with Sorbitol but their colonies are diverse and easy to
differentiate from E. coli.
Because the failure in the fermentation of sorbitol by
some strains on E. coli no-enterotoxigenic and the atypi-
cal colony production by some enterohaemorrhagic ones
it is recommended the use of some others media in con-
comitance with the MacConkey Agar with Sorbitol and to
confirm the suspect colonies by serological, biochemical
or molecular techniques.
MacConkey w/o Salt Agar
Ref. 01-320
Specification
A selective and differential medium for the detection,
isolation and enumeration of enterobacteria, especially
Proteus sp. from the clinical specimens.
Formula (in g/L)
Peptone ................................................ 20,000
Lactose ................................................. 10,000
Bile Salts #3 ........................................... 1,500
Neutral red .............................................. 0,075
Agar ...................................................... 15,000
Final pH 7,4 ± 0,2
Directions
Add 46,5 g of powder to 1 L of distilled water. Bring to
the boil and distribute in suitable containers. Esterilize by
autoclaving at 121°C for 15 minutes.
MacConkey WHO Agar
Ref. 01-121
Specification
Differential medium with moderate selectivity, without
crystal violet, according to WHO recommendation, for
the isolation of enterobacteria.
Formula (in g/L)
Peptone ................................................ 20,000
Lactose ................................................. 10,000
Bile Salts mixture .................................... 2,500
Sodium chloride ...................................... 5,000
Neutral Red ............................................ 0,075
Agar ...................................................... 15,000
Final pH 7,4 ± 0,2
 MacConkey Media
Directions
Suspend 53 g of powder in a litre of distilled water. Bring
to the boil, distribute in suitable containers and autoclave
at 121°C for 15 minutes.
Description
The MacConkey media are well known and popular
enrichment system for coliform bacteria.
At the beginning of the last century, MacConkey made
the original formulation and included ox bile as inhibitor
of the gram-positive bacteria and the litmus as the indi-
cator of the acid production from lactose sugar. Lately
the litmus has been substituted by phenol red indicator
to make interpretations easier and more precise.
The media have been adapted to facilitate the coliform
detection with the advancement of knowledge of the
bacterial physiology. The most significant modification to
the original formulation has been the substitution of the
ox bile by purified bile salts that improve the selectivity
and avoid the inherent turbidity which is due to the fat
material of the bile. The effectivity of the inhibition of the
bile salts is variable and depends on the relative concen-
tration of cholate and taurocholate.
Another important modification was the inclusion of
supplementary inhibitors such as crystal violet and/or
brilliant green, that are the most popular formulations in
America, but not in Europe where the lower selectivity is
prefered.
In the 60’s the toxicity of neutral red on the stressed
cells of coliforms was demonstrated, especially on
some strains of E. coli, and then the pH indicator was
changed to the Bromcresol purple, being less aggressive
than the neutral red.
However the most extensive use is in the liquid form,
nevertheless there are solids formulations with agar, for
every
modification.
At the moment, the several formulations available of the
MacConkey media offer a wide range from a low to high
selectivity and the lactose positive bacteria grown on this
medium form red colonies due to acid production due to
the lactose fermentation and thus E. coli colonies can be
easily distinguished as they also form a small precipita-
tion zone of bile salts around them.
Eventually,some enterococci can also grow, but they
are easy to distinguish from the coliforms, as they form
smaller colonies and the absence of precipitation zone.
Medium can be used as Presumptive medium for E.coli
(by fluorescent reaction) if before sterilization MUG (Ref.
06-102CASE) is added. In the medium with MUG the E.
coli colonies show a light blue fluorescence under the
UV illumination. The formulation without salt offers a low
electrolyte content that almost suppresses the swarming
growth of Proteus.
Technique
From a decimal dilution bank, 1 mL samples are inocu-
lated into empty sterile petri dishes in duplicate. Then ,15
mL of molten medium at 45°C is poured into every plate
and mixed carefully . After the solidification, a second
layer of another 5 mL of sterile medium is poured into
every plate to seal the surfaceand improve enumera-
tion of the colonies.
For the enumeration, after an incubation of 24 h. at
35°C, select plates with 30-300colonies. The character-
istic colonies must be confirmed for coliform identity by
gas production from lactose in a broth culture.
The MacConkey broth (code 2-118) can be used for the
enumeration of coliforms by the MPN technique, select-
ing the positive tubes that shows turbidity, colour turned
to red purple and with gas production.
The same above characteristics, but turning into a yellow
color are valid if the MacConkey broth code 2-120 is
used.
If the MUG medium is used a supplementary reading
under UV illumination must be carried out.
References
ADAMS, S. (1991) Screening for verotoxin-producing E.
coli. Clin Lab. Science 4:1:19-20.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc. London.
CLESCERI, L.S., A.E. GEENBERG & A.D. EATON
(1998) Standard Methods for the Examination of Water
and Wastewater. 20th ed. APHA-AWWA-WEF. Washing-
ton DC. USA
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Foods.4th. ed.
APHA. Washington
EUROPEAN PHARMACOPOEIA (2007) 5th ed.
(Supp.5.7) EDQM. Council of Europe. Strasbourg.
HITCHINS, A.D., P. FENG, W.D. WATKINS, S.R. RIPEY
y C.A. CHANDLER (1998). E. coli and coliform bacteria.
Bacteriological Analytical Manual. 8th ed. AOAC Interna-
tional. Gaitherburg. MD. USA
HORWITZ, W. (2000) Official Methods of Analysis.
AOAC Intl. Gaithersburg. MD. USA
ISO 9308-2:1990 Standard. Water Quality. Detection and
enumeration of coliforms, thermotolerant coliforms and
presumptive E. coli. MPN Method.
ISO 21150:2006 Standard. Cosmetics – Detection of
Escherichia coli
ISO 21567. Standard (2004) . Horizontal method for the
detection of Shigella ssp.
MARCH, S.B. y S. RATMANN (1986) Sorbitol-McConkey
Medium for detection of E. coli O157:H7 associated with
hemorrhagic colitis. J. Clin. Microbiol. 23:869-872
McCONKEY, A.T. (1905) Lactose-fermenting Bacteria in
faeces. J. Hyg 5:333.
MURRAY, P.R., E.J. BARON, M.A. PFALLER, F.C. TEN-
OVER, y R.H. YOLKEN (Eds) (1995) Manual of Clinical
Microbiology 6th ed. A.S.M. Washington D.C. USA
RAPPAPORT, F. y E. HENING (1952) Media for the iso-
lation and differentiation of pathogenic E. coli (serotypes
O111 and O55) J. Clin. Pathology 5:361-362
USP 29-NF 25. (2006) <61> Microbial Limit Tests US
Pharmacopoeial Corp. Inc. Rockville. MD, USA
VANDERZANT, C. y D.F. SPLITTOESSER (Eds) (1992)
Compendium of methods for the microbiological exami-
nation of foods. 3rd ed. A.P.H.A. Washington D.C.
97
 MacConkey Media

VARNAM, A.H. y M.G. EVANS (1991) Foodborne patho-

gens. Manson Publishing Ltd. London. U.K.
WHO (1963) International Standards for Drinking Wa-
ters, 7th ed., Churchill Ltd. London
WINDLE-TAYLOR, E.(1958) The Examination of Water
and Wastewater Supplies, 7th ed. Churchill Ltd. London

Malachite Green Broth

Ref. 02-467 by the German StandardizationInstitute (DIN) and other

Specification
Liquid culture medium for the selective enrichment of
Pseudomonas aeruginosa in water, according the DIN
38411 standard.
Formula (in g/L)
Meat peptone ......................................... 15,00
Meat extract .............................................. 9,00
Malachite green (Oxalate) ........................ 0,03
Dipotassium phosphate ............................ 1,10
Final pH 7,0 ± 0,2
Directions
To prepare concentrated broth: Dissolve 25 g of pow-
der in 1 L of distilled water and distribute into suitable
containers.
To prepare diluted broth: Dissolve 8,3 g of powder in 1 L
of distilled water and distribute into suitable containers.
In both the cases, sterilize by autoclaving at 121ºC for
15 minutes.
Description
Habs and Kirschner first described this medium in 1948
based on the resistance of Pseudomonas aeruginosa
to malachite green as a selective factor. Lately, in 1974
Schubert and Blum modified the medium composition
and proposed it as an enrichment step of Pseudomonas
in very polluted water since malachite green oxalate
at the concentration as above formulation inhibited the
growth of almost all gramnegative microorganisms but it
did not affect Pseudomonas growth.
Years after the proposal of Schubert and Blum, the
medium was adopted officially as an enrichment medium
legal bodies for the microbiological analysis of water and
food.
Technique
If the product to be examined is not restricted to specific
standard (as DIN 38411 for water), it is suggested that
the final malachite green concentration for enrichment
should not exceed 0,01 g/L. Thus the concentrated or
diluted broth should be used in function of the size or
volume of the sample.
Carry out the incubation at 35±2ºC for 24-48 hours.
Cultures that show turbidity due to growth should be
selected for the later confirmation for the presence of
Pseudomonas aeruginosa.
References
BUNDESGESUNDHEITSAMT: Amtliche Sammlung von
Untersuchungverfahren nach 35 LMBG Beuth Verlag
Berlin Köln.
DEUTSCHE EINHEITSVERFAHREN sur Wasser-,
Abwasser- und Schlammuntersuchung. VCH Verlags-
gesellschaft D-6940 Weinheim.
DIN 38411; Teil 6: Mikrobiologische Verfahren (Gruppe
K):Nachweis von Pseudomonas aeruginosa (K8)
HABS, H., K.H. KIRSCHNER (1943) Der Pyocyaneus
Meerschweinchenhautversuch zur Prüfung von Haut
desinfektionsmiteln. Z. Hyg. 124:557-578.
SCHUBERT, R., U.BLUM (1974) Zur Frage der Eignun
der Malachitgrün-Bouillon nach Habs u. Kirschner als
Anreicherungsmedium fur Pseudomonas aeruginosa
aus dem Wasser. Zbl. Bakt. Hyg. I Orig. B 158:583-587

98
 Malt Extract Media
Media based on malt extract may be considered as gen-
eral growth substrates due to their richness and nutrient
balance. They are very suitable for the cultivation of
fastidious microorganisms. Classically, with acidic pH,
they are used for the isolation, cultivation and mainten-
ace of moulds and yeast, but with pH near to neutrality,
they support bacterial growth of bacteria with special or
fastidious nutritional needs.
The SCHARLAU manufacturing program covers all the
published formulations, and included in this manual are
some of the most commonly used. We propose that the
technicians choose the formulation most suited to their
requirements, or, alternatively, ask us for more details or
possible modifications.
See also YM Media (Refs. 1-219, 2-219) and Wort Agar
(Ref. 01-132)
Malt Extract Agar No. 1
Ref. 01-111
Specification
Culture medium for moulds and yeast.
Formula (in g/L)
Malt extract ............................................... 13,0
Dextrine ...................................................... 2,5
Gelatin peptone .......................................... 5,0
Agar .......................................................... 15,0
Final pH 5,5 ± 0,2
Directions
Suspend 35,5 g of powder in 1 L of distilled water and
heat gently with constant stirring until the boiling. Dis-
pense in suitable containers and sterilize by autoclaving
at 115°C for 15 minutes. Avoid overheating since the low
pH of the medium may hydrolize the agar.
Description
Malt Extract Agar is a classic culture medium for moulds
and yeast. Malt extract has enough sugar (maltose, glu-
cose, sucrose) to allow a copious growth, and in extreme
cases, additional necessary growth factors are provided
by the gelatin peptone.
Technique
Malt Extract Agar has been widely used in maintenance,
isolation and identification of fungi, and it is also pro-
posed in several pharmacopiea as a medium for the
control of sterility in pharmaceutical products, though it is
mostly used for comparative morphological studies.
Should more selectivity be desired, you can add a few
millilitres of 10% Lactic Acid, or 5% Tartaric Acid, but
then this will make the solidification of agar more difficult.
When acidification is below pH 5,0 do not remelt the
agar since the solidifying agent will be hydrolized.
Malt Extract Agar No. 2
Ref. 01-573
Specification
Solid medium for the isolation and enumeration of fungi.
Formula (in g/L)
Malt extract ............................................... 30,0
Soy peptone ............................................... 3,0
Agar .......................................................... 15,0
Final pH 5,6 ± 0,2
Directions
Suspend 48 g of powder in 1 L of distilled water and let it
soak. Bring to the boil. Distribute into suitable containers
and sterilize in the autoclave at 121°C for 15 minutes.
Description
Malt Extract Agar may support the growth of almost all
of the fungi very well, because of its balanced composi-
tion, and restrains most of the bacteria due to the strong
acidity.
Should more selection against the bacterial growth be
desired, readjust the pH to 3,5 by adding a sterile solu-
tion of 10% lactic acid or 5% tartaric acid to the molten
medium. After the additions do not reheat the medium.
Malt Extract Agar No. 3
Ref. 01-574
Specification
Solid medium for the isolation and enumeration of fungi.
Formula (in g/L)
Malt extract ............................................... 30,0
Mycological peptone ................................... 5,0
Agar .......................................................... 15,0
Final pH 5,4 ± 0,2
Directions
Suspend 50 g of powder in 1 L of distilled water and let it
soak. Heat to the boil and distribute into suitable contain-
ers. Sterilize in the autoclave at 121°C for 15 minutes.
99
 Malt Extract Media

Description
The balanced and rich nutrient composition of the me-
dium makes it suitable for morphogenetic and structural
studies of fungi. Due to its low pH it restrains the bacte-
rial growth to a greater extent, but the total supression
can be achieved by adding to the melted medium at
55°C, 20 mL of sterile solution of 10% Lactic acid or 5%
tartaric acid, making the pH reduces to 3,5. In these
conditions, do not heat the medium to avoid the hydroly-
sis of agar.
Malt Extract Broth No. 1
Ref. 02-111
Specification
Liquid culture medium for the moulds and yeasts.
Formula (in g/L)
Malt extract ............................................... 13,0
Dextrine ...................................................... 2,5
Gelatin peptone .......................................... 5,0
Final pH 5,5 ± 0,2
Directions
Dissolve 20,5 g of powder in 1 L of distilled water, heat-
ing up if necessary. Distribute into suitable containers
and sterilize in the autoclave at 121°C for 15 minutes.
Description
Malt Extract Broth is a classic culture medium for the
moulds and yeasts. Malt extract has sufficient sugar
(maltose, glucose, sucrose) to allow a copious growth,
and in more demanding cases, the necessary growth
factors are provided by the gelatin peptone.
Description
This formulation of classic Malt Extract Broth is ac-
cording to Reiss’ modification in order to achieve better
results for the cultivation of Aspergillus flavus.
Technique
Malt Extract Broth has been widely used in mainte-
nance, isolation and identification of fungi, and it is also
proposed in several pharmacopeia as a medium for the
control of sterility in pharmaceutical products, though it is
mostly used for comparative morphological studies.
Should more selectivity be desired, you can add a few
millilitres of 10% Lactic Acid, or 5% Tartaric Acid, but this
makes the solidification of agar very difficult. When acidi-
fication is below pH 5,0, do not remelt the agar since the
solidifying agent is hydrolized below pH 5,0.
References
FDA (1998). Bacteriological Analytical Manual. 8th ed.
Revision A AOAC International Gaithersburg) MD.
DOWNES, F.P & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 4th ed.
APHA. Washington.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press,.
BALLOWS, HAUSLER, HERMAN, ISENBERG &
SHADOMY (eds.) (1991) Manual of Clinical Microbiol-
ogy. ASM. Washington.
REIS, J. (1972) Ein selektives kulturmedium für der
Nachweiss von Aspergillus flavus. Zbl. Bakt. Hyg. I. Abt.
Orig. 220:564-566.
RAPP, M. (1974) Indikator-Zusätze zur Keimdifferen-
zierung auf Würze und Malzextrakt Agar Milchwiss.
29:341-344.

Malt Extract Broth No. 2

Ref. 02-491

Specification
Liquid culture medium for the moulds and yeasts.

Formula (in g/L)

Mycological peptone ................................... 3,0
Malt extract ............................................... 17,0
Final pH 5,4 ± 0,2

Directions
Dissolve 20 g of powder in 1 L of distilled water, heat-
ing up if necessary. Distribute in suitable containers and
sterilize by autoclaving at 121ºC for 15 minutes. Do not
overheat, since a browning by Maillard reaction can be
produced.

100
 Mannitol Salt Agar (Chapman Agar)

Ref. 01-116
Specification
Selective medium for the isolation of staphylococci
according USP and ISO standard.
Formula (in g/L)
Meat extract ............................................ 1,000
Casein peptone ...................................... 5,000
Meat peptone ......................................... 5,000
Sodium chloride .................................... 75,000
D-Mannitol ............................................ 10,000
Phenol red .............................................. 0,025
Agar ...................................................... 15,000
Final pH 7,4 ± 0,2
References
ATLAS, R.M. & L.C.PARKS (1993) Handbook of Micro-
biological Media. CRC Press. BocaRaton. Fla. USA
CHAPMAN (1945) The significance of sodium chloride in
studies of staphylococci. J. Bact 50:201
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Foods.4th ed.
APHA. Washington DC. USA
FDA (1995) Bacteriological Analytical Manual. 8th ed.
Revision A. AOAC Internacional Inc. Gaithersburg. Md.
USA
ISO 22718:2006 Standard. Cosmetics – Detection of
Staphylococcus aureus.
USP 29- NF 25(2005) <61>Microbial Limit Tests. US
Pharmacopoeial Convention Inc. Rockville. Md. USA

Directions
Suspend 111 g of powder in 1 L of distilled water and
bring to the boil. Dispense in tubes or flasks and sterilize
by autoclaving at 121°C for 15 minutes.

Description
Mannitol Salt Agar is a classical medium for the detec-
tion and enumeration of staphylococci. It was described
by Chapman and has been adopted by many official or-
ganisations. Several modifications have been developed
from it with more or less similar effectivity.
This medium uses the advantage of high tolerance of
staphylococci to salinity, to use sodium chloride as a se-
lective agent, since only the staphylococci and halophilic
enterobacteria are able to grow freely at this concentra-
tion of salt employed in this medium while other bacteria
are inhibited. It also exploits the correlation between
the pathogenic and fermentative capacity of mannitol
of staphylococci, to establish a presumptive diagnosis.
Mannitol fermentation with an accumulation of acid prod-
ucts is shown by the phenol red indicator turning yellow, Staphylococcus aureus ATCC 25923
that produces a yellow halo surrounding the presumptive
pathogen colonies, meanwhile the rest of the medium
remains orange in colour.

Technique
A massive surface inoculation and an incubation at 37°C
for 36 hours or at 32°C for 3 days is recommended.
The typical appearance of the colonies after the correct
incubation is as follows: Presumptive pathogenic staphy-
lococci (coagulase +) are mannitol positive and are big
colonies with a yellow halo. Non-pathogenic Staphyloco-
cci (coagulase -) are usually mannitol negative and are
small colonies without halo or change in colour.
In any case, coagulase presence must be tested by the
classical technique, after a pure culture in the liquid me-
dium is obtained, in order to establish its true pahogenic
potential.

101
 Maximum Recovery Diluent

Ref. 02-510 Technique

According to the ISO method, the sample is diluted in a
Specification ratio 1:10 with the Maximum Recovery Diluent and ho-
Isotonic diluent for the maximal recovery of stressed mogenized by a vortex mixer or stomacher. After a short
microorganisms according ISO standard. period (10-15 minutes) of rest, a decimal dilution bank
with the same diluent is released following the standard
procedures. Plates are inoculated from the different
Formula (in g/L)
concentration of the dilution bank.
Peptone .................................................... 1,00
Sodium chloride ........................................ 8,50
Final pH 7,0 ± 0,2 Reference
ISO/DIS 6649 Meat and Meat Products. Detection and
Enumeration of Clostridium perfringens
Directions
ISO 21149:2006 Cosmetics – Enumeration and detec-
Disolve 9,5 g of powder in 1 L of distilled water and dis-
tion of aerobic mesophilic bacteria.
tribute into suitable containers. Sterilize by autoclaving at
ISO 21150:2006 Standard. Cosmetics – Detection of
121ºC for 15 minutes.
Escherichia coli
ISO 22717:2006 Standard. Cosmetics – Detection of
Description Pseudomonas aeruginosa.
This formulation combines the osmotic pressure of the ISO 22718:2006 Standard. Cosmetics – Detection of
physiological saline solution with the protective action Staphylococcus aureus.
of the peptone to obtain a good recovery of stressed
microorganisms.
The sodium chloride assures the isotonic conditions and
the low concentration of the peptone does not allow the
cellular growth in the short period (2-4 hours) of time
required for the preparation of the dilution bank of the
sample.

102
 Mayeux Agar
Ref. 01-223
Specification
Solid culture medium for the detection of Leuconostoc in
fermentation starters of mixed flora.
Formula (in g/L)
Peptone ................................................ 10,000
Yeast extract ........................................... 5,000
Sucrose .............................................. 100,000
Dextrose ................................................. 5,000
Sodium citrate ........................................ 1,000
Gelatine .................................................. 2,500
Sodium azide .......................................... 0,075
Agar ...................................................... 15,000
Final pH 6,0 ± 0,2
Directions
Suspend 138,5 g of powder in 1 L of distilled water and
heat up in boiling water bath at 50-55°C, till the complete
liquefaction of the gelatine is obtained. Heat to boiling
and dispense into suitable containers. Sterilize in the
autoclave at 121°C for 15 minutes. Avoid overheating,
since it may affect the solidification.
Description
This differential and selective medium for Leuconostoc
was originally described by Mayeux in 1961, and was
later modified by the same author to this formulation.
This allows a very specific separation of microorganisms
in the lactic fermentation starters with mixed flora.
Citrate, glucose and gelatine helps for the growth of Leu-
conostoc, and the large amount of sucrose allows a co-
pious production of dextrane polymer by L.dextranicum.
Sodium azide avoids the growth of undesired flora, and
at the same time, hampers the colonial development of
lactic streptococci. Other authors state that the addition
of little amounts of tetracycline (15 mcg/mL) produces
the inhibition of lactic streptococci without affecting
Leuconostoc.
Technique
The plates are inoculated by surface inoculation, and
then incubated at 21°C for 4 days. Most of the strepto-
coccal strains, including Streptococcus lactis, S.cremoris
and S.diacetilactis do not grow or grow a little after the
third day of incubation. In those cases, their colonies are
small, opaque and cream or yellow coloured. Leucon-
ostoc colonies have a bigger and earlier growth. Leu-
conostoc citrovorum form the colonies of 0,5 to 1 mm
diameter, which are translucent and iridescent.
L. dextranicum form big colonies (1-5 mm), which are
transparent and mucosal.
References
MAYEUX and COLMER (1961) J. Bact. 81:1.009.
MAYEUX, SANDINE and ELLIKER (1962) J. Dairy Sci.
45:665.
McDONOUGH, HARDGROVE and TITSLER (1962) J.
Dairy Sci. 45:656.
FIL-IDF Standard 149A (1997) Dairy starters of lactic
acid bacteria culture. Composition standard.
103
 Meat Liver Agar
Ref. 01-562
Specification
Solid medium for the cultivation of anaerobic microor-
ganisms
Formula (in g/L)
Meat Extract ........................................... 10,00
Liver Extract ........................................... 10,00
Dextrose ................................................... 0,75
Soluble starch ........................................... 0,75
Sodium sulfite ........................................... 1,20
Ammonium Iron (III) citrate ....................... 0,50
Agar ........................................................ 13,00
Final pH 7,6 ± 0,2
Directions
Suspend 36,2 g of powder in 1 L of distilled water and
had to boiling with constantly stirring. Distribute in suit-
able containers and sterilize in the autoclave at 121ºC
for 15 minutes.
Description
The mixture of meat and liver extract is a highly reduc-
ing nutrient basis that provides the supply of nitrogen for
the growth of anaerobes. The energy source is provided
by the dextrose and the starch acts only as a metabolic
detoxifier. The sulphite present in the culture medium is
104
reduced to H2S by some Clostridium spp and reacts with
the iron from ammonium iron citrate producing a dark
precipitate that blackening the medium.
Technique
The meat liver agar can be used in petri dishes or
in tubes. When the inoculum is in poured plates, the
reducing power of the medium permits the growth of the
anaerobes. If spreading plates are used the incubation in
an anaerobic environment is compulsory, but some times
is enough cover the plate with Sealing Anaerobic Agar
(Ref. 01-174). Temperature and time of the incubation
must be suitable to the sample, but it is recommended
more than 48 hours at 35-37ºC
References
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press Inc. London.
CORETTI, C. (1962) Prüfung eines leberpulvers auf
Eignung zur Herstellung von Leberbrühe und Leberagar
zur Anaerobenzuchtung. Berl. Münch. Tierärztl. Wissch.
75:205.
VANDERZANT, C. & D.F. SPLITTSTOESSER (1992)
Compendium of methods for the microbial examination
of Foods. 3rd Ed. APHA. Washington.
 Methyl Red Voges Proskauer Media (MRVP Media)
Methyl Red Voges Proskauer
Broth (MRVP Broth)
(Clarks Lubs Medium)
Ref. 02-207
Specification
Classic liquid medium for differential tests (Voges
Proskauer and Methyl Red) in Enterobacteria according
ISO standards 6579 and 6585 and FIL - IDF 93 stand-
ard.
Formula (in g/L)
Peptone ...................................................... 7,0
Dextrose ..................................................... 5,0
Potassium phosphate ................................. 5,0
Final pH 7,0 ± 0,2
Directions
Dissolve 17 g of powder in 1 L of distilled water, heating
up only if necessary. Dispense in tubes and sterilize by
autoclaving at 121°C for 15 minutes.
Description
The classical Clark and Lubs medium which is used to
perform the tests of Methyl Red and Voges Proskauer,
that together with Indole and Citrate tests (IMViC) allow
the differentiation within the coliform group of bacteria.
The fundamentals of these reactions are as follows:
Methyl Red Test (M.R. test)
Among the Enterobacteriaceae, the E.coli biotype fer-
ments glucose by the mixed acid pathway, accumulating
acid, which reduces the initial pH. It can be detected by
the methyl red indicator, which turns yellow above the
pH 5,1 and becomes red below pH 4,4.
Voges Proskauer Test (V.P. test)
Enterobacteria of Klebsiella-Enterobacter biotype
ferment the glucose by the 2-3-butanediol pathway.
Although the acids are produced in this way, the neutral
or alkaline products are also formed and at the end the
reaction is neutral or alkaline. Due to this, the incubation
must be extended up to 3 days. After this period, the
methyl red reaction is negative.
Nonetheless, Voges Proskauer test is complementary
to Methyl Red test in some ways. It shows the 2-3-bu-
tanediol and acetoin production, that are substances
difficult to find in the mixed acid pathway. It exploits the
fact that these two products, in alkaline medium, oxidize
themselves to diacetyl, which reacts with guanidine and
produces visibly coloured compounds.
Methyl Red Voges Proskauer
Modified Broth for Bacillus
(MRVP Modified Broth)
Ref. 02-572
Specification
Liquid culture media to perform the Voges-Proskauer
test in Bacillus cultures according FIL-IDF 181 standard.
Formula (in g/L)
Tryptone .................................................. 7,00
Dextrose ................................................... 5,00
Dipotasium phosphate .............................. 5,00
Sodium chloride ........................................ 5,00
Final pH 7,0 ± 0,2
Directions
Dissolve 22 g of powder in 1 L of distilled water, heating
if it is necessary. Distribute in suitable containers (5 mL
in test tubes of 16 x 160 mm) and sterilize in autoclave
at 121ºC for 15 minutes
Description
This medium is produced according the FIL-IDF formula-
tion to perform the test of acetil-metil-carbinol production
in Bacillus cereus and other species of Bacillus.
Methyl Red Voges Proskauer
Saline Broth
(MRVP Saline Broth)
Ref. 02-456
Specification
Liquid culture medium for the Mehyl Red and Voges
Proskauer tests.
Formula (in g/L)
Peptone ................................................... 7,00
Dextrose ................................................... 5,00
Sodium chloride ...................................... 30,00
Dipotassium phosphate ............................ 5,00
Final pH 7,4 ± 0,2
Directions
Dissolve 47 g of powder in 1 L of distilled water, heating
up only if necessary. Distribute into suitable containers
and sterilize by autoclaving at 121ºC for 15 minutes.
105
 Methyl-red Voges Proskauer Media (MRVP Media)
Description
This medium is used to perform the tests of Methyl Red
and Voges Proskauer, that, together with Indole and Cit-
rate tests allow the differentiation within the enteric bac-
teria. The fundamentals of these reactions are described
in the Methyl-Red Voges-Proskauer Broth (Ref. 02-207)
Technique
There are several techniques to carry out these tests.
One of them is as follows:
The tube with medium is inoculated with the microorgan-
ism to be studied and incubated at 30°C for at least 3
days and up to 5 days maximum. Just before reading,
culture is separated in two portions, one for each test.
1) Methyl Red Test.
Add 4-5 drops of Methyl Red Reagent (Ref. 06-007) to
the culture, and shake in order to homogenize. Observe
for the colour development in the medium. The test is
considered positive if it turns to red and negative if it
remains yellow.
Positive (red colouration): E.coli, Edwardsiella, Shigella,
Salmonella, Citrobacter, Proteus, Klebsiella ozoe-
nae, Klebsiella rhinoscleromatis, Yersinia.
Negative (yellow colouration): Enterobacter, Hafnia, Ser-
ratia, Klebsiella pneumoniae.
With Erwinia, this reaction has no significance since it
gives variable reactions.
2) Voges Proskauer Test
Add Barrit’s Reagent to the medium (Ref. 06-027) until it
gets a milky appearance and then add O’Meara’s Rea-
gent (Ref. 06-006) until milky appearance disappears.
Shake vigorously.
Test is positive if the medium acquires a pink-violet col-
our, forming at the top of the tube. If the test is negative,
there is no colour formation. Relative amounts of each
reagent depend on initial volumes of the medium. Never
incubate above 30°C.
Positive (pink-intense red): Enterobacter, Hafnia, Kleb-
siella pneumoniae, Serratia.
Negative (no colour change): Escherichia, Edwardsiella,
Citrobacter, Salmonella, Shigella, Yersinia, Kleb-
siella ozonae, Klebsiella rhinoscleromatis.
With Proteus and Erwinia types, this reaction has no
significance since it gives variable reactions.
106
Voges Proskauer test may be performed in a quicker
way, using very little volumes of medium and massive in-
oculum. This allows the readings with short incubations
(18-20 hours), and also the readings may be accelerated
by gently heating the culture almost to boiling point after
adding the reagents. However, erroneous results are
more likely by using this method.
Refer to the FIL-IDF Standard for the specific technique
of MRVP Modified Broth.
References
VOGES, O, B. PROSKAUER (1898) Beitragzur
Ernährungsphysiologic und zur Differentialdiagnose der
hämorrhagischen Septicäemie. Z. Hyg.
CLARK, W., H. LUBS. (1915) The differentiation of bac-
teria of the colon-aerogenes family by the use of indica-
tors. J. Inf. Dis. 17:160-173
BARRIT, M. (1936) The intensification of the Voges
Proskauer reaction by the addition of alpha-naftol. J.
Path. Bact. 42:441-452
O’MEARA, R. (1931) A simple delicate and rapid
methods of detecting the formation of acetylmethyl/car-
binol by bacteria fermenting carbohidrats. J. Path. Bact.
34:401-406
MOLLÄNDER, R., J. BÖHMANN, B. GREWING (1982)
Die Verstörkung der Voges-Proskauer Reaktion durech
fumarat. Zbt. Bakt. Hyg. I Alet. Orig. A 252:316-323.
SCHWEIZERISCHES LEBENSMITTELBUCH 5th Ed.
Ch. 56A. Berna.
FDA (1998) Bacteriological Analytical Manual. 8th ed.
Rev. A. AOAC International. Gaithersburg. MD.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc.,London.
PASCUAL ANDERSON, MªRª (1992) Microbiología
Alimentaria. Diaz de Santos, S.A.,Madrid,.
ISO Standard 6579 (2002) Microbiology of Food and ani-
mal feeding stuffs- Horizontal method for the detection of
Salmonella species.
FIL-IDF (1998) International Provisional Standard 181.
Dried Milk Products: Enumeration of Bacillus cereus:
Most Probable Number Technique.
FIL-IDF (2001) Milk and Milk products Detection of
Salmonella.
ISO 6585 standard (2001) Milk and Milk products - De-
tection of Salmonella.
 Microbial Content Test Agar (TSA Lecithin Polysorbate)

Ref. 01-613

Specification
Solid medium for sampling of surfaces of sanitary impor-
tance with RODAC plates technique.

Formula (in g/L)

Tryptone ................................................. 15,00
Soy peptone ............................................. 5,00
Sodium chloride ....................................... 5,00
Lecithin ..................................................... 0,70
Polysorbate 80 ......................................... 5,00
Agar ........................................................ 15,00
Final pH 7,3 ± 0,2

Directions
The dehydrated medium has a characteristic “brown
sugar” appearance and may seem moist.
Suspend 45,7 g of powder in 1 L of distilled water and
let it soak. Bring to the boil and dDistribute in suitable
containers and sterilize in autoclave at 121ºC for 15
minutes.

Description
This medium is a modification of the classical TSA for
the surface sampling by the RODAC (Replicate Organ-
ism Detection and Counting) plate technique. Collection
of samples from identical areas (replicate) “before and
after” treatment with disinfectant yields data useful in
evaluating cleaning procedures in environmental sanita-
tion.
Lecithin is incorporated to neutralize quaternary ammo-
nium compounds and polysorbate 80 is used to neutral-
ize phenolic disinfectants, hexachlorophene, formalin
and, with lecithin, ethanol.

References
HICKEY, P.J., C.E. BECKELHEIMER, & T. PARROW
(1992) Microbiological tests for equipment, containers,
water and air. In R.T. Marshall (Ed.) Standard Methods
for the examination of Dairy Products 16th ed. APHA
Washington.
EVANCHO, G.M., W.H. SVEUM, LL. J. MOBERG & J.F.
FRANK (2001) Microbiological Monitoring of the Food
Processing Environment. In Downes & Ito (Eds) Compen-
dium of Methods for the Microbiological Examination of
Foods. 4th ed. APHA. Washington DC.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Culture Media. CRC Press. Boca Ratón, Fla.

107
 Milk Agar

Ref. 01-514

Specification
Solid culture medium for the plate count test in dairy
products.

Formula (in g/L)

Peptone .................................................... 5,00
Yeast extract ............................................. 3,00
Powdered milk .......................................... 1,00
Agar ........................................................ 15,00
Final pH 7,2 ± 0,2

Directions
Suspend 24 g of powder in 1 L of distilled water and let
it soak. Bring to the boil and distribute into suitable con-
tainers. Sterilize by autoclaving at 121ºC for 15 minutes.

Description
Milk Agar is approved by the European Commission and
it is formulated according to the recommendations of the
European Association of Ice-Cream Producers (EuroGla-
ce) for the microbiological examination of ice-creams.

Technique
Suggested technique is the standardized count on mass-
inoculated plates. Inoculum is obtained from a decimal
dilution bank of the sample. Once inoculated, the plates
should be left undisturbed for 1-3 hours and then incu-
bated at 30ºC for 3 days.

References
KLOSE, J. (1968) Harmonisierung des speisesrechtes
under EWC-Sübwaren 14:778-780.
KLOSE, J. (1968) Entwarf einer Riehlinie zar Aufleichung
der Rechtvorschiften fur Speiseeis unden Mitfliedsstaaten
der EWG. Sübwaren 14:780-782
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press. Boca Raton. Fla.

108
 Motility Media
Motility Indole Ornithine Fluid
Medium (MIO)
Ref. 03-422
Specification
Medium for the demonstration of motility, indole produc-
tion and ornithine decarboxylase activity of enterobacte-
ria.
Formula (in g/L)
Yeast extract ............................................. 3,00
Dextrose ................................................... 1,00
Gelatin peptone ...................................... 10,00
Casein peptone ...................................... 10,00
L-Ornithine HCl ......................................... 5,00
Bromocresol purple .................................. 0,02
Agar .......................................................... 2,50
Final pH 6,6 ± 0,2
Directions
Suspend 31,5 g of powder in 1 L of distilled water and let
it soak. Bring to the boil and distribute in tubes. Sterilize
by autoclaving at 121°C for 15 minutes.
Technique
Remove all the dissolved air in the medium by heating
up the tubes in boiling water bath and cooling them upto
room temperature. Taking the growth of the primary
isolation as the inoculum, inoculate the tubes by a single
deep stab. Incubate aerobically at 35±2°C for a 18-24
hours period.
Motility can be observed by the diffuse growth at the
upper side of the stab, meanwhile the immotile bacteria
grow along the stab, producing a clear streak.
Ornithine decarboxylation is indicated by the presence of
a dark purple colour throughout the tube. Negative reac-
tion produces only a single purple band at the top, and
the rest of the tube changes to yellow.
Indole production is verified after the addition of a few
drops of Kovac’s Reagent (Ref. 06-018) (shake gently).
The presence of a red ring signifies the positive reaction,
and if the colour is yellow, then the reaction is negative.
References
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press,Boca Raton,Fla.
EDERER, G.M. and M. CLARK (1970) Motility-Indol-Or-
nithine Medium. Appl. Microbiol. 2:849.
FDA (1998) Bacteriological Analytical Manual 8th ed.
REvision A. AOAC International. Gaithersburg. MD.
Motility Nitrate Medium
Ref. 03-612
Specification
Medium for the motility determination and nitrate reduc-
tion of clostridia in food products acc. ISO 7937.
Formula (in g/L)
Meat extract ................................................ 3,0
Peptone ...................................................... 5,0
Potassium nitrate ........................................ 1,0
Disodiun phosphate .................................... 2,5
Galactose ................................................... 5,0
Agar ............................................................ 5,0
Final pH 7,3 ± 0,2
Directions
Suspend 21,5 g of powder in 1 L of distilled water con-
taining 5 mL of glycerol and heat to boiling. Dispense
into suitable containers and sterilize by autoclaving at
121°C for 15 minutes.
Description
This semisolid medium has been made according to the
rules suggested by the US Food and Drug Administra-
tion for the identification of Clostridium perfringens in
food.
Technique
Prepared tubes regenerate themselves if kept in boiling
water bath for 10 minutes to eliminate the dissolved oxy-
gen. Let them get cooled to 70-80°C and then inoculate
them by stabbing the centre. Take a black colony grown
on TSN Agar (Ref. 01-195) as the inoculum. Incubate
the tubes at 37°C for 18-20 hours without sealing nor in
anaerobic chamber.
In this medium, if the growth is stopped at 5-7mm. from
the surface, signifies that there is anaerobiosis. Non-mo-
tile is evident as the growth is observed only inside the
stab.
To verify the nitrate reduction to nitrite, pour a few drops
of Nitrate A Solution (Ref. 06-003) and Nitrate B Solution
(Ref. 06-004) on the surface of the medium. If a pink or
red colour appears, reaction is positive.
Clostridium perfringens is an anaerobic , non-motile and
reducer of nitrate to nitrite microorganism.
References
FDA (1998) Baceriological Analytical Manual. 8th ed.
Revision A. AOAC International. Gaithersburg.
ISO 7937 Standard (2004). Microbiology of food and ani-
mal feeding stuffs. Horizontal methods for the enumera-
tion of Clostridium perfringens. Colony count technique.
109
 MRS Media
MRS Agar
Ref. 01-135
Specification
Solid culture medium for lactobacilli, according to de
Man, Rogosa and Sharpe and ISO standards 9332 and
15214.
Formula (in g/L)
Peptone Proteose ................................... 10,00
Meat extract .............................................. 8,00
Yeast extract ............................................. 4,00
D(+)Glucose ........................................... 20,00
Sodium acetate ........................................ 5,00
Triammonium citrate ................................. 2,00
Magnesium sulfate ................................... 0,20
Manganese sulfate ................................... 0,05
Dipotassium phosphate ............................ 2,00
Polysorbate 80 ......................................... 1,00
Agar ........................................................ 14,00
Final pH 6,2 ± 0,2
Directions
Suspend 66 g of powder in 1 L of distilled water. Bring to
the boil slowly with gentle stirring until complete dissolu-
tion. Dispense into suitable containers and sterilize by
autoclaving at 121°C for 15 minutes.
MRS Broth
Ref. 02-135
Specification
Liquid culture medium for lactobacilli, according to de
Man, Rogosa and Sharpe and ISO standards 9332 and
15214.
Formula (in g/L)
Peptone Proteose ................................... 10,00
Meat extract .............................................. 8,00
Yeast extract ............................................. 4,00
D(+)-Glucose .......................................... 20,00
Sodium acetate ........................................ 5,00
Triammonium citrate ................................. 2,00
Magnesium sulfate ................................... 0,20
Manganese sulfate ................................... 0,05
Dipotassium phosphate ............................ 2,00
Polysorbate 80 ......................................... 1,00
Final pH 6,2 ± 0,2
110
Directions
Suspend 52 g of powder in 1 L of distilled water. Heat up
to complete dissolution and dispense into suitable con-
tainers. Sterilize by autoclaving at 121°C for 15 minutes.
Description
MRS Agar and Broth are a modification of the previously
used media for the cultivation of lactobacilli, all of them
based on tomato juice’s nourishing properties. The ad-
dition of magnesium, manganese and acetate, together
with the Polysorbate, has provided an improved medium
for the growth of lactobacilli, including that of very fastidi-
ous species such as Lactobacillus brevis and Lactoba-
cillus fermenti.
On the other hand, the quality of the peptones in addition
to the meat and yeast extracts, combine together all the
necessary growth factors that make the MRS media one
of the best media for the cultivation of lactobacilli.
Nevertheless, these media selectivity is low and the con-
taminants tend to grow in these media, which signifies
a higher selectivity is needed. We therefore suggest the
use of subculture in solid medium, on double layer and
broth. In many cases,the growth is encouraged by a CO2
enriched atmosphere in the medium.
MRS media is particularly recommended for the enumer-
ation and maintenance of lactobacilli either by the MPN
technique (in broth) or on the plate by massive inocula-
tion, overlaying it with a second layer of molten medium.
This technique overcomes the need of a CO2 enriched
atmosphere.
References
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Foods.4th Ed.
APHA. Washington DC. USA
FIL-IDF Standard 146 (2003) Yoghurt. Identification of
characteristic micro-organisms.
IFU Method No 5 (1996) Lactic Acid Bacteria Count Pro-
cedure. Schweizerischer Obstverband. CH-6302 Zug
IFU Method No 7 (1998) Sterility testing of aseptic filled
products, commercial sterile products and preserved
products. Schweizerischer Obstverband. CH-6302 Zug
IFU Method No 9 (1998) Microbiological examination of
potential spoilage micro-organisms of tomato products.
Schweizerischer Obstverband. CH-6302 Zug
ISO Standard 9232 (2003) Yoghurt – Identification of
characteristic microorganisms (Lactobacillus delbrueckii
subsp bulgaricus and Streptococcus thermophilus)
ISO Standard 15214 (1998) Horizontal method for the
enumeration of mesophilic lactic acid bacteria – Colony
count technique at 30ºC
MAN, J.C. de, ROGOSA, M. y SHARPE, M. Elisabeth
(1960) A médium for the cultivation of lactobacilli. J.
Appl. Bact.; 23:130.
 Mueller Hinton Media
Mueller Hinton Agar
Ref. 01-136
Specification
Widely recommended medium for antibiotic and sulfona-
mide susceptibility testing, according to the Kirby-Bauer
and the Ericsson methods.
Formula (in g/L)
Peptone .................................................... 17,5
Beef infusion solids .................................... 2,0
Starch ......................................................... 1,5
Agar .......................................................... 17,0
Final pH 7,3 ± 0,2
Directions
Add 38 g of powder to 1 L of distilled water and let it
soak. Bring to the boil to dissolve the medium complete-
ly. Sterilize by autoclaving at 121°C for 15 minutes.
Description
The Mueller Hinton Agar was originally designed for the
primary isolation of meningococci and gonococci. With
the addition of blood it becomes an optimal medium
for the growth of Neisseria. It is also more effective if
reheated and turned into a Chocolate Agar. It should
never be remelted or reheated once the blood has been
added to it.
Mueller Hinton Broth
Ref. 02-136
Specification
Liquid version of the agar with the same name, recom-
mended for the studies about MIC of antibiotic.
Formula (in g/L)
Peptone .................................................... 17,5
Starch ......................................................... 1,5
Solids of meat infusion ............................... 2,0
Final pH 7,3 ± 0,2
Directions
Add 21 g of powder to 1 L of distilled water and dissolve
it completely. Distribute in suitable containers. Sterilize
by autoclaving at 121°C for 15 minutes.
Description
Mueller Hinton Broth is the liquid version of the agar with
the same name, and can be used in parallel with the
agar when comparative studies are desired as well as
when a broth with a high nutritive capacity is required.
It is especially suggested for inoculum preparation for
sensitivity assays.
In this medium, and in its solid version, presence of the
starch is very important, since it acts as a detoxifying
agent against the toxic substances if present in the sam-
ple and it also acts as the cell regenerator.
Technique
For the culture of Neisseria the best results are obtained
if incubation is carried out in a humid chamber with a
CO2 enriched atmosphere, if an anaerobic jar is not
available. This environment can be obtained by placing
the plates in a hermetically sealed air-tight container,
a dessicator for instance, together with a cotton swab
soaked in water and a lighted candle end. Once the con-
tainer is full the flame consume oxygen and by the time it
is extinguished, the atmosphere inside the container has
got 5 to 8% CO2 enrichment.
The Mueller-Hinton Agar has proved to be one of the
most efficient medium in the anti-bacterial susceptibil-
ity testing. Without the addition of blood it can even
be used for sulfonamide sensitivity testing since it is
free from most of its antagonists (nucleotides, etc.). If
this type of assay is conducted, the zones of inhibition
should be examined just after 12-18 hours, before the
usual overgrowth occurs, since after 24 hours it tends to
interfere with the examination of sulfonamides sensitivity.
For this purpose, a small inoculum will help the early
formation of zones of inhibition. It should amount to a
100 to 300 times smaller inoculum than that of the cor-
responding strain which is used in the antibiotic sensitiv-
ity testing.
In 1970 the WHO proposed this medium for antibacte-
rial sensitivity testing, and it has been widely used since
then.
Sensitivity testing can be conducted by a variety of
techniques, both on solid and liquid media. The most
commonly used method in routine work is that derived
from Kirby-Bauer and recommended by the American
Association of Clinical Pathologists. It provides informa-
tion on growth around a disk impregnated with antibacte-
rial substance.
The Bauer-Kirby method is more precise and is semi-
quantitative by category. It uses the Mueller-Hinton Agar
and disks with high antibiotic concentration. The inocu-
lum is first standardized with a Mac-Farland nephlom-
eter. Then the plate is inoculated with a swab dipped in
the standardized suspension, and finally the disks are
arranged properly and at the equidistance from each
other on the plate and then incubated.
Some authors suggest that the inoculum should be
modified by introducing a double layer of mass inoculat-
ed medium. This system undoubtedly provides sharper
and more defined zones of clearing or inhibition. Plates
are incubated at 37°C
111
 Mueller Hinton Media
overnight and then the zones of inhibition are measured.
Results are reported in terms of Resistant, Moderately
Resistant and Sensitive strains (Table above).
The Ericsson technique, which has been adopted in
most European countries has already standardized the
culture medium (Mueller-Hinton) and the quantity per
plate (25 mL on 9 cm diameter plates). It has also stand-
ardized the inoculum concentration.
The fresh culture suspension to be examined (incubated
for 18 hours in liquid medium) must be diluted enough,
so as to ensure the presence of confluent growth on the
agar.
Suggested Dilutions:
Enterobacteria- Pseudomonas: dilution of 1/300.
Staphylococcus - Enterococcus: dilution of 1/300.
Streptococcus - Haemophilus: dilution of 1/10.
The plate is seeded by flooding its surface. The ex-
cess inoculum is removed with a sterile pipette and the
antibiotic disks are arranged properly on the plate. Allow
a pre-diffusion period of 30-60 minutes before incuba-
tion so that the antibiotic can slowly diffuse before the
growth. After the incubation at 37°C for 12-18 hours,
measure the zones of inhibition and refer to the Assay
Regression Curves. Results are reported in terms of
112
Sensitive, Resistant or as Minimum Inhibitory Concentra-
tion (MIC).
This latest technique undoubtedly offers more precision
and reliability than the previous ones. Nevertheless, the
Kirky method, which is semiquantitative, is much more
simple and easy to adopt in everyday practice. On the
other hand, the Ericsson technique is highly recom-
mended for the effectivity and the sensitivity studies.
The Mueller-Hinton medium plates can be stored refrig-
erated in plastic bags for a month without affecting their
results of sensitivity testing. However, they should not be
used if the medium shows any dehydration.
The Mueller Hinton Agar Scharlau fulfills the WHO re-
quirements for the conducting microbial sensitivity tests
and the basic characteristics are verified in every batch.
Nevertheless some variation in the results between
batches can be observed and technicians claims about
the origin of these variability. At this point must keep in
mind a lot of factors that are a source of variability:
1. Since the nutritional requirements of organisms vary,
some strains may be encountered that fail to grow or
grow poorly on these media.
 Mueller Hinton Media
2. Numerous factors can affect results: inoculum size,
rate of growth, medium formulation and pH, length of
incubation and incubation environment, disk con-
tent and drug diffusion rate, and measurement of
endpoints. Therefore, strict adherence to protocol is
required to ensure reliable results.
3. Disk diffusion susceptibility testing is limited to rapidly
growing organisms. Drug inactivation may result
from the prolonged incubation times required by slow
growers.
4. Media containing excessive amounts of thymidine or
thymine can reverse the inhibitory effects of sulfona-
mides and trimethoprim, causing zones of growth
inhibition to be smaller or less distinct.
5. Variation in the concentration of divalent cations,
primarily calcium and magnesium, affects results of
aminoglycoside, tetracycline, and colistin tests with
Pseudomonas aeruginosa isolates. A cation content
that is too high reduces zones sizes, whereas a
cation content that is too low has the opposite effect.
6. When Mueller Hinton Medium is supplemented with
blood, the zone of inhibition for oxacillin and methicil-
lin may be 2 to 3 mm smaller than those obtained
with unsupplemented agar. Conversely, sheep blood
may markedly increase the zone diameters of some
cephalosporins when they are tested against ente-
rococci. Sheep blood may cause indistinct zones or
a film of growth within the zones of inhibition around
sulfonamide and trimethoprim disks.
7. Mueller Hinton Medium deeper than 4 mm may cause
false-resistant results, and agar less than 4 mm deep
may be associated with a false-susceptibility report.
8. A pH outside the range of 7,3±0,1 may adversely
affect susceptibility test results. If the pH is too low,
aminoglycosides and macrolides will appear to lose
potency; others may appear to have excessive activ-
ity. The opposite effects are possible if the pH is too
high.
9. When Mueller Hinton Medium is inoculated, no drop-
lets of moisture should be visible on the surface or on
the petri dish cover.
10. Mueller Hinton Medium should be inoculated within
15 minutes after the inoculum suspension has been
adjusted.
11. The zone of inhibition diameters of some drugs, such
as the macrolides, aminoglycosides and tetracy-
clines, are significantly altered by CO2. Plates should
not be incubated in increased CO2 atmosphere.
For further information on the performance of the anti-
biotic disk susceptibility test refer to the M2-A6 NCCLS
Monograph.
References
BAUER A.L., W.M.M. KIRBY, J.C.SHERRIS &
M.TURCK (1966) Antibiotic susceptibility testing by a
standardized single disc method. Am. J. Clin. Pathol 45:
493.
BARRY, A.L., M.D. COYLE, C. THORNBERRY, E.H.
GARLACH & R.W. HAWKINSON (1979) Methods of
measuring zones of inhibition with Bauer-Kirby disk sus-
ceptibility test. J. Clin. Microbiol. 10:885-889.
ERICSSON & SHERRIS (1971) Antibiotic sensitivity
testing. Report of an International Collaborative Study.
Acta Pathol. Microbiol. Scand Suppl. 217 p: 90.
HINDLER, J. (1998) Antimicrobial Susceptibility Testing
In Essential Procedures for Clinical Microbiology. ASM
Press. Washington D:C.
MUNRO, S. (1995) Disk Diffusion Susceptibility Testing.
In Clinical Microbiology Procedures Handbook. H.D.
Isenberg (Ed) APHA Whasington D.C.
MILLER, J.M., C. THORNBERRY & C.N. BAKER (1984)
Disk diffusion susceptibility test troubleshooting guide.
Lab. Med. 15:183-185.
NCCLS Standard M2-A6 (1997) Performance standards
for antimicrobial disk susceptibility tests. 6th ed. National
Committee for Clinical Laboratory Standards. Vilanova.
PA.
THORNSBERRY, C., W.G. GAVAN, E.H. GERLACH &
J.C. SHERRIS (1977) Cumitech 6. ASM. Washington.
WHO (1977) Requeriments for antibiotic susceptibility
tests. Technical Report Series No 610. Geneva.
WOODS, G.L. & J.A. WASHINGTON (1995) Antibacteri-
al susceptibility tests: dilution and disk diffusion methods.
In P.R. Murray, E.J. Baron, M.A. Pfaller, P.C. Tenover
and R.H. Yolken (Eds.) Manual of Clinical Microbiology.
6th ed. APHA. Washington, D.C.
CFR (1972) Rules and Regulations. 37: 20525.
NEUMAN, M.A., D.F. SAMM, C. THORNSBERRY, I.E.
McGOWAN (1991) New developments in antimicrobial
agent susceptibility testing: A practical guide. ASM.
Washington, D.C.
113
 Mycological Agar
Ref. 01-131
Specification
Solid culture medium for the maintenance, enumeration
and chromogenesis of fungi.
Formula (in g/L)
Soy peptone ............................................. 10,0
Dextrose ................................................... 10,0
Agar .......................................................... 17,0
Final pH 7,0 ± 0,2
Directions
Suspend 37 g of powder in 1 L of distilled water and heat
to boiling. Dispense in flasks or tubes and sterilize in the
autoclave at 121°C for 15 minutes. Should a selective
medium by the acidic pH be desired, adjust the pH to
4,0 with a sterile solution of 10% lactic acid, and do not
reheat the medium afterwards.
114
Description
Mycological Agar is a general medium that provides
enough nutrients for the development of most yeasts
and moulds.
It is employed in plates for the colonial isolation and
characterization, as it aids chromogenesis.
In the slants it is used for the maintenance of strains,
because its low content of glucose yields very slow acid
formation.
References
AJELLO, GEORG, KAPLAN and KAUFFMAN (1963)
CDC Lab Manual for Medical Mycology. PHS Pub. N°
994, Washington DC.
ATLAS, M.R., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, London.
VANDERZANT & SPLITTSTOESSER (1992). Compen-
dium of Methods for the Microbiological Examination of
Food. 3rd. Ed. APHA. Washington.
CTFA Microbiological Guidelines (1993) The Cosmetic
Toiletry and Fragance Association. Washington DC.
 Neutralizing Fluid Eur. Phar.

Ref. 02-512 Description

Specification
Liquid medium for neutralizing the antimicrobials accord-
ing to the European Pharmacopoeia.
Formula (in g/L)
Peptone .................................................... 1,00
L-Histidine HCl ......................................... 1,00
Lecithin ..................................................... 3,00
Monopotassium phosphate ...................... 3,60
Disodium phosphate ................................. 7,20
Sodium chloride ........................................ 4,30
Final pH 7,0 ± 0,2
Neutralizing Fluid is formulated according to the Euro-
pean Pharmacopeia formulation for the microbiological
examination of non sterile products. Its composition is
the same as the general diluting solution for biological
assays with the addition of polysorbate and lecithin as
non toxic neutralizing agents.
However, the European Pharmacopoeia lets the tech-
nician to increase the concentration of polysorbate if
the original is not enough or to add other agents when
thepreservative type to be neutralized is not known.
This way, the European Pharmacopeia suggests the
compounds shown in the table below, which have to be
always aseptically added to the fluid once sterilized and
cooled to 50ºC or below.

Directions
Dissolve 20,1 g of powder in 1 L of distilled water con-
References
European Pharmacopoeia (2002) 4th ed. Supplement
taining 30 mL of Polysorbate 80 (Ref. 06-088). Distribute
4.2.2.6.13. Tests for specified microorganisms. Council
into suitable containers and sterilize by autoclaving at
of Europe.Strasbourg.
121ºC for 15 minutes. Cool to 50ºC and homogenize the
solution.

115
 Nickerson Agar (BiGGY)
Ref. 01-137
Specification
Solid medium for the isolation and identification of Can-
dida sp.
Formula (in g/L)
Yeast extract ............................................... 1,0
Dextrose ................................................... 10,0
Glycine ..................................................... 10,0
Sodium sulfite ............................................. 3,0
Ammonium Bismuth Citrate ........................ 5,0
Agar .......................................................... 15,0
Final pH 6,8 ± 0,2
Directions
Suspend 44 g of powder in 1 L of distilled water and
heat to boiling. Dispense in tubes or dishes, stirring the
precipitate before pouring. Do not autoclave. Avoid
overheating.
Description
Nickerson Agar is suitable for the isolation and identi-
fication of yeast of the Candida type. Medium is made
according to the general principles of Bismuth-Sulfite
Agar, as inhibitor and differential agent, and completely
selective with the high concentration of glycine. This
medium is highly inhibitory, and does not allow bacterial
growth, however most Candida grow freely and quickly.
In some occasions, tiny colonies of the bacteria or highly
repressed moulds may appear. Bacterial development
may be totally prevented by adding neomycin sulfate to
the medium before pouring it into Petri dishes and its
concentration in the medium in this case must be around
2 mcg/mL, so that the antibiotic will not affect the devel-
opment or appearance of yeast.
116
The appearance of the colonies in this medium after an
incubation of 48-72 hours at 30-35°C is as follows:
Candida albicans: Creamy colonies, very convex, circu-
lar with very slight mycelial border and black or
dark brown colour. Neither it has metallic sheen
nor the diffused pigment, even after 72 hours of
incubation.
Candida tropicalis: Acuminated colonies, creamy, irregu-
lar and with slight mycelial borders. Dark brown
with black centre. After 72 hours of incubation it
may take on a metallic sheen and produce a dif-
fussed zone of pigment.
Candida krusei: Big and plain colonies, with irregular
borders. Brown colour, darker in the centre. A yel-
low halo appears around the colony.
Candida parakrusei: Plain colonies, average size, irregu-
lar. Dark red centre and light red borders. Yellow
mycelial border.
Candida pseudotropicalis: Big and plain colonies, dark
red colour. Mycelial border.
Candida stellatoidea: Average size plain colonies, dark
brown colour, without mycelial development.
Rhodotorula: Creamy convex colonies, with irregular
border and colours ranging from pink to orange.
Moulds in general: Restricted colonial growth and cot-
tony appearance.
To maintain these colony characteristics it is important
that the medium is freshly prepared and not reheated or
overheated.
References
NICKERSON, W.J. (1953) Reduction of inorganic
substance by yeast I. Extracellular reduction of sulfite by
species of Candida. J.Inf.Dis 93:43.
 Nitrate Broth
Ref. 02-138
Specification
A liquid culture medium, according to ISO 7932 stand-
ard, to determine the ability of enterobacteria to reduce
the nitrate to nitrites or free nitrogen gas.
Formula (in g/L)
Meat extract ................................................ 3,0
Peptone ...................................................... 5,0
Potassium nitrate ........................................ 1,0
Final pH 7,0 ± 0,2
Directions
Dissolve 9 g of powder in 1 L of distilled water, heating
up only if necessary to help the dissolution. Distribute
into final containers and sterilize by autoclaving at 121°C
for 15 minutes.
Description
The Nitrate Broth is prepared according to classical
formula for the assay of nitrate reduction by enterobacte-
ria, although it can also be used with aerobic bacilli and
other bacterial types.
Nutrient Media
Nutrient Agar (APHA)
Ref. 01-144
Specification
Solid culture medium for the general purposes according
ISO standard.
Formula (in g/L)
Peptone ...................................................... 5,0
Meat extract ................................................ 3,0
Agar .......................................................... 15,0
Final pH 7,0 ± 0,2
Directions
Suspend 23 g of powder in 1 L of distilled water and heat
to boiling. Dispense into suitable containers and sterilize
in the autoclave at 121°C for 15 minutes.
Technique
Inoculate 2-3 tubes of broth with one loop of pure culture
and incubate at 37°C, reading after 18-24 hours, 2 days
and 5 days in each tube, adding some drops of Nitrate
A Reagent (Ref. 06-003) and of Nitrate B Reagent (Ref.
06-004). If the first two readings are negative, it is rec-
ommended to investigate with the third one for the pres-
ence of nitrate by the method of zinc powder in order to
have quick nitrate reduction reaction .
References
DOWNES, F.P. & K. ITO (2001). Compendium of Meth-
ods for the Microbiological Examination of Food.4th ed.
APHA. Washington.
F.D.A. (1998) Bacteriological Analytical Manual 8th ede.
Rev. A. AOAC International, Gaithersburg.MD
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc.,London.
ISO standard 7932 (1993) General guidance for the
enumeration of B. cereus. Colony count at 30ºC.
Nutrient Agar (Brit. Phar.)
Ref. 01-140
Specification
Solid culture medium for general purposes and less
fastidious organisms acc. EN 12780:2002
Formula (in g/L)
Meat extract ................................................ 1,0
Yeast extract ............................................... 2,0
Peptone ...................................................... 5,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,4 ± 0,2
Directions
Suspend 28 g of powder in 1 L of distilled water and
bring to the boil to dissolve completely. Sterilize by auto-
claving at 121°C for 15 minutes.
Description
The Nutrient Agar is a simple medium in the range of
meat infusions, complemented by a formulation which
reinforces its nutrient qualities as well as its growth fac-
tors by adding yeast extract. It is most suitable for gen-
eral routine work and can support the growth of common
117
 Nutrient Media
organisms, even those considered mildly fastidious with
regard to nutrient elements. Besides this, by incorporat-
ing sodium chloride it allows the addition of blood, even
though it is not an optimal medium for it.
Nutrient Broth (APHA)
Ref. 02-144
Specification
Liquid medium for the cultivation of non fastidious micro-
organisms according ISO standard.
Formula (in g/L)
Peptone ...................................................... 5,0
Meat extract ................................................ 3,0
Final pH 7,0 ± 0,2
Directions
Dissolve 8 g of powder in 1 L of distilled water heating up
only if necessary. Dispense into suitable containers and
sterilize by autoclaving at 121°C for 15 minutes.
Description
Nutrient Broth is a modern version of the classical
general culture medium based on meat infusion. It is a
simple medium that may be used in general purposes
(i.e. maintenance of strains) as well as a base for other
specialized media. However, in this way, there are other
media with more nutrient capacity and better perform-
ance.
Nutrient broth is the liquid version of the Nutrient Agar,
and it is a classical medium for normal tasks with non
fastidious microorganisms. It is the ideal medium for
the subculture of general bacteria, especially staphyloco-
cci, to carry out later the coagulase and other biochemi-
cal tests. It may also be used to determine the Phenol
Coefficient by following the technique and microorgan-
isms suggested by the AOAC.
Nutrient Broth (Brit. Phar.)
Ref. 02-140
Specification
A general purpose liquid culture medium for the less
fastidious microorganisms.
Formula (in g/L)
Meat extract ................................................ 1,0
Yeast extract ............................................... 2,0
Peptone ...................................................... 5,0
Sodium chloride .......................................... 5,0
Final pH 7,4 ± 0,2
118
Directions
Dissolve 13 g of powder in 1 L of distilled water, heating
if necessary to help dissolve the medium. Distribute into
final containers and sterilize by autoclaving at 121°C for
15 minutes.
Description
The Nutrient Broth is the liquid version of the solid me-
dium which bears the same name. It is a classical broth
in the range of meat infusions. It is useful for the routine
laboratory purposes since its yeast extract supplement
allows the growth of most common organisms. It is also
suitable for the preparation of inocula and for the ef-
ficiency testing of bactericides, as well as for determina-
tion of the Phenol Coefficient and others.
Nutrient Broth No. 2
Ref. 02-561
Specification
Liquid medium for general purposes.
Formula (in g/L)
Meat Extract .......................................... 10,00
Peptone .................................................. 10,00
Sodium chloride ........................................ 5,00
Final pH 7,5 ± 0,2
Directions
Dissolve 25 g of the powder in 1 L of distilled water,
heating if necessary. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes.
Description
This medium in the classical way of the meat infusions,
presents a specially rich nutritional characteristics that
facilities the growth of very low inocula, even with fastidi-
ous microorganisms. Its formulation is according the
BSI for the determination of Rideal-Walker Coefficient of
disinfectants, where it is used at double concentration.
Nutrient DEV Agar
Ref. 01-451
Specification
Solid culture medium for the total enumeration of micro-
organisms in water, according to the German legislation
and ISO standard.
 Nutrient Media
Formula (in g/L)
Meat peptone ........................................... 10,0
Meat extract .............................................. 10,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 18,0
Final pH 7,3 ± 0,2
Directions
Suspend 43 g of powder in 1 L of distilled water. Bring
to the boil with constant stirring . Distribute into suitable
containers and sterilize in the autoclave at 121°C for 15
minutes.
Description
This medium is formulated according to the German
legislation but it differs from the other Anglo-Saxon
media with the same name in the concentration of its
compounds. This change intends to aid the recovery
and growth of damaged microorganisms.
Technique
German standards state a deep inocule of the water
sample, following the mass seed technique, directly in
the Petri plate. Incubation is performed at 20±2°C for
44±4 hours in most of the cases, but incubations at
37±1°C for the same period of time are also allowed. If
the water is chlorinated, the incubation time must last up
to 72 hours.
References
APHA (1948) Standard Methods for the Examination of
Dairy Products. Washington.
BRITISH PHARMACOPOEIA (1968), 357.
BRITISH STANDARD 541 (1934). Determining the Ri-
deal-Walker Coefficient of Disinfectants. BSI London 9.
Nutrient Gelatin Media
Nutrient Gelatin
Ref. 03-088
Specification
Culture medium for determination of gelatin liquefaction.
Formula (in g/L)
Meat extract ................................................ 3,0
Gelatin peptone .......................................... 5,0
Gelatin .................................................... 120,0
Final pH 6,7 ± 0,2
Directions
Suspend 128 g of powder in 1 L of cold distilled water
and heat gently in boiling bath up to 50°C. Keep at that
temperature until total dissolution of gelatin. Dispense in
tubes or flasks and sterilize in the autoclave at 121°C for
15 minutes.
DOWNES F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food.4th ed
APHA. Washington.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc. London.
EUROPEAN STANDARD EN 12780:2002 Water Quality.
Detection and enumeration of Pseudomonas aeruginosa
by membrane filtration
BUNDESGESUNDHEITAMT: Amtliche Sammlung von
Untersuchungsverfahren nach §35 LMBG. Beuth Verlag
Berlin- Köln.
VERORDNUNG von 12/12/1990 über Trinkwasser und
über Wasser fur Lebensmittelbetriebe. Bundesgesetz-
blatt: Teil I:2613-2629.
DEUTSCHE EINHEITSVERFAHREN zur Wasser-,
Abwasser- Und Schlammuntersuchung. VCH Verlags-
gesellchaft, D-6940 Weinheim.
ISO 8523 standard (1991) General guidance for the
detection of enterobacteriaceae with pre-enrichment.
ISO 6785 standard (2001) Milk and milk-products - De-
tection of Salmonella spp.
ISO 6340 standard (1995) Water Quality - Detection of
Salmonella species.
ISO 6579 standard (2002) Horizontal method for the
detection of Salmonella spp.
ISO 10273 standard (1994) General guidance for the
detection of presumptive pathogenic Yersinia enteroco-
litica.
ISO 21567. Standard (2004) . Horizontal method for the
detection of Shigella ssp.
ISO 16266:2006 Standard. Water Quality.– Detection
and enumeration of Pseudomonas aeruginosa. Method
by membrane filtration
Description
Gelatin Nutrient is used, essentially, to identify pure
cultures that have no specific nutritional requirements.
On the other hand, gelatin liquefaction is considered
very important to differentiate enteric bacilli on the basis
of their proteolysis. Gelatin was one of the first solidify-
ing agents employed in bacteriology, and helped in the
development of the Plate Count Technique, performed
by Koch. Nonetheless, nowadays it is not used in that
way, since it was replaced by Agar, which bears incuba-
tions at higher temperatures and was not so attacked or
degraded as gelatin.
However, the Plate Count Method is still used with
gelatin. Standard Methods still recommend it for aerobic
counting at 20-22°C.
119
 Nutrient Gelatin Media
Technique
The gelatin liquefaction test may be performed in plates
or tubes, that are inoculated by stabbing,and incubated
at 20-22°C or at the optimum temperature for the micro-
organism to assay. Because gelatin is liquid above 20°C,
it is necessary to put the plates or tubes in the refrigera-
tor before reading.
High temperature assays (35-37°C) eliminate wrong
results from bacteriolysis, but they require more care in
readings and also inclusion of a control (uninoculated) in
order to verify effect of heat on the medium.
Gelatin tube readings must be carried out carefully, since
often they have diagnostic value. In the case of using
several plates, Stone’s reaction may be used to include
different strains in the same dish, seeding them by paral-
lel streak. After incubation period, cool in the refrigerator
and then, over each streak, put a few drops of saturated
solution of Ammonium sulfate or Sulfosalicilic acid 20%
solution (freshly prepared).
Liquefaction (positive test) is shown by the presence
of a clear halo or zone around the growth, 10 minutes
after reagent addition. Incubation periods for gelatinase
activity assay vary, ranging from a few days to weeks,
at 20-22°C. Some Klebsiella and Enterobacter strains
take up to 3 weeks before they show activity. Though
incubation times are not standarized, it is recommended
a maximum of 14 days with regular intermittent readings
every 3 days.
A modern version of this methodology is the usage of
photographic film strips, not exposed, for the liquid me-
dia, but this te chnique has a drawback that sometimes
photosensitive particles included in the film are toxic to
the microorganisms, and then they give false - negative
results.
References
APHA/AWWA (1995) Standard Methods for the Exami-
nation of Water and Wastewater. 19th. Ed. APHA Inc.
New York. 596-597.
ASM (1981) Manual of Methods for General Bacteriol-
ogy, ASM, Washington, D.C.
120
Nutrient Gelatin DEV
Ref. 03-453
Specification
Culture medium for the enumeration of total bacteria
in not very polluted waters, according to the German
legislation.
Formula (in g/L)
Gelatin .................................................. 120,00
Meat extract ............................................ 10,00
Meat peptone ......................................... 10,00
Sodium chloride ........................................ 5,00
Final pH 7,3 ± 0,2
Directions
Suspend 145 g of powder in 1 L of cold distilled water
and heat up, in boiling water bath, to 50-60°C. Maintain
this temperature until total dissolution. Distribute into
containers and sterilize in the autoclave at 121°C for 15
minutes.
Description
This medium has the same applications as the Gelatin
Nutrient (Ref. 03-088) recommended by the APHA,
AWWA and Standard Methods. The only difference is the
concentration of nutrients and the inclusion of sodium
chloride.
Technique
German legislation states that water samples are inocu-
lated by deep inoculum (in mass) in Petri plates. Incuba-
tion is performed at 20±2°C for 44±4 hours. After the
incubation, carry out the counting of total bacteria.
If water is chlorinated, incubation must be 24 hours more
in order to let damaged cells recover and form visible
colonies.
References
DEUTSCHE EINHEITSVERFAHREN zur Wasser-,
Abwasser- Und Schlammuntersuchung. VCH Verlags-
gesellschaft D-6940 Weinheim.
 Oxidation-Fermentation Fluid Medium (O/F Medium)
Ref. 03-037
Specification
Fluid medium according to the Hugh and Leifson formu-
lation, for determining the oxidative and/or fermentative
metabolism of gram-negative bacilli.
Formula (in g/L)
Casein peptone ........................................ 2,00
Sodium chloride ........................................ 5,00
Dipotassium phosphate ............................ 0,20
Bromothymol Blue .................................... 0,08
Agar .......................................................... 2,50
Final pH 7,1 ± 0,2
Directions
Suspend 9,8 g of powder in 1 L of distilled water and
bring to the boil. Add sugar in the desired concentration
and distribute in fermentation tubes. Add the vaseline
seals or vaspar to half of them. Sterilize by autoclaving
at 121°C for 15 min.
Description
Hugh and Leifson obtained a clear differentiation of
gram-negative bacteria with this medium. They classi-
fied them into three categories: fermentative, oxidative
and inactive. The strain to be studied is inoculated in two
long narrow tubes (12x120 mm) by deep stab and one
is covered with a vaseline layer to induce an anaerobic
environment that forces the strain to carry out the fer-
mentative metabolism.
Fermentative organisms give a copious production of
acid in both the tubes, and it is indicated by the yel-
P Medium Agar
Ref. 01-500
Specification
Solid culture medium used as seed agar in the inhibitory
substances test in milk.
Formula (in g/L)
Meat extract .............................................. 3,00
Peptone .................................................... 5,00
Soy peptone ............................................. 0,30
Dextrose ................................................... 5,25
Polysorbate 80 ......................................... 1,00
Sodium chloride ........................................ 0,50
Dipotassium phosphate ............................ 0,25
Bromcresol purple .................................... 0,06
Agar ........................................................ 15,00
Final pH 7,8 ± 0,2
low colouration of the bromothymol blue indicator. The
bacteria following the oxidative metabolism carry out this
reaction only in the tube without the vaseline but in the
other one, which is closed, they change insignificantlly
or simply do not grow. Inactive strains do not use sugars
and therefore do not induce any change in both the
tubes. However, some times there is a slight blue col-
ouration in the open tube, probably due to alkalinization
by peptone degradation.
Some authors have proposed the usage of just one
tube for this assay, but in that case the medium must be
modified to be the solid (with 1,5% Agar) and with yeast
and/or cystine extract. In these tubes the stab must be,
at least, 8 cm.
Hugh and Leifson recommend simultaneous assay with
glucose, lactose and sucrose of 1% concentration, add-
ing the sterilized sugars to the medium by filtration.
References
HUGH, R. and E. LEIFSON, (1953) The taxonomic
significance of fermentative vs. oxidative metbolism of
cabohidrates by various gram negative bacteria J.Bact.
66:24
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological media. CRC Press, Inc.London
FDA (1998) bacteriological Analitycal Manual. 8th ed.
Rev. A. AOAC International. Gaithersburg. MD
DOWNES, F.P. & K. ITO (2001) Compendium of meth-
ods for the microbiological examination of food. 4th ed.
APHA Washington
ISENBERG, H.D. (1992) Clinical Microbiology Proce-
dures Handbook Vol I ASM Press. Washington
Directions
Suspend 30,36 g of powder in 1 litre of distilled water
and bring to the boil. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes.
Description
The present formulation is according the Food and Drug
Administration Bacteriological Analytical Manual for the
investigation of Inhibitory substances (Antibiotics and
preservatives) in milk. This medium that in previous edi-
tions of the BAM was called “PM Indicator Agar”, is used
as seed agar with B. stearothermophilus spores in the
qualitative method or disk assay.
References
MATURIN, L.J. (1998) Inhibitory substances in milk.
Qualitative Method II: B. stearothermophilus disk assay.
In FDA Bacteriological Analytical Manual. 8th Ed. Revi-
sion A. AOAC International Inc. Gaithersburg MD.
121
 Peptone Agar

Ref. 01-570
Specification
Solid culture medium used for the enumeration of
contaminants in dairy products, according the FIL-IDF
standard
Formula (in g/L)
Casein Peptone ........................................ 7,50
Gelatine Peptone ...................................... 7,50
Sodium chloride ........................................ 5,00
Agar ........................................................ 15,00
Final pH 7,5 ± 0,2
Directions
Suspend 35 g of powder in 1 L of distilled water and
bring to the boil. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes.
Description
Peptone Agar is produced according the formulation of
the FIL-IDF for its use in the detection and enumera-
tion of non-lactic contaminants in butter, fresh cheese
and fermented milk. All the components of the medium
are sugar-free and the pH is adjusted to 7,5 that’s is the
used for cheese and butter. If the sample is fermented
milk, the medium will be adjusted to 8.00 with 0,1N
NaOH sterile solution after the sterilization.
Technique
The inoculum or its dilution (in duplicate) is deposited
over the surface of the medium in the plate in volumes
of 0,1 mL. The inocula are spreaded quickly with a
Drigalski rod (Ref. 5-010) and let stand 15 minutes to
be absorbed in the medium. The plates are incubated
at 30ºC for 72±2 hours. Select plates with less than 150
colonies to the count. In the counting the needle-bite
colonies are not considered because they are probably
lactic bacteria. The contaminant must be confirmed by
its active catalase.
For the sampling, processing and dilution of the products
refer to the corresponding FIL-IDF standard.
References
FIL-IDF (1991) Provisional Standard 13: Butter, fresh
cheese and fermented milk. Enumeration of non-lactic
contaminants. Plate Count at 30ºC technique.

Peptone Water Phosphate-Buffered

Ref. 02-568 References

BEKERS, H.J. (1987) Studies with salmonellae. J. Appl.
Specification Bact. 62:97-112
Liquid non-selective pre-enrichment medium for entero- SCHWEIZERISCHES LEBENSMITTELBUCH (1992) 5th
bacteria ed. Chapter 56A

Formula (in g/L)

Meat Peptone ......................................... 10,00
Sodium chloride ........................................ 5,00
Disodium phosphate ................................. 3,50
Monopotasium phosphate ....................... 1,50
Final pH 7,2 ± 0,2

Directions
Dissolve 20 g of powder in 1 L of distilled water, heating
if necessary. Distribute in suitable containers and steri-
lize in autoclave at 121ºC for 15 minutes.

Description
This medium is produced according the formulation of
the Schwezerisches Lebensmittelbuch and is recom-
mended as non-selective pre-enrichment for sub-lethally
damaged cells of the enterobacteria group in food or in
others samples.

122
 Phenol Red Broth Base
Ref. 02-032
Specification
Liquid culture media,suitable for the sugar and other
substrate fermentation studies according ISO 10273
standard.
Formula (in g/L)
Casein peptone .................................... 10,000
Sodium chloride ...................................... 5,000
Phenol red .............................................. 0,018
Final pH 6,8 ± 0,2
Directions
Dissolve 15 g of powder in 1 L of distilled water. Add
sugar in the desired concentration and distribute into
suitable containers with Durham’s tubes. Sterilize in
the autoclave at 121°C for 10 minutes. Heat up the
autoclave before putting the tubes into it to avoid sugar
caramelization. Addition of some kinds of sugars may
need a pH adjustment.
Description
Phenol Red Base Broth is a liquid version of the agar
base for the fermentation studies, which is preferred by
many authors to use with Durham’s tubes inclusion, to
verify the gas production.
Phenylalanine Agar (PPA)
Ref. 01-083
Specification
Culture medium for Enterobacteria, according Ewing et
al. formulation.
Formula (in g/L)
Yeast extract ............................................... 3,0
DL-Phenylalanine ....................................... 2,0
Di-sodium phosphate ................................. 1,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,3 ± 0,2
Directions
Suspend 26 g of powder in 1 L of distilled water and heat
to boiling. Dispense in tubes or flasks and sterilize in the
autoclave at 121°C for 15 minutes.
Description
This formulation corresponds to the solid form, proposed
by Ewing et al. which is a modification of the medium
developed by Buttiaux et al. in order to achieve the
green colour that confirms the positive reaction which
lasts longer.
Capacity to deaminate the phenylalanine oxidatively to
convert it in phenylpiruvic acid is property of the Proteus
type in enterobacteria. Phenylalanine is revealed by
Sugar addition can be done in sterile solution after autoclav-
ing the medium, or by adding impregnated discs to 10 mL of
medium. Addition of some sugars may cause the acidifica-
tion of the medium, in which case the original pH must be
maintained by adding a few drops of 0,1 N NaOH.
Should you be working with anaerobics, it is advisable to
use a freshly prepared medium, or put the medium in boiling
water bath for a few minutes, in order to eliminate dissolved
oxygen. Many authors recommend the addition of 0,04%
agar for these purposes to avoid convection streams and
subsequent incorporation of the air.
To study the sugar fermentations of enterobacteria, Brom-
cresol Purple Base Broth (Ref. 02-031) is more suitable, as
it is a better indicator of choice which is less toxic than the
phenol red.
References
ISO 10273 Standard (1994) General guidance for the detec-
tion of presumptive pathogenic Yersinia enterocolitica.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press,Boca Raton,Fla.
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 4th ed.
APHA. Washington.
FDA (1998) Bacteriological Analytical Manual 8th ed.
Rev. A. AOAC International. Gaithersburg. MD
the presence of a characteristic greenish colour in the
medium when it reacts with iron. Nowadays, this test and
the urease production test, have a lot of importance in
the taxonomy of Proteus type .
Technique
A recommended technique is the following:
Inoculate the slant surface with plenty of inoculum, and
incubate it for 12-16 hours. Add 0,2 mL of 10% ferric
chloride solution so that the solution floods all over the
growth.
Phenylpiruvic acid presence (positive test) is shown by
the presence of a characteristic green-blue colour on the
surface, after approximately 1 minute.
References
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc.London
BUTTIAUX,R., R. OSTEUX, R. FRESNOY & J. MO-
RIAMEZ (1954) Les propietés biochimiques du genre
Proteus.Ann.Inst. Pasteur 87:357-386
EDWARDS and EWING (1973). Identification of Entero-
bacteriaceae. Burges Pub.Cod. Minneapolis.
ISENBERG, H.D. (1992) Clinical Microbiology Proce-
dures Handbook. Vol I ASM Press Washington.
123
 Plate Count Media
Plate Count Agar (PCA)
Ref. 01-161
Specification
Medium for the aerobic plate count by surface inocula-
tion method (Standard Plate Count Agar) according ISO
4833 and 17410 standards.
Formula (in g/L)
Casein peptone .......................................... 5,0
Yeast extract ............................................... 2,5
Dextrose ..................................................... 1,0
Agar .......................................................... 15,0
Final pH 7,0 ± 0,2
Directions
Suspend 23,5 g of powder in 1 L of distilled water.
Dissolve by bringing to the boil with frequent stirring. Dis-
tribute into final containers and sterilize by autoclaving at
121°C for 15 minutes.
Description
The Plate Count Agar follows the directions given by
Buchbinder et al. in their study about media for the plate
count of microorganisms.
The original formulation of the standardized agar for
dairy microbiology has been modified in order to avoid
the addition of milk. This new composition allows the
growth of most microorganisms without any further ad-
ditions.
This medium’s formulation is equivalent to that pre-
scribed by the ‘Standard Methods for the Examination
of Dairy products’, the USP’s ‘Tryptone Glucose Yeast
Agar’, the ‘Deutsche Landswirtchaft’ and to the APHA
and AOAC’s Plate Count Agar. Nowadays this is the
medium selected for the plate count of any type of the
sample.
Plate Count Modified Agar
Ref. 01-329
Specification
Modification of Plate Count Agar (Ref. 01-161), with a
lesser amount of agar, especially recommended for the
aerobic enumeration in plates, by the poured plates
method.
Formula (in g/L)
Casein peptone .......................................... 5,0
Yeast extract ............................................... 2,5
Dextrose ..................................................... 1,0
Agar ............................................................ 9,0
Final pH 7,0 ± 0,2
124
Directions
Dissolve 17,5 g of powder in 1 L of distilled water. Heat
to the boiling by constant stirring. Distribute in the suit-
able containers and sterilize in the autoclave at 121°C
for 15 minutes.
Description
Plate Count Modified Agar follows the same specifica-
tions as Plate Count Agar, with the exception that of the
agar concentration has been reduced. This modification
provides a better growth of colonies if massive inocula-
tion method is used, as the medium is softer and hence
the colony expansion is improved.
Plate Count Skim Milk Agar
Ref. 01-412
Specification
Solid medium for the plate counts of milk and dairy prod-
ucts, according to DIN and FIL/IDF standards.
Formula (in g/L)
Casein peptone .......................................... 5,0
Yeast extract ............................................... 2,5
Skimmed milk ............................................. 1,0
Dextrose ..................................................... 1,0
Agar .......................................................... 10,5
Final pH 7,0 ± 0,2
Directions
Suspend 20 g of powder in 1 L of distilled water and let
it soak . Bring to the boil with constant stirring. Distribute
into suitable containers and sterilize in the autoclave at
121°C for 15 minutes.
Description
This medium, with the added milk, has a major nutri-
ent richness than other standard media, however, the
opalescence of the medium makes early observations
sometimes difficult.
Due to its lesser agar concentration, it may be used by
the pouring plate method or by the surface inoculation
method.
Technique
Prepare a decimal dilution bank of the sample and take
1 mL in duplicate from each dilution and put them in
sterile Petri plates. Pour 20 mL approx. of sterile cooled
medium (around 47°C) in each of the plates. Mix gently
by moving the plate in eight (8) shape. Leave the plates
undisturbed to solidify and incubate in inverted position.
Time and temperature of incubation depend on the type
of microorganism under study. For a general aerobic
count, incubate for 3 days at 30°C, by observing also
after 24 and 48 hours.
The plate count method proposed by the APHA consists
of a massive inoculum by pouring the molten agar at
50°C on plates containing the diluted samples. The final
 Plate Count Media
count is carried out after 48 hours of incubation at 32-
35°C.
As for the microorganisms with other temperature
requirements, the following incubations have been sug-
gested: 2 days at 32-35°C, 2-3 days at 45°C, 2 days at
55°C, 3-5 days at 20°C, 7-10 days at 5-7°C.
Sample dilutions are prepared with solutions of 1/4 of
Ringer solution (Ref. 06-073), 1% of Peptone Water(Ref.
03-156) or 0,1M of phosphate buffer at pH 7,0; depend-
ing on their nature.
The poured plate count method is preferred to the
surface inoculation method, since it gives higher results.
Nevertheless, the latter gives a more appropriate isola-
tion of the colonies.
References
MARSHALL, R.T. (1992) Standard Methods for the
Examination of Dairy Products, 16th Ed. APHA. Wash-
ington.
CLESCERI. L.S., A.E. GREENBERG, and A.D. EATON
(1998) Standard Methods for the Examination of Water
and Wastewater, 20th ed.. APHA, AWWA, WEF. Wash-
ington.
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 4th. Ed.
APHA. Washington.
HORWITZ, W. (2000) . Official Methods of Analysis.
AOAC International. Gaithersburg
DIN 10192 Standard. Prüfungesbestimmungen für Milch
und Milcherzeugnisse (Deutsche Landwirtsachft, Fach-
bereit und Ernahrung). 1971.
FIL/IDF Standards 3 (1958), 100 (1981), 101 (1981),
109 (1982) and 132 (2004).
PASCUAL ANDERSON, MªRª (1992) Microbiología
Alimentaria. Diaz de Santos, S.A.,Madrid,SPAIN.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc.,Boca Raton,Fla.
BUCHBINDER, L., Y. BARIS, L. GOLDSTEIN (1953)
Further studies on new milk-free media for the stand-
ard plate count of dairy products. Am. J. Public Health
43:869-872.
ISO 4833 Standard (2003) Microbiology of food and ani-
mal feeding stuffs. Horizontal method for the enumera-
tion of microorganisms. Colony count technique at 30°C.
ISO 17410 Standard (2001) Horizontal Method for the
enumeration of psychrotrophic microorganisms.
ISO 8552 Standard (2004) Milk - Estimation of psychro-
trophic microorganisms - Colony-count technique at
21°C (Rapid method).
125
 Potato Dextrose Media
Potato Dextrose Agar
Ref. 01-483
Specification
Solid culture medium for the detection and enumeration
of yeast and moulds in food, specially recommended in
butter and other dairy products.
Formula (in g/L)
Potato peptone ......................................... 4,00
Glucose .................................................. 20,00
Agar ........................................................ 15,00
Final pH 5,6 ± 0,2
Directions
Suspend 39 g of powder in 1 L of distilled water and
bring to the boil. Distribute into suitable containers and
sterilize in the autoclave at 121°C for 15 minutes. Do not
overheat.
Description
Potato Dextrose Agar is a weakly selective medium for
fungi due to its high sugar content and acidic pH. The
pigment production and aerial mycelium development
is enhanced by the potato peptone, specially in the
Fusarium, Aspergillus and Penicillium species.
The selectivity can be increased by adding antibacterial
antibiotics like chloramphenicol or tetracyclines, or by
simply decreasing the pH to an acidic level. At pH 3,5
the bacterial growth is almost totally inhibited without
significant effect on fungi. This acidification can be
obtained by the aseptic addition of an adequate amount
of organic acid to the medium after sterillization: 10-15
mL/L of a 10% sterile solution of tartaric or lactic acid is
usually sufficient.
After its acidification the medium should not be over-
heated or reheated since it can hydrolyze the agar and
hence there can be a loss in solidification property of the
medium.
Technique
Distribute the diluted samples into sterile petri plates.
Pour the molten agar melted cooled to 45-50°C and gen-
tly mix to homogenize the mixture. After the solidification,
plates are incubated for 5-7 days at 20-25°C to permit
the complete development of the fungal colonies.
The weak consistency of the agar due to its original
acidity makes this medium inadequate for streaking.
126
Potato Dextrose Broth
Ref. 02-483
Specification
Liquid culture medium for the maintenance and multipli-
cation of yeast.
Formula (in g/L)
Potato peptone ........................................... 4,0
Glucose .................................................... 20,0
Final pH 5,6 ± 0,2
Directions
Dissolve 24 g of powder in 1 L of distilled water, heating
up only if necessary. Distribute into suitable containers
and sterilize by autoclaving at 121ºC for 15 minutes.
Description
Potato Dextrose Broth is the liquid version of the agar
with the same name. This broth is mainly used to detect
and enumerate yeast and moulds, since it does not
contain any solidifying agent it may be acidified without
altering its physical properties.
At pH 3,5, the bacterial growth is totally inhibited without
significant influence on fungi. This acidification may be
achieved by the aseptic addition of an adequate amount
of organic acid to the medium after sterilization:10-15
mL/L of a 10% sterile solution of tartaric or lactic acid.
This addition may also be made before sterilization, but
it must be considered that in acidic conditions Maillard
reactions are strong and hence the medium may turn
slightly brownish.
References
ATLAS, R.M. & PARKS,L.C. (1995) Handbook of
Microbiological Media for the Examination of Food. CRC
Press, London.
RICHARDSON, G. H. (1985) Standard Methods for the
examination of dairy products.15th Ed. APHA Washing-
ton.
DOWNES, F.P. & K. ITO (2001) Compendium of meth-
ods for the microbiological examination of food. 4thEd.
APHA Washington
US PHARMACOPOEIA (2002) 25th ed. <61> Microbial
Limit Test. US Pharmacopoeial Convention Inc. Ltd.
Rockville. MD
FDA (1998) Bacteriological Analitycal Manual. 8th ed.
Rev. A. AOAC International. Gaithersburg. MD.
 R2A Agar (Eur. Phar. Medium S)

Ref. 01-540 Technique

The water sample must be processed as quickly as
Specification possible. If it is no possible within the first 6 hours, the
Solid medium for the enumeration of heterotrophic micro sample must be refrigerate, but not for more than 30
organisms in treated waters hours: then the sample is rejected.
R2A Agar is used with pour plates, streak plates or
filtration but must be keep in mind that the pour plates
Formula (in g/L)
method can affect the recovery capacity of the medium
Yeast Extract .......................................... 0,500
because the thermal shock. The incubation period at
Proteose peptone ................................... 0,500
35ºC is of 3-5 days but is more effective a incubation
Casein hidrolysate .................................. 0,500
temperature of 20-28ºC an a time of 5-7 days. In any
Glucose .................................................. 0,500
case the plates must be protected against an excessive
Starch ..................................................... 0,500
drying.
Dipotassium hydrogen phosphate ......... 0,300
The fast-growing or non-stressed microorganisms in
Magnesium sulphate, anhydrous .......... 0,024
these conditions of incubation produce different and
Sodium pyruvate ................................... 0,300
minute colonies than in the rich media.
Agar ...................................................... 15,000
Final pH 7,2 ± 0,2
References
ATLAS, R.M. (1995) Handbook of Media for Environ-
Directions
mental Microbiology. CRC Press. Boca Raton USA.
Suspend 18,1 g of powder in 1 L of distilled water and
EATON, A.D., A.E. GREENBERG and L.S. CLESCERI
bring to the boil with constant stirring. Distribute into suit-
(1995). Standard Methods for the Examination of Water
able containers and sterilize by autoclaving at 121ºC for
and Wastewater. 19ª Ed. APHA Washington D.C. USA .
15 minutes.
EUROPEAN PHARMACOPOEIA 4th Ed. Suppl. 4.6
(2004) 2.6.13 Test for specified Microorganisms (pg
Description 2621)
The R2A Agar was proposed in 1979 by Reasoner and GREENBERG, A.E., R.R TRUSSELL and L.S.
Geldenreich and few years later accepted by the APHA CLESCERI (1985). Standard Methods for the Examina-
as an alternative medium for stressed cells in treated tion of Water and Wastewater. 16ª Ed. APHA-AWWA-
potable water. WPCF Washington D.C. USA
The use of nutrient rich media like PCA or TSA allows to REASONER, D.J. and E.E. GELDREICH (1979) A new
the growth of normal microbiota, but do not permits the medium for the enumeration and subculture of bacteria
recuperation of the stressed or chlorine resistant biota. from potable water. Abstracts of Annual Meeting . ASM
By the use of a medium like R2A of low nutrients in com- 79th Meeting. Paper #N7.
bination with a lower temperature and longer incubation Van SOETSBERGER, A.A. and C.H. LEE (1969) Pour
time it is possible induce the resuscitation of this dam- plates or streak plates? Appl. Microbiol 18:1092-1094.
aged cells.
In the R2A Agar the source of nitrogen is the peptone
and the Yeast Extract supplies the vitamins and growth
factors. The source of carbon is the dextrose and mag-
nesium sulphate and potassium phosphate maintains
the osmotic pressure. The starch is a detoxifier and
sodium piruvate increases the recuperations of stressed
cells. The agar acts as gelling agent.

127
 Rappaport Vassiliadis Media
Rappaport Vassiliadis Broth
Ref. 02-379
Specification
Liquid medium for the selective enrichment of Salmo-
nella in foodstuffs and other materials.
Formula (in g/L)
Soy peptone ........................................... 4,500
Sodium chloride ...................................... 7,200
Monopotassium phosphate .................... 1,260
Dipotassium phosphate .......................... 0,180
Magnesium chloride ............................ 13,580
Malachite green ...................................... 0,036
Final pH 5,2 ± 0,2
Directions
Dissolve 26,8 g of powder in 1 L of distilled water, heat-
ing if necessary to help dissolve the powder. Dispense
into test tubes or flasks and sterilize by autoclaving at
121°C for 15 minutes.
Description
The Rappaport Vassiliadis medium complies with the
recommendations of the APHA for the examination of
food.
This culture medium is the modificaction of the R10 me-
dium (from Rappaport et cols) or RV broth (from Vas-
siliadis et cols.)by van Schothort & Renaud. The modi-
fications are an adjustement in the magnesium chloride
concentration and a buffered reaction of the medium.
It shows a higher selectivity towards Salmonella and
produces better yields than other similar media, espe-
cially after preliminary enrichment and at an incubation
temperature of 43°C.
Malachite green and magnesium chloride inhibit the
growth of the microorganisms normally found in the in-
testine but do not affect the proliferation of most Salmo-
nellae. Malachite green inhibits the growth of Shigella.
Soy peptone improve the growth of Salmonella. The low
pH of the medium increases the selectivity.
Technique
Inoculate the culture medium with the sample or mate-
rial from a pre-enriched culture in Buffered Peptone
Water (Ref. 02-277) and incubate for up to 18-24 hours
at 41±1°C. Streak the sample material from the resulting
cultures onto selective culture media.
References
ATLAS, R.M., LC. PARKS (1993) Handbook of Microbio-
logical Media. CRC Press Inc.,London
VASSILIADIS,P (1983) The Rappaport-Vassiliadis(RV)
enrichment medium for the isolation of salmonellas: An
overview. J.Appl.Bact.54;54, 69-76.
128
VASSILIADIS, P, PATERAKI, EPAPAICONOMOU,N
, PAPADAKIS, J.A. A. TRICHOPOULOS, D (1976)
Nouveau procédé d’enrichissement de Salmonella. Ann.
Microbiol. (inst. Pasteur) 127B (195-200)
RAPPAPORT, F. N. KONFORTI & B. NAVON (1956) A
new enrichment medium for certain salmonellae. J. Clin
Pathol. 9:261-266
VAN SCHOTHORST M. & A.M. RENAUD (1983)
Dynamics of salmonellae isolation with modified Rappa-
port’s medium (R10) J.Appl. Bact. 54: 209-215
FIL-IDF Standard 93B:1995. Milk and Milk products.
Detection of Salmonella. Brussels
FDA (1998) Bacteriological Analytical Manual 8th ed.
Rev A. AOAC International. Gaithersburg. MD.
HORWITZ, W. (2000) Official Methods of Analysis.
AOAC International. Gaithersburg. MD
DOWNES, F.P. & K. ITO (2001) Compendium of meth-
ods for the microbiological examination of foods. 4th ed.
APHA Washington.
Rappaport Vassiliadis Modified
Semi-Solid Medium Base (MSRV)
Ref. 03-376
Specification
Semi-solid medium for the isolation of mobile strains of
Salmonella.
Formula (in g/L)
Tryptose ................................................. 4,590
Casein Peptone ...................................... 4,590
Sodium chloride ...................................... 7,340
Mono-Potassium phosphate .................. 1,470
Magnesium chloride ............................. 10,930
Malachite green ...................................... 0,037
Agar ........................................................ 2,700
Final pH 5,2 ± 0,2
Directions
Suspend 31,6 g of powder in 1 L of distilled water. Heath
in a water bath until boil and complete dissolution. Cool
to 50ºC an add 20 mg/L of Novobiocin. Without autoclav-
ing nor reheating, homogenize and pour plates. Keep
plates in a fresh place to settle the gel (1 hour minimum)
and handle it with care because the medium is only
semi-solid. It is recommended to keep MSRV plates in a
cooler at 2-8ºC at the dark.
Description
The Modified Semi-Solid Rappaport-Vassiliadis Medium
Base is formulated according DeSmedt and cols. That
shows its higher efficiency over the traditional enrich-
ment methodology.
 Rappaport Vassiliadis Media
The rapid migration of mobile strains of Salmonella in
the semisolid medium allows to the early detection by
the production of an halo of growth around the inocula-
tion zone.
The other competitive mobile organisms are inhibited
by the novobiocin, the malachite green and the high
concentration of magnesium chloride.
The low concentration of agar produces a very soft and
fragile gel but, at the temperature of incubation (42ºC), it
is an special environment in which the mobile strains of
Salmonella moves easy and quickly.
Technique
1. Three drops (~0,1 mL) of a pre-enrichment culture
are inoculated in a three different spots on the dry
surface of the medium in a room-temperate plate.
2. Incubate the plates aerobically in an upright position
for no longer than 24 hours at 42ºC.
3. The formation of a turbid or opaque halo around the
initial inoculation zone shows the presence of mobile
salmonellae.
4. To confirm the purity of the isolation and to follows
with the identification tests, samples of the external
border of the halo can be used.
Reinforced Clostridial Media
Reinforced Clostridial Agar
Ref. 01-289
Specification
Solid medium for the cultivation and enumeration of
clostridia and other anaerobic bacteria.
Formula (in g/L)
Casein peptone ........................................ 10,0
Yeast extract ............................................... 3,0
Meat extract .............................................. 10,0
Dextrose ..................................................... 5,0
Sodium chloride .......................................... 5,0
Sodium acetate .......................................... 3,0
Soluble starch ............................................. 1,0
L-Cysteine HCl ........................................... 0,5
Agar .......................................................... 15,0
Final pH 6,8 ± 0,2
Directions
Suspend 52,5 g of powder in 1 L of distilled water and
heat to boiling with constant stirring. Distribute into suit-
able containers and sterilize in the autoclave at 121°C
for 15 minutes.
5. To prevent false negatives results due to the ab-
sence of mobile strains of Salmonella is convenient
to performs simultaneously a traditional enrichment
in liquid medium.
References
De SMEDT, J.M., R. BOLDERDJIK, H. RAPPOLD and
D. LAUTENSCHLAEGER (1986). Rapid Salmonella
detection in foods by motility enrichment on a Modi-
fied Semi-Solid Rappaport-Vassiliadis Medium. J. Food
Protect. 49:510-514
De SMEDT, J.M. and R. BOLDERJIK (1987) Dynamics
of Salmonella isolation with Modified Semi-Solid Rappa-
port-Vassiliadis Medium. J. Food Protect. 50:658-661
HOLBROOCK, R., J.M. ANDERSON, A.C. BAIRD-
PARKER, L.M. DODDS, D. SAWHNEY , S.H. STRUCH-
BURY and D. SWAINE (1989) Rapid detection of Salmo-
nella in food: A convenient two-day procedure. Lett. Appl.
Microbiol. 8:139-142
Reinforced Clostridial Medium
(RCM) (Eur. Phar. Medium P)
Ref. 03-289
Specification
Fluid medium for the cultivation and enumeration of
clostridia by the MPN method.
Formula (in g/L)
Casein peptone ........................................ 10,0
Yeast extract ............................................... 3,0
Meat extract .............................................. 10,0
Dextrose ..................................................... 5,0
Sodium chloride .......................................... 5,0
Sodium acetate .......................................... 3,0
Soluble starch ............................................. 1,0
L-Cysteine HCl ........................................... 0,5
Agar ............................................................ 0,5
Final pH 6,8 ± 0,2
Directions
Suspend 38 g of powder in 1 L of distilled water and heat
to boiling with constant stirring. Distribute into suitable
containers and sterilize in the autoclave at 121°C for 15
minutes.
129
 Reinforced Clostridial Media

Description
Reinforced Clostridial Agar was originally described by
Hirsch and Grinstead to initiate the growth of small in-
oculums and get a higher Clostridial count. Later, Barnes
and Ingram used the medium to develop vegetative cells
in assays of Clostridium perfringens. Barnes also used
this medium to count clostridia in food, moreover other
authors used this medium in enumeration assays of Cl.
thermoscharolyticum in sugar, study of intestinal flora,
and bacterial count in human or animal faeces, etc.
For the enumeration by the MPN method, the liquid ver-
sion is the preferred one.
Technique
Material to be examined is ground in a Turmix or Stom-
acher, and a decimal dilution bank is prepared. From
each of the dilutions, take an aliquote to Petri plates or
tubes, and pour the molten medium at 50°C over them.
Let it solidify. Incubate at 30-55°C (depending on the
microorganism that is anticipated to be found) for 1-10
days. An anaerobic environment can be achieved if
tubes are used and they are covered with Sealing Anaer-
obic Agar (Ref. 01-174) immediately after the Reinforced
Clostridial Medium is solidified. If the plates are used,
they have to be incubated in the anaerobic jars.
Muñoa and Parés added a filter sterilized solution of
Nalidixic acid 0,02 g/L, Polymyxin 0,025 g/L, Kanamy-
cin sulfate 0,05 g/L, Sodium iodoacetate 0,025 g/L and
triphenyl-tetrazolium HCl 0,025 g/L to obtain a selective
and differential medium for bifidobacteria in water and
wastewater.
References
ATLAS, R.M., LC. PARKS (1993) Handbook of Microbio-
logical Media. CRC Press, Inc.,Boca Raton,Fla.
INGRAM, M. and BARNES, E.M. (1956) A Simple Modi-
fication of the Deep Shake Tube for Counting Anaerobic
Bacteria. Lab. Practise 5, 4:145.
HIRSCH, A. and GRINSTEAD, E. (1954) Methods for
the Growth and Enumeration of Anaerobic Sporeformers
from Cheese, with Observations on the Effect of Nisin.
J.Dairy Res. 21:101.
MUÑOA, F.J., R. PARÉS (1988) Selective medium for
isolation and enumeration of Bifidobacterium spp. Appl.
Environm. Microgiol 54:1715-1718.
EUROPEAN PHARMACOPOEIA,(2002) 4th ed. Suplle-
ment 4.2 Chap. 2.6.13.Test for specified micro-organ-
isms. Council of Europe. Strasbourg.

Rinse Fluid K

Ref. 03-109 Technique

Specification
Medium for rinsing the membrane filters according to the
USP and European Pharmacopoeia specifications.
Formula (in g/L)
Meat peptone ............................................. 5,0
Meat extract ................................................ 3,0
Final pH 6,9 ± 0,2
Directions
Dissolve 8 g of powder in 1 L of distilled water with 10
mL of Polysorbate 80 (Ref. 06-088). Distribute into suit-
able containers and sterilize in the autoclave at 121°C
for 15 minutes.
After the filtration, wash the membrane by passing 3
volumes of 100 mL of solution through it.
When the sample has a high concentration of fats or
sugars it is recommended to double the concentration of
polysorbate
(2 mL/L).
References
US PHARMACOPOEIA (2002) 25th ed.<71> Sterility
Tests. US Pharmacopoeial Convention Inc. Rockville.
MD
European Pharmacopoeia. (2002) 4th Ed. V.2.18 Control
of microbial contamination in no sterile products

Description
This nutrient solution which is formulated according to
the USP (Rinsing Fluid K) and European Pharmacopoe-
ial specifications, removes all the fat and carobohydrate
residues, due to the surfactant effect of Polysorbate,
and at the same time, it avoids the osmotic shock to the
microorganisms.

130
 Rogosa Agar Base
Ref. 01-300
Specification
Selective solid medium for the isolation and enumeration
of lactobacilli.
Formula (in g/L)
Glucose ................................................ 20,000
Tryptone ............................................... 10,000
Sodium acetate .................................... 10,000
Monopotasium phosphate ...................... 6,000
Yeast extract ........................................... 5,000
Ammonium citrate .................................. 2,000
Sorbitan monooleate .............................. 1,000
Magnesium sulphate .............................. 0,575
Manganese sulphate .............................. 0,120
Ferrous sulphate .................................... 0,034
Agar ...................................................... 15,000
Final pH 5,5 ± 0,2
Directions
Suspend 69,7 g of powder in 1 L of distilled water and
add 1,32 mL of glacial acetic acid and the complemen-
tary amount of sodium acetate to fit standard. Heat with
gently stirring until boiling. Pour plates without autoclav-
ing nor overheating.
Description
Rogosa Agar was developed in 1951 for the isolation
and enumeration of oral and faecal lactobacilli, but with
changes in the acetate concentration it was used for
several kinds of samples, including foods.
The low pH and high acetate concentration, that can be
adjusted to the sample confers to this medium a high
selectivity for the lactobacilli. Nevertheless, it is no suit-
able for the dairy samples, for which is recommended
the addition of 20% sterile tomato serum (Ref. 06-092)
or using the MRS Agar (Ref. 01-135) that offers a best
performance with the dairy lactobacilli.
Rogosa Agar, due to its high acidity, is not suitable for
the maintenance of the microorganisms.
References
ROGOSA, M., J.A. MITCHELL & R.F. WISEMAN (1951)
A selective medium for the isolation and enumeration of
oral and faecal lactobacilli. J. Bacteriol. 62:132.
ROGOSA, M., J.A. MITCHELL & R.F. WISEMAN (1951)
A selective medium for the isolation and enumeration of
oral and faecal lactobacilli. J. Dental Res. 30:682
DOWNES, F.P. & K. ITO (Eds) (1991) Compendium of
methods for the microbial examination of foods. 4th ed.
APHA. Washington D.C.
MACFADDIN J.D. (1985) Media for isolation-cultivation-
identification-maintenance of medical bacteria. William &
Wilkins, Baltimore. MD.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press. London.
131
 Rose Bengal Agar

Ref. 01-301 T Technique

Specification
Solid and selective medium for the isolation of yeast and
moulds from the environment and food products.
Formula (in g/L)
Peptone .................................................... 5,00
Dextrose ................................................. 10,00
Potassium phosphate ............................... 1,00
Magnesium sulfate ................................... 0,50
Rose bengal ............................................. 0,05
Chloramphenicol ...................................... 0,10
Agar ........................................................ 15,00
Final pH 7,2 ± 0,2
Directions
Suspend 32 g of powder in 1 L of distilled water and heat
to boiling with constant stirring. Distribute in suitable
containers and sterilize in the autoclave at 121°C for 15
minutes.
R-45
After making a dilution bank, take 0,1 mL from each dilu-
S-53-45 tion and inoculate with a Drigalsky Loop (Ref. 5-010) or
glass spreader on Rose Bengal Agar plates. Should the
massive seed method be preferred, take 1 mL from each
dilution and put it in an empty plate. Pour the molten
medium at 50°C and homogenize it by gently moving the
plate in an eight (8) shape. Incubate at 22°C for 5 days
and proceed to enumerate the fungi.
References
ATLAS, R.M., & PARKS, L.C. (1993) Handbook of Micro-
biological Media. CRC Press, Inc.,Boca Raton,Fla.
DOWNES F.P. & K. ITO (2001) Compendium of Methods
for the Microbiological Examination of Food. 4th Ed.
APHA. Washington.
MARSHALL, R.T. (1993) Standard Methods for the ex-
amination of dairy products. 16th ed. APHA Washington.
APHA-AWWA-WEF (1998) Standard Methods for the
examination of water and wastewater. 20th. ed. APHA
Washington.

Description
Rose Bengal Agar is a selective medium to detect
and enumerate moulds and yeast in food samples.
Apart from the nutritional requirements for moulds and
yeast,this medium also contains Rose Bengal, which
apart from tainting the yeast with a pink colour, also fa-
cilitates their count, avoiding massive growth of moulds
such as Rhizopus and Neurospora, therefore it is easier
to detect other moulds with slower growth.
Chloramphenicol and also the Rose bengal, restrains
bacterial growth, but does not interfere with fungi growth.

Yeast and moulds colonies after 5 days at 22ºC

132
 Sabouraud Media
Sabouraud Chloramphenicol
Agar
T new formula helps to maintain the morphological aspects
Ref. 01-166
R-45
S-53-45
Specification
Solid culture medium for the isolation of fungi.
Formula (in g/L)
Casein peptone .......................................... 5,0
Meat peptone ............................................. 5,0
D(+) Glucose ............................................ 40,0
Chloramphenicol ........................................ 0,5
Agar .......................................................... 15,0
Final pH 5,6 ± 0,2
Directions
Suspend 65,5 g of powder in 1 L of distilled water and
bring to the boil. Distribute into final containers and
sterilize by autoclaving at 121°C for 15 minutes. Do not
overheat or reheat the medium since it will affect the
solidification.
Description
This culture medium differs from the classical Sabour-
aud Agar only in the addition of Chloramphenicol. This
thermostable antibiotic has a wide antibacterial spectrum
which ensures the selective isolation of fungi from highly
contaminated samples, such as eudates, faeces, nails
and hair.
Sabouraud Dextrose Agar
(Eur. Phar. Agar Medium C)
Ref. 01-165
Specification
Medium for the enumeration and cultivation of fungi.
Formula (in g/L)
D(+) Glucose ............................................ 40,0
Casein peptone .......................................... 5,0
Meat peptone ............................................. 5,0
Agar .......................................................... 15,0
Final pH 5,6 ± 0,2
Directions
Dissolve 65 g in 1 L of distilled water and bring to the
boil with frequent stirring. Distribute into final containers
and sterilize by autoclaving at 121°C for 15 minutes. Do
not overheat the medium as its acidic pH may partially
hydrolize the agar. Alternatively,if the European Pharma-
copoeia formulation is desired, add before sterilization
50 mg/L of chloranphenicol (Ref. 06-118CASE)
Description
Sabouraud Dextrose Agar is a modification of the clas-
sical Sabouraud medium for the cultivation of fungi. This
of fungi and thus permits a reliable cultivation and dif-
ferentiation.
Its selectivity is due to a low pH and a high glucose con-
centration, which together with incubation at a relatively
lower temperature (25-30°C) favours the growth of fungi
while discouraging that of bacteria. Besides, the com-
position of this peptone has been studied to provide the
fungi with all their nitrogenated nutrient requirements.
Since the Sabouraud medium’s strong acid reaction
partially hydrolyzes the agar, only the required amount
should be prepared and it should not be remelted. Any
overheating will considerably diminish its gelling capac-
ity.
Should a higher selectivity be required, a variety of in-
hibitors or selective agents may be added after steriliza-
tion, while the medium is still in the molten form. It can
even be made differential by adding the indicator agents.
Some of the inhibitory and differential mixtures most
commonly used are listed below:
Penicillin: at 20,000 units/litre, encourages the selectivity
of the medium by inhibiting most of the bacteria.
Penicillin and Streptomycin: at 20,000 u/L and 40,000
u/l each, favours the isolation of Histoplasma in
dogs.
Penicillin and Neomycin: at 20,000 u/L and 40 mg/L
each, is used for the isolation of yeast.
Streptomycin and Chloramphenicol: at 40 mg/L and 500
mg/L each, for the isolation of Trichophyton ver-
rucosum .
Colistin, Novobiocin and Cycloheximide: at 8 mg/L, 0.1
mg/L and 30 mg/l each, for the isolation of Can-
dida albicans .
Potassium Tellurite: at 150 mg/L, is used for the primary
isolation of fungi from scales and scabs.
Cupric Sulfate, Crystal Violet and Brilliant Green: at 500
mg, 2 mg and 5 mg each, achieves considerable
bacterial inhibition.
Triphenyltetrazolium chloride (TTC): at 100 mg/L, it is the
basis of a Pagano-Levin medium for the isola-
tion of Candida albicans, unpigmented, among
other pathogenic yeast which form pink coloured
colonies.
Sabouraud Broth
Ref. 02-165
Specification
Liquid medium for the sterility control.
133
 Sabouraud Media

Formula (in g/L)

Casein Peptone .......................................... 5,0 Sabouraud Oxytetracycline
Meat Peptone ............................................. 5,0
D (+) Glucose ........................................... 20,0
Agar Base (OGYEA)
Final pH 5,8 ± 0,2
Ref. 01-275
Directions
Dissolve 30 g of powder in 1 L of distilled water, heating Specification
up only if necessary. Dispense into suitable containers Solid culture medium for the total enumeration of moulds
and sterilize in preheated autoclave for 15 minutes at and yeasts.
121°C. Avoid overheating, since it may caramelize the
glucose. Formula (in g/L)
Glucose .................................................... 20,0
Description Yeast extract ............................................... 5,0
This medium is especially adapted to fungi and acido- Agar .......................................................... 20,0
philic bacteria culture. Final pH 7,0 ± 0,2
Sabouraud USP Broth is according to the formulations of
US Pharmacopeia, US NF and 21 CFR guidelines. In the Directions
latest editions of these books it is also mentioned and Suspend 45 g of powder in 1 L of distilled water and let
allowed to use Tryptone and Soy Broth for the sterility it soak for a few minutes. Distribute into suitable contain-
checking in parenteral pharmaceutical products. This for- ers and sterilize by autoclaving for 10 minutes at 115ºC.
mulation is similar to the Antibiotic Medium 13 by Grove Cool to 50°C and then add oxytetracycline (Ref. 06-
and Randall and 21 CFR. 115CASE) to reach a 0,1 mg/mL concentration.
This medium is not a selective one, but the strong acidic
pH notably inhibits the growth of non acidophilic microor- Description
ganisms. Nonetheless, special measures must be taken This formulation differs with others as it has no peptone
while reconstituting and heating the medium due to this and has a neutral reaction or pH. Unlike the others, it
strong acid reaction and the high content of glucose. It is has a high oxytetracycline concentration that makes it
important to preheat the autoclave and thereby reach the almost impossible for the growth of bacteria.
sterilization temperature as soon as possible and in a
regular way, since otherwise, glucose becomes caramel-
Technique
ized turning the medium dark and effectiveless.
Some authors suggests an inoculum of 1 mL in each
dilution, in duplicate and in mass. Perform an incubation
Technique at 22-25°C for 5 days with the intermittent observations
It has been recommended to use this medium in many or readings after 3 days of incubation.
tests and assays, but for a long time it has been the me-
dium of choice for the verification of sterility of the sterile
References
pharmaceutical products.
AJELLO, L.(1957) Cultural Methods for Human Patho-
Efficacy of the medium and absence of fungistatic prod-
genic Fungi J. Chron. Dis. 5:545-551.
ucts is verified by checking if there is a growth from a
PAGANO, J., LEVIN,J.D. and TREJO, W.(1957-58)
loop of inoculum of Candida albicans, from a 1:1000 di-
Diagnostic Medium for Differentiation of Species of Can-
lution of a fresh 24 hours grown culture. Sterility assay or
dida. Antibiotics Annual, 137-143.
test is carried out in controlled and verified medium. To
SABOURAUD, R.(1910) Les Tignes. Masson, Paris.
check the fungistatic activity of any product, prepare an
HANTSCHKE, D.(1968) Mykosen, 11:769-778.
inoculum as mentioned above and inoculate two series
EUROPEAN PHARMACOPOEIA (2002), 2.6.13 Tests
of tubes with the same medium as follows:
for specified micro-organisms Supplement 4.2, 4th Ed.,
a) Add to one batch the specified amount of product.
EDQM. Council of Europe, Strasbourg.
This is the test series.
US PHARMACOPOEIA (2002) 25th Ed. <51>Antimicro-
b) Add to the another batch only the inoculum and
bial efectiveness Testing; <61> Microbial Limit Tests. US
simultaneously incubate with the test series. This is
Pharmacopoeial Convention Inc. Rockville. MD
the control series.
ISO 13681 Standard (1995) Enumeration of yeasts and
Incubation of both the series must be carried out at 22°C
moulds - Colony count technique.
for 10 days. After this period compare the results or
observations of both the series. If the assay series has a
lesser growth than the control one, product has the fun-
gistatic activity. If the growth is equal or more, then it has
not any fungistatic properties. For the quantitative assay
of the fungistatic activity, perform the assay with several
series of different concentrations (one lower than the
previous) until reaching an equal growth in both control
and test series.

134
 Salmonella Shigella Agar (SS Agar)

Ref. 01-171 medium, such as Brilliant Green Agar (Ref. 01-203) or

MacConkey Agar (Ref. 01-118).
Specification Incubate the inoculated plates at 37°C for 18-24 hours.
Solid and highly selective medium for the isolation of The suspicious colonies should then be subcultured
Salmonella and some Shigella species. on differential media to be identified biochemically or
serologically.
Appearance of the colonies after 24 hours on SS Agar:
Formula (in g/L) Shigella: Colourless, transparent and flat.
Meat extract ........................................ 5,00000
Salmonella (Non H2S producers): Colourless, transpar-
Peptone .............................................. 5,00000
ent and flat.
Lactose ............................................. 10,00000
Salmonella (H2S producers): Black or black centered,
Bile salts ............................................. 8,50000
flat, with transparent borders.
Sodium citrate .................................. 10,00000
Proteus: Similar appearance as Salmonella colonies, but
Sodium thiosulfate .............................. 8,50000
smaller in size.
Ferric citrate........................................ 1,00000
Escherichia coli: If they grow, they are small, convex and
Brilliant green ..................................... 0,00033
pink or red coloured.
Neutral red .......................................... 0,02500
Coliforms (in general): Big, opaque, smooth and col-
Agar .................................................. 15,00000
oured in white or pink shade.
Final pH 7,0 ± 0,2
References
Directions LEIFSON, E.(1935) New culture media based on sodium
Suspend 63,1 g of the dehydrated medium in 1 L of
deoxycholate for the isolation of intestinal pathogens and
distilled water. Slowly bring to the boil, stirring until com-
for the enumeration of colon bacilli in milk and water. J.
plete dissolution. Boil for 2 minutes. Do not autoclave.
Pathol. Bacteriol., 40.581.
Cool to 50°C and pour into sterile Petri dishes.Do not
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
overheat.
ods for the Microbiological Examination of Food. 4th Ed.
APHA. Washington DC.
Description HORWITZ,W.(2000). Official Methods of Analysis 17th
The SS Agar is a highly selective agar for the isolation of ed. AOAC International. Gaithersburg. MD.
Salmonella and Shigella species from very contaminated ATLAS, R.M.,and L.C. PARKS (1993) Handbook of
samples. Microbiological Media. CRC Press, London
Selectivity is obtained by a high concentration of bile GRAY, L.D. (1995) Escherichia, Salmonella, Shigella
salts and brilliant green, which inhibits the growth of and Yersinia. In Murray, Baron, Pfaller Tenover & Yolken
gram-positive bacteria. As for the other gram-negative (eds) Manual Clinical Microbiology. 6th ed. ASM Wash-
flora, its growth is highly repressed by the presence of ington DC.
citrate and thiosulfate. Nevertheless, some coliforms
may still grow on this medium. In such case, differentia-
tion between pathogenic species and coliforms becomes
evident by the colour change of the pH indicator neutral
red. Lactose fermenters produce a pink or red colored
medium and colonies, while non-fermenting species
form colourless colonies and turn the medium yellow.
Should any species eventually produce H2S, it will be
easily detected by the ferrous sulfide’s black precipitate,
which turns the colonies black.
The peptone and the meat extract are usually capable of
inducing the growth of most pathogenic species, nev-
ertheless some Shigella are very fastidious and grow
poorly.

Technique
While using the samples suspected of being exposed
to the treatments that might have damaged the viability
of microorganisms (processed food, faeces from the
patients under antibiotic treatment, etc.) it is advisable to
proceed with a previous enrichment in Selenite Cystine
Broth Base (Ref. 02-602) or Tetrathionate Base Broth
(Ref. 02-033/Ref. 02-335). Afterwards, inoculate SS
Agar plates heavily with the specimen and proceed in Salmonella typhimurium ATCC 14028
the same way with other specimens of a less selective

135
 Schaedler Media
Schaedler Agar
Ref. 01-231
Specification
Solid medium with high reducing and nutrient capacity
for the cultivation of fastidious anaerobic microorgan-
isms.
Formula (in g/L)
Casein peptone ........................................ 5,60
Soy peptone ............................................. 1,00
Meat peptone ........................................... 5,00
Yeast peptone ........................................... 5,00
Glucose .................................................... 5,80
Sodium chloride ........................................ 1,70
Dipotassium phosphate ............................ 0,80
Tris buffer ................................................. 3,00
L-Cysteine HCl ......................................... 0,40
Hemine ..................................................... 0,01
Agar ........................................................ 15,00
Final pH 7,6 ± 0,2
Directions
Suspend 43,3 g of powder in 1 L of distilled water and
heat to boiling. Dispense into suitable containers and
sterilize in the autoclave at 121°C for 15 minutes. Pour
into sterile plates just before the use.
Schaedler Broth
Ref. 02-231
Specification
Liquid version of the agar with the same name, especial-
ly suitable for fastidious anaerobic microorganisms.
Formula (in g/L)
Casein peptone ........................................ 5,60
Soy peptone ............................................. 1,00
Meat peptone ........................................... 5,00
Yeast peptone ........................................... 5,00
Glucose .................................................... 5,80
Sodium chloride ........................................ 1,70
Dipotassium phosphate ............................ 0,80
Tris buffer ................................................. 3,00
L-Cysteine HCl ......................................... 0,40
Hemine ..................................................... 0,01
Final pH 7,6 ± 0,2
136
Directions
Dissolve 28,3 g of powder in 1 L of distilled water, heat-
ing up only if necessary. Distribute into suitable contain-
ers and sterilize by autoclaving at 121°C for 15 minutes.
Description
These media viz. Schaedler Agar and Broth, were
developed to create the selective conditions to allow
the growth of fastidious anaerobic microrganisms from
a mixed flora, like gastrointestinal tract, where there are
many antagonistic activities
between fast growing facultatives and the delicate fas-
tidious anaerobic organisms. For this aspect, the media
with thioglycolate are widely used, but this compound
seems to inhibit some delicate anaerobic organisms. On
the other hand, Schaedler media have L-Cystine as a
reducing agent, thus some gramnegative do not grow.
Effective separation or isolation in several biotypes is
achieved with the addition of selective agents to the nu-
trient base. For example, this medium can be rendered
selective for lactic bacteria by adding 10 g/L of sodium
chloride and 0,002 g/L of neomycin.
For the selection of Clostridium and Bacteroides, it is
more advisable to add 2 g/L of placenta powder and
0,002 g/L of neomycin. Should a selective medium for
Flavobacterium be desired, add 7 mL of alcoholic solu-
tion of tyrothricin 0,5% to 1 L of medium base. In any
case, incubation must be carried out at 37°C and in an
anaerobic atmosphere.
References
SCHAEDLER, R.W., DUVOS, R. and COSTELLO, R.
(1965) The development of the bacterial flora in the gas-
trointestinal tract of mice. J. Exp. Med. 122:59.
ATLAS, R.M., LC. PARKS (1993) Handbook of Microbio-
logical Media. CRC Press, Inc.,London
STALONS, D.R., C.THORNSBERRY and V.R. DOWELL
(1974) Effect of culture medium and CO2 concentration
of growth of anaerobic bacteria commonly encountered
in clinical specimens. Appl. Microbiol 27:1098-1104.
ISENBERG H.D. (1992) Clinical Microbiology Proce-
dures Handbook. ASM. Washington DC.
MARSHALL, R.T. (1992) Standard Methods for the ex-
amination of Dairy Products. APHA. Washington
MacFADDIN, J.F. (1985) Media for Isolation-Cultiva-
tion- Identification and Maintenance of Medical bacteria.
William & Wilkins. Baltimore, MD, USA.
WILKINS, T.D. and S. CHALGREN (1976) Medium for
use in the susceptibility testing of anaerobic bacteria.
Antimicrob. Agents. Chemother 10:926:928.
 Sealing Anaerobic Agar
Ref. 01-174
Specification
Solid substrate with high reducing capacity to cover and
seal the cultures anaerobically.
Formula (in g/L)
L-Cysteine HCl ......................................... 0,30
Sodium Thioglycolate .............................. 0,70
Resazurine ............................................... 0,01
Agar ........................................................ 20,00
Final pH 7,0 ± 0,2
Directions
Suspend 21 g of powder in 1 L of distilled water and
bring to the boil. Dispense into suitable containers and
sterilize in the autoclave at 121°C for 15 minutes.
Selenite Media
Selenite Brilliant Green
Broth Base (SBG Broth Base)
Ref. 02-603
Specification
Selective enrichment medium for Salmonella types.
Formula (in g/L)
Gelatine peptone .................................... 5,000
Yeast extract ........................................... 5,000
D-Mannitol .............................................. 5,000
Sodium taurocholate .............................. 1,000
Potassium phosphate ............................. 3,500
Sodium sulfapyridine .............................. 0,500
Brilliant green ......................................... 0,005
Final pH 7,2 ± 0,2
Directions
Dissolve 20 g of powder. in 1 L of distilled water and add
4 g of sodium biselenite (Ref. 06-615). Homogenize and
bring to the boil. Distribute in suitable containers. Termo-
labile medium: Use immediately. Do not autoclave.
Description
SBG Broth is a modification to the classical Osborne and
Stockes medium for enrichment of Salmonella from eggs
and egg derivative products.
The medium is maintained at a neutral pH, in spite of the
acid products are liberated from the mannitol fermenta-
tion, due to the strong phosphate buffer. On the other
hand, inhibitor effect of sulfamide in gram-negative
bacteria is helped by the classical selective agents for
salmonellae like brilliant green, selenite and bile salts.
Notwithstanding, presence of these substances, makes
the medium thermolabile and thus autoclaving must be
Xn Description
This solid substrate, without the capacity to support any
R-22-43
S-24-37-46 growth but with strong reducing power, is made to cover
the tubes for anaerobic growth. Once liquified, let it cool
to 50-55°C and pour into the tubes, taking care not to
mix it with the medium.
A minimum column of 1,5 cm over the medium is recom-
mended to assure an anaerobic cover.
Should cultures be stored for a long period of time, it is
advisable to put a sterile vaseline layer over the cap to
avoid
desiccation.
References
SANCHO, J. (1977) Personal communication.
avoided. Is not advisable to store the prepared medium
for more than eight days, since it loses its selectivity
notably.
Selenite Broth Base
Ref. 02-598
Specification
Liquid medium for Salmonella and Shigella enrichment.
Formula (in g/L)
Peptone .................................................... 5,00
Lactose ..................................................... 4,00
Potassium phosphate ............................. 10,00
Final pH 7,0 ± 0,2
Directions
Dissolve 19 g of powder. in 1 L of distilled water and add
4 g of sodium biselenite (Ref. 06-615). Homogenize and
bring to the boil. Distribute in suitable containers. Termo-
labile medium: Use immediately. Do not autoclave.
Description
Selenite Broth is formulated according to an original
formulation by Leifson for selective enrichment of Salmo-
nellae from very contaminated samples.
Enrichment is especially effective during the first 12
hours of cultivation, since in this period it seems that
only Salmonellae, some Proteus and some strains of
Pseudomonas grow easily. For this reason, it is advis-
able not to extend the enrichment phase and go quickly
for the selective medium, either liquid or solid. According
to Bänffer, the efficacy of the medium is improved nota-
bly if enrichment is performed at 43°C. Presence of a red
precipitate in the medium before inoculation, indicates
137
 Selenite Media

that there was a overheating in which case the selective When starting material is urine, the best procedure is to
properties of the medium are reduced. use Selenite Cystine Broth in double concentration, and
to inoculate it with an equal volume of urine. Anyway,
Selenite Cystine Broth Base subculturing must always be done after 6 hours of incu-
bation but before 24 hours. Most authors recommend
the simultaneous use of another enrichment broth, such
Ref. 02-602 as Tetrathionate Base Broth (Ref. 02-033).

Specification References
Liquid enrichment medium for Salmonella sp. acc. USP
US PHARMACOPOEIA (2002) 25th ed Chapter <61>
and ISO 6785 and 6340 standards
“Microbial Limit Tests” The U.S. Pharmacopoeial Con-
vention. Rockville MD.
Formula (in g/L) DOWNES F.P. & K. ITO (2001) Compendium of Meth-
Peptone .................................................... 5,00 ods for the Microbiological Examination of Food. 4th ed.
Lactose ..................................................... 4,00 APHA. Washington.
Potassium phosphate ............................. 10,00 FDA (1998) Bacteriological Analytical Manual 8th ed.
L-Cystine .................................................. 0,01 Rev. A. A.O.A.C.International.Gaitherburg VA.
Final pH 7,0 ± 0,2 LEIFSON, E. (1936) “A new Selenite Selective Enrich-
ment media for the Isolation of Typhoid and Paratyphoid
Directions (Salmonella) Bacilli” Am.J.Hyg. 24:423-432.
Dissolve 19,01 g of powder. in 1 L of distilled water and US FDA (1962) “The determination of Salmonellae in
add 4 g of sodium biselenite (Ref. 06-615). Homog- Food”.
enize and bring to the boil. Distribute in suitable con- BÄNFFER, J.R. (1971) Comparison of the isolation of
tainers. Termolabile medium: Use immediately. Do not Salmonellae from human faeces by enrichment at 37ºC
autoclave. and 43ºC Zbl. Bakt. I Orig. 217:(35-40)
Description STOCKES, J.L. and OSBORNE, W.W. (1955) A Selenite
Selenite Cystine Broth has been developed according Brilliant Green Medium for isolation of Salmonella. Appl.
to Leifson’s formulation with the addition of L-Cystine to Microbiol 3-4:217-227.
comply with FDA specifications, since it was proved that ATLAS, R.M., LC. PARKS (1993) Handbook of Microbio-
the medium was better in reduced CO2 atmosphere. logical Media. CRC Press, Inc London
Essencially, it is an enrichment medium for Salmonella DIN - Standard 10160: Untersuchung von Fleisch u.
coming from food or pathological materials, such as fae- Fleischerzneugissen. Nachweiss von Salmonella (Ref-
ces or urine, as a previous step to isolation in selective erenzverfahren).
media plates such as SS Agar (Ref. 01-171) or Hektoen ISO 6785 Standard (2002) Milk and Milk products - De-
Agar (Ref. 01-216). tection of Salmonella spp.
ISO 6340 Standard (1995) Water Quality Detection of
Salmonella spp.
Technique
For normal assays or experiments, an incubation at
37°C for a period not exceeding 18 hours is recommend-
ed, since within this period a good nutrition of coliforms
and an enhancement of pathogens is achieved, but after
24 hours this effect seems to disappear and the growth
of accompanying organisms may mask the growth of
Salmonella.
Appearance of red precipitate before inoculation is the
indication of overheating the medium, in which case the
selective properties are significantly reduced. Presence
of abundant sample residues may also inactivate the
selective property of the medium, if the sample is e.g. Ref. 02-602
faeces and or egg powder. In those cases, it is better Selenite Cystine
to make a dilution of 1:10 and let the bigger particles Broth Base
separate by settling down the dilution tube, and then
inoculate Selenite Cystine Broth with an aliquot portion
of it. Maintain a proportion of 1:10 between the sample
and the medium.
It has been demonstrated that when it is desired to iso-
late Salmonella from faeces,the results are better if the
enrichment medium is incubated at 43°C. However this
procedure does not work with the isolation of Salmonella
Left: Salmonella typhimu-
typhi.
rium ATCC 14028; right:
control.
138
 Sellers Agar
Ref. 01-175
Specification
Solid differential medium for gram-negative non ferment-
ing coccobacilli.
Formula (in g/L)
Gelatin peptone .................................... 20,000
Sodium chloride ...................................... 2,000
Sodium nitrate ........................................ 1,000
Sodium nitrite ......................................... 0,350
D-Mannitol .............................................. 2,000
L-Arginine ............................................... 1,000
Yeast extract ........................................... 1,000
Magnesium sulfate ................................. 1,500
Dipotassium phosphate .......................... 1,000
Bromothymol Blue .................................. 0,040
Phenol red .............................................. 0,008
Agar ...................................................... 13,500
Final pH 6,7 ± 0,2
Directions
Suspend 43,5 g of powder in 1 L of distilled water and
bring to the boil. Distribute into tubes and sterilize by
autoclaving at 121ºC for 15 minutes. Let it solidify in
slanted position with short slant and good butt.
Just before the inoculation, allow 2 drops of sterile glu-
cose 10% solution to slide down on the opposite side of
the slant.
Description
Sellers Agar is formulated to differentiate the gram nega-
tive bacilli that do not produce characteristic acidification
by the fermentation in the usual diagnostic media, such
as TSI (Ref. 01-192) or Kligler Iron Agar (Ref. 01-103).
Criteria for the differentiation among the microorganisms
that can be obtained from this medium are as follows:
- Fluorescence production enhanced by the magnesium
sulfate and mannitol.
- Glucose oxidation and pH changes that can be fol-
lowed with the colour variation of the pH indicators
included (phenol red and bromo thymol blue)
- Nitrogen gas release, indicated by the bubbles which
may eventually break the agar.
Technique
The medium is inoculated with a pure culture by streak-
ing on the surface and also by stabbing deeply. Incu-
bation is performed at 35-37ºC for 24-48 hours. After
the incubation period, observe the fluorescence under
Wood’s light and also observe the change in pH and
gas production. The yellow band on the surface of the
medium due to glucose oxidation may disappear within
24 to 48 hours.
On the slant, fluorescence may appear under UV light
in some species of the fluorescent Pseudomonas group,
especially Ps. aeruginosa. On the surface of the me-
dium, most of Acinetobacter species produce a yellow
band due to glucose oxidation. This band may disappear
after 24 hours, and this phenomenon is very common
with some strains of A. calcoaceticus.
A blue colour at the bottom of the tube indicates ar-
ginine-dehydrolase positive or any other reaction like
anaerobic degradation of nitrate or the mannitol utilisa-
tion by some strains of Alcaligens faecalis. Nitrogen gas
released in the form of small bubbles or splitting of agar
indicates denitrification reaction from nitrate or from
nitrite. In the table below all these typical reactions are
tabulated.
References
ATLAS, R.M. and R.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press, London
MacFADDIN, J. (1985) Media for isoltion-cultivation-iden-
fication-maintenance of medical bacteria. Vol I. William &
Wilkins. Baltimore,MD.
SELLERS, W. (1964) J. Bact. 87:46
139
 SF Medium
Ref. 02-072
R-45-22-32-52/53
Specification
Liquid selective medium for faecal streptococci detection
in several materials.
Formula (in g/L)
Casein peptone .................................... 20,000
Dextrose ................................................. 5,000
Di-potassium phosphate ......................... 4,000
Sodium azide .......................................... 0,500
Sodium chloride ...................................... 5,000
Brom cresol purple ................................. 0,032
Monopotassium phosphate .................... 1,500
Final pH 6,9 ± 0,2
Directions
Dissolve 36 g of powder in 1 L of distilled water. Dis-
pense in tubes or flasks with Durham´s tubes and steri-
lize in the autoclave at 121°C for 15 minutes.
Description
The efficacy of this diagnostic medium for detecting the
faecal streptococci (enterococci) from several materials
has been widely proved since its publication by Hajna
and Perry, who utilized the advantage of the azide effect
and combined it with the fermentative ability of strepto-
SIM Medium
Ref. 03-176
Specification
Differential fluid medium for detecting the motility, H2S
production and indole formation.
Formula (in g/L)
Yeast extract ............................................. 10,0
Casein Peptone ........................................ 10,0
Meat peptone ............................................. 6,0
Ferric-ammonium sulfate ............................ 0,2
Sodium thiosulfate ...................................... 0,2
Agar ............................................................ 3,7
Final pH 7,3 ± 0,2
Directions
Suspend 30 g of powder in 1 L of distilled water and let it
soak for a few minutes. Heat to boiling and dispense into
suitable containers. Sterilize by autoclaving at 121°C for
15 minutes.
Description
This classical medium was originally developed to dis-
tinguish several types of enterobacteria, on the basis of
motility test , detection of indole and H2S production.
It is a semisolid or fluid medium, and so the motile micro-
organisms can move freely. At the same time, richness
of sulfur containing amino acids and presence of thiosul-
140
T cocci to ferment glucose at 44,5°C. Inhibition of accom-
panying bacteria is achieved by the high concentration of
S-53-45-7-61 azide, which does not affect the growth of streptococci.
Enterococci growth is indicated by the indicator brom
cresol purple turning to yellow, when the tubes are incu-
bated at 44,5°C.
In this medium, the indicator turning yellow in pres-
ence of enterococci is evident after 18-20 hours, but to
proceed for the isolation, a supplementary incubation in
petri plates is recommended.
If the MPN method is used, do not excessively dilute the
medium with the sample.
References
HAJNA, A.A. and PERRY, C.A. (1943) Comparative
study of presumptive and confirmativemedia for bacteria
of the coliform group an fecal streptococci. Am.J.Pub.
Hlth.,33:550.
ATLAS, R.M. and L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press, Inc.,London
APHA-AWWA-WPCF (1998) Standard Methods for the
examination of Water and Wastewater,20th Ed.,APHA,
Washington.
DOWNES, F.P. and K. ITO (2001)Compendium of Meth-
ods for the Microbiological Examination of Food,4th Ed.
American Public Health Association,Washington D.C.
fate allow those microorganisms that are able to produce
sulfides, and then this reacts with iron and produces
black precipitates which in turn make the medium darker.
The amount of thiosulfate present in medium does not
affect the motility mechanisms, instead it assures H2S
production by those microorganisms that are not able to
produce it from cystine or cysteine.
Finally, the medium allows the production of indole from
Tryptophan present in the peptone, which can be easily
detected with the addition of Kovac’s Reagent (Ref.
06-018) (directly or with extraction) or with paper stripes
impregnated with the reagent.
These three characteristics are common for enterobac-
teria, and on the basis of these properties, more differen-
tial or even selective media can be used.
Technique
Recommended technique is to inoculate by deep stab
from a pure culture (or from an isolated colony). After an
incubation period of 16-18 hours at 37°C, observe for the
clarity in stab. Immotile microorganisms produce growth
only in the stab, whereas motile ones may be easily
detected by their displacement which is indicated by the
turbidity in the medium.
H2S production is indicated by the general blackening of
the medium when there is a great amount of FeS pro-
duced or by blackening of the stab when there is a little
amount of FeS produced.
 SIM Medium
Indole formation is the last test to be performed if the
soaked or impregnated paper stripes are not used.
Despite the fact that many authors suggest a previ-
ous extraction of indole by stirring on the surface of the
culture medium with chloroform. If Kovacs’ Reagent
(Ref. 06-018) is employed then this is not necessary and
the observations can be done by pouring a few drops of
reagent on the surface of the medium. A positive test will
produce the interphase in the medium turning to pur-
ple red colour, whereas a negative test will produce no
colour change. Many a times, chloroform extraction may
give erroneous results, since the appearance of colour
must be observed immediately after the reagent addi-
tion. However if it is delayed by more than 30 seconds,
the test must be considered negative.
Simmons Citrate Medium
Ref. 01-177
Specification
Medium for verifying the citrate utilization by enterobac-
teria according ISO 10273 standard.
Formula (in g/L)
Magnesium sulfate ................................... 0,20
Monoammonium phosphate ..................... 1,00
Dipotassium phosphate ............................ 1,00
Sodium citrate .......................................... 2,00
Sodium chloride ........................................ 5,00
Brom thymol blue ..................................... 0,08
Agar ........................................................ 15,00
Final pH 6,8 ± 0,2
Directions
Dissolve 24 g of powder in 1 L of distilled water. Bring to
the boil. Dispense in tubes and sterilize by autoclaving at
121°C for 15 minutes. Allow to solidify with long slant.
Description
Simmons Citrate Agar is the solid version of the classical
Koser citrate medium, and it can be used in the plates as
well as in slanted tubes. Slanted tubes can be inoculated
by surface streaking or by deep stab.
Although, originally, it was described as an isolation and
identification medium for certain fungi, Edwards and
Ewing had recommended it for the IMViC test since it
has the advantage over the Koser’s medium that the
readings can be made by the indicator colour change,
instead of turbidity of the medium, which is sometimes
difficult to detect.
References
HARRIGAN, W.F. and McCANCE, M.E. (1966) Labora-
tory Methods in Microbiology. Academic Press, .
BLAZEVIC, D.J. (1968) Improved Motility Indole Me-
dium. Appl. Microbiol. 16, (4),668.
BULLMASH, J.M. and FULTON, M (1964). Discrepant
Test for Hydrogen Sulfide. J.Bact. 88(6)1813.
Technique
The techique is simple and one has to take care in us-
ing an inoculum as small as possible and the medium
should be freshly prepared, because if it is very dry, false
turning (colour change) may appear, even before the
inoculation, especially at the bottom of the slant.
The basis of this medium is in the capacity of microor-
ganisms to use citrate as carbon source and ammonium
compounds as the nitrogen source for their growth.
Among enterobacteria, these properties are possessed
by the following genera: Enterobacter, Klebsiella, Ser-
ratia, Citrobacter and some species of Salmonella as
S.schottumelleri, S.typhimurium, S.arizona etc. whereas
Escherichia, Shigella, Salmonella typhi and S.paratyphi
are unable to grow.
Although, the test result must be read as the growth pro-
duced, the presence of an indicator makes it easy, as the
citrate degradation yields an alkaline reaction, which is
indicated by the turning of the indicator to intense blue.
This is evident even when the growth rate is high.
References
SIMMONS J.S. (1926) A culture medium for differentiat-
ing organisms of typhoid-colon aerogenes group and for
isolatig certain fungi.J.Inf.Dis. 39:209
FDA (1998) Bacteriological Analytical Manual. 8th Ed.
Revision A. AOAC International. Gaithersburg.
APHA-AWWA-WEC (1998) Standard Methods for the
examination of water and wastewater. APHA. Washing-
ton DC.
HORWITZ, W. (2000) Official Methods of Analysis. 17th
Ed. AOAC International. Gaithersburg. MD.
ISO 10273 Standard (1994) General guidance for the
detection of presumptive pathogenic Yersinia enteroco-
litica.
141
 Slanetz Bartley Media
Slanetz Bartley Agar
Xn
Ref. 01-178
R-22-32-52/53
S-7-46-61
Specification
Differential and selective medium for the enumeration of
enterococci,
Formula (in g/L)
Tryptose .................................................... 20,0
Yeast extract ............................................... 5,0
Dextrose ..................................................... 2,0
Potassium phosphate ................................. 4,0
Sodium azide .............................................. 0,4
TTC............................................................. 0,1
Agar .......................................................... 12,0
Final pH 7,0 ± 0,2
Directions
Suspend 43,5 g of powder in 1 L of distilled water and
bring to the boil. Cool to 50°C and distribute into sterile
petri plates immediately. Do not autoclave or overheat.
Description
The Slanetz Bartley Agar is recommended for the
determination of faecal streptococci by the membrane
filtration technique. Although originally this medium was
described by the authors only for the above technique, it
gives such excellent results that it is now recommended
for viable enumeration of enterococci from food samples.
Slanetz Bartley Agar Base
Xn
Ref. 01-579
R-22-32-52/53
S-7-46-61
Specification
Differential and selective medium for the detection and
enumeration of enterococci acc. ISO Standard 7899-
2:2000
Formula (in g/L)
Tryptose .................................................... 20,0
Yeast extract ............................................... 5,0
Dextrose ..................................................... 2,0
Potassium phosphate ................................. 4,0
Sodium azide .............................................. 0,4
Agar .......................................................... 12,0
Final pH 7,0 ± 0,2
Directions
Suspend 43,4 g of powder in 1 L of distilled water and
bring to the boil. Sterilize by autoclaving at 121ºC for
15 minutes. Cool to 50°C and add 10 mL/L of 1% TTC
sterile solution (Ref. 06-023). Mix well and distribute into
sterile petri plates immediately.
142
Description
This formulation, without TTC allows its sterilization by
autoclaving without the development of the pink colour
due to the formazan which is formed as a result of the
partial-thermal reduction of TTC. This modification is
more tedious in its preparation but provides a colour-
less medium, making the results easier to read and the
colonies are more sharply defined.
Technique
For the membrane filtration technique, take 100 mL of
water sample which is previously homogenized, and
pass it through a sterile membrane filter. Then wash
it with 30 mL of sterile dilution water to wipe away the
things left on the funnel of the filtering system.
By using sterile forceps, transfer the membrane asepti-
cally on to the culture medium contained in a Petri dish,
making sure that the filter surface faces upwards. Close
the lid and invert the plate. lncubate at 37°C for 48
hours.
All the developed colonies which appear red or purple
coloured must be considered as enterococci, since those
bacteria reduce Triphenyltetrazolium-HCl to an insolu-
ble formazan which is red in colour. The secondary or
accompanying gram negative bacteria are inhibited by
sodium azide.
As regards, food samples from a decimal dilution bank
of the product to be examined, incoculate 0,1 mL on the
surface of the petri dishes using the Drigalski technique.
lncubation and examination is carried out as same as in
the membrane filtration technique.
References
SLANETZ L.W. & BARTLEY, C.H. (1957). Numbers of
Enterococci in Water, Sewage and Faeces Determined
by The
Membrane Filter technique with an lmproved Medium.
J.Bact., 74:591-596.
ATLAS, R.M., & LC. PARKS (1993) Handbook of Micro-
biological Media,CRC Press Inc.,London
ISO Standard 7899-2:2000 Water Quality. Detection
and enumeration of enterococci by membrane filtration
method.
LACHICA, L.V.F. & P.A., HARTMAN (1968) Two im-
proved media for isolating and enumerating enterococci
in certain frozen foods
J. Appl. Bact. 31:151-156
Ref. 01-579 Slanetz-
Bartley Agar Base + Ref.
06-023 TTC 1% Sterile
Solution.
Enterococcus faecalis
ATCC 29212.
 SOB Broth

Ref. 02-523 The addition of 4 g/L of dextrose to this medium pro-

Specification
Liquid medium for the cultivation of recombinant strains
of Escherichia coli.
Formula (in g/L)
Tryptone .............................................. 20,000
Yeast Extract .......................................... 5,000
Sodium Chloride .................................... 0,500
Magnesium sulphate ............................. 2,400
Potasium chloride ................................... 0,186
Final pH 7,0 ± 0,2
duce the SOC Medium that is used in the final phase of
transformation and in the recovery of electroporated E.
coli cells.
References
HANAHAN, D. (1983) Studies on transformation of Es-
cherichia coli wth plàsmids. J. Mol. Biol. 166:557
SAMBROOK, J., E.F. FRITSCH & T. MANIATIS (1989)
Molecular cloning: a laboratory manual. 2nd Ed. Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

Directions
Dissolve 28 g of powder in 1 L of distilled water heating
if it is necessary. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes.

Description
This medium was developed by Hanahan in 1983 as a
nutritionally rich base for growth preparation and trans-
formation of competent cells that allows the introduction
of foreign DNA onto cells..

Sporulating Agar (AK Agar) (USP Antibiotic Medium 32)

Ref. 01-069 in 10 mL of Ringer Solution the growth from a slant. With

Specification
This medium is according to the Arret and Kirshbaum
formulation, adopted by US FDA, for the preparation
of spore suspension for antibiotic assay. It is the same
medium as the Antibiotic Medium 32.
Formula (in g/L)
Gelatin peptone .......................................... 6,0
Casein peptone .......................................... 4,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Dextrose ..................................................... 1,0
Manganous sulfate ..................................... 0,3
Agar .......................................................... 15,0
Final pH 6,6 ± 0,2
Directions
Dissolve 30,8 g of powder in 1 L of distilled water. Bring
it to the boil with constant stirring . Dispense in suitable
containers and sterilize at 121°C for 15 minutes.
this suspension, inoculate on the surface a Roux flask
(bottle) containing 300 mL of solidified and controlled
Sporulation Agar.
The roux flask must be incubated at 35°C for 5 days.
Growth is harvested with 50 mL of sterile Ringer So-
lution, helping with a Drigalsky loop or sterile crystal
balls (pearls) if necessary. The suspension obtained is
carefully centrifuged at 5000 rpm for 15 minutes. Discard
the supernatant liquid and suspend the pellet in a fresh
volume of Ringer Solution. Put it in a boiling water bath
at 70°C for 30 minutes. This suspension is active upto 6
months if stored refrigerated. Depending on the objective
of the experiment,it can be standardized by turbidimetry.
References
ARRET and KIRSHBAUM (1959) J.Milk. Food Tech.
22:329
SANCHO, GUINEA, PARES (1980) Microbiología
Analítica Básica. Ed. JIM. Barcelona,
US PHARMACOPOEIA (2002) 25th ed. <81> Antibiotic
Microbial Assays. US Pharmacopoeial Convention Inc.
Rockville. MD

Description
Sporulating Agar is made according to the original for-
mulation of Arret and Kirshbaum, and later adopted by
FDA for the preparation of Bacillus subtilis ATCC 6633
spores suspension. To prepare this suspension, suspend

143
 SPS Agar
Ref. 01-050
Specification
Solid medium for the detection of Clostridium perfrin-
gens in food.
Formula (in g/l)
Sodium sulfite ........................................... 0,50
Polymyxin (B) sulfate ................................ 0,01
Sodium sulfadiazine ................................. 0,12
Casein peptone ...................................... 15,00
Yeast extract ........................................... 10,00
Ferric citrate.............................................. 0,50
Sodium thioglycolate ................................ 0,10
Polysorbate 80 ......................................... 0,05
Agar ........................................................ 15,00
Final pH 7,0 ± 0,2
Directions
Suspend 41,3 g of powder in 1 L of distilled water and
bring to the boil. Distribute into tubes or screw-cap
containers and sterilize by autoclaving at 121°C for 15
minutes. Cool the sterilized medium quickly by placing in
the refrigerator or in cold water.
Description
The SPS Agar (Sulfite-Polymyxin-Sulfadiazine) is a
modification of the original Wilson & Blair medium for the
detection of clostridia. The present medium excels the
formulation of Mossel and also the later modification of
Angelotti et al. It achieves a higher selectivity for Cl. perf-
ringens with the addition of sulfadiazine and polymyxin.
On the other hand, the nutritional substrates constituted
by the tryptone and the yeast extract are complemented
by the Polysorbate, which also allows the recovery of
the most delicate cells. The anaerobic conditions are im-
proved by the thioglycolate, which permits the use of the
medium on the plates without the Miller-Prichett tubes,
used by Mossel and Wilson-Blair.
Clostridium perfringens ATCC 13124
144
The differential system consists of sodium sulfite and
ferric citrate. It allows the detection of sulfite-reducing
organisms, which form black colonies due to the ferrous
sulfide precipitate.
Technique
The usual technique for the use of this medium is as
follows:
The samples to be examined are ground or homog-
enized with a vortex in a stomacher and then a decimal
dilution bank is prepared. Take a sample aliquot from
each one of these dilutions and place in the petri dishes.
The medium, molten and cooled to 50°C, is now poured
in the dishes and allowed to solidify. The dishes are incu-
bated in an anaerobic system at 35°C for 24-36 hours.
Usually, 90% of the black colonies which are formed can
be attributed to the Clostridium perfringens. Neverthe-
less, and since the medium is not extremely selective,
it is advisable to verify the black colonies formed by
gram-positive sporulated immotile organisms incapable
of reducing the nitrates to nitrites.
The Indole Nitrite Fluid Medium (Ref. 03-101) is suitable
for such purposes, still a small quantity of agar has to be
occasionally added in it.
Most clostridia are sulfite reductors. Among them are Cl.
perfringens and the Cl. botulinum which along with Cl.
bifermentans are the species most frequently involved
in food poisoning.
References
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food,4th Ed.
APHA,Washington.
FDA (1998) Bacteriological Analytical Manual, 8th ed.
Revision A. AOAC International. Gaithersburg. MD
control
 Staphylococcus 110 Agar
Ref. 01-587
Specification
Selective medium for the isolation and presumptive iden-
tification of staphylococci.
Formula (in g/L)
Casein Peptone ...................................... 10,00
Yeast extract ............................................. 2,50
Gelatin .................................................... 30,00
Sodium chloride ...................................... 75,00
Di-potassium phosphate ........................... 5,00
Lactose ..................................................... 2,00
D-Mannitol .............................................. 10,00
Agar ........................................................ 15,00
Final pH 7,2 ± 0,2
Directions
Suspend 149,5 g of powder in 1 L of distilled water and
heat up to 55-60°C in boiling water bath until total dis-
solution of gelatin. Bring to the boil. Dispense in suitable
containers and sterilize by autoclaving at 121°C for 15
minutes. Before pouring in the petri dishes, gently shake
to suspend possible precipitate. Should a blood addi-
tion be desired, cool to 45°C and add sterile defibrinated
blood and homogenize before pouring in the plates.
Description
This medium combines the basic criteria of staphylococci
identification i.e.: halotolerance, pigmentation, mannitol
fermentation and gelatin liquefaction as were marked by
Stone and Chapman. Smucker and Appleman suggests
the addition of sodium azide (5mg/L) to improve the
inhibition of Bacillus spp.
It is widely known that pathogenic staphylococci are co-
agulase positive, pigment former, highly halotolerant and
able to liquefy gelatin and produce acids by fermenting
mannitol. They are also strongly hemolytic.
In the 110 medium, the following reactions can be
observed: pigmentation, mannitol fermentation, gelatin
liquefaction and saline tolerance. From this medium,
hemolysis and coagulase production can also be proved
afterwards.
Technique
The sample is inoculated in the petri dish by wire loop to
obtain isolated colonies, and it is incubated for 48 hours
at 30°C.
At 37°C early reactions or results are obtained, but
pigmentation is not good,as the pigmented colonies form
a characteristic golden yellow colour, which in this case
are white.
Acid production from mannitol is verified by adding a few
drops of Bromthymol Blue 0,04% solution on suspected
colonies. Positive reaction produces a yellow coloura-
tion.
Gelatin hydrolysis (Stone’s reaction) is visualised by a
clear halo surrounding the colony which forms 10 min-
utes after a few drops of 20% aq. soln.of Sulfosalicylic
acid or sat. aq. solution of Ammonium sulfate are added
over them.
These last two reactions are helped by placing Oxford
cups around the discrete colonies, isolating them from
affecting the other colonies. Identification is completed
by verifying hemolysis tests, subculturing on Blood Agar
plates, and verifying plasmacoagulase activity on rabbit
serum.
Although the subcultures can be done from isolated
colonies in 110 medium, results are always better if a
short (4-5 hours) incubation on Tryptic Soy Broth (Ref.
02-200) is carried out.
References
STONE, R.V. (1935) A cultural method for classifying
staphylococcias of the “food poisoning” type. Proc.Soc.
Exptl.Biol Med.33:185-187
CHAPMAN, G.H. (1945) The significance of sodium
chloride in studies of staphylococci. J. Bacteriol 50:201-
205
FDA (1998) Bacteriological Analytical Manual. 6th ed.
Revision A. AOAC International. Gaithersburg
145
 Starch Media

Starch Agar

Ref. 01-283

Specification
Solid medium to detect the starch hydrolysis by microor-
ganisms,

Formula (in g/L)

Meat extract ................................................ 3,0
Peptone ...................................................... 5,0
Soluble starch ............................................. 2,0
Agar .......................................................... 15,0
Final pH 7,2 ± 0,2

Directions
Suspend 25 g of powder into 1 L of distilled water and
let it soak. Heat to boiling and distribute into suitable
containers. Sterilize in the autoclave at 121°C for 15
minutes.

Description
Although this medium was initially formulated to perform
the test for the identification of Bacillus cereus, it can
be applied to any kind of microorganism where starch
hydrolysis activity is required to be analyzed.

Technique
Over the medium plates with Starch Agar, inoculate in
straight streaks the strains to be examined, (maximum
four per plate). Incubate at 30-35°C for 48 hours if the
strains are of Bacillus cereus and up to 5 days for dubi-
ous cases.
After the incubation, flood the plates with an alcoholic
iodine solution 2%. Starch hydrolysis is seen by the ap-
pearance of a clear halo surrounding the growth streak,
whereas the rest of the medium in the plate acquire a
dark blue colour.
The bigger the clear zone, the starch activity is consid-
ered higher of the strain under study.

References
COLLINS, C.H., LYNE, P.M. (1976) Microbiological
Methods. 4th Ed. Butterworths,London.
ISENBERG H.D. (1992) Clinical Microbiology Proce-
dures Handbook. ASM Washington.
ATLAS, R.M., & LC. PARKS (1993) Handbook of Micro-
biological Media. CRC Press,Inc.,London

146
 Stuart-Ringertz Transport Medium
Xn
Ref. 03-454
R-22-43
S-24-37-46
Specification
Medium for the conservation and transport of pathologi-
cal specimens or fastidious microorganisms.
Formula (in g/L)
Sodium glycerophosphate .................... 10,000
Sodium thioglycolate .............................. 1,000
Calcium chloride ..................................... 0,100
Methylene blue ....................................... 0,002
Agar ........................................................ 8,000
Final pH 7,4 ± 0,2
Directions
Suspend 19 g of powder in 1 L of distilled water and
bring to the boil. Distribute in tubes or flasks close with
air tight cap in such a way that the medium forms a
vertical column of 7-10 cm. Sterilize in the autoclave
at 121°C for 15 minutes and cool quickly in the vertical
position.
Description
The growth of microorganisms in this medium is restrict-
ed by the total lack of nitrogen, but they remain alive and
inactive for a long period. Thanks to the buffering and
protective effect of glycerophosphate. Thioglycolate pro-
vides a reducing environment which is aided and main-
tained by to the low concentration of agar, that avoids
convection streams and restricts oxygen difussion.
Progressive oxidation of the medium can be seen by
the change of the methylene blue, which acts as an Eh
(redox) indicator.
Technique
Sample is placed directly inside the tube, taking care that
it is beneath the blue band. If the sample is taken with a
swab, it is advisable to impregnate it with a suspension
of active carbon (activated charcoal) before puting it into
the transport medium.
The sample must always be in the centre of the medium
and beneath the blue band that indicates oxidation. If
the depth of the blue band is bigger than the half of the
medium, do not use the tube.
References
STUART, R.D. (1959) Transport medium for specimens
in public health bacteriology. Publ. Hlth. Rep. 74:431-
438.
RINGERTZ, O. (1960) A modified Stuart medium for the
transport of gonococcal specimens. Acta Path. Microbiol.
Scand. 48:105-112
147
 TCBS Media
TCBS Agar
Ref. 01-190
Specification
TCBS Agar is specially recommended for the selective
isolation of Vibrio that causes cholera or dysenteric diar-
rhoea, and for the examination of food that might have
Vibrio.
Formula (in g/L)
Casein peptone ........................................ 5,00
Meat peptone ........................................... 5,00
Yeast peptone ........................................... 5,00
Sodium citrate ........................................ 10,00
Sodium thiosulfate .................................. 10,00
Ox bile ...................................................... 5,00
Sodium Cholate ........................................ 3,00
Sucrose .................................................. 20,00
Sodium chloride ...................................... 10,00
Ferric citrate.............................................. 1,00
Thymol blue .............................................. 0,04
Bromo thymol blue ................................... 0,04
Agar ....................................................... 14,00
Final pH 8,6 ± 0,2
Directions
Suspend 88 g of powder in 1 L of distilled water and
bring to the boil. Boil it for 1 minute. Cool to 45-50°C and
pour it into sterile petri plates. Do not autoclave it.
TCBS Modified Agar
Ref. 01-567
Specification
TCBS Agar is specially recommended for the selective
isolation of Vibrio parahaemolyticus according ISO 8914
standard.
Formula (in g/L)
Proteosa Peptone ................................... 10,00
Yeast Extract ............................................ 5,00
Sucrose .................................................. 20,00
Sodium citrate ........................................ 10,00
Sodium chloride ...................................... 10,00
Ox bile ...................................................... 8,00
Sodium thiosulphate ............................... 10,00
Iron citrate ................................................ 1,00
Thymol Blue ............................................. 0,04
Bromthymol blue ...................................... 0,04
Agar ........................................................ 14,00
Final pH 8,6 (± 0,2)
148
Directions
Suspend 88 g of powder in 1 L of distilled water and
bring to the boil. Boil it for 1 minute. Cool to 45-50°C and
pour it into sterile petri plates. Do not autoclave it.
Description
Nowadays TCBS Agar is universally accepted as the
medium of choice for differential isolation of enteropatho-
genic Vibrio, and it achieves a great inhibition of all the
accompanying organisms. This formulation allows a
high growth of Vibro cholerae and V.parahaemolyticus.
V.alginoliticus and NAG vibrios also grow well.
Enterobacteria are strongly inhibited by high concentra-
tions of citrate, thiosulfate, bile and sodium chloride.
Although some enteric bacteria may also grow in this
medium, their colony morphology is quite different than
Vibrios.
The organisms that may be confused with vibrios are
some biotypes of Proteus and Pseudomonas. There are
some resistent enterococci which may form exception-
ally small and yellow colonies on this medium.
Usually, in this medium, colonies are selected or chosen
and then identified with primary tests (oxidase reactions
in Kliger Iron Agar (Ref. 01-103), MRVP Broth (Ref.
02-207), and antibiogram) before performing serological
identification and phage typing.
Due to its high selectivity, the medium allows massive
inoculation of pathological material. Once solidified and
cooled, the medium is turbid, but the observations are
not affected.
This medium is very thermolabile and so it must not be
autoclaved, overheated or remelted.
Colonial appearance on TCBS Agar after 24 hours at
37°C:
Vibrio alginolyticus: Big, yellow
Vibrio cholerae and sucrose fermentative strains of Vi-
brio parahaemolyticus: Average size, dirty yellow
with yellow halo in the center
Non sucrose fermentative strains
of Vibrio parahaemolyticus: Small, yellow, without halo
and with a green core.
Streptococcus faecalis: Very small and convex, yellow
with yellow halo
Enterobacteria: Small and transparent
Pseudomonas, Aeromonas, Proteus: Average size and
blue.
References
KOBAYASHI, T., ENOMOTO, S. SAKAZARI, R. and
KUWAHARA, S. (1963) A new selective medium for
pathogenic vibrios TCBS, (modified Nakanishi Agar)Jap.
J.Bact.18:387.
FDA (1998) Bacteriological Analytical Manual 8th ed.
Revision A. AOAC INTERNATIONAL. Gaithersburg
ISO 8914 Standard (1990) General guidance for the
detection of Vibrio parahemolyticus.
 TCBS Media

DOWNES, F.P. & K. ITO (2001) Compendium of Meth- KP DAS (1993). Outbreak of Vibrio cholerae non-01 in
ods for the Microbiological Examination of Food,4th Ed. India and Bangladesh. Lancet, 341:1346-1347
American Public Health Association,Washington D.C. HORWITZ, W. (2000) Official Methods of Analysis of
PASCUAL ANDERSON, MªRª (1992) Microbiología AOAC International 17 ed. Gaithersburg. MD
Alimentaria. Diaz de Santos, S.A.,Madrid,.
BHATTACHARYA, M.K., S.K. BATTACHARYA, S.
GARG, P.K. SAHA, D.DUTTA, G.B. NAIR, B.C. DEB &

Ref. 01-567
TCBS Modified Agar

Proteus mirabilis
Vibrio alginolyticus
ATCC 14273

control

Terrific Broth

Ref. 02-474 Description

Specification
Liquid medium for the cultivation of recombinant strains
of Escherichia coli.
Formula (in g/L)
Casein peptone ...................................... 12,00
Yeast extract ........................................... 24,00
Dipotassium phosphate ............................ 9,40
Monopotassium phosphate ...................... 2,20
Final pH 7,3 ± 0,2
Directions
Dissolve 47,6 g of powder in 1 L of distilled water, heat-
ing up only if necessary. Distribute in suitable containers
and sterilize by autoclaving at 121ºC for 15 minutes.
Terrific Broth was developed by Tartoff and Hobbs to
improve yield in plasmid bearing E. coli. This medium
supports a high cellular density and mass and maintains
the growth in the logarithmic phase for a long time. Due
to this fact, it provides greater yields of recombinant
proteins and plasmid DNA. On many occasions it sub-
stitutes the classical LB Broth (Ref. 02-385, 2-384 and
2-406) therefore usually increases yields of plasmid DNA
and recombinant proteins.
References
SAMBROC, J., E.F. FRITSCH, T. MANIATIS (1989) Mo-
lecular Cloning: A laboratory Manual. 2ª Ed.,Cold Spring
Harbor Press. Cold Spring Harbor,USA.
TARTOFF, K.D. & C.A. HOBBS (1987) Improved media
for growing plasmids and cosmid clones. Bethesda Re-
search Laboratoires Focus 9:12

149
 Tetrathionate Media
Tetrathionate Broth Base
Ref. 02-033
Specification
Medium for the selective enrichment of Salmonellae
(AOAC 17th, ICMSF 1968, USP 25th)
Formula (in g/L)
Meat peptone ............................................. 2,5
Casein peptone .......................................... 2,5
Bile salts ..................................................... 1,0
Calcium Carbonate ................................... 10,0
Sodium Thiosulfate ................................... 30,0
Directions
Suspend 46 g of powder to 1 L of distilled water, heat to
boiling and cool to 40-45°C. Add 20 mL of iodine-iodide
solution and 2 vials of the Selective Supplement of Bril-
liant Green-Novobiocin ref. 06-017CASE and distribute
in sterile tubes.
Do not heat after adding the iodine solution. Medium
must be used immediately. Without the iodine solution,
medium can be stored in refrigeration for some days.
The appearance of white medium precipitate is normal,
and it comes from calcium carbonate.
Description
This is the version originally used, which has been modi-
fied or improved the Muller-Kauffmann formulation, since
the latter one has more efficacy.
Muller-Kauffmann Medium Base
Ref. 02-335
Specification
Medium for selective enrichment of Salmonellae, acc.
ISO standard.
Formula (in g/L)
Bile salts #3 .............................................. 4,78
Meat extract .............................................. 4,30
Casein peptone ........................................ 8,60
Sodium chloride ........................................ 2,60
Calcium carbonate ................................. 38,70
Sodium thiosulfate .................................. 47,80
Directions
Add 107 g of powder to 1 L of distilled water. Heat to
boiling and let it cool to 40-45°C. Add 20 mL of iodine-
iodide solution and 2 vials of the Selective Supplement
of Brilliant Green-Novobiocin Ref. 06-017CASE and
distribute into sterile tubes.
Do not heat after adding the iodine solution. Complete
medium must be used immediately; the base, without
iodine, may be stored in the refrigerator for some days.
150
White precipitate is due to calcium carbonate and it must
be considered as normal.
Description
Tetrathionate Broth is a classic medium for the enrich-
ment of enteric or intestinal pathogens, and for all the
members of Salmonella type, from very polluted sam-
ples, like faeces, urine, waste water and others.
During the preparation, when iodine is added, tetrathion-
ate is produced from the sulfate, and this salt together
with the bile salts in the medium, provoke a strong inhibi-
tion to most of the normal intestinal bacteria, except for
those which are capable of reducing tetrathionate, e.g.
Salmonellae. Reduction reaction liberate sulfuric acid,
which is neutralized by the carbonate, avoiding a de-
crease of the pH, which is harmful even for Salmonellae.
However, many Proteus species resist the bile salts
concentration and moreover, they may even reduce
tetrathionate. So, many authors recommend the addition
of other inhibitors simultaneously, such as 0,1% Brilliant
Green Solution (10 mL/L) which at the same time inhibits
gram-positive flora, or Novobiocin in a concentration
between 4 and 40 mg/L.
Basal medium can be kept indefinitely in the refrigerator,
but after the addition of inhibitors, efficacy of the medium
decreases with time.
Sorting the inhibitors depending on their stability if they
are kept in the refrigerator produce the following list: bril-
liant green, novibiocine, which is effective for 2 months
in the refrigerator but only 48 hours at 37°C, and finally
iodine solution, which is only effective for 40 hours once
the inhibitor is added to the medium.
Technique
It is recommended to prepare the Base Broth, distribute
it into tubes, sterilize and cool it. Add the Brilliant Green
 Tetrathionate Media
solution and store it in the refrigerator. If you are going
to use the medium before 60 days, you may also add
novobiocin. Iodine-iodide solution has to be added just
before its use. Do not reheat the medium after one of
these additions.
Usual technique consists of adding the sample to the
medium (1:10) and then homogenizing it well. Incubate
at 37°C for a period not longer than 48 hours, since
after this time the medium loses its selectivity and the
suppressed flora may also grow. Some authors suggest
to incubate at 43°C and perform observations after 18,
24 and 48 hours, but one can get better results if the
sample from the surface of the broth is taken after 30-36
hours.
Take aliquotes with a loop and inoculate on the surface
of the selective media like SS Agar (Ref. 01-171) or
Hëktoen Enteric Agar (Ref. 01-216), etc...
References
DIN Standard 10160 Untersuchung von Fleisch und
Fleischerzeugnissen: Nachweis von Salmonellen. Ref-
erenzverfahren.
DIN Standard 10181 Mikrobiologische Milchuntersuc-
hung: Nachweis von Salmonellen. Referenzverfahren.
DOWNES, F.P. & K.ITO (2001) Compendium of meth-
ods fort he microbiological examination of foods. 4th ed.
APHA. Washington DC. USA
FDA (1998) Bacteriological Analytical Manual. 8th ed.
Revision A. AOAC International. Gaithersburg. Md. USA.
Thioglycollate Media
Thioglycollate Broth (USP
Alternative Thioglycollate
Medium)
Ref. 02-186
Specification
A medium for sterility test and the cultivation of microaer-
ophilic and anaerobic organisms. It is specially used for
viscous or turbid samples.
Formula (in g/L)
Peptone from casein ................................ 15,0
Yeast extract ............................................... 5,0
Dextrose ..................................................... 5,5
Sodium chloride .......................................... 2,5
Sodium thioglycollate ................................. 0,5
L-Cystine .................................................... 0,5
Final pH 7,1 ± 0,2
FIL-IDF Standard 93. (2001) Milk and milk products:
Research of Salmonella.
HORWITZ, W. (2000) Official Methods of Analysis. 17th
ed. AOAC International. Gaithersburg. Md.USA
ISENBERG, H.D. (1992) Clinical Microbiology Proce-
dures Handbook. Vol. 1. APHA. Washington DC. USA
ISO Standard 6579 (2002) Microbiology of food and ani-
mal feeding stuffs – Horizontal method fort he detection
of Salmonella spp.
ISO Standard 6785 (2001) Milk and Milk Products – De-
tection of Salmonella spp.
ISO (1975) Standard 3565. Meat Products: Reference
Method for detection of Salmonellae..
U.S. PHARMACOPEIA. (2002) 25th ed. <61> Microbial
Limits Test. US Pharmacopoeial Convention Inc. Rock-
ville. Md. USA.
KAUFFMAN, F. (1931) Ein Kombiniertes Anreicherungus
verfahren für Typhus und Paratyphus Bazillen.Zblt. Bakt
Microbiol. Hyg Abt. I. Orig. 119:148
MARSHALL, R.T. (1993) Standard methods for the ex-
amination of dairy products. 16th ed. APHA Washington
DC. USA.
MULLER, L. (1923) Un nouveau milieu
d’enrichiessement pour la recherche du bacille typhique
est des partyphyques. Comp. Rend. Soc. Biol. 89:434-
437.
Directions
Dissolve 29 g of powder in 1 L of distilled water, heating
if necessary to help dissolution. Distribute into suitable
containers and sterilize by autoclaving at 121°C for 15
minutes. This culture medium should always be freshly
Xi
prepared or heated at 100°C for 10 minutes before use.
R-43
S-24-37
Description
The Thioglycolate broth is a standard medium, named
also Alternative Thioglycollate Medium, formulated and
recommended by USP, NF, NIH and FDA.
It is used for sterility testing of biological products or
samples of turbid appearance where Fluid Thioglycol-
late Medium (Ref.3-187) is not suitable because of its
viscosity.
The formula of Thioglycollate broth is the same as
Thioglycollate USP Fluid Medium without resazurin and
agar.
Media must be freshly prepared, boiled, sterilised,
cooled and used within 4 hours for its inoculation.
151
 Thioglycollate Media
References
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc. London
DOWNES, F.P. & K. ITO. (2001) Compendium of Meth-
ods for the Microbiological Examination of Foods. 4th
Ed. APHA. Washington DC. USA
HORWITZ, W. (2000) Official Methods of Analysis. 17th
ed. AOAC International. Gaithersburg. Md. USA
US PHARMACOPOEIA (2002) <71> Sterility Test. 25th
ed. US Phamacopoeial Convention Inc. Rockville. Md.
USA.
Thioglycollate Fluid Medium
Xi
Ref. 03-187
R-43
S-24-37
Specification
Fluid medium for sterility testing acc. to Eur. Phar., USP,
FDA, and ISO 7937:2004 for the cultivation of microaer-
ophilic and anaerobic organisms. It is specially used for
viscous or turbid samples.
Formula (in g/L)
Peptone from casein ............................ 15,000
Yeast extract ........................................... 5,000
Dextrose ................................................. 5,500
Sodium chloride ...................................... 2,500
Sodium thioglycollate ............................. 0,500
L-Cystine ................................................ 0,500
Resazurine ............................................. 0,001
Agar ........................................................ 0,750
Final pH 7,1 ± 0,2
Directions
Dissolve 30 g of powder in 1 L of distilled water, slowly
bring to the boil, stirring until complete dissolution. Dis-
tribute into final containers and sterilize by autoclaving
at 121°C for 15 minutes. Store in dark at room tempera-
ture for not more than one month. If after the storage the
medium is pink coloured (sign of oxidation) for more than
1/3 of its depth, recreate anaerobic conditions by heating
at 100°C for 10 minutes.
Ref. 03-187 Thioglycollate
Fluid Medium
Left: anaerobical growth; center: control; right:
fermentative growth
152
Description
The Thioglycollate Fluid Medium is a standard medium
formulated and recommended by European Pharmaco-
poeia, USP, NIH and FDA.
The reducing agents thioglycollate and L-cystine ensure
an anaerobiosis which is adequate even for fastidious
anaerobes. The sulfhydryl groups of these substances
also inactivate arsenic, mercury and other heavy metal
compounds. The Thioglycollate media are thus suitable
for the examination of materials which contain heavy
metals or heavy metal preservatives.
The higher viscosity of the fluid thioglycollate medium
prevents rapid uptake of oxygen. Any increase in the
oxygen content is indicated by the redox indicator so-
dium resazurine which changes its colour to pink.
Technique
Inoculate the culture medium with the sample material
taking care that the sample reaches the bottom of the
tubes.
Incubate for at least 14 days at the optimal temperature.
Anaerobes grow in the lower part of the culture medium
container.
References
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media, CRC Press Inc., London
BREWER J.H (1940) Clear liquid medium for the
“aerobic” cultivation of anaerobes. J. Amer. Med. Assoc.
113:598-600
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 3rd ed.,
A.P.H.A., Washington D.C.
EUROPEAN PHARMACOPOEIA, (2005) § 2.6.1 Steril-
ity. 5th Ed., EDQM Council of Europe. Strasbourg.
FDA (1998).Bacteriological Analytical Manual, 8th ed.
Revision A., AOAC International. Gaithersburg. MD
HORWITZ, W., (2000) Official Methods of Analysis, 17th
ed., A.O.A.C. International. Gaithersburg. MD
ISENBERG, H.D. (Ed), (1998) Essential Procedures for
Clinical Microbiology. ASM., Washington. USA
MacFADDIN, J.F. (1985) Media for Isolation-cultivation-
identification-maintenance of medical bacteria. Vol. I.,
Williams & Wilkins. Baltimore. MD. USA
US PHARMACOPOEIA (2005) 25th Ed. § <71> Steril-
ity Test., US Pharmacopoeial Convention Inc., Rockville
MD.
 Triple Sugar Iron Agar (TSI Agar) (Eur. Phar. Agar
Medium M)
Ref. 01-192

Specification
Differential medium for identification of enterobacteria,
according ISO standard.

Formula (in g/L)

Peptone ................................................ 20,000
Meat extract ............................................ 3,000
Yeast extract ........................................... 3,000
Lactose ................................................. 10,000
Sucrose ................................................ 10,000
Dextrose ................................................. 1,000
Sodium chloride ...................................... 5,000
Ferric ammonium citrate ......................... 0,500
Sodium thiosulfate .................................. 0,300
Phenol red .............................................. 0,025
Agar ..................................................... 12,000
Final pH 7,4 ± 0,2

Directions
Dissolve 64,8 of powder in 1 L of distilled water and
bring to boiling. Dispense into tubes and sterilize at
121°C for 15 minutes. Leave to solidify with short slant
and good butts.

Description
TSI Agar is a modification of the classical Kliger’s agar.
1% of sucrose has been added to this medium to dif-
ferentiate Proteus and Hafnia (sucrose positive) from
Salmonella and Shigella (sucrose negative).
Sugar degradation with acid formation is detected by the
turning of an indicator (phenol red) to yellow, whereas
if there is alkalinization, it turns to purple. When there is
only glucose degradation, the acid production is weak
and is evaporated on the surface, so indicator may be
reoxidised producing an alkaline surface (red) and an
acid butt (yellow). If lactose or sucrose are degradated,
acid production is intense and then all of the medium
(surface and depth) turns yellow. Gas production is
detected by the formation of bubbles and occasionally
cracks in the agar.
Hydrogen sulfide production, from thiosulfate or sulfured
aminoacids of peptones, is detected by the formation
of black FeS precipitate when medium reacts with iron
salts.
Use the medium in slanted tubes with good depth EDWARD, S.P. and EWING, W.H. (1962). Identification
and short slant. Inoculate by streaking on surface and of Enterobacteriaceae. Burgess. Pub. Co. Minneapolis.
stabbing deeply. It is advisable to use tubes with cotton EUROPEAN PHARMACOPOEIA.(2005) Supp. 5.8 §
plugs, in order to allow a reoxidation of the indicator. If 2.6.13 Test for specified micro-organisms. EDQM. Stras-
screw caps are used, they must be loose. boug E.U.
Following will find the table of reading (observations) and FIL-IDF (1991) International Standard 93A. Milk and Milk
interpretation of results in TSI Agar. Products. Detection of Salmonella species.
HAJNA, A.A. (1945) Triple Sugar-Iron medium for the
References identification of the intestinal group of bacteria. J.Bact.
DOWNES, F.P. & K. ITO (2001) Compendium of Meth- 49:516-517
ods for the microbiological examination of Foods. 4th ed. HORWITZ, W. (2000) Official Methods of Analysis. 17th
APHA. Washington DC. USA ed. AOAC International. Gaithersburg. Md. USA.

153
 Triple Sugar Iron Agar (TSI AGAR) (Eur.Phar. Agar
Medium M)
ISO 3560 Standard. (1975) Reference Method for the
Detection of Salmonella in meat and meat products.
ISO 6579 Standard. (2002) Microbiology of foods and
animal feeding stuffs - Horizontal method for the detec-
tion of Salmonella spp.
ISO 6785 Stanbdard (2001) Milk and milk Products
– Detection of Salmonella spp.
ISO 10272 Standard (1995) Microbiology of foods and
animal feeding stuffs - Horizontal method for the detec-
tion of thermotolerant Campylobacter.
ISO 21567. Standard (2004) . Horizontal method for the
detection of Shigella ssp.
KRUMWIEDE, C. & L. KOHN (1917) A triple sugar modi-
fication of the Russell Double Suigar Medium. J. Med.
Res. 37:225-229
US PHARMACOPOEIA (2002)<61> Microbial Limit
Tests. 25th ed. US Phamacopeial Convention Inc. Rock-
ville. Md. USA

Todd Hewitt Broth

Ref. 02-191 as an alternative type in epidemiologic studies of group A

streptotocci as well as pathogenic microorganisms.
Specification With the addition of 15g/L of agar, medium can be solidi-
Liquid culture medium for the propagation of beta-hemo- fied and then it is an excellent substrate for the produc-
lytic streptococci and for studies about serologic typing. tion of capsules in streptococci.

Formula (in g/L) References

Meat extract .............................................. 10,0
Casein peptone ........................................ 20,0
Glucose ...................................................... 2,0
Sodium bicarbonate ................................... 2,0
Sodium chloride .......................................... 2,0
Disodium phosphate ................................... 0,4
Final pH 7,8 ± 0,2
Directions
Dissolve 36,4 g of powder in 1 L of distilled water and
sterilize by autoclaving at 121°C for 15 minutes.
TODD, E.W. and HEWITT, L.F. (1932) A new culture
medium for the production of antigenic streptococcol
haemolysin.J. Path. Bact. 35:973-974
MacFADDIN, J. (1985) Media for isolation-cultivation-
identification-maintenance of medical bacteria. Vol. 1.
Williams & Wilkins. Baltimore. USA.
ATLAS, R.M. and L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press Inc.London
ISENBERG H.D. (1992) Clinical Microbiology Proce-
dures Handbook. ASM. Washington DC.

Description
This classical medium formulation has been modified
to achieve optimal results in the growth and production
of hemolysins, which are not inhibited due to the high
buffer composition of the medium. Many official organi-
sations, such as APHA, have recommended this medium

154
 Tryptic Soy Media
Tryptic Soy Agar (TSA)
(Eur. Phar. Agar Medium B)
(Casein Soybean Digest Agar)
Ref. 01-200
Specification
General purpose solid medium containing animal and
plant peptone according ISO 9308-1 standard.
Formula (in g/L)
Casein peptone ........................................ 15,0
Soy peptone ............................................... 5,0
Sodium chloride .......................................... 5,0
Agar ......................................................... 15,0
Final pH 7,3 ± 0,2
Directions
Mix 40 g of powder in 1 L of distilled water. Let it soak
and bring to the boil to dissolve the agar. Sterilize by
autoclaving at 121°C for 15 minutes.
Description
TSA is a widely used medium containing two peptones
which support the growth of a wide variety of organisms,
even that of very fastidious ones such as Neisseria,
Listeria, Brucella, etc. It is frequently used for routine
diagnostic purposes due to its reliability and its easily
reproducible results.
The following list includes some of its most common
applications:
1.- Sensitivity testing either by the Kirbky-Bauer system
or by following the WHO guidelines. Both the sys-
tems recommend the use of the Mueller Hinton Agar
(Ref. 01-136) for verification purposes.
2.- The medium provides, with added blood,perfectly de-
fined hemolysis zones, while preventing the lysis of
the erythrocytes due to its sodium chloride content.
3.- It can be used for the preparation of an exceptionally
nutrient ‘chocolate´ agar, thanks to the richness of its
peptones.
4.- In a reducing environment or with a CO2 enriched
atmosphere, its plates provides an excellent medium
for the isolation of Brucella and Neisseria . It may be
made selective by using certain additives.
5.- Most streptococci grow in this medium though clear
differences can be observed from one species to
another.
6.- The Tryptic Soy Agar is the selective medium for the
count of urine samples although the differentiation
must be done on selective differential media.
7.- Several tests for the differentiation and identification
of staphylococci can be obtained in this medium,
provided with suitable additives.
8.- Yeast, particularly Candida species, can grow in this
medium forming very characteristic colonies.
9.- Chromogenic pseudomonads frequently produce
pigmentation on the TSA and are therefore easily
recognized.
10.- It is widely used for testing contaminated samples.
A vast bibliography documents its applications in the
food industry.
11.- It has been frequently used in the Health industry to
produce antigens, toxins,etc...
12.- Its simple and inhibitors-free composition makes it
suitable for the detection of antimicrobial agents in
food and other products.
13.- A balanced and highly nutrient value together with a
lack of fermentable carbohydrates make this medium
one of the most recommended for the strain mainte-
nance.
Tryptic Soy Broth (TSB)
(Eur. Phar. Broth Medium A)
(Casein Soybean Digest Broth)
Ref. 02-200
Specification
Highly nutrient liquid medium for the general purposes,
formulated according to USP, FDA and Eur. Phar. regula-
tions.
Formula (in g/L)
Casein peptone ........................................ 17,0
Soya peptone ............................................. 3,0
Sodium chloride .......................................... 5,0
Dipotassium phosphate .............................. 2,5
Dextrose ..................................................... 2,5
Final pH 7,3 ± 0,2
Directions
Dissolve 30 g of powder in 1 L of distilled water and
sterilize by autoclaving at 121°C for 15 minutes.
Description
The Tryptic Soy Broth was initially developed for the
cultivation of very fastidious microorganisms without the
addition of serum, blood or any other enrichment agent.
As a general purpose culture medium it supports the
growth of most organisms, both aerobes and faculta-
tives, even if their requirements are high. Due to its high
vitamin content the development of Brucella, Pasteurel-
la and Streptococcus is perfectly viable, moreover a
CO2 enriched atmosphere can further favour it.
In anaerobic conditions this broth will easily bear the
growth of Bacteroides and Clostridium species. For this
purpose, the best results can be obtained by adding
0.3% agar and 0.05% sodium azide for Clostridium.
The Tryptic Soy Broth’s superior growth-promoting prop-
erties makes it particularly suitable for the tube dilution
method for antibiotic sensitivity testing. It also achieves
good results in the detection of gram-positive cocci. The
broth can be used for bile solubility testing in pneumo-
155
 Tryptic Soy Media
cocci, and also used for catalase and coagulase assays
and for the preparation of hypersaline broths.
It is a most suitable medium for the preparation of anti-
gens and toxins in bacteria, moulds and yeasts.
TSB is used as a primary enrichment medium for food
examination. In the dairy industry it is employed for test-
ing resazurine reduction.
The medium is not suitable for maintenance purposes
since carbohydrate fermentation liberates many acids
which may threaten the organisms’ viability. Therefore,
though it allows the growth of streptococci and Neisse-
ria, these species tend to die if repeatedly subcultured in
this medium. Such fastidious organisms are best main-
tained on Cystine Tryptone Fluid Medium (CTA) (Ref.
03-045) or even TSA (Ref. 01-200) if it is not suitable or
convenient to the use of solid media.
References
US PHARMACOPOEIA (2002) 25th ed. <61> Microbial
Limit Tests. US Phsarmacopoeial Convention. Rockville.
MD.
EUROPEAN PHARMACOPOEIA (2002) 2.6.13 Tests
for Specified Microorganisms. 4th Ed. Suppl.4.2 EDQM
Council of Europe Strasbourg.
ATLAS, R.M., & LC. PARKS (1993) Handbook of Micro-
biological Media. CRC Press, Inc.London
DOWNES, F.P. & K. ITO (2001). Compendium of Meth-
ods for the Microbiological Examination of Food, 4th
ed,ASM,Washington,D.C.
PASCUAL ANDERSON, MªRª (1992) Microbiologia
Alimentaria. Diaz de Santos, S.A.Madrid,.
FDA (1998) Bacteriological Analytical Manual. 8th ed.
Revision A. AOAC International. Gaithersburg. MD
ISO 9308-1 Standard (2000) Water Quality - Detection
and enumeration of E.coli and coliform bacteria. Mem-
brane filtration method.
156
Tryptic Soy Broth w/o Dextrose
(TSB w/o Dextrose)
Ref. 02-227
Specification
Liquid culture medium for the massive production of
spores of B. stearothermophilus for the inhibitory sus-
tances in food acc. FDA-BAM
Formula (in g/L)
Tryptone ................................................. 17,00
Soy peptone ............................................. 3,00
Sodium chloride ........................................ 5,00
Dipotasium phosphate .............................. 2,50
Final pH 7,3 ± 0,2
Directions
Disolve 27,5 g of powder in 1 L of distilled water, heat-
ing if necessary. Distribute in suitable containers and
sterilize in autoclave at 121ºC for 15 minutes.
Description
TSB w/o Dextrose is produced according the formulation
from Bacteriological Analitycal Manual of Food and Drug
Administration for the massive production of spores of
Bacillus stearothermophilus used to determine the pres-
ence of inhibitory sustances in milk and dairy products.
This medium is not recommended for sugars fermenta-
tion studies because the great amount of fermentable
carbohidrates in the soy peptone.
References
MATURIN, L.J. (1998) Inhibitory substances in milk.
Qualitative Method II: B. stearothermophilus disk assay.
en FDA Bacteriological Analitical Manual. 8th Ed. Revi-
sion A. AOAC International Inc. VA.
 Tryptone Bile Agar

Ref. 01-526 The indol producer microorganisms other than E. coli are
inhibited by the bile salts and the incubation tempera-
Specification ture, but in sugar-rich samples the indol production can
Selective solid medium for the rapid enumeration of be inhibited due to the sugar concentration interferes the
Escherichia coli according ISO 9308-1 standard. tryptophanase synthesis.

Formula (in g/L) Technique

Tryptone ................................................. 20,00 A cellulose acetate membrane of 0,45 ìm pore is extend-
Bile salts #3 .............................................. 1,50 ed on the surface of the medium. A 0,5 mL of the sample
Agar ........................................................ 15,00 dilution (with Tryptone Water Ref. 03-156) inoculum is
Final pH 7,2 ± 0,2 spreaded on the membrane and the plates are incubate
in upright position at 44±1ºC for 18-24 hours.
After incubation the membrane is immersed in indole
Directions reactive using the petri dish cover as container and
Suspend 36,5 g of powder in 1 L distilled water and bring
exposed the direct sunlight for 5 minutes or to the Wood
to the boil. Distribute into suitable containers and steri-
lamp for 10 minutes. The indole positive colonies turns
lize at 121ºC for 15 minutes.
to the reddish colours (pink to deep red). The results are
expressed as number of Escherichia coli per g or mL of
Description sample.
This medium is formulated according the Anderson &
Baird-Parker Direct Plating Method (DPM) for the rapid
References
enumeration of Escherichia coli in raw meats. The
ANDERSON, J.M. & A.C. BAIRD-PARKER (1975) Appl.
method is based on the characteristically production of
Bact. 39:111-117
indol from tryptophane when the bacteria growth at 44ºC
International Commission on Microbiological Specifica-
over a cellulose acetate membrane in the surface of the
tions for Foods (1979) Can. J. Microbiol. 25:1321-1327
Tryptone bile agar.
ISO 9308-1 Standard (2000) Water Quality - Detection
The International Commission on Microbiological Stand-
and enumeration of E.coli and coliform bacteria. Mem-
ards for Food noticed that he DPM was lesser variable
brane filtration method.
and offer a better recovery and rapidity than the MPN
method for frozen samples of meat. The ISO Standard
6391:1988 also uses this medium for the E. coli enu-
meration.

157
 Tryptone Glucose Extract Media

Tryptone Glucose Extract Agar APHA-AWWA-WEF (1998) Standard Metods for the
Examinationof Water and Wastewater. 20th ed. APHA.
(TGE Agar)
Washington
HORWITZ, W. (2000) Official Methods of Analysis.
Ref. 01-082 AOAC International. Gaitherburg. MD

Specification Tryptone Glucose Extract Broth

Plate count medium for the milk and dairy products, ac-
cording to Standard Methods for Dairy Products.
for Membrane Filtration (m-TGE
Broth)
Formula (in g/L)
Meat extract ................................................ 3,0 Ref. 02-082
Casein peptone .......................................... 5,0
D (+) Glucose ............................................. 1,0 Specification
Agar .......................................................... 15,0 Non-selective liquid medium for enumerating microor-
Final pH 7,0 ± 0,2 ganisms by membrane filtration method.

Directions Formula (in g/L)

Add 24 g of powder in 1 L of distilled water. Heat to the Tryptone ................................................ 10,00
boil by constantly stirring. Dispense in suitable contain- Meat extract .............................................. 6,00
ers and sterilize by autoclaving at 121°C for 15 minutes. Dextrose ................................................... 2,00
Final pH 7,0 ± 0,2
Description
Solid medium Tryptone Glucose Extract was adopted Directions
long ago as an alternative to Nutrient Agar acc. APHA Dissolve 18 of powder in 1 L of distilled water, heating
(Ref. 01-144) and Nutrient Agar acc. British Pharmaco- if it is necessary. Distribute in suitable containers and
poeia (Ref. 01-140) for milk bacteria enumeration, being sterilize in autoclave at 121ºC for 15 minutes.
a complement to Plate Count Agar (Ref. 01-161).
Description
Technique This medium is a liquid nutritive substrate that can be
For the enumeration purposes, it is suggested to use the used in the colony count by the membrane filtration
poured plate method, and incubation at 30-32°C for 48 method for the absorbent pad impregnation The broth
hours. If the dilutions in the plate is more than 10% it is has the same formulation as Tryptone Glucose Extract
advisable to add milk to the medium. To do this, prepare Agar , except that the broth contains no agar and the
the supension of skimmed milk (Ref. 06-019) separately, ingredients are at twice the concentration.
and sterilize it for 10 minutes at 118°C. Autoclaving must
be as short as possible. Homogenize with the culture
Technique
medium which is sterilized and cooled to 50°C. The use
The sample is filtered through a membrane. In a plate
of natural milk is not recommended due the wide varia-
the absorbent pad is impregnated with the medium
tion .
avoiding any excess. The membrane is transferred on
Medium must be quickly poured into Petri dishes be-
the pad and the complete system is incubated at 35±2ºC
cause if it remains hot for too long, floccules and abnor-
for 18-24 hours. After this time total colonies are counted
mal precipitates may appear. If the sample under study
and results recorded.
is not diluted or the volume in the plate is more than 2
mL, it is not necessary to add the skimmed milk (Ref. 06-
019) because it is assumed that the sample provides the References
required growth factors. DOWNES, FP. & K. ITO (2001) Compendium of methods
for the microbiological examination of foods 4th ed APHA
Washington
References APHA-AWWA-WEF (1998) Standard Methods for the
FDA.(1998) Bacteriological Analytical Manual 8th ed
examination of water and wastewater.
Revision A. AOAC International,Gaitherburg MD
DOWNES, F.P. & K. ITO (2001) Compendium of meth-
ods for the Microbiological Examination of Foods, 4th
Ed. APHA, Washington.
MARSHAL, R.T.. (Ed.) (1992) Standard Methods for the
Examination of Dairy Products. 16th Ed. APHA. Washing-
ton.

158
 Tryptone Phosphate Water (Buffered Peptone Water)
Ref. 02-277
Specification
Dilution and nonselective pre-enrichment liquid medium
acc. to ISO 6579, 8523, 8261 and 6785 standards.
Formula (in g/L)
Peptone .................................................... 10,0
Sodium chloride .......................................... 5,0
Disodium phosphate ................................... 9,0
Potassium phosphate ................................. 1,5
Final pH 7,0 ± 0,2
Directions
Dissolve 25,5 g of powder in 1 L of distilled water.
Distribute into suitable containers and sterilize in the
autoclave at 121°C for 15 minutes.
Description
This formulation of Tryptone Phosphate Water has the
advantages of the two classical diluents for food sam-
ples: it has the property of revitalization of the peptoned
Tryptone Sulfite Neomycin Agar (TSN Agar)
Ref. 01-195
Specification
Solid selective medium for Clostridium perfringens isola-
tion.
Formula (in g/L)
Casein peptone ...................................... 15,00
Sodium sulfite ........................................... 1,00
Neomycin sulfate ...................................... 0,05
Polymyxin B .............................................. 0,02
Yeast extract ........................................... 10,00
Ferric citrate.............................................. 0,50
Agar ........................................................ 13,50
Final pH 7,2 ± 0,2
Directions
Suspend 40 g of powder in 1 L of distilled water and
bring to the boil. Dispense in suitable containers and
sterilize by autoclaving at 121°C for 15 minutes.
To obtain better results, add 20 mL/L of a solution con-
taining 1 g/L dipotassium phosphate, 0,5 g/L sodium car-
bonate and 1 g/L sodium thioglycollate just before use.
Description
This culture medium was formulated by taking the
advantage of the tolerance of Cl. perfringens to the
high concentration of sulfite, which apart from being an
inhibitor agent, provides a strong reducing environment.
Selection of Cl.perfringens is almost complete when it
is incubated at 46°C, since neomycin and polymyxin
water and the pH change absorbing capacity of the
phosphate buffer.
The composition of this diluent is made according the
specification of the ISO Standard 6579 for the detection
of Salmonella in foods.
References
ATLAS, R.M.,& L.C. PARKS (1993) Handbook of Micro-
biological Media,CRC Press, Inc.,London
PASCUAL ANDERSON, MªRª (1992) Microbiologia
Alimentaria. Diaz de Santos, S.A.,Madrid,.
ISO 6579 Standard (2002). Microbiology of food and
animal feeding stuffs. Horizontal method for the detec-
tion of Salmonella spp.
ISO 8523 Standard (1991) General guidance for the
detection of Enterobacteriaceae with pre-enrichment.
ISO 8261 Standard (2001) Milk and milk products -
General guidance for the preparation of test samples for
microbiological examination.
ISO 6785 Standard (2001) Milk and milk products - De-
tection of Salmonella spp.
included in the medium restrain the development of
Cl.bifermentans and all the accompanying gram nega-
tive bacteria.
The medium is especially suitable for the investigation of
food products, and it may be used in tubes as well as in
plates. If the incubation is not performed in an anaerobic
jar, thioglycolate buffered solution must be added or the
inoculated surface must be covered with a sterile layer of
medium.
Colonies of Cl.perfringens form very characteristic black
colonies that, if exposed to air, become decolourised by
oxidation. TSN have a very short storage period once
prepared, so it is advisable to rehydrate or reconstitute it
in small amounts and use it on the day of its preparation.
References
MARSHALL, R.S., STEENBERGEN, J.F., MacCLUNG,
L.S. (1955) Rapid Technique for the enumeration of
Clostridium perfringens. Appl. Microbiol. 13:559-563.
MOSSEL, D.A.A (1959) Enumeration of sulfite reducing
clostridia occurring in nfoods. J. sci. Food Agr. 10:662-
669
MacFADDIN, J.F. (1985) Media for Isolation-Cultivation-
Identification-Maintenance of Medical Bacteria,Williams
& Wilkins. Baltimore,USA.
ATLAS, R.M., & L.C. PARK (1993) Handbook of Micro-
biological Media,CRC Press Inc.,London
159
 Tryptone Water (Peptone Water)
Ref. 03-156
Specification
Substrate with low nutrient capacity, for the research of
indole production in coliform micro-organisms according
ISO 7251 standard.
Formula (in g/L)
Casein peptone ........................................ 10,0
Sodium chloride .......................................... 5,0
Final pH 7,2 ± 0,2
Directions
Dissolve 15 g of powder in 1 L of distilled water and dis-
pense into suitable containers. Sterilize by autoclaving at
121°C for 15 minutes.
Description
The standard protocol requires to reinoculate one loop
from each suspected tube in 10 mL of Tryptone Water.
Incubate for 48 hours at 44°C before investigating the
indole production with the Kovacs’ Reagent for indole
(Ref. 06-018).
Tryptone Yeast Extract Agar
Ref. 01-590
Specification
Solid medium for the enumeration of water microorgan-
isms acc. ISO Standard 6222.
Formula (in g/L)
Tryptone ..................................................... 6,0
Yeast extract ............................................... 3,0
Agar .......................................................... 15,0
Final pH 7,2 ± 0,2
Directions
Suspend 24 g of powder in 1 L of distilled water and
bring to the boil. Distribute into containers and sterilize
by autoclaving at 121°C for 15 minutes.
Description
This medium, formulated according to ISO Standard
6222, is for the enumeration of heterotrophic microor-
ganisms from water.
Technique
From the water sample, obtained according the ISO
Standard 5667-2 and 5667-3, make a decimal dilution
bank (see ISO Standard 6887) with Ringer Solution (Ref.
6-073) and take aliquots to 2 parallel series of plates.
Pour the Tryptone Yeast Extract Agar melted and cooled
to 45°C, and homogenize with sample (see ISO Stand-
ard 8199). Once solidified, incubate one of the series at
160
As an alternative method, the Ehrlich’s Reagent (Ref.
06-024) could also show indole production. After 48
hours of incubation at 37°C, take 0,5 mL of growth and
mix it with 0,5 mL of Ehrlich’s Reagent. Let them settle
a few minutes. If it takes on a pink colour,then the test is
positive. Colour appearance is accelerated if a few drops
of saturated solution of potassium persulfate is added.
Other authors prefer extraction and concentration of in-
dole with 1 mL of ether, and then observe on the extract
with any of the reagents mentioned above.
References
ATLAS, R.M.& L.C. PARKS (1993) Handbook of Micro-
biological Media,CRC Press Inc.,London
DOWNES, F.P. & K. ITO (2001). Compendium of Meth-
ods for the Microbiological Examination of Food. 4th Ed.
APHA,Washington
APHA-AWWA-WEP (1998) Standard Methods for the
examination of water and wastewater. 20th ed. APHA.
Washington, DC.
ISO 7251 Standard (1993) General guidance for the
enumeration of E.coli by the MPN technique.
36±2°C for 44±2 hours and the other one at 22°C for 3
days (68±4 hours).
In order to achieve a good count, select plates with
30-300 colonies. Express the results as number of
colony forming units per milliliter (cfu/mL) of the sample
for each temperature of incubation. If there are no colo-
nies with the undiluted sample express the results as “no
detected in one mL”. If there are more than 300 colonies
in the highest dilution express the results as “>300/mL”
References
ISO Standard 6222 Watewr Quality – Enumeration of
culturable microorganisms. Colony count by inoculation
in a nutrient agar culture.
ISO Standard 5667-2 (1991) Water Quality-Sampling
– Guidance on sampling techniques
ISO Standard 5667-3 (1996) Water Quality – Sampling.
–Guidance on the presevation and handling of samples
ISO Standard 6887 (1999) Microbiology- General
– Guidance for the preparation of dilutions for microbio-
logical examination.
ISO Standard 8199 (1988) Water Quality – General
guide to the enumeration of microorganisms by culture.
 Tryptophan Broth
Ref. 02-418
Specification
Liquid medium for the indole production according to
ISO standard.
Formula (in g/L)
Meat peptone ........................................... 10,0
L-Tryptophan .............................................. 1,0
Sodium chloride .......................................... 5,0
Final pH 7,2 ± 0,2
Directions
Dissolve 16 g of powder in 1 L of distilled water. Distrib-
ute into suitable containers and sterilize in the autoclave
at 121°C for 15 minutes.
Description
This broth allows the indole production from the tryp-
tophan, and therefore it is suitable for the differentiation
and identification of coliforms from water and food.
Its formulation is according to the German standards for
waters and foods.
Tryptose Media
Tryptose Agar
Ref. 01-197
Specification
Solid medium for isolation, cultivation and differentiation
of Brucella, streptococci and fastidious pathogens.
Formula (in g/L)
Tryptose ................................................ 20,000
Dextrose ................................................. 1,000
Sodium chloride ...................................... 5,000
Thiamine HCl .......................................... 0,005
Agar ...................................................... 15,000
Final pH 7,2 ± 0,2
Directions
Suspend 41g of powder in 1 L of distilled water and heat
to boiling. Dispense in tubes or flasks and sterilize it in
the autoclave at 121°C for 15 minutes.
To obtain better results, add to the molten medium, 20
mL/L of a solution composed by dipotassium phosphate
1 g/L, sodium carbonate 0,5 g/L and sodium thioglyco-
late 1 g/L just before using the medium.
Technique
Medium is inoculated with the previously isolated culture,
and then incubated at 30-32°C for 24-48 hours.
Indole production is observed adding a few drops of
Kovacs’ Reagent (Ref. 06-018) over the broth (with or
without previous extraction) and shaking gently. Forma-
tion of a red ring indicates indole presence.
References
VERORDNUNG über Trinkwasser und über Wasser für
Lebensmittelbetriebe vom 12-12-1990. Bundesgesatbl. I.
2613-2619.
BUNDESGESMELHEITSAMT: Amtliche Samnulung von
Untersuchungs verfahren nach #35LMBG. Beuth Verlag.
Berlin-Köln.
ISO 9308-1 Standard (2000) Water Quality. Detection
and enumeration of Escherichia coli and coliform bacte-
ria. Part 1: Membrane filtration method.
ISO 6785 Standard (2001) Milk and milk products. De-
tection of Salmonella spp.
ISO 21567. Standard (2004) . Horizontal method for the
detection of Shigella ssp.
Tryptose Broth
Ref. 02-197
Specification
Liquid culture medium for massive culturing of fastidious
microorganisms.
Formula (in g/L)
Tryptose ................................................ 20,000
Dextrose ................................................. 1,000
Sodium chloride ...................................... 5,000
Thiamine HCl .......................................... 0,005
Final pH 7,3 ± 0,2
Directions
Dissolve 26 g of powder in 1 L of distilled water, heating
up if necessary. Dispense into suitable containers and
sterilize by autoclaving at 121°C for 15 minutes.
Description
Tryptose culture media are suitable, essencially for the
growth of fastidious strains, such as Listeria, Pasteurella,
Brucella, etc. despite the fact that nowadays it seems
there is a trend towards the use of more defined media
such as Brucella Agar (Ref. 01-042) and Broth (Ref.
161
 Tryptose Media

02-042), which provide better results. Tryptose Agar and Description

Broth have been recommended by several National and
International Organisations for Brucelosis Control and for
the maintenance, propagation and cultivation of stand-
ardized strains. They may be successfully used to dif-
ferentiate the several types of Brucella with the addition
of suitable indicators. In this case, its use is analogous to
the Brucella Broth (Ref. 01-042).
With the adequate additives (crystal violet, sodium azide
etc.) the media may become very selective and efficient,
and their nutrient conditions may be improved if citrate
and blood are added, although glucose is not very suit-
able for observing hemolysis.
References
CASTAÑEDA, M.R. (1947) A practical method for rutine
blood cultures in brucellosis. Proc. Soc. Exp.Biol. Med.
64:114-115
HAUSSLER, W.J. (1976) Standard Methods for the
Examination of Dairy Products,9th Ed. APHA.
MARSHALL, R.T.. (1992) Standard Methods for the Ex-
amination of Dairy Products. 16th Ed. APHA. Washington.
RENNER, E.D., K.J. McMAHUN (1981) Brucellosis in Di-
agnostic Procedures for Bacterial Mycotic and Parasitic
Infections. 6th Ed.,APHA, Washington.DC
ATLAS, R.M. & LC. PARKS (1993) Handbook of Micro-
biological Media. CRC Press Inc.,London.
DOWNES,F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 4rdEd.
APHA.Washington
FDA (1998) Bacteriological Analytical Manual. 6th ed.
Revision A. AOAC International. Gaithersburg. MD
MURRAY,P.R., E.J. BARON, M.A PFALLER, F.C. TENO-
VER, & R.H. YOLKEN (1995) Manual of Clinical Microbi-
ology. 6th ed. APHA. Washington DC .
Tryptose Phosphate Broth is a recommended medium
for the cultivation and propagation of microorganisms
that have strong needs, such as streptococci, menin-
gococci, and Brucella. It has also been also used to
determinate antibiotic sensitivity testing by the dilution in
tube method.
This medium has been used as primary diluent and
emulsifier in dairy products for determination of Brucella,
but it is really effective for the cultivation of many strepto-
cocci and to test the bile solubility of these organisms.
When it is used to isolate streptococci, it is suggested to
add 0,1% Agar to render it into a fluid medium. Should a
very selective medium be desired, add 2,5% of sodium
azide. To get a solid medium, add 1,5% of Agar.
References
GINSBERG, H.S. (1955) Tryptose Phosphate Broth as
Supplementary Factor for Maintenance of Hella Cell Tis-
sue Cultures. Proc. Soc. Exper. Biol. Med. 89(1):66-71.
WAISBREN, B.A. (1951). The Tube Dilution Method of
Determining Bacterial Sensitivity to Antibiotics. Am. J.
Clin. Path 21:884.
BALOWS, A., W.J. HAUSSLER (1981) Diagnostic Pro-
cedures for Bacterial Mycotic and Parasitic Infections.
6thEd,APHA. Washington.
ATLAS, R.M., & J.W. SNYDER (1995) Handbook of Me-
dia for Clinical Microbiology. CRC Press. London
FDA (1998) Bacteriological Analytical Manual. 8th ed.
Revision A. AOAC International. Gaithersburg. MD.

Tryptose Phosphate Broth

Ref. 02-199

Specification
Liquid culture medium, with glucose and buffer, for
cultivation of fastidious microorganisms and for tissues
culture media.

Formula (in g/L)

Tryptose .................................................... 20,0
Dextrose ..................................................... 2,0
Sodium chloride .......................................... 5,0
Disodium phosphate ................................... 2,5
Final pH 7,3 ± 0,2

Directions
Dissolve 29,5 g of powder into 1 L of distilled water. Dis-
pense into suitable containers and sterilize by autoclav-
ing at 121°C for 15 minutes.

162
 Tryptose Lauryl Sulfate Media
Tryptose Lauryl Sulfate Broth
Ref. 02-108
Specification
Liquid medium for the detection and enumeration of
coliform bacteria according IDF-FIL 73B and ISO 4831
and 7251 standards.
Formula (in g/L)
Tryptose .................................................. 20,00
Sodium Lauryl sulfate ............................... 0,10
Lactose ..................................................... 5,00
Dipotassium phosphate ............................ 2,75
Monopotassium phosphate ...................... 2,75
Sodium chloride ........................................ 5,00
Final pH 6,8 ± 0,2
Directions
Dissolve 35,6 g of powder in 1 L of distilled water. Dis-
tribute into tubes or containers fitted with the inverted
Durham tubes (for gas). Sterilize at 121°C for 15 min-
utes. As for the double concentration medium, dissolve
71,2 g/L and proceed as indicated above.
Preferably store the broth at room temperature, and use
screw-capped bottles to prevent evaporation of water.
Refrigerated broth generally becomes cloudy or forms
precipitates but clears at incubation at room tempera-
ture. However, clarity is not important as only the gas
production is significant criterion.
Description
Laurylsulfate broth is used for MPN Presumptive Test
of coliforms in water and sewage, confirmatory test of
lactose fermentation with gas production for milk and
detection of coliforms in food.The high nutrient quality
and the presence of phosphate buffer in this medium en-
sures rapid growth and increased gas production, even
by slow lactose-fermenting coliforms.
This medium can be used as Presumptive broth for
E.coli (by fluorescent reaction) if before sterilization
MUG (Ref. 06-102CASE) is added.
Technique
If the volume of sample to inoculate is substantial, then
reconstitute the medium at such a concentration which
would remain normal, once the sample has been added
to it.
Incubate at 37°C for 24-48 hours. Lactose fermenta-
tion within 48 hours, shown by the appearance of gas in
the Durham tubes , indicates the presence of coliform
bacteria.
Verification can be done by the isolation and identifica-
tion of coliforms on an appropiate medium.
References
F.D.A. (1998) Bacteriological Analytical Manual. 8th ed.
Revision A. AOAC International Gaitherburg, MD.
FIL-IDF Standard 73B (1998) Milk and milk products.
Enumeration of coliforms. IDF. Brussels.
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 4th. ed.
APHA. Washington.
MARSHALL R.T. (1992) Standard Methods for the ex-
amination of dairy products. 16th ed. APHA. Washington
APHA-AWWA-WPCF (1995) Standard Methods for the
examination of water and wastewater. APHA. Washing-
ton
HORWITZ, W. (2000) Official methods of Analysis.17th
ed. AOAC International. Gaithersburg. MD.
ISO 4831 Standard (1991) General guidance for the
enumeration of coliforms - MPN technique.
ISO 7251 Standard (1993) General guidance for enu-
meration of E.coli by MPN technique.
Tryptose Lauryl Sulfate
Mannitol Tryptophan Broth
Ref. 02-460
Specification
Liquid medium for the production of indole and gas in
a single tube, according to the ISO 9308-1 and 9308-2
standards.
Formula (in g/L)
Tryptose .................................................. 20,00
Mannitol .................................................... 5,00
Sodium chloride ........................................ 5,00
Monopotassium phosphate ...................... 2,75
Dipotassium phosphate ............................ 2,75
Sodium Lauryl sulfate ............................... 0,10
L-Tryptophan ............................................ 0,20
Final pH 6,8 ± 0,2
Directions
Dissolve 35,8 g of powder in 1 L of distilled water,
heating up if necessary. Distribute in tubes containing
Durham´s tubes and sterilize by autoclaving at 121ºC for
15 minutes. Do not overheat.
Description
This broth is proposed in the ISO 9308-1 standard as an
alternative medium for the production of indole and gas
in a single tube and to confirm the presence of thermo-
tolerant coliforms and the presumptive presence of E.coli
in the water sample.
163
 Tryptose Lauryl Sulfate Media

Technique
Tubes with medium are inoculated from suspicious
colonies on the already incubated membrane and then
are incubated at 44ºC for 24 hours. Gas production, that
appears in the Durham tubes, confirms the presence of
thermotolerant coliforms.
If after the addition of 0,2-0,3 mL of Kovacs Reagent
(Ref. 06-018) a cherry red colour appears on the top
surface of the medium (Indole +), a presumptive pres-
ence of E.coli is considered and it should be confirmed
with other tests.

References
ISO Standard 9308-1 (1990) Water Quality Detection
and Enumeration of coliform organisms, thermotolerant
coliform organisms and presumptive E.coli. Part 1. Mem-
brane filtration method. Part 2. MPN method.

164
 Tryptose Sulfite Cycloserine Agar Base (TSC Agar Base)
Ref. 01-278
Specification
Solid selective and differential medium for isolation and
presumptive identification of Clostridium perfringens ac-
cording to ISO 7937 Standard.
Formula (in g/L)
Tryptose .................................................. 15,00
Soya peptone ........................................... 5,00
Yeast extract ............................................. 5,00
Sodium metabisulfite ................................ 1,00
Ferric ammonium citrate ........................... 1,00
Agar ....................................................... 20,00
Final pH 7,6 ± 0,2
Directions
Suspend 47 g of powder in 1 L of distilled water and let it
soak . Heat to boiling and distribute into suitable contain-
ers, but not more than 250 mL in each one. Sterilize in
the autoclave at 121°C for 15 minutes. Let it cool to 60°C
and add 1 flask of CycloserineSelective Supplement
(Ref. 06-116CASE) to every 250 mL of medium. Mix well
and pour it into plates. If it is desired to include egg yolk,
then add Egg Yolk Sterile Emulsion (Ref. 06-016) in a
concentration of 80 mL/L, simultaneously to the antibi-
otic,
Description
The medium is a modification of the classical TSN Agar
(Ref. 01-195) in which the traditional antibiotics, polymix-
in and neomycin have been replaced by cycloserine.
Cycloserine has been found more selective for Clostrid-
ium perfringens, and it seems also to reduce the trend to
produce diffuse blackening. On the other hand, Clostrid-
ium perfringens is more resistant to cycloserine than to
sulfadiazine, polymixin and neomycin, which permits a
better dosage.The medium has sodium metabisulfite and
ferric ammonium citrate to manifest the reducing capac-
ity of sulfite, and in this way, three
differential characteristics of this anaerobic species may
be verified with just one assay. These characteristics are
Left and center: Clostridium perfringens
ATCC 13124; right: control.
sufite reduction, growth at 46°C and cycloserine resist-
ance.
Nevertheless, it has to be noted that cycloserine does
not tolerate temperatures above 100°C and its stability
in a solution is very restrained, even then it is used in
alkaline media. Therefore, it is advisable to prepare the
exact number of plates that are going to be used.
Anyway, if it is desired, an active solution of cycloserine
in phosphate buffer at pH 8,0 may be prepared (Dipotas-
sium phosphate 16,73 g/L and monopotassium phophate
0,52 g/L) and if it is maintained refrigerated, it can be
used for approx. 5 days.
Technique
The standard procedure recommends surface inocula-
tion of the samples or their dilutions, and once absorbed,
to pour a second layer as cover and seal for anaerobio-
sis. After an incubation at 46°C for 18-20 hours, proceed
to enumerate the black colonies that appear in the plate.
References
SMITH, L.D. (1981) Clostridial Anaerobic Infections, in
Diagnostic Procedures for Bacterial,Mycotic and Para-
sitic Infections. 6thEd. APHA,Washington.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press Inc.,London
DOWNES, F:P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food. 4rd ed.
APHA Washington
ISO 7937 Standard (2004) Microbiology of food and
animal feeding stuffs- Horizontal method for enumeration
of Clostridium perfringens - Colony-count technique.
DIN Standard 10165. Referenz Verfahren fur Bes-
timmung von Clostridium perfringens. Fleisch und
Fleischerzeugnissen.
FDA (1998) Bacteriological Analytical Manual 8th ed.
Revision A. AOAC International. Gaithersburg. MD.
ISO 6461-2:1986 Standard Water Quality - Detection
and enumeration of the spores of sulfite - reducing
anaerobes (Clostridia) - Part 2: Method by MF.
165
 Urea Media
Urea Agar Base
acc. to Christensen
Ref. 01-261
Specification
Solid medium for detection of urea lysis, according to
ISO 6579, 6340 and 6785 standards and DIN 10160
standard.
Formula (in g/L)
Gelatin peptone ...................................... 1,000
Dextrose ................................................. 1,000
Sodium chloride ...................................... 5,000
Monopotassium phosphate .................... 2,000
Phenol red .............................................. 0,012
Agar ...................................................... 15,000
Final pH 7,0 ± 0,2
Directions
Suspend 24 g of powder in 950 mL of distilled water and
bring to the boil. Sterilize in the autoclave at 121°C for
15 minutes. Let it cool to 50-55°C. Add 50 mL of Urea
Sterile Solution 40% (Ref. 06-083) and mix well. Distrib-
ute aseptically in tubes and let them solidify in slanted
position.
Description
Urea Agar complies with Christensen’s specifications,
and it is recommended for the detection of urolytic or
urea degrading microorganisms, especially Enterobacte-
riaceae, although it can be used also with gram positive
bacteria.
Technique
Pure culture is inoculated by surface streaking, and then
incubated at 37°C. Generally, organisms with strong
urease activity can be read after 3-5 hours.
Reaction is evident as the medium changes its colour. It
turns form orange to pink-fuchsia, due to a strong alkali-
nization produced by ammonia release.
References
CHRISTENSEN, W.B. (1946) Urea decomposition as
means of differentiating Proteus and Paracolon cultures
from each other and from Salmonella and Shigella types.
J.Bact. 52:461.
EDWARDS and EWING (1962) Identification of Entero-
bacteriaceae. Burgess Pub. Co.
ATLAS, R.M., & L.C. PARK (1993) Handbook of Micro-
biological Media. CRC Press Inc.London
DOWNES, F.P. & K. ITO (2001) Compendium of meth-
odscfor the Microbiological Examination of Foods 4th ed.
APHA. Washington
MARSHALL, R.T. (1992) Standard Methods for the ex-
amination of Dairy Products. 16th ed APHA. Washington
DC
ISO 6785 Standard (2001) Milk and milk products - De-
tection of Salmonella spp.
166
ISO 6340 Standard (1995) Water Quality - Detection of
Salmonella spp.
ISO 6579 Standard (2002) Microbiology of food and ani-
mal feeding stuffs. Horizontal method for the detection of
Salmonella spp.
DIN Standard 10160. Untersuchung von Fleisch und
Fleischerzeugnissen. Nachweiss von Salmonellen. Ref-
erenzverfahren.
FIL-IDF 93 Standard (2001) Detection of Salmonella.
Urea Broth Base
Ref. 02-202
Specification
Liquid diagnostic medium according to Rustigian and
Stuart formulation.
Formula (in g/L)
Monopotassium phosphate ...................... 9,10
Disodium phosphate ................................. 9,50
Yeast extract ............................................. 0,10
Phenol red ................................................ 0,01
Final pH 6,8 ± 0,2
Directions
Dissolve 19 g of powder into 950 mL of distilled water
and sterilize by autoclaving at 121°C for 15 minutes. Let
it cool to 50-55°C and then add 50 mL of Urea Sterile
Solution 40% (Ref. 06-083). Mix well and dispense in
hemolysis tubes (3,0 mL/tube).
Description
According to Rustigian and Stuart, this Urea Broth is
excellent for diagnosing enterobacteria, since within this
family, only Proteus may alkalinize the medium over pH
8,1. Despite the fact that some authors prefer a buffer of
potency 10 or 100 times lower to obtain faster results for
saving the time (about 2 hours) does not compensate for
the instability of the medium.
Urease production is shown by the indicator turning to
dark pink, produced by strong alkalinization by ammo-
nium. With plenty of inoculum (2-3 loops in 3-5 mL of
medium), Proteus produces the colour change after 6-8
hours, meanwhile other positive enterobacteria need up
to 24-48 hours.
References
RUSTIGIAN, R., STUART, C.A. (1941) Decomposition of
urea by Proteus. Proc. Soc. Exp. Biol. Med. 47:108
DOWNES, FP & K ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food, 4rd ed.
APHA,Washington.
FDA (1998) Bacteriological Analytical Manual. 8th ed.
Revision A. AOAC International. Gaithersburg. MD
PASCUAL ANDERSON, MªR. (1992) Microbiología
Alimentaria. Diaz de Santos, S.A.,Madrid.
 Violet Red Bile Media
Violet Red Bile Agar (VRB Agar)
Ref. 01-164
Specification
Selective and differential agar medium for the detection
and enumeration of coliforms in milk and other dairy
products, according to APHA, ICMSF, FIL-IDF and ISO
standard.
Formula (in g/L)
Yeast extract ........................................... 3,000
Gelatin peptone ...................................... 7,000
Bile salts #3 ............................................ 1,500
Lactose ................................................. 10,000
Sodium chloride ...................................... 5,000
Neutral red .............................................. 0,030
Crystal violet ........................................... 0,002
Agar ...................................................... 13,000
Final pH 7,4 ± 0,2
Directions
Suspend 39,5 g in 1 L of distilled water. Bring to the
boil and distribute into final containers. Sterilization at
121°C for 15 minutes is optional, but If the medium is
to be used on the same day of preparation it need not
be sterilized. No sterilization improves the recovery of
stressed microorganisms.
Description
The Violet Red Bile Agar corresponds to the classic
formulation of standardized media for the screening of
coliforms in milk and other dairy products.This medium
has been adopted for the enumeration of coliforms as
well as for differentiating between lactose fermenting and
non-lactose fermenting organisms, due to its contents of
crystal violet and bile salts, whose inhibiting or selecting
properties have been widely confirmed.
This medium can be used as Presumptive medium for
E.coli (by fluorescent reaction) if before sterilization
MUG (Ref. 06-102CASE) is added.
Technique
The recommended procedure is the massive inocula-
tion directly on Petri dishes, with the molten agar cooled
to 47°C. Observations can be read after 24 hours of
incubation at 37°C.
The size of the colonies ranges from 2 to 5 mm, depend-
ing on the amount per plate. The enterococci that might
eventually develop will appear small in size and pink
coloured. Lactose fermenting enterobacteria acquire a
dark red colour with a clearing zone around them, while
lactose non-fermenting ones form colourless colonies.
References
DOWNES, F.P. & K. ITO (2001). Compendium of Meth-
ods for the Microbiological Examination of Food. 4rd ed.
APHA, Washington. DC
MARSHALL, R.T. (1992) Standard Methods for the Ex-
amination of Dairy Products,16thEd. APHA, Washington.
DC
ICMSF (1978). Microorganisms in Food, University of
Toronto Press.
ISO (1986) Standard 5541-1 Milk and Milk Products.
enumeration of coliforms. Colony count technique at
30ºC
FIL-IDF. (1998) Standard 73B. Enumeration of coliform
bacteria.
PASCUAL ANDERSON, MªR. (1992) Microbiología
Alimentaria. Diaz de Santos, S.A.,Madrid,.
Violet Red Bile Dextrose Agar
(VRBD Agar)
Ref. 01-295
Specification
Solid medium for the enumeration of enterobacteria ac-
cording ISO 8523 standard.
Formula (in g/L)
Yeast extract ........................................... 3,000
Gelatin peptone ...................................... 7,000
Bile salts #3 ............................................ 1,500
D (+) Glucose ....................................... 10,000
Sodium chloride ...................................... 5,000
Neutral red .............................................. 0,030
Crystal violet ........................................... 0,002
Agar ...................................................... 13,000
Final pH 7,4 ± 0,2
Directions
Suspend 39,5 g in 1 L of distilled water and let it soak.
Bring to the boil and sterilize by autoclaving at 121°C for
15 minutes. If the medium is to be used on the same day
of preparation it need not be sterilized. Prolonged heat-
ing in thermostatic bath could cause slight precipitates.
Description
This medium is a modification of the Violet Red Bile Agar
(Ref.1-164) and the MacConkey Agar (Ref.1-118) as
described by Mossel et al. These authors proved that the
addition of glucose to the Violet Red Bile Agar favoured
both the growth of the most fastidious enterobacteria
and the recovery of those having suffered from adverse
conditions. Later on, Mossel himself realized that by
removing the lactose and keeping the glucose, the
medium’s efficiency remained stable. Furthermore, an
economic improvement occurred since the same amount
of product allows the reconstitution of more litres of the
medium.
167
 Violet Red Bile Media
This medium can be used as presumptive medium for
E.coli (by fluorescent reaction) if before sterilization
MUG (Ref. 06-102CASE) is added.
Technique
The Violet Red Bile Dextrose Agar is widely used in the
analysis of food, medicines and cosmetics. It is particu-
larly indicated for the recovery of bacteria which have
been damaged during preparation. In such cases, a
progressive enrichment is recommended in TSB (Ref.
02-200) first and in EE Broth (Ref. 02-064) next. Once
the enriched culture is ready it can be inoculated by
profound inoculation in tubes or by isolation in Violet Red
Bile Dextrose Agar plates.
For the count of enterobacteria, the technique to use will
be the massive inoculum described for the Violet Red
Bile Agar.
Observations can be read after 24 hours of incubation
at 31°C. Enterobacterial colonies form an intense purple
colouring surrounded by a clearer zone . If enterococci
colonies eventually develop, then they will be small and
pink coloured.
Violet Red Bile Lactose
Dextrose Agar (VRBLD Agar)
(Eur. Phar. Medium F)
Ref. 01-220
Specification
Solid selective medium for the detection of Enterobacte-
riaceae according the European Pharmacopoeia.
Fórmula (in g/L)
Yeast extract ........................................... 3,000
Peptone .................................................. 7,000
Sodium chloride ...................................... 5,000
Bile salts # 3 ........................................... 1,500
Lactose monohydrate ........................... 10,000
Glucose monohydrate .......................... 10,000
Neutral red .............................................. 0,030
Crystal violet ........................................... 0,002
Agar ...................................................... 15,000
Final pH 7,4 ± 0,2
Directions
Suspend 51.5 g of powder in 1 L of distilled water and
heat to the boil. Pour into Petri dishes inmediately. Do
not sterilize in autoclave nor overheat.
Description
This medium developed in 1962 by Mossel et al. as
more effective than MacConkey Agar for the detection of
Enterobacteriaceae in foods, has been officially adopted
by the European Pharmacopoeia for the microbiological
examination of non-sterile products. The medium is spe-
168
cially used in the recovery of process-stressed bacteria
using a progressive enrichment technique.
This medium can be used as presumptive medium for
E.coli (by fluorescent reaction) if before sterilization
MUG (Ref. 06-102CASE) is added.
Technique
Sample is diluted 1:10 in Lactose Broth (Ref. 02-105)
and incubate 2-5 hours at 35-37 ºC. Then a volume of
this pre-enrichment is ten fold dilute in EE Broth (Ref.
02-064) and incubate at 35-37ºC for 18-24 hours. From
this enrichment the surface of several plates of VRBDL
Agar are inoculated. The product passes the test if after
18-24 hours of incubation at 35-37ºC there is no growth
of gram negative bacteria in any plate.
In the surface of the VRBDL Agar the Enterobacteriace-
ae colonies are deep purple in colour surrounded by a
clearing zone. Sometimes are present little colonies from
Pseudmonas or Aeromonas that can be easy differenti-
ated by the oxidase test.
References
MOSSEL, D.A.A., MENGERINK and SCHOLTS H.H.
(1962) Use of a modified MacConkey Agar medium for
the selective growth and enumeration of all Enterobacte-
riaceae. J. Bact. 84:381.
MOSSEL, D.A.A., VISER, M. and CORNELISSEN,
A.M.R. (1963) The examination of food for Enterobacte-
riaceae using a test of the type generally adopted for the
detection of Salmonellae. J. Appl. Bact.(26) 444-452.
MOSSEL, D.A.A. (1985) Media for Enterobacteriaceae.
Int. J. Food Microbiol. 2:27-35
ISO 5552 Standard (1997) Meat and Meat Products.
Detection and enumeration of Enterobacteriaceae
without resuscitation. MNP technique and colony-count
technique.
PASCUAL ANDERSON, MªR. (1992) Microbiología
Alimentaria. Diaz de Santos, S.A.,Madrid.
MOSSEL, D.A.A. and M.A. RATTO (1970) Rapid detec-
tion of sub-lethally impaired cells of Enterobacteriaceae
in dried foods Appl. Microbio¡ 20: 273-275.
EUROPEAN PHARMACOPOEIA 3ª Edición (Suppl.
1999) Cap. 2.6.13 Microbiological examination of non
sterile products. Tests for specified organisms. Council
of Europe. Strasbourg
ISO 8523 Standard (1991) General guidance for the
detection of Enterobacteriaceae with pre-enrichment.
 Ref. 01-164 Violet Red Bile Agar
Escherichia coli ATCC 25922
control
Vogel Johnson Agar (VJ Agar)
Ref. 01-206
Specification
Solid and very selective medium for isolation and identifi-
cation of staphylococci according ISO 22718 standard.
Formula (in g/L)
Casein Peptone .................................... 10,000
Yeast Extract .......................................... 5,000
Mannitol ................................................ 10,000
Dipotassium phosphate .......................... 5,000
Litium chloride ........................................ 5,000
Glycine ................................................ 10,000
Phenol Red ............................................. 0,025
Agar ...................................................... 15,000
Final pH 7,2 ± 0,2
Directions
Suspend 60 g of powder in 1 L of distilled water and
bring to the boil. Dispense in suitable containers and
sterilize at 121°C for 15 minutes. Cool it to 50°C approx.
and add aseptically 20 mL of Potassium Tellurite Solu-
tion 1% (Ref. 06-089) or 6,0 mL of Potassium Tellurite
Solution 3.5% (Ref. 06-011). Do not reheat after tellurite
addition.
Description
VJ Agar is a selective medium for detection and enu-
meration of pathogenic staphylococci.
The medium’s strong selective action is due to lithium
chloride, glycine and potassium tellurite presence. They
inhibit almost all the accompanying organisms, mean-
while staphylococci are not affected. Although staphy-
Salmonella typhimurium
ATCC 14028
lococci may reduce tellurite to tellurium, lithium may per-
form some action that is compensated by glycine.
Moreover a high correlation between tellurite reduction
and mannitol fermentation has been proved, and this is
shown in the medium by the indicator turning to yellow
due to the amount of acid produced.
The medium’s selectivity avoids, in the first 24 hours, the
development of any other bacteria, so massive inocu-
lation is permited. Nonetheless, after this period, it is
possible that other bacteria may appear like micrococci,
which produce tiny colonies, and staphylococci that
ferment mannitol and coagulase negative, therefore it is
recommended to verify this last test separately.
Due to reduced tellurite, staphylococci generally appear
as black colonies over red medium (if they do not fer-
ment mannitol) or yellow medium (if they do, and these
are presumptive pathogen). Saprophytic staphylococci
(S.epidermidis, S.saprophiticus and S.intermedius) have
a grey-black colour and are mannitol negative. Complete
medium may be stored up to 1 week in the refrigerator.
Do not remelt it after tellurite is added.
References
VOGEL and JOHNSON (1960) A modification of the
tellurite-glycine medium for the use in the identification of
Staphylococcus aureus. Pub. Health. Lab. 18:131-133.
US PARMACOPOEIA (2002) 25th ed. <61> Microbial Limit
Tests. Pharmacopoeial Convention. Rockville. MD
ATLAS, R.M.,& L.C. PARK (1993) Handbook of Microbio-
logical Media. CRC Press Inc.,London
FDA (1998) Bacteriological Analytical Manual. 8th ed.
Revision A. AOAC International. Gaithersburg. MD
ISO 22718:2006 Standard. Cosmetics – Detection of
Staphylococcus aureus.
169
 Wilkins-Chalgren CN Modified Fluid Medium
(WCCN Modified Fluid Medium)

Ref. 03-408 Directions

Suspend 87,5 g of powder in 1 L of distilled water. Bring
Specification to the boil. Sterilize in the autoclave at 121°C for 10
Fluid medium for the cultivation and enumeration of minutes.
anaerobic bacteria by the impedometry technique. Prepared medium takes on a dark colour due to the high
concentration of sugar. Do not reheat.
Formula (in g/L)
Tryptone ............................................. 10,0000 Description
Gelatin peptone .................................. 10,0000 This medium is a modification of the Wilkins-Chalgren
Gelatin .................................................. 8,0000 Anaerobic Medium for impedometric methods.
Dextrose ............................................. 41,0000
Yeast extract ......................................... 5,0000 References
Sodium chloride .................................... 5,0000 WILKINS, T.D. and S. CHALGREN (1976) Medium for
Ammonium sulfate ................................ 5,0000 use in antibiotic susceptibility testing of anaerobic bacte-
L-Arginine ............................................. 1,0000 ria. Antimicrob. Agents Chemorther 10:6:926.
Sodium pyruvate .................................. 1,0000
Sodium bicarbonate ............................. 1,0000
Ferrous sulfate ..................................... 0,1000
Calcium chloride ................................... 0,1000
Hemine ................................................. 0,0050
Menadione ............................................ 0,0005
Agar ...................................................... 0,3000
Final pH 7,1 ± 0,2

WL Nutrient Media

WL Nutrient Agar Directions

Suspend 80 g of the powder in 1 L of distilled water.
Ref. 01-210 Mix thoroughly. Heat with frequent agitation and boil for
one minute. If a final pH of 6,5 is desired, the pH may be
adjusted with one percent aqueous sodium carbonate,
Specification using about 30 mL per litre of medium.
Solid medium for the cultivation and enumeration of
Dispense and sterilize the medium by autoclaving at
yeast and bacteria for microbiological control in brewing
121°C for 15 minutes.
and other fermentation industries.
The WL Differential Agar has the same formula as the
WL Nutrient Agar with the addition of 2 flasks/L of Cy-
Formula (in g/L) cloheximide Selective Supplement Ref. 06-022CASE.
Yeast extract ......................................... 4,0000
Tryptone ............................................... 5,0000
Description
Dextrose ............................................. 50,0000
WL Nutrient Agar was formulated by Green and Gray in
Monopotassium phosphate .................. 0,5500
the Wallerstein Laboratory for use in the control of indus-
Magnesium sulfate ............................... 0,1250
trial fermentations, particularly the processing of beer. It
Calcium chloride ................................... 0,1250
is recommended for examination of worts, beers, liquids
Potassium chloride ............................... 0,4250
containing yeast and other materials.
Iron (III) chloride ................................... 0,0025
WL Nutrient Agar has a pH of 5,5 which is optimal for
Manganese sulfate ............................... 0,0025
the enumeration of brewers´yeast . If bakers or distiller´s
Bromcresol green ................................. 0,0220
yeast is to be examined, the pH should be adjusted to
Agar .................................................... 20,0000
6,5 (better yields). When cultivating the microorganisms
Final pH 5,5 ± 0,2
from an alcoholic mash, tomato juice should be added to
the medium.

170
 WL Nutrient Media
WL Differential Agar contains cycloheximide to suppress
yeast and any other moulds which may be present; this
medium allows reliable counting of all bacteria which
may be encountered in the tests performed in brewery
laboratories.
Technique
Dilute the sample material and spread 0,1 mL onto WL
Nutrient Agar or WL Differential Agar.
The WL Nutrient Agar and WL Differential agar are used
together, one plate with nutrient agar and two with the
differential agar.
The WL Nutrient Agar plate is incubated aerobically to
obtain a total count, mainly of yeast colonies. One WL
Differential Agar plate is incubated aerobically for growth
of acetic acid bacteria, Flavobacterium, Proteus, and
other organisms; the second plate is incubated anaerobi-
cally for detection of such organisms as lactic acid bacilli
and Pediococcus species.
Plates prepared with both the media are generally incu-
bated at 25°C, if brewing materials are being studied,
and at 30°C for baker´s yeast and alcohol mash sam-
ples. Incubation may be continued for a week, or even
for ten days to two weeks, depending upon the flora
present. Counts are made at the intervals during the
incubation period.
WL Nutrient Broth
Ref. 02-210
Specification
Liquid medium for the microbial control of industrial fer-
mentations and massive cultivation of yeast.
Formula (in g/L)
Yeast extract ......................................... 4,0000
Tryptone ............................................... 5,0000
Dextrose ............................................. 50,0000
Monopotassium phosphate .................. 0,5500
Magnesium sulfate ............................... 0,1250
Calcium chloride ................................... 0,1250
Potassium chloride ............................... 0,4250
Ferric chloride ....................................... 0,0025
Manganous sulfate ............................... 0,0025
Bromcresol green ................................. 0,0220
Final pH 5,5 ± 0,2
Directions
Dissolve 60 g of powder in 1 L of distilled water and dis-
pense into suitable containers. Sterilize by autoclaving at
121°C for 15 minutes. Should a pH 6,5 be desired, ad-
just it by adding 30 mL of Sodium carbonate solution 1%.
To obtain 1 L of WL Differential Broth just add, asepti-
cally, 2 flasks of Cycloheximide Selective Supplement
(Ref. 06-022CASE) to 1 L of WL Nutrient Broth, after
sterilization.
Description
WL Medium was developed in the Wallerstein Labora-
tories for industrial uses, since it allows to differentiate
between beer yeast and wild yeasts contaminants.
Adjusting pH to 6,5 is very advisable to enumerate bread
and alcohol yeast. The medium also allows bacterial
growth and it is possible then to count the contaminant
bacteria of fermented liquors, but it is recommended to
use WL Differential Broth for this assay, since it inhibits
yeast growth.
The WL Nutrient Broth is useful to enumerate cells by
the MPN technique. Alternatively, it can be used as an
enrichment broth previous to the colony plate count.
Technique
Usual the technique is to inoculate one plate of WL Nutri-
ent Broth and two plates of WL Differential. Incubate all
the plates at 25°C for 5-15 days. It is advisable to incu-
bate one of the plates with differential agar anaerobically
to enhance development of contaminants that produce
lactic acid. Green and Gray stated that to perform the
observations of viable yeast in bread, WL Nutrient broth
at pH 5,5 may be used, but to do it in distilleries, the pH
has to be adjusted to 6,5.
Analogously, time and temperature of incubation vary
depending on material to be analysed. Beer samples
are incubated at 35°C, but bread, and alcohol fermenta-
tion ones are incubated at 30°C. Incubation time varies
between 2 and 7 days, and in some cases, depend-
ing on the flora found, it may be up to 14 days. When
Differential type (with cycloheximide) is employed for a
bacterial count, it should be incubated anaerobically to
detect cocci in beer and lactic bacilli, and it should be
incubated aerobically to detect acetic acid bacteria and
thermobacteria.
References
GREEN, S.R. & GRAY, P.P. (1950) A differential proce-
dure applicable to bacteriological investigation in brew-
ing. Wallerstein Lab. Comm. 13:357
GREEN, S.R. & GRAY, P.P. (1950) Paper read at
Am.Soc. of Brewing Chemists Meeting; Wallerstein Lab.
Comm. 12:43
GREEN, S.R. & GRAY, P.P. (1951) A differential proce-
dure for bacteriological studies useful in the fermentation
industries. Wallerstein Lab. Comm. 14:289
GRAY, P.P. (1951) Some advances in microbiological
control for beer quality. Wallerstein Lab. Comm. 14:169
ATLAS, R.M., & L.C. PARK (1993) Handbook of Micro-
biological Media for the examination of Food. CRC Press
Inc. Boca Ratón,Fla.
MASTERS BREWERS ASSOCIATION OF THE AMERI-
CAS (2002) The Practical Brewer 3rd ed. St. Paul. Min-
nesota
171
 Wort Media

Wort Agar Directions

Suspend 37 g of powder in 1 L of distilled water and
Ref. 01-132 add 2-3 mL of glycerol and bring to the boil to dissolve
completely. Distribute into final containers and sterilize
by autoclaving at 121°C for 15 minutes.
Specification
Solid medium for the cultivation, isolation and enumera-
tion or enrichment of fungi, especially of yeast. Description
It is especially designed to propagate the multiplication
of yeast, and often it has been employed as a semise-
Formula (in g/L) lective or enrichment medium, due to its high acidity,
Malt extract ............................................... 15,0
which makes it inhibitory for bacteria. This effect may
Casein Peptone .......................................... 1,0
be more enhanced by adding, before sterilization, 10
Maltose ..................................................... 12,5
mL/L of a 10% solution of lactic or tartaric acid. To avoid
Dextrine ...................................................... 2,5
precipitate it is recommended to sterilize by filtration.
Dipotassium hydrogen phosphate .............. 1,0
Ammonium chloride .................................... 1,0
Agar .......................................................... 17,0 References
Final pH 4,8 ± 0,2 SCARR, M.P. (1959) Selective media used in the micro-
biological examination of sugar products. J. Sci. Food
Agric. 10:678-681
Directions RAPP, M (1974) Indikator-zusätze zur Keimdifferen-
Suspend 50 g of powder in 1 L of distilled water and
zierung auf Würze und Malzextrakt-Agar. Milchwiss
add 2-3 mL of glycerol and bring to the boil to dissolve
29:341-344
completely. Distribute into final containers and sterilize
ATLAS, R.M.,& L.C. PARKS (1993) Handbook of Micro-
by autoclaving at 121°C for 15 minutes. Do not over-
biological Mediafor the examination of Food. CRC Press
heat. Prolonged heating will diminish the gelling strength
Inc.London
of the medium.
MASTERS BREWERS ASSOCIATION OF THE AMERI-
CAS (2002) The Practical Brewer. 3rd ed. St. Paul. Min-
Description nesota
Wort Agar is used for the cultivation, isolation and enu-
meration of yeast and moulds.
It is particulary well adapted for counting osmophilic
yeast in butter, sugar and syrups, in lemonade and more
generally in sweet or soft drinks.
For a more selective utilization it is possible to adjust the
pH to 4,5 or 3,5. Never heat the medium after adding
acid, in order to prevent the loss of solidifying properties
of the agar. The acid pH inhibits the growth of bacteria
and favours that of yeast.

Wort Broth

Ref. 02-132

Specification
This medium is the liquid version of the classical Wort
Agar (Ref. 01-132).

Formula (in g/L)

Malt extract ............................................... 15,0
Casein Peptone .......................................... 1,0
Maltose ..................................................... 12,5
Dextrine ...................................................... 2,5
Dipotassium hydrogen phosphate .............. 1,0
Ammonium chloride .................................... 1,0
Final pH 4,8 ± 0,2

172
 Xylose Lysine Deoxycholate Media
Xylose Lysine Deoxycholate
Agar (XLD Agar) (Eur. Phar.
Agar Medium K)
Ref. 01-211
Specification
Solid medium for the isolation of enteropathogenic
species, especially Salmonella according to ISO 6340
standard.
Formula (in g/L)
Xylose ....................................................... 3,50
L-Lysine .................................................... 5,00
Lactose ..................................................... 7,50
Sucrose .................................................... 7,50
Sodium chloride ........................................ 5,00
Yeast extract ............................................. 3,00
Phenol red ................................................ 0,08
Sodium Deoxycholate .............................. 2,50
Sodium thiosulfate .................................... 6,80
Ammonium ferric citrate............................ 0,80
Agar ........................................................ 15,00
Final pH 7,4 ± 0,2
Directions
Suspend 56,68 g of powder in 1 L of distilled water. Heat
up constantly with stirring until boiling. Pour it immediate-
ly into plates. Do not autoclave and avoid remelting.
Description
Xylose Lysine Deoxycholate Agar is a differential me-
dium, slightly selective, very suitable for the detection
of pathogenic enterobacteria, especially Shigella. Gram
negative flora is inhibited by the low amount of deoxy-
cholate, but Shigella grows easier in this medium than in
any other selective media.
Xylose, lactose or sucrose fermentation produce the
acidification of the medium, and this is seen by an
indicator turning to yellow, surrounding the colonies. This
colour disappears after 24 hours, so observations must
be carried out between 18 and 20 hours.
Hydrogen sulfide production from thiosulfate is easily de-
tected because colonies become darker, due to the ferric
sulfure precipitate. Lysine decarboxylation to cadaverine
may also be observed in the medium, since it produces
alkalinization and consequently the indicator turns to red.
All these reactions allow a good differentiation of Shig-
ella, which besides Edwardsiella and Proteus inconstans
are the single enterobacteria that do not ferment xylose
and therefore show negative fermentation reaction.
Salmonella type members do ferment xylose, but it is
consumed quickly and then alkalinization of the medium,
due to lysine decarboxylation, may mask the reaction.
The difference between Shigella and Salmonella is that
with the latter colonies become darker due to ferrous
sulfure precipitates, and this is a common property with
Edwardsiella. The other types of enterobacteria do not
suffer this phenomenon, since acid acumulation due to
lactose and sucrose fermentation is so high that it avoids
pH reversion by decarboxylation and even ferrous sul-
fure precipitate in the first 24 hours.
In the table below, typical colonial appearances on XLD
medium after 24-36 hours of incubation at 37°C are
described.
References
TAYLOR, W.J. (1965) Isolation of Shigella. I. Xylose
Lysine Agars: New media for isolation of enteric patho-
gens. Am. J.Clin. Path 44:471-475
DOWNES, F.P. & K. ITO (2001) Compendium of Meth-
ods for the Microbiological Examination of Food,4th ed.
APHA,Washington.
ICMSF (1978) Microorganisms in Food 1. Univ. Toronto
Press.
FDA (1998) Bacteriological Analytical Manual. 8th ed.
Revision A. AOAC International Gaithersburg. MD.
ATLAS, R.M.,& L.C. PARK (1993) Handbook of Micro-
biological Mediafor the examination of Food. CRC Press
Inc. London
PASCUAL ANDERSON, MªR. (1992) Microbiología
Alimentaria. Diaz de Santos, S.A. Madrid, .
EUROPEAN PHARMACOPOEIA, (2002) 2.6.13 Test
for specified micro-organisms 4th Ed.,Suppl. 4.2 EDQM
Council of Europe, Strasbourg,
US PHARMACOPOEIA (2002) <61> Microbial Limit
Tests. 25th Ed. US Pharmacopoeial Convention. Rocville.
MD.
ISO 6340 Standard (1995) Water Quality - Detection of
Salmonella spp.
Xylose Lysine Deoxycholate
Modified Agar (XLD Modified
Agar)
Ref. 01-552
Specification
Medium for isolation of enteropathogenic species, espe-
cially Salmonella in food and animal feeding stuffs, acc.
ISO Standard 6579:2002
Formula (in g/L)
Xylose ...................................................... 3,75
L-Lysine ................................................... 5,00
Lactose .................................................... 7,50
Sucrose ................................................... 7,50
Sodium chloride ....................................... 5,00
Yeast extract ............................................ 3,00
Phenol red ............................................... 0,08
Sodium Deoxycholate .............................. 1,00
Sodium thiosulfate ................................... 6,80
Ammonium iron(III) citrate ....................... 0,80
Agar ....................................................... 15,00
Final pH 7,4 ± 0,2
173
 Xylose Lysine Deoxycholate Media

Directions precipitates, and this is a common character with Edwar-

Suspend 55,43 g of powder in 1 L of distilled water. Heat
up constantly stirring until boiling. Pour it immediately
into plates. Do not sterilize and avoid remelting.
Description
Xylose Lysine Deoxycholate Agar is a differential me-
dium, slightly selective, very suitable for the detection
of pathogen enterobacteria in food, especially Shigella,
with a modification in the original formulation of Tay-
lor to perform the specifications of the ISO standard
6579:2002
Gram negative flora is inhibited by the low amount of
deoxycholate, but Shigella grows easier than in other
selective media.
Xylose, lactose or sucrose fermentation produce medium
acidification, and this is shown by an indicator turning to
yellow, surrounding the colonies. This colour disappears
after 24 hours, so readings must be carried out between
18 and 20 hours.
Sulfhydric production from thiosulfate is easily detected
because colonies become darker, due to the ferric
sulfide precipitate. Lysine decarboxylation to cadaverine
may also be observed in the medium, since it produces
alkalinization and consequently the indicator turns to red.
All these reactions allow a good differentiation of Shig-
ella, which besides Edwarsiella and Proteus inconstans
are the single enterobacteria that do not ferment xylose
and therefore show negative fermentation reaction.
Salmonella-type members do ferment xylose, but it is
consumed quickly and the medium alkalinization, due
to lysine decarboxylation, may hide the reaction. The
difference between Shigella and Salmonella is that with
the latter colonies become darker due to ferrous sulfide
siella. The other types of enterobacteria do not suffer this
phenomenon, since acid accumulation due to lactose
and sucrose fermentation is so big that it avoids pH
reversion by decarboxylation and even ferrous sulfide
precipitate in the first 24 hours.
In the table below, typical colonial appearances on XLD
Agar after 24-36 hours of incubation at 37°C are de-
scribe.
References
TAYLOR, W.J. (1965) Isolation of Shigella. I. Xylose
Lysine Agars: New media for isolation of enteric patho-
gens. Am. J. Clin. Path 44:471-475
VANDERZANT & SPLITTSTOESSER (1992). Compen-
dium of Methods for the Microbiological Examination of
Food. 3rd. Ed. APHA. Washington.
ICMSF, ( 1978) Microorganisms in Food 1. University of
Toronto Press.
FDA (1990) Bacteriological Analytical Manual AOAC
International Arlington. VA. USA.
ATLAS, R.M., L.C. PARK (1993) Handbook of Microbio-
logical Media for the examination of Food. CRC Press
Inc. Boca Ratón.
PASCUAL ANDERSON, MOR. (1992) Microbiología
Alimentaria. Diaz de Santos, S.A. Madrid.
ISO Standard 6579 (2002) Microbiology of foods and
animal feeding stuffs. Horizontal method for the detec-
tion of Salmonella spp.

Colony appearance Microorganism

Red colonies, transparent
Red colonies, transparent with black core.
Orange and slightly opaque colonies
Red and translucent colonies, without halo.
Yellow and opaque
Yellow, opaque, mucose and with black core.
Yellow, transparent and with black core.
Yellow, opaque and without halo
Shigella sp., Proteus incontans, Salmonella paratyphi A.,
sometimes S.cholerasuis and S. Pullorum
Edwardsiella and most of biotypes of Salmonella
Salmonella typhi
Pseudomonas, Proteus rettgeri.
Escherichia when it grows, Enterobacter, Aeromonas, Citro-
bacter.
Klebsiella, Citrobacter intermedius when it grows
Most of Proteus mirabilis, P.vulgaris.
Serratia, Hafnia.

Ref. 01-552
XLD Modified Agar

control Salmonella typhimurium ATCC 14028

174
 Yeast Extract Media
Yeast Extract Agar
Ref. 01-465
Specification
Solid medium for the enumeration of microorganisms
from water.
Formula (in g/L)
Tryptone .................................................... 5,0
Yeast extract ............................................... 3,0
Agar .......................................................... 15,0
Final pH 7,2 ± 0,2
Directions
Suspend 23 g of powder in 1 L of distilled water and
bring to the boil. Distribute into suitable containers and
sterilize by autoclaving at 121ºC for 15 minutes.
Description
This medium, formulated according to Windle Taylor, is
the most used in the UK for the enumeration of hetero-
trophic microorganisms from water. Distinction between
bacteria, yeast and filamentous fungi must be carried out
by morphology after differential incubations at 35º and
20ºC.
Technique
From the water sample, make a decimal dilution bank
with Ringer Solution (Ref. 06-073) and take aliquotes to
2 parallel series of plates. Pour the Yeast Extract Agar,
molten and cooled to 45ºC, and homogenize with sam-
ple. Once solidified, incubate one of the series at 35ºC
for 24 hours and the other one at 20ºC for 3 days.
In order to achieve a good count, select the plates with
30-300 colonies.
References
WINDLE TAYLOR, E. (1958) The examination of water
and water supplies. 7th Ed. Churchill Ltd. London.
ATLAS, R.M., & L.C. PARKS (1993) Handbook of micro-
biological media, CRC Press, London
Yeast Extract Peptone Dextrose
Agar (YPD Agar)
Ref. 01-473
Specification
Solid medium for the cultivation of yeast in molecular
biology procedures.
Formula (in g/L)
Peptone .................................................... 20,0
Yeast extract ............................................. 10,0
Dextrose ................................................... 20,0
Agar .......................................................... 20,0
Final pH 6,8 ± 0,2
Directions
Suspend 70 g of powder in 1 L of distilled water and
bring to the boil. Distribute into suitable containers and
sterilize by autoclaving at 121ºC for 15 minutes.
Yeast Extract Peptone Dextrose
Broth (YPD Broth)
Ref. 02-473
Specification
Liquid medium for the cultivation of yeast in molecular
biology procedures.
Formula (in g/L)
Peptone .................................................... 20,0
Yeast extract ............................................. 10,0
Dextrose ................................................... 20,0
Final pH 6,8 ± 0,2
Directions
Dissolve 50 g of powder in 1 L of distilled water, heating
up if necessary. Distribute into suitable containers and
sterilize by autoclaving at 121ºC for 15 minutes.
Description
These media support the growth of most heterotrophic
microorganisms, but due to their simple composition
they have been adopted as the basal media for the rou-
tine cultivation of yeasts for molecular biology studies.
References
SHERMAN, F. (1991) Studies on the phenotype switch-
ing with Candida albicans. Meth. Enzimol 194:3-17.
MARTINEZ, J.P., M.L. GIL, M. CASANOVA, J.L. LOPEZ-
RIBOT, J. GARCIA DE LOMAS, R. SENTANDREU
(1990)
Wall mannoproteins in the cells from colonial phenotypic
variants. J. Gen. Microbiol. 136:2421-2432.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-
biological Media. CRC Press. London
AUSUBEL, F.M., R.BRENT,R.E. KINGSTON, D.D.
MOORE, J.G. SEIDMAN, J.A. SMITH & K. STRUHL
(1994) Current Protocols in Molecular Biology. Current
Protocols. Brooklyn. N.Y.
175
 Yeast Malt Media

Yeast Malt Agar (YM Agar) Yeast Malt Broth (YM Broth)

Ref. 01-219 Ref. 02-219

Specification Specification
Solid medium for the cultivation of fungi and actinomyc- Liquid medium for the cultivation of fungi and actinomyc-
ete. etes.

Formula (in g/L) Formula (in g/L)

Dextrose ................................................... 10,0
Peptone ...................................................... 5,0
Malt extract ................................................. 3,0
Yeast extract ............................................... 3,0
Agar .......................................................... 20,0
Final pH 6,2 ± 0,2
Directions
Suspend 41 g of powder in 1 L of distilled water and
let it soak. Bring to the boil and distribute into suitable
containers. Sterilize in the autoclave at 121°C for 15
minutes.
Dextrose ................................................... 10,0
Peptone ...................................................... 5,0
Malt extract ................................................. 3,0
Yeast extract ............................................... 3,0
Final pH 6,2 ± 0,2
Directions
Dissolve 21 g of powder in 1 L of distilled water. Distrib-
ute into suitable containers and sterilize by autoclaving
at 121°C for 15 minutes.
Description
This is a classical culture medium for the cultivation of
moulds, yeasts and acidophilic actinomycetes. Medium
may become selective to one or other group of micro-
organisms by adding antibiotics when the medium is at
50°C.

References
ATLAS, R.M.,& L.C. PARK (1993) Handbook of Micro-
biological Mediafor the examination of Food,CRC Press
Inc.London.
SAMSOM, R.A., E.S. HOEKSTRA, J.C. FRISVAD, O.
FILTENBORG (2002) Introduction to food- and airborne
fungi. 6th. Ed. CBS. Utrech.

176
Culture Media Ingredients
 Culture Media Ingredients

Definitions Production Process

EXTRACTS are concentrated preparations of liquid, The general production process is eschematized in the
solid or intermediate consistency, usually obtained from following flow-chart.
dried vegetable or animal matter. For some preparations,
the matter to be extracted may undergo a preliminary
treatment, for example, inactivation of enzymes, grind-
ing or defatting. Extracts are prepared by maceration,
percolation or other suitable, validated methods, using
water, ethanol or another suitable solvent. After extrac-
tion unwanted matter is removed if necessary.

PEPTONES are complex water-soluble mixes of free

amino acids, peptides, sugars, mineral salts and other
components obtained by acid, alkaline or enzymatic
hydrolysis of protein substrates. Their very variable char-
acteristics depend on:
- The nature of the substrate(s).
- The nature of the hydrolysis: enzyme(s), alkali(s),
acid(s), term of hydrolysis.
- The technique of purification (filtration, ultrafiltration,
…).
- The other operating conditions used in the production
process.

The term peptone is more commonly applied to the

hydrolisates obtained by enzymatic digestion. The en-
zymes more frequently used are:

Papain that acts on the links adjacent to arginine, lysine,

phenylalaline and glycine. Bromelain and Ficin are
also used because are similar to papain but from
other plants and with pH-range and temperature-
range different.
Pepsin, that acts on the links adjacent to phenylalanine
or leucine.
Pancreatin (a variable mixture of trypsin and chimo-
trypsin) that acts on the links adjacent to arginine,
lysine, tyrosine, tryptophane, phenylalanine and
leucine.
Microbial Proteases are obtained from microbial
cultures as extracellular enzymes and used for the
peptones production. Acid and Neutral microbial pro-
teases works in similar way as papain and alkaline
microbial proteases as pancreatin.
The papain is obtained from plant material and pepsin
and pancreatin, that are animal enzymes, are of swine
origin.

179
 Culture Media Ingredients

Warranty of health and origin Series in Nutrition and Food. Section G. Vol III. CRC
All the animal tissue raw materials used in the elabora- Press. Cleveland
tion of the Scharlau Microbiology Peptones come from SYKES, J. (1956) Constituents of Bacteriologic Culture
approved slaughterhouses and are covered by certifi- Media. Cambridge University Press. Cambridge.
cates obtained from the veterinary authorities.
The country of origin of bovine animal tissues and casein
used in the manufacture of each batch of peptone is
specified in the health certificate.
These documents certify that animals from which tissues
have been taken were in good health and fit for human
consumption. They are used by Scharlau Microbiology
Quality Control Department to edit a health certificate for
each batch of product manufactured: a copy of this certif-
icate is submitted to our customers upon each delivery.

Analytical data and control

methods
All data figuring in the following documentation result
from the analysis of a significant number of batches of
every product. These data may be:
Typical data, which are in fact average values (com-
parative tables and technical data sheets).
Norms of acceptability (Technical data sheets).

Bacteriological controls as well as most general physico-

chemical controls are carried out each batch system-
atically. The other characters are verified according to
routine.
For the control methodology, a norm is defined spe-
cifically for each character. When a pharmacopoeial
monograph (Eur. Phar. 4th ed. 2002; USP 25th ed 2002)
is available, it is adopted as routine method. If it is not
possible an other documented methodology is accepted.
The typical data are show in the following Tables 1 to
6 and in the specific description of any product. The
information contained in this publication is based in own
research and development work and is the best of our
knowledge true and accurate. Users should however
conduct their own tests to determine the suitability of our
products for their own purpouses. Statements contained
herein should not be considered as a warranty of any
kind, expressed or implied, and no liability is accepted
for the infringement of any patents.

References
ATLAS, R.M. & L.C. PARKS ( 1993) Handbook of Micro-
biological Media CRC Press London.
BRYDSON, E.Y. (1978) Natural and Synthetic Culture
Media for Bacteria. In M. Rechcigl Jr. (ed) Handbook

180
Culture Media Ingredients

181
 Culture Media Ingredients

182
Culture Media Ingredients

183
 Culture Media Ingredients

184
Culture Media Ingredients

185
 Culture Media Ingredients

186
 Agar-Agar

Ref. 07-490
Agar is the dried, hydrophilic, colloidal substance ex-
tracted from the algae known as Agarophytes (several
species and genera of the Class Rodophyceae). It con-
sists of two polysaccharides, agarose and agaropectine,
in a variable proportion depending on the geographical
origin zone.

The Agar-Agar is a solidifying agent with the same gel-

ling strength as the Agar Technical but with an inferior
grade of purification, and shows a greater opacity and
a higher salts content. Its use among the culture media
is recommended only when the brightness and clarity is
not a critical requirement.

The most important characteristics are shown in the fol-

lowing tables. Data are average values, which may vary
from batch to batch.

Agar Bacteriological

Ref. 07-004
Agar is the dried, hydrophilic, colloidal substance ex-
tracted from the algae known as Agarophyites (several
species and genera of the Class Rodophyceae). It con-
sists of two polysaccharides, agarose and agaropectine,
in a variable proportion depending on the geographical
origin zone.

The Agar Bacteriological is a solidifying agent selected

and prepared by mixing different agar from several
zones of origin and is especially recommended for
gelling the microbiological culture media where a great
transparency and brightness is required.

The most important characteristics are shown in the fol-

lowing tables. Data are average values, which may vary
from batch to batch.

187
 Agar Technical

Ref. 07-521
Agar is the dried, hydrophilic, colloidal substance ex-
tracted from the algae known as agarophyites (several
species and genera of the Class Rodophyceae). It con-
sists of two polysaccharides, agarose and agaropectine,
in a variable proportion depending on the geographical
origin zone.

The Agar Technical is a solidifying agent with a gel

strengh higher than Agar Bacteriological, especially sug-
gested when the culture medium does not require a total
brightness, since it shows a slight turbidity.

The most important characteristics are shown in the fol-

lowing tables. Data are average values, which may vary
from batch to batch.

Beef Extract

Ref. 07-515
For a long time beef extract has been the basic com-
ponent of culture media, and initially it substituted meat
infusions due to its easy usage. Now, there is a trend to
substitute it by peptones and different mixtures with a
more defined composition, because they allow a greater
reproducible result.
Scharlau Microbiology Beef Extract is obtained from free
tendons and fat beef muscle, enzymatically predigested.
Its production also includes the elimination of fermenta-
ble sugars.
The totally desiccated version is easier to use than the
paste form, and require less quantity in order to obtain
the same effects. Beef extract solutions are clear, slightly
coloured and with pH near to neutral. In culture media
they are used in concentrations varying from 0,3-0,5%.
The most important characteristics are shown in the fol-
lowing figures and tables and in Tables 1 to 6 at the beg-
gining of this chapter. Data are average values, which
may vary from batch to batch.

Among the raw materials and auxiliaries used in its

preparation, the bovine constituents belong to the cat-
egory 4 of the WHO classification. The bovine tissues
are sourced from New Zealand, and come from herds
free from Bovine Spongiform Encephalopathy virus and
foot-and-mouth disease after examination by the Veteri-
nary Authorities. The enzymatic preparation is of porcine
origin.

The product does not contain and is not derived from

specified risk material as defined in the European Com-
mission Decision 97/534/EC.
The manufacturing process includes boiling at 100ºC for
a minimum of 5 minutes and instantaneous heating at
200ºC on spray drying.

188
 Beef Extract

07-515 Beef Extract 07-515 Beef Extract

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

Bile

Ref. 07-039 Dry powder has a colour varying form yellow-beige to

yellow-green, and it allows transparent 5% solutions in
Ox bile powder is obtained by spray drying the fresh bile water, that have a pH between 6 and 7,5. In alcoholic
at high temperature, assuring the maintenance of the solutions (ethanol 84%) it provides less than 0,1% of
most important characteristics or properties of fresh bile. insoluble substances.
1g of ox bile powder is corresponds to approx. 10 g of
fresh bile. The most important characteristics are shown in the fol-
lowing tables and in Tables 1 to 6 at the beggining of this
The bile used in its preparation is naturally of bovine chapter. Data are average values, which may vary from
origin. It comes from animals which are raised and batch to batch.
slaughtered in Holland, and are in good health and suit-
able for human comsumption on ante- and post- mortem
examination by the Veterinary Authorities. These animals
come from herds free from Bovine Spongiform Encepha-
lopathy and Foot-and-Mouth disease.

The manufacturing process includes the rapid heating at

200ºC on spray drying.

In the culture media it is employed at concentrations

varying from 1-2%. Ox bile act as a non enteric micro-
bial flora inhibitor, and it is used as selective agent in
enterobacterial isolation media. Ox bile solutions at low
concentrations are clear and have a slight colour, but
at higher concentrations they are opalescent and have
darker colours.

189
 Bile Salts #3

Ref. 07-525 The Scharlau Bile Salts #3 is standardized batch by

The bile salts for microbiological applications are ob-
tained from fresh animal (sheep, pig) bile by precipitation
with chlorhidric acid, in a process that removes pigments
and other toxic substances and concentrate the bile
salts. Nevertheless the standardization of the results is
very difficult because the composition of the final mixture
depends not only on the process but on the raw material
that is very variable in origin.
In the normal preparation of bile salts several compo-
nents like gluconate, taurocholate, cholate and deoxy-
cholate, and others, can be identified, and the inhibitory
character of the mixture depends on the relative rate
between all these substances. Usually, in the micro-
biological culture media, the bile salts are dosed in a
concentration of 0,5% (w/v) to inhibit the growth of gram-
positive bacteria.
batch intending to supply a product as homogeneous as
possible.
Dry powder has a flowable blurred white aspect, with a
bitter in odour and taste, but provides clean, transparent
and pale yellow 2% solutions in water, which present
an alkaline reaction (pH 8,0) and therefore its addition
to culture media may require pH adjustment. When this
product is used as culture media component it is advis-
able do nod exceed never the 0,3 % (w/v) concentration.
The most important characteristics are shown in the fol-
lowing tables and in Tables 1 to 6 at the beggining of this
chapter. Data are average values, which may vary from
batch to batch.

There are other type of preparations of mixture o bile

salts, with a higher level of purification and concentration
of actives substances. These ones are used, in micro-
biological applications, in lower concentration, that is,
a third o the concentration of the normal preparations.
Because its efficacy, they are called Bile Salts #3 and its
usual concentration in culture media is 0,15%.

Brain Extract

Ref. 07-076
For a long time animal brain infusions have been one of
the basic components of some culture media for fastidi-
ous microorganisms, and nowadays, in most cases they
are still necessary.

Scharlau Microbiology Brain Extract produces clear,

clean and stable solutions at 121ºC, and provide the cul-
ture medium with very complex nutrients. It is obtained
from brains of healthy pigs with ante- and post-mortem
sanitary certification.

Among the raw materials and auxiliaries used in its prep-

aration, the bovine constituents belong to the category 4
of the WHO classification. The product does not contain
and is not derived from specified risk material as defined
in the European Commission Decision 97/534/EC. All
the constituents are of swine origin.

The manufacturing process includes boiling at 100ºC for

a minimum of 5 minutes and instantaneous heating at
200ºC on spray drying.

The most important characteristics are shown in the fol-

lowing tables and in Tables 1 to 6 at the beggining of this
chapter. Data are average values, which may vary from
batch to batch.

190
 Casein Acid Hydrolysate

Ref. 07-151 The most important characteristics are shown in the fol-
lowing figures and tables and in Tables 1 to 6 at the beg-
Casein Acid hydrolysate is a protein hydrolysate ob- gining of this chapter. Data are average values, which
tained by acid digestion, where all the casein com- may vary from batch to batch.
pounds reduced to their aminoacids, except tryp-
tophane which almost disappears. Vitamines also almost
disappear due to the acid digestion process.

Among the raw materials and auxiliaries used in its prep-

aration the bovine constituents belong to the category
4 of the WHO classification. The lactic casein from cow
milk is sourced from New-Zealand, and come from herds
free from Bovine Spongiform Encephalopathy and foot-
and-mouth disease after examination by the Veterinary
Authorities.
The product does not contain and is not derived from
specified risk material as defined in the European Com-
mission Decision 97/534/EC.
The manufacturing process includes boiling at 100ºC for
a minimum of 5 minutes and instantaneous heating at
200ºC on spray drying.

07-151 Casein Acid Hydrolisate 07-151 Casein Acid Hydrolisate

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

191
 Casein Pancreatic Peptone

Ref. 07-154 The manufacturing process includes boiling at 100ºC for

Casein Pancreatic Peptone is a protein hydrolysate,
obtained by digestion with pancreatic extracts. It differs
from Tryptone (Ref. 07-119) only in the way it is ob-
tained, which produces a different aminoacid composi-
tion and a lesser molecular size. It is the most used type
of peptone in industrial fermentation operations.
a minimum of 5 minutes and instantaneous heating at
200ºC on spray drying.
The most important characteristics are shown in the fol-
lowing figures and tables and in Tables 1 to 6 at the beg-
gining of this chapter. Data are average values, which
may vary from batch to batch.

Among the raw materials and auxiliaries used in its

preparation the bovine constituents belong to the cat-
egory 4 of the WHO classification. The Milk casein from
cow milk is sourced from New-Zealand, and come from
herds free from Bovine Spongiform Encephalopathy and
foot-and-mouth disease after examination by the Veteri-
nary Authorities.

The enzymatic preparation is of porcine origin.

The product does not contain and is not derived from
specified risk material as defined in the European Com-
mission Decision 97/534/EC.

07-154 Casein Pancreatic Peptone 07-154 Casein Pancreatic Peptone

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

192
 Casein Trypsic Peptone (Tryptone)

Ref. 07-119 The manufacturing process includes boiling at 100ºC for

The Casein Trypsic Peptone or Tryptone, is a protein
hydrolysate obtained by digestion of casein with an
especially tripsin-enriched pancreatic enzymatic prepa-
ration.
Both, its nitrogen content and balanced amino-acid com-
position makes it more suitable for the manufacturing of
culture media, producing exceptionally clear solutions.
a minimum of 5 minutes and instantaneous heating at
200ºC on spray drying.
The most important characteristics are shown in the fol-
lowing figures and tables and in Tables 1 to 6 at the beg-
gining of this chapter. Data are average values, which
may vary from batch to batch.

Among the raw materials and auxiliaries used in its

preparation the bovine constituents belong to the cat-
egory 4 of the WHO classification. The lactic casein from
cow milk is sourced from New-Zealand, and come from
herds free from Bovine Spongiform Encephalopathy
and foot-and-mouth disease after examination by the
Veterinary Authorities. The enzymatic preparation is of
porcine origin.

The product does not contain and is not derived from

specified risk material as defined in the European Com-
mission Decision 97/534/EC.

07-119 Casein Trypsic Peptone 07-119 Casein Trypsic Peptone

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

193
 Gelatin Pancreatic Peptone

Ref. 07-153 gining of this chapter. Data are average values, which
may vary from batch to batch.
Gelatin peptone is a cream coloured powder, with
characteristic odour, obtained by pancreatic digestion of
gelatin.

The gelatin is of porcine origin. None of the raw materi-

als and auxiliaries used in its preparation are of bovine
origin. The manufacturing process includes boiling at
100ºC for a minimum of 5 minutes and instantaneous
heating at 200ºC on spray drying.

It has a low content of Tryptophan, and has no fermenta-

ble sugars. Their solutions, even at high concentrations
(10%), take a light colours, without precipitate, due to
their elaborated manufacturing process. It produces a
slight acid reaction (after sterilization) and has no indole.
Its nutritional capacity is low, but it may be used for non
fastidious microorganisms, and complies with the pep-
tone specifications for fermentation studies.

The most important characteristics are shown in the fol-

lowing figures and tables and in Tables 1 to 6 at the beg-

07-153 Gelatin Pancreatic Peptone 07-153 Gelatin Pancreatic Peptone

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

194
 Heart Extract

Ref. 07-077 gining of this chapter. Data are average values, which
may vary from batch to batch.
Bovine heart extract has been widely used as an alterna-
tive to meat extract where very special nutrient require-
ments are needed. Scharlau Microbiology Heart Extract
is obtained from the bovine cardiac muscle of healthy
animals with explicit sanitary certification.

Among the raw materials and auxiliaries used in its

preparation, the bovine constituents belong to the cat-
egory 4 of the WHO classification. The bovine tissues
are sourced from New Zealand, and come from herds
free from Bovine Spongiform Encephalopathy and foot-
and-mouth disease after examination by the Veterinary
Authorities.

The product does not contain and is not derived from

specified risk material as defined in the European Com-
mission Decision 97/534/EC. The manufacturing process
includes boiling at 100ºC for a minimum of 5 minutes
and instantaneous heating at 200ºC on spray drying.

The most important characteristics are shown in the fol-

lowing figures and tables and in Tables 1 to 6 at the beg-

07-077 Heart Extract 07-077 Heart Extract

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

195
 Lecithin

Ref. 07-342
Phosphatidylcholine, a mixture of diglycerides of the
stearic, palmitic and oleic acids, linked to the cholic ester
of the phosphoric acid.

The SCHARLAU Lecithin is a clear brown powder,

obtained from soy beans by extraction. It is especially
treated to be included in culture media as an emulsifier
or as a nutrient factor for fastidious microorganisms.

The most important characteristics are shown in the fol-

lowing tables. Data are average values, which may vary
from batch to batch.

Liver Peptone

Ref. 07-614 gining of this chapter. Data are average values, which
may vary from batch to batch.
Liver peptone is a proteic hydrolized by enzymatic diges-
tion of fresh swine liver, followed by a careful desiccation
process to maintain its fundamental characteristics.

Liver peptone is very employed in culture media for Tri-

chomonas and other fastidious protozoa, and for some
pathogen and saprofitic fungi, micoplasms and anaero-
bic bacteria.

Liver peptone provides clear solutions of a dark colour,

and is perfectly compatible with other medium com-
ponents. It accepts sterilization and does not loose its
characteristics. Generally it uses to replace peptones, at
the same weight. In culture media for protozoa, its con-
centration uses to be high (25-30 g/L), but for bacteria
it uses to vary from 0,5 to 1 %, except for anaerobics
enrichment media, where concentration may be higher.

The most important characteristics are shown in the fol-

lowing figures and tables and in Tables 1 to 6 at the beg-

196
 Liver Peptone

07-614 Liver Peptone 07-614 Liver Peptone

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

Malt Extract

Ref. 07-080
Malt extract is used in the culture media for fungi, as
much as enrichment as a true nutritive base, because
very often it substitutes the peptone. It is obtained by
extraction of soluble fraction of malted barley, followed
by a drying proccess at low tempertature so that there
is only minimal alteration in its nitrogenated composition
and high sugar content, especially maltose. All the raw
materials and auxiliaries are of plant origin. It has no
diastatic activity. Very hygroscopic product.

Malt extract solutions are usually opalascent or turbid.

Should clear solutions are required, filtration is neces-
sary.

The most important characteristics are shown in the fol-

lowing figures and tables and in Tables 1 to 6 at the beg-
gining of this chapter. Data are average values, which
may vary from batch to batch.

197
 Meat Extract

Ref. 07-075 The totally desiccated (dried) version is easier to use

Meat extract has been the basic component of culture
media for a long time, and initially it substituted meat
infusions due to its easy usage. Now, there is a trend to
substitute it by peptones and different mixtures with a
more defined composition, because they allow a greater
reproductivity result.
Meat extract is obtained from free tendons and fat
tissues of the animals (sheep and pork), which are
enzymatically predigested. Its production involves the
elimination of fermentable sugars.
and requires less quantity in order to obtain the same
effects. Meat extract solutions are clear, slightly coloured
and with pH near to neutrality. In the culture media they
are used in concentrations varying from 0,3-0,5%.
The most important characteristics are shown in the fol-
lowing figures and tables and in Tables 1 to 6 at the beg-
gining of this chapter. Data are average values, which
may vary from batch to batch.

Among the raw materials and auxiliaries used in its

preparation the bovine constituents belong to the cate-
gory 4 of the WHO classification. The bovine tissues are
sourced from New-Zealand, and come from the herds
free from Bovine Spongiform Encephalopathy and foot-
and-mouth disease after examination by the Veterinary
Authorities.
The product does not contain and is not derived from
specified risk material as defined in the European Com-
mission Decision 97/534/EC. The other constituents are
of porcine origin. The manufacturing process includes
boiling at 100ºC for a minimum of 5 minutes and instan-
taneous heating at 200ºC on spray drying.

07-075 Meat Extract 07-075 Meat Extract

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

198
 Meat Peptone

Ref. 07-152 The manufacturing process includes boiling at 100ºC for

Meat peptone is an hydrolysate obtained by a partial
digestion of meat by pepsine. It complies with the USP/
NF25 and Eur. Phar. 4th. Ed. specifications for the peptic
digestion of animal tissues.
It is a fine powder, cream or brown coloured, that gives
very clear and light solutions and is specially prepared
for using in the culture media.
a minimum of 5 minutes and instantaneous heating at
200ºC on spray drying.
The most important characteristics are shown in the fol-
lowing figures and tables and in Tables 1 to 6 at the beg-
gining of this chapter. Data are average values, which
may vary from batch to batch.

Among the raw materials and auxiliaries used in its

preparation the bovine constituents belong to the cat-
egory 4 of the WHO classification. The bovine tissues
are sourced from New-Zealand, and come from the
herds free from Bovine Spongiform Encephalopathy and
Foot-and-Mouth Disease after the examination by the
Veterinary Authorities.
The product does not contain and is not derived from
specified risk material as defined in the European Com-
mission Decision 97/534/EC. The other constituents are
of porcine origin.

07-152 Meat Peptone 07-152 Meat Peptone

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

199
 Peptone from Casein

Ref. 07-489 The digestion process by an exceptionally trysin-rich

enzymatic preparation produces a high content of tryp-
The Peptone from casein is a protein hydrolysate ob- tophane and assures the absence of fermentable sugars
tained from trypsin-digested casein according to the USP and enzymatic activity.
specifications. Both its nitrogen rate and its balanced
amino-acid composition provide an adequate support for The most important characteristics are shown in the fol-
the production of culture media, producing exceptionally lowing figures and tables and in Tables 1 to 6 at the beg-
clear solutions. gining of this chapter. Data are average values, which
may vary from batch to batch.
Among the raw materials and auxiliaries used in its
preparation the bovine constituents belong to the cat-
egory 4 of the WHO classification. The bovine tissues
are sourced from New-Zealand, and come from the
herds free from Bovine Spongiform Encephalopathy and
Foot-and-Mouth Disease after the examination by the
Veterinary Authorities.
The product does not contain and is not derived from
specified risk material as defined in the European Com-
mission Decision 97/534/EC.

07-489 Peptone from Casein 07-489 Peptone from Casein

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

200
 Proteose Peptone

Ref. 07-213 The manufacturing process includes boiling at 100ºC for

This peptone is obtained after a partial enzymatic (pep-
tic) digestion process of animal tissues. It is obtained in
such a way that there is a high proportion of peptides
of low molecular weight, with free amino acids and
other growth factors. Although all these things make its
definition very difficult, it has a high nutritive capacity
that makes it suitable for obtaining toxins and as a basic
growth support for very fastidious microorganisms.
a minimum of 5 minutes and instantaneous heating at
200ºC on spray drying.
The most important characteristics are shown in the fol-
lowing figures and tables and in Tables 1 to 6 at the beg-
gining of this chapter. Data are average values, which
may vary from batch to batch.

Among the raw materials and auxiliaries used in its pro-

duction, the bovine constituents belong to the category
4 of the WHO classification. The bovine tissues are
sourced from New-Zealand, and come from herds of cat-
tle which are free from Bovine Spongiform Encephalopa-
thy and foot-and-mouth disease after examination by the
Veterinary Authorities.
The product does not contain and is not derived from
specified risk material as defined in the European Com-
mission Decision 97/534/EC. The other constituents are
of porcine origin.

07-213 Proteose Peptone 07-213 Proteose Peptone

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

201
 Soy Peptone

Ref. 07-155
Soy peptone is a proteic hydrolysate obtained by papaic
digestion of soy flour. It complies with the USP/NF25 and
Eur. Phar. 4th. Ed. specifications for these type of prod-
ucts, and it is a useful compound in laboratory culture
media. However, due to its high content of sugar it is not
recommendable for fermentation assays.

The most important characteristics are shown in the fol-

lowing figures and tables and in Tables 1 to 6 at the beg-
gining of this chapter. Data are average values, which
may vary from batch to batch.

07-155 Soy Peptone 07-155 Soy Peptone

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

202
 Tryptose

Ref. 07-197 The manufacturing process includes boiling at 100ºC for

a minimum of 5 minutes and instantaneous heating at
Tryptose is a mixed peptone with high nutrient properties 200ºC on spray drying.
that make it appropiate for the use in culture media for
very fastidious microorganisms. The most important characteristics are shown in the fol-
lowing figures and tables and in Tables 1 to 6 at the beg-
Among the raw materials and auxiliaries used in its gining of this chapter. Data are average values, which
preparation the bovine constituents belong to the cat- may vary from batch to batch.
egory 4 of the WHO classification. The bovine tissues
are sourced from New-Zealand, and come from herds
free from Bovine Spongiform
Encephalopathy and foot-and-mouth disease after ex-
amination by the Veterinary Authorities.

The product does not contain and is not derived from

specified risk material as defined in the European Com-
mission Decision 97/534/EC. The other constituents are
of porcine origin.

07-197 Tryptose 07-197 Tryptose

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

203
 Yeast Extract

Ref. 07-079
A water soluble extract of fresh autolyzed yeast cells.
Prepared and standardized for use in microbiological
culture media.

It is commonly added to culture media in concentrations

between 0.2% and 1%.

The most important characteristics are shown in the fol-

lowing figures and tables and in Tables 1 to 6 at the beg-
gining of this chapter. Data are average values, which
may vary from batch to batch.

07-079 Yeast Extract 07-079 Yeast Extract

Molecular Weigth Distribution Amino Acids (Free/Total) x 100

204
Additives
 Dextrose Powder (D(+)- Glucose Powder)

Ref. 06-048 Specifications

Specific rotation ([α]20ºC/D, c=10, H O) ...... +52,6 - +53,2 º
2

Presentation Molecular weight .............................. 180,16 g/mol

500 g Flask Acidity/alkalinity ................................ passes test
Insoluble in water ................................. max. 0,01 %
Chlorides (Cl)........................................ max. 0,01 %
Description
Sulfates (SO4) ...................................... max. 0,02 %
Purified and standardized carbohydrate for the use in
Sulfite (as SO2) .................................... max. 0,001 %
microbiological cultrure media as energy source for
Arsenic (As) .......................................... max. 0,0001 %
bacteria. Carbohydrate are adapted to the suitable basic
Calcium (Ca)......................................... max. 0,02 %
media.
Heavy metals (as Pb) ........................... max. 0,0005 %
Scharlau carbohydrates are pure and without mixtures,
Iron (Fe) ................................................ max. 0,0005 %
and this important characterístic is assured in order to
Lead (Pb) .............................................. max. 0,00005%
get always right results.
Sulfated ash ......................................... max. 0,1 %
Water (K.F.) .......................................... max. 1 %
Physical Data Foreign sugars,starchs,dextrines ..... passes test
Bulk density............................................... ~ 630kg/m3 Residual solvents (Eur. Phar./ICH) ...... excluded
Solubility in water (20ºC) .......................... ~ 470g/L
Melting point ............................................. ~ 146ºC
Ignition temperature ................................. ~ 500ºC
pH (100 g/L H2O, 20ºC)................................ 6-7

Egg’s Yolk Sterile Emulsions

Egg’s Yolk Sterile Emulsion

The Egg’s Yolk Sterile Emulsion has been widely used
Ref. 06-016 as bactericide neutralizer on sampling liquids. Such an
effect can be considerably intensified by adding a bit of
Polysorbate with the yolk in the formulation. 1% (v/v) is
Presentation usually enough.
100 mL Flask
Egg’s Yolk Tellurite Sterile
Description
Sterile egg’s yolk emulsion stabilized for use in bacte- Emulsion
riology, especially with Bacillus cereus Agar Base (Ref. Xn
01-262), Bacillus cereus Selective Agar Base (Ref. 01- Ref. 06-026
487) and Tryptose Sulfite Cycloserine Agar Base (Ref. R-22
S-46
01-278). Presentation
100 mL Flask
It is also used for the detection of lecithinase in spe-
cies such as Bacillus, Clostridium and Staphylococcus,
Description
and in all processes related with this enzyme which are
This emulsion has been especially formulated for its ad-
present in dairy microorganisms and most psychrotroph-
dition to the Baird Parker Agar Base (Ref. 01-030)
ics.
Aseptically add 50 mL of Egg’s Yolk Tellurite Sterile
Lecithinase Assay
Emulsion to 1 litre of sterile Baird Parker Agar Base
The medium is prepared by aseptically adding 0,5-1,0
(Ref. 01-030), melted and cooled down to approximately
mL of yolk emulsion to 10 mL of sterile melted solid
55-60°C. Mix uniformly avoiding bubbles and foam, and
medium, cooled to 55-60°C. Tryptic Soy Agar (Ref. 01-
pour into Petri dishes.
200), Nutrient Agar (APHA) (Ref. 01-144) and Nutrient
The presumptive Staphylococcus aureus colonies show
Agar (B. Ph.) (Ref. 01-140) are very adequate for these
the lecithinase activity by the halo digestion around the
purposes. The solid medium is inoculated with the assay
colonies and simultaneously a black center due to the
strain and incubated at 35-37° C for 5 days. If there is
tellurite reduction.
lecithinasic activity the broths will turn opalescent and
solid media will present an opaque zone of clearing
around the colonies.
Bacillus cereus, with a strong lecithinase, produces vis-
ible results in just a few hours.

207
 Lactose Powder

Ref. 06-051 Specifications

Specific rotation ([α]20ºC/D, c=10, H O) ...... +54,4 - +55,9 º
2

Presentation Molecular weight .............................. 360,32 g/mol

500 g Flask Acidity/alkalinity ................................ passes test
Appaerance of solution (10% water) ....... passes test
Proteins ............................................ passes test
Description
Arsenic (As)...........................................max. 0,00005 %
Purified and standardized carbohydrate for the use in
Copper (Cu) .......................................... max. 0,0025 %
microbiological cultrure media as energy source for
Heavy metals (as Pb) ........................... max. 0,0005 %
bacteria. Carbohydrate are adapted to the suitable basic
Lead (Pb)...............................................max. 0,00005 %
media.
Zinc (Zn) ............................................... max. 0,0025 %
Scharlau carbohydrates are pure and without mixtures,
Sulfated ash ......................................... max. 0,1 %
and this important characterístic is assured in order to
Water (K.F.)............................................4,5 - 5,5 %
get always right results.
Residual solvents (Eur. Phar./ICH)...........excluded
Physical Data
Bulk density.............................................~ 500 kg/m3
Solubility in water (20ºC) ............freely soluble
Melting point ...........................................~ 223 ºC
pH (50 g/L H2O, 20ºC) ................................4-6

Maltose Powder

Ref. 06-052 Physical Data

Presentation
500 g Flask
Description
Purified and standardized carbohydrate for the use in
microbiological cultrure media as energy source for
bacteria. Carbohydrate are adapted to the suitable basic
media.
Scharlau carbohydrates are pure and without mixtures,
and this important characterístic is assured in order to
get always right results.
Bulk density.............................................~ 320 kg/m3
Solubility in water (20ºC) ............freely soluble
Melting point ..................................~ 160 - 165 ºC
pH (50 g/L H2O, 20ºC) ..........................4,5-6,0
Specifications
Specific rotation ([α]20ºC/D, c=10, H O) ...... +137 - +139
2
º
Molecular weight .............................. 342,31 g/mol
Acidity/alkalinity ................................ passes test
Appaerance of solution (10% water) ....... passes test
Proteins ............................................ passes test
Arsenic (As).......................................max. 0,00005 %
Copper (Cu).......................................max. 0,0005 %
Heavy metals (as Pb)........................max. 0,002 %
Barium (Ba).......................................max. 0,0005 %
Calcium (Ca)......................................max. 0,005 %
Sulfated ash.......................................max. 0,1 %
Water (K.F.)........................................4,5 - 5,5 %
Residual solvents (Eur. Phar./ICH) ...... excluded

208
 Mannitol Powder
Ref. 06-050
Presentation
500 g Flask
Description
Purified and standardized carbohydrate for the use in
microbiological cultrure media as energy source for
bacteria. Carbohydrate are adapted to the suitable basic
media.
Scharlau carbohydrates are pure and without mixtures,
and this important characterístic is assured in order to
get always right results.
Physical Data
Bulk density...................................~ 400 - 500
Spec. density.................................................1,49
Solubility in water (25ºC)...........................213
Melting point ...................................~ 164 -169
Boiling point (4 hPa) ......................~ 290 - 295
pH (100 g/L H2O, 20ºC) ..............................5-7
Polysorbate 80 (Polyoxyethylene sorbitan monooleate)
Ref. 06-088
Presentation
100 mL Flask
1 L Flask
Description
Under the name of Polysorbate 80 or polyoxyethylene
sorbitanmonooleate are included a serie of derivates of
polyose-1,2-ethanodiol sorbitan-mono-9-octodecenoate.
The product supplied by SCHARLAU is verified to be a
nutrient in some cases and an emulsionant in others, but
it is always compatible with the rest of components of
the culture medium.
It is a very thick liquid, amber colour, and density 1,08
approx. It is very soluble in water, it has an average solu-
bility in organic diluents but it is not soluble in mineral
lipids.
Specifications
Assay (iodometric) ............................ min. 98 %
Acidity/alkalinity ................................ passes test
Appaerance of solution (20% water) ....... passes test
Conductivity (20ºC, 20% water)........ passes test
Specific rotation ([α]20ºC/D, c=8, Na B O ) ... +23 - +25 º
2 4 7
Molecular weight ............................. 182,17 g/mol
Chlorides (Cl)........................................ max. 0,005 %
Sulfates (SO4) ....................................... max. 0,01 %
Arsenic (As) .......................................... max. 0,0001 %
Copper (Cu) .......................................... max. 0,001 %
Heavy metals (as Pb) ........................... max. 0,0005 %
Lead (Pb)...............................................max. 0,00005 %
Nickel (Ni) ............................................. max. 0,0001 %
Zinc (Zn) ............................................... max. 0,0025 %
Related substances (sorbitol) ............... max. 2 %
kg/m3 Red. impurities (as glucose) ................. max. 0,05 %
g/m3 Sulfated ash ......................................... max. 0,1 %
g/L Loss on drying (105ºC, 4h) ................... max. 0,3 %
ºC Residual solvents (Eur. Phar./ICH)...........excluded
ºC
The incorporation of Polysorbate 80 to culture me-
dia may slightly affect the final pH, if the SCHARLAU
medium is not originally formulated to be composed by
Polysorbate.
Although it may bear sterilization in the autoclave when
it is more than 1% in the medium, it is usual to homog-
enize the medium after the sterilization, since with the
autoclaving sometimes the polysorbate is separated
from the medium.
Polysorbate is a tensioactive agent that makes decrease
the superficial tension of the cell, modifying at the same
time the cellular exchange speed. The response use to
be a quicker growht or the increase of some bacterial
activities.
209
 Potassium Tellurite Solutions

Potassium Tellurite Solution 1% It is used in media such as Giolitti Cantoni Broth (Ref.
02-230), Vogel-Johnson Agar (Ref. 01-206) and other
Xn
Ref. 06-089 selective media for staphylococci. This solution is also
R-22 contained in selective media for corynebacteria, strepto-
S-46
cocci and vibrios.
Presentation
100 mL Flask There is high relation between the ability to reduce po-
tassium tellurite to tellure and the staphylococci’s patho-
Potassium Tellurite genity. Therefore, the presence of potassium tellurite in
Solution 3,5% a medium helps to determine staphylococci of clinical
interest, together with other tests.
Xn
Ref. 06-011 The Potassium Tellurite Solution should be stored at
R-22
S-46 room temperature, since low temperatures will cause
Presentation the crystallization and later precipitation of the product.
100 mL Flask Should this occur, intense agitation will help redissolve
the precipitate. Due it its thermolabile qualities, the po-
Description tassium tellurite is supplied sterile filtered.
Aquouse solutions of potassium tellurite at 1% or 3,5%,
sterilized by filtration and suitable to be used as an
inhibitor additive in culture media.

The Potassium Tellurite Solution is added to culture me-

dia as an inhibitor. Its purpose is to prevent the growth of
most gram-negatives and of those gram-positives unable
of reducing it.

Ringer Powder

Ref. 06-073 Description

Ringer saline solution is an isotonic medium which is
Presentation more balanced than the simple sodium chloride saline
500 g Flask solution, and its formulation permits the autoclaving
100 g Flask without producing any precipitation.

For the routine work with bacteria the solution is diluted

Specification
Isotonic solution for the cellular suspensions.
Formula (in g/L)
Sodium chloride ...................................... 2,250
Potassium chloride ................................. 0,105
Calcium chloride ..................................... 0,120
Sodium bicarbonate ............................... 0,050
Directions
To obtain an isotonic solution for eukaryotic cells, dis-
solve 10 g of powder in 1 L of distilled water. To obtain
an isotonic solution for prokaryotic cells, dissolve 2,5 g of
powder in 1 L of distilled water.
Distribute into suitable containers and sterilize in the
autoclave at 121°C for 15 minutes.
one fourth (to the fourth part) (Ringer 1/4), and is em-
ployed to get cell suspensions or to prepare the dilution
banks.
Nonetheless, to dilute the food samples or substances
that have undergone thermal treatment, it is more advis-
able to use Peptone Water (Ref. 3-156) for the dilutions,
since the Peptone Water acts as a revitalizer also.
References
DAVIS, J.G. (1956) Laboratory Control of Dairy Plant.
Dairy Industries Ltd. London.
ANONYMOUS (1937) Bacterial Tests for Graded
Milk. Memo 139-Food. Dept. of Health and Social
Security,London.

210
 Skimmed Milk

Ref. 06-019 Skimmed milk may be use alone or as an additive to

other culture media. It is a very suitable medium for the
Presentation culture of acid lactic bacteria and for identification in
500 g Flask general, in base to its capacity to coagulate o peptonize
milk. With the additon of suitable indicators as brom-
cresol purple at 0,004% the pH variations in the transfor-
Description mation may be screened. It also accepts oxid-reduction
Powder of skimmed milk for bacteriology is obtained
indicators like methylen blue, resazurine or TTC (Ref.
after a depurated atomization process that keep it free
6-023) to verify microorganisms development.
from thermophil organismsm that use to interfere with its
use.
Skimmed milk may be added to media as Tryptone Soy
Agar (Ref. 01-200) or Nutrient Agar (Ref. 01-144 and 01-
100 g of powder produce 1 L of skimmed milk. Water
140) to detect caseolytic activity.
addition must be gradual, until getting an homogene-
ous paste. Then fullfil with water to the desired volume.
Fat ............................................................. 0,5 %
Sterilization may be under fluent vapor for 30 minutes
Protein ..................................................... 33,0 %
and three consecutive days or in the autoclave at 121°C
Ash ............................................................ 8,0 %
for 15 minutes or at 114°C for 15-20 minutes (this last
Moisture ..................................................... 5,0 %
way is the better). Anycase, do not overheat prepared
Lactic acid .................................................. 1,5 %
milk since natural sugars may become caramelizated
Antibiotic test .....................................Negative
and produce toxic compounds.

Sodium Biselenite

Ref. 06-615 T N The intended use of this product is to complete the fol-
R-23/25-33-50/53
lowing culture media by adding the specified amounts:
Presentation S-20/21-28-45-60-61

100 g Flask 02-602 Selenite Cystine Broth Base .....................4 g/L

02-603 Selenite Brilliant Green Broth Base ...........4 g/L
02-598 Selenite Broth Base ..................................4 g/L
Description
Chemical compound to be added to selenite based
The use of this product is restricted to technically quali-
culture media.
fied personnal. Keep attention to the job limitations in
inexpert personnal.
The toxic and potetially theratogenicity of this product
recommend its exclusion from the dehydrated powder
mixture of culture media, to minimize the hazard of acci-
dental inhalation or contact. The supply of this product in
a separate form from the base medium, with all the risk
considerations, enhances its safe and responsible use.

211
 Sucrose Powder (D(+) Saccharose Powder)
Ref. 06-049
Presentation
500 g Flask
Description
Purified and standardized carbohydrate for the use in
microbiological cultrure media as energy source for
bacteria. Carbohydrate are adapted to the suitable basic
media.
Scharlau carbohydrates are pure and without mixtures,
and this important characterístic is assured in order to
get always right results.
Physical Data
Bulk density....................................~ 800 - 950
Solubility in water (25ºC) ............freely soluble
Melting point ...................................~ 169 -170
pH (100 g/L H2O, 20ºC) ..............................~ 7
212
Specifications
Specific rotation ([α]20ºC/D, c=26, H O) ...... +66,3 - +67,0 º
2
Molecular weight .............................. 342,30 g/mol
Acidity/alkalinity ................................ passes test
Appaerance of solution (50% water) ....... passes test
Conductivity ..................................... max. 35 μS/cm
Chlorides (Cl)........................................ max. 0,0035 %
Sulfites (as SO3) ................................... max. 0,0015 %
Sulfates (SO4) ....................................... max. 0,005 %
Dextrines .......................................... passes test
Glucose and invert sugar ................. passes test
Calcium (Ca)......................................... max. 0,001 %
Heavy metals (as Pb) ........................... max. 0,0005 %
Lead (Pb) .............................................. max. 0,00005
%
Sulfated ash ......................................... max. 0,01 %
kg/m3 Organic volatile impurities (NF) ......... passes test
Residual solvents (Eur. Phar./NF) ....... max. 0,5 %
ºC
 TTC Sterile Solution 1%

Ref. 06-023 The general structure is the following:

Presentation
100 mL Flask

Description
Sterile solution at 1% of 2-3-5-triphenyl-2H-tetrazolium
chloride. It is used as an additive for culture media to
show biological activity, since the colourless form gets
hydrogenizated or reduced to a red insoluble pigment:
triphenylformazan, which may be easily observed.

Despite of TTC decomposes at 243°C, it is not advis-

able to incorporate it to culture media before steriliza-
tion, because it lose efficacy. Very good results may be
achieved when the addition is carried out asseptically
with cold medium at 60°C maximum. TTC is photolabile
and becomes yellow by the effect of light, therefore keep
This product is especially produced to be added to
it in the refrigerator and avoid direct light.
the following media: Chapman TTC Agar Base (Ref.
01-053), KF Media (Ref. 01-294 and Ref. 02-294) and
Concentration of use vary depending on the medium, but
Slanetz Bartley Agar Base (Ref. 01-579).
generally it goes between 0,3 and 1% (v/v).

Urea Sterile Solution 40%

Ref. 06-083 It is supplied to be used with the dehydrated media Urea

Agar acc. to Christensen (Ref. 01-261) and Urea Broth
Presentation (Ref. 02-202). It must be added to these media after the
100 mL Flask sterilization and with the media cooled below 55°C.

Once it is added, do not reheat the media.

Description
Aquose urea solution 40%, sterilized by filtration and
suitable to be used as an additive in culture media.

Vaseline Sterile

Ref. 06-077 incorporation of air to the medium. To seed the tubes,

use a capilar pipette or inoculate them previously to the
Presentation vaseline addition.
100 mL Flask
Vaspar is another method used to achieve an hermetic
lock. It is prepared melting vaseline and solid parafine
Description toghether in equal parts.
Liquid media may be kept in anaerobic conditions if
Vaseline sterile is especially suggested for the addition
sterile compounds, as vaseline, are used to grant an
to O/F Medium (Ref. 03-037). For solid media is more
hermetic lock.
suitable to use the Sealing Agar (Ref. 01-174).
To achieve an hermetic lock in tubes with liquid medium,
heat them up in boiling bath for 10 minutes to remove
the oxygen and add after the vaseline to avoid the

213
Supplements
 Improved NEW presentation
PRESENTATION: 10 VIALS INSIDE A RESISTANT CASE
REFERENCE DESCRIPTION
06-012CASE SC Selective Supplement for Clostridium
VIAL CONTENTS: 120 mg of Sodium azide, 90 mg of Neomycin
sulphate and solvent.
Each vial is sufficient to supplement 500 ml of Blood
Columbia Agar Base (Ref. 01-034) or Blood Agar Base
(Ref. 01-352) in order to prepare Clostridium spp. Selective Agar.
06-013CASE CP Gram-positive cocci in Blood Agar Selective Supplement
VIAL CONTENTS: 5 mg of Colistin sulphate, 7,5 mg of Nalidixic acid
and solvent.
Each vial is sufficient to supplement 500 ml of Blood Columbia
Agar Base (Ref. 01-034) or Blood Agar Base (Ref. 01-352) and
obtain Staphylococcus and Streptococcus Selective Agar.
06-017CASE Brilliant Green + Novobiocin Selective Supplement
VIAL CONTENTS: 5 mg of Brilliant green, 20 mg of Novobiocin
and solvent.
Each vial is sufficient to supplement 500 ml of Tetrationate
Base Broth (Ref. 02-033) and Muller-Kauffmann Medium
(Ref. 02-335).
06-021CASE Polymyxin B Sulphate Selective Supplement
VIAL CONTENTS: 50 mg Polymyxin B sulphate and solvent.
Each vial is sufficient to supplement 500 ml of Bacillus cereus
Agar Base (Ref. 01-262).
06-022CASE Cycloheximide Selective Supplement
VIAL CONTENTS: 2 mg of Cycloheximide and solvent.
Each vial is sufficient to supplement 500 ml of WL Nutrient
Agar (Ref. 01-210) or WL Nutrient Broth (Ref. 02-210)
and converted in WL Differential Agar or Broth.
06-025CASE Brucella Selective Supplement
VIAL CONTENTS: 50 mg of Cycloheximide, 3000 u.i. of
Polymyxin B Sulphate, 12500 u.i. of Bacitracin sulphate
and solvent.
Each vial is sufficient to supplement 500 ml of Brucella Selective
Agar (Ref. 01-042) and Brucella Selective Broth (Ref. 02-042).
06-085CASE Rosolic Acid Selective Supplement
VIAL CONTENTS: 50 mg of Rosolic acid and solvent.
Each vial is sufficient to supplement 500 ml of Faecal Coliforms
Agar (m-FC Agar) (Ref. 01-287) or Faecal Coliforms Broth
(m-FC Broth) (Ref. 02-287).
06-091CASE CPB Selective Supplement for Campylobacter
VIAL CONTENTS: 2,5 mg of Trimethoprim, 5 mg of Vancomycin,
7,5 mg of Cephalotin, 1250 u.i. of Polymyxin B sulphate,
1 mg of Amphotericin B and solvent.
Each vial is sufficient to supplement 500 ml of Blood Columbia
Agar Base (Ref. 01-034) and obtain 500 ml of Campylobacter
Selective Agar acc. Blaser-Wang.
06-102CASE MUG Supplement. Fluorescent Agent for Escherichia coli
VIAL CONTENTS: 50 mg of MUG
(4-methilumbeliferil-ß-D glucuronide) and solvent.
Each vial is sufficient to supplement 500 ml of Coliforms Agar
or Broth.
06-106CASE Listeria Selective Supplement for Primary Enrichment (UVM I)
VIAL CONTENTS: 10 mg of Nalidixic acid, 6 mg of Acriflavine and
solvent.
Each vial is sufficient to supplement 500 ml of Listeria Enrichment
Broth Base (UVM) (Ref. 02-472) in order to prepare 500 ml
of Listeria Primary Enrichment Medium (UVM I formulation).
06-107CASE Listeria Selective Supplement for Enrichment acc. FDA
and IDF/FIL
VIAL CONTENTS: 20 mg of Nalidixic acid, 25 mg of Cycloheximide,
7,5 mg of Acriflavine and solvent.
Each vial is sufficient to supplement 500 ml of Listeria
Enrichment Broth Base acc. Lovett (Ref. 02-498).
06-109CASE Oxford Agar Selective Supplement for Listeria
VIAL CONTENTS: 5 mg of Phosphomycin, 1 mg of Sodium
Cephotaxim, 10 mg of Colistin, 200 mg of Cycloheximide,
2,5 mg of Acriflavine and solvent.
Each vial is sufficient to supplement 500 ml of Oxford Agar
Base (Ref. 01-471) in order to prepare Listeria Selective Agar
(Oxford formulation).
06-110CASE Palcam Agar Selective Supplement for Listeria
VIAL CONTENTS: 5 mg of Polymyxin B sulphate, 10 mg of Sodium
ceftazidime, 2,5 mg of Acriflavine and solvent.
Each vial is sufficient to supplement 500 ml of Palcam Agar
Base (Ref. 01-470) in order to prepare Listeria Selective Agar
(Palcam formulation).
216
REFERENCE DESCRIPTION
06-111CASE Listeria Selective Supplement for Secondary Enrichment
(UVM II/Fraser)
VIAL CONTENTS: 10 mg of Nalidixic acid, 12,5 mg of Acriflavine
and solvent.
Each vial is sufficient to supplement 500 ml of Listeria
Enrichment Broth Base (UVM) (Ref. 02-472) in order to prepare
500 ml of Listeria Secondary Enrichment Medium (UVM II
formulation); or to supplement 500 ml of Listeria Enrichment
Broth acc. Fraser (Ref. 02-496) in order to prepare 500 ml
of Fraser Broth.
06-112CASE Ferric Ammonium Citrate for Bacteriology
VIAL CONTENTS: 250 mg of Ferric ammonium citrate and solvent.
Each vial is sufficient to supplement 500 ml of Listeria
Enrichment Broth Base acc. Fraser (Ref. 02-496)
06-113CASE Ferric Ammonium Citrate for bacteriology
VIAL CONTENTS: 312 mg of Ferric ammonium citrate and solvent.
Each vial is sufficient to supplement 500 ml of Lactose Sulphite
Broth Base (Ref. 02-519).
06-114CASE Disodium disulphite Selective Supplement for bacteriology
VIAL CONTENTS: 375 mg of Disodium disulphite and solvent.
Each vial is sufficient to supplement 500 ml of Lactose Sulphite
Broth Base (Ref. 02-519).
06-115CASE Oxytetracycline Selective Supplement
VIAL CONTENTS: 50 mg of Oxytetracycline and solvent.
Each vial is sufficient to supplement 500 ml of Sabouraud with
Oxytetracycline Agar Base (OGYEA) (Ref. 01-275).
06-116CASE Cycloserine Selective Supplement
VIAL CONTENTS: 100 mg of Cycloserine and solvent.
Each vial is sufficient to supplement 250 ml of Tryptose Sulphite
Cycloserine (TSC) agar Base for Clostridium perfringens
Ref. 1-278).
06-118CASE Chloramphenicol Selective supplement
VIAL CONTENTS: 25 mg of Chloramphenicol and solvent.
Each vial is sufficient to supplement 500 ml of Sabouraud
Dextrose Agar (Eur. Phar. Agar Medium C), (Ref. 01-165).
06-124CASE Nalidixic Acid Selective Supplement
VIAL CONTENTS: 7,5 mg of Nalidixic acid and solvent.
Each vial is sufficient to supplement 500 ml of CN Selective
Agar Base for Pseudomonas (Ref. 01-609).
06-125CASE m-CP Selective Supplement
VIAL CONTENTS: 200 mg of D-Cycloserine, 12,5 mg of Polymyxin
B sulphate, 30 mg of 3-indoxyl-ß-D-glucopyranoside, 50 mg
of Phenolphthalein diphosphate, 45 mg of Iron III Chloride,
and solvent.
Each vial is sufficient to supplement 500 ml of m-CP Agar Base
(Clostridium perfringens Agar) (Ref. 01-513).
Storage conditions:
Should be stored at 2-8ºC in the dark.
Shelf life:
18-24 months depending the reference. Larger vial-larger variety of supple-
ments.
MORE INFORMATION
AVAILABLE IN OUR WEB
AND CD-ROM CATALOGUE
 Selective Supplements

Culture media supplements in a practical presentation:

an extemporaneous solution.
Main advantages:

• fast
• simple
• easy
• safe
• ready to add
• easy storage
• longer shelf life
• less risk of contamination

With this 10 vial-case format you no longer have to worry

about things like sterile solvents, sterile syringes, sterilis-
ing the supplements that must be added to the medium
by filtration ...
With one simple pressure on the lid you obtain the sterile
supplement solution, ready to add to the medium base.
Selective supplements should be stored at 2-8°C in
the dark. When stored as directed the reagents remain
stable until the expiry date shown on the label.

Method of use: 4 simple steps

1 2
Press on the cap brea- The solid then falls
king the container that into the solvent. Shake
holds vigorously
the solid. for total dissolution.

3 4
The supplement is now Homogenize and
ready. Open the vial distribute into the suitable
aseptically, close to a fla- container: flasks, tubes or
me or in a safety cabinet. plates.
Pour over the medium
base, which has been
cooled down to 45-50°C.

217
 Basic Fuchsin 200 Selective Supplement

Ref. 06-617CASE F
Precautions
R-11 • This product should be for laboratory use only.
Contents S-7-16 • Do not use beyond stated expiry date.
The box contains 10 vials. Each vial is sufficient to sup-
plement 250 mL of Endo LES (Ref. 01-604). Applicable media
Ref. 01-604 Endo LES Agar Base
Vial contents
Necessary amount for 250 mL of medium.
Basic Fuchsin ............................................ 200 mg
Ethanol .......................................................... 5 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 250 mL of Endo LES Agar Base.
Note: Don’t heat the media once the supplement has
been added.

Basic Fuchsin 250 Selective Supplement

Ref. 06-607CASE F

R-11 Precautions
Contents S-7-16 • This product should be for laboratory use only.
The box contains 10 vials. Each vial is sufficient to sup- • Do not use beyond stated expiry date.
plement 500 mL of Endo Agar Base (Ref. 01-589), Endo
DEV Agar Base (Ref. 01-606) and only 250 mL of Endo Applicable media
Base Broth (Ref. 02-605). Ref. 01-589 Endo Agar Base
Ref. 01-606 Endo DEV Agar Base
Vial contents
Necessary amount for 500 mL of solid medium or 250
mL of liquid broth.
Basic Fuchsin ............................................ 250 mg
Ethanol .......................................................... 5 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to the boiled medium. Homogenize and use as per each
monography medium. Do not remelt the solid media.

218
 Brilliant Green + Novobiocin Selective Supplement

Ref. 06-017CASE Precautions

• This product should be for laboratory use only.
Contents • Do not use beyond stated expiry date.
The box contains 10 vials. Each vial contains inhibitors
sufficient to add to 500 mL of Tetrathionate Broth Base Applicable media
Ref. 02-033 or 02-335 Muller Kauffmann Medium. Ref. 02-033 Tetrathionate Base Broth
Ref. 02-335 Muller-Kauffmann Medium
Vial contents
Necessary amount for 500 mL of medium.
Brilliant Green ................................................ 5 mg
Novobiocin, sodium salt .............................. 20 mg
Distilled water ................................................ 5 mL

Directions
Mix the liquid with the powder by pressing down on
the cap. Shake to dissolve and aseptically add the vial
contents to 500 mL of boiled broth base cooled to 50°C.
Homogenize and use as per each monography medium.
Note: Don’t heat the media once the supplements have
been added.

219
 Chloramphenicol Selective Supplement
T
Ref. 06-118CASE Precautions
R-45 • This product should be for laboratory use only.
S-53-45
Contents • Do not use beyond stated expiry date.
The box contains 10 vials. Each vial is sufficient to
supplement 500 mL of Sabouraud Dextrose Agar (Ref. Applicable media
01-165). Ref. 01-165 Sabouraud Dextrose Agar (Eur. Phar. Agar
Medium C)
Vial contents
Necessary amount for 500 mL of medium.
Chloramphenicol ......................................... 25 mg
Distilled water ................................................ 5 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500 mL of sterile agar base cooled to 50°C. Ho-
mogenize and distribute the complete medium into the
containers.
Note: Don’t heat the media once the supplement has
been added.

m-CP Selective Supplement

Ref. 06-125CASE Precautions

• This product should be for laboratory use only.
Contents • Do not use beyond stated expiry date.
The box contains 10 vials. Each vial is sufficient to
supplement 500 mL of m-CP Agar Base, Ref. 01-513, Applicable media
in order to prepare 500 mL of m-CP (Clostridium perfrin- Ref. 01-513 m-CP Agar Base
gens) Agar.

Vial Contents
Necessary amount for 500 mL of complete medium.
D-Cycloserine .......................................200,0 mg
Polymixin B sulfate .................................12,5 mg
3-Indoxyl-ß-D-Glucopyranoside .............30,0 mg
Pehnolphthalein di-phosphate ................50,0 mg
Iron III Cloride .........................................45,0 mg
Distilled water ...........................................5,0 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500 mL of sterile agar base cooled to 50ºC.
Note: Don’t heat the media once the supplement has
been added.

220
 CP Gram-positive cocci in Blood Agar Selective
Supplement
Ref. 06-013CASE Precautions
• This product should be for laboratory use only.
Contents • Do not use beyond stated expiry date.
The box contains 10 vials. Each vial is sufficient to sup-
plement 500 mL of Blood Columbia Agar Base Ref. 01-
034 or Blood Agar Base Ref. 01-352 in order to prepare Applicable media
500 mL of Staphylococcus and Streptococcus selective Ref. 01-034 Blood Agar Base (Columbia)
blood agar. Ref. 01-352 Blood Agar Base

Vial contents
Necessary amount for 500 mL of complete medium.
Colistin sulfate ........................................ 5,00 mg
Nalidixic Acid, sodium salt ...................... 7,50 mg
Distilled Water ........................................ 5,00 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500mL of blood agar. Homogenize and use as per
each monography medium.
Note: Don’t heat the media once the supplements have
been added.

221
 Cycloheximide Selective Supplement

Ref. 06-022CASE Precautions

• This product should be for laboratory use only.
Contents • Do not use beyond stated expiry date.
The box contains 10 vials. Each vial is sufficient to
supplement 500 mL of WL Nutrient Agar or Broth Ref. Applicable media
01-210 or 2-210 in order to prepare 500 mL of WL Dif- Ref. 01-210 WL Nutrient Agar
ferential Agar or Broth. Ref. 02-210 WL Nutrient Broth

Vial contents
Necessary amount for 500 mL of complete medium.
Cycloheximide ............................................. 2 mg
Sodium chloride ........................................... 8 mg
Distilled water .............................................. 5 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500 mL of sterile agar base cooled to 50°C.
Note: Don’t heat the media once the supplement has
been added.

Cycloserine Selective Supplement

Ref. 06-116CASE Precautions

• This product should be for laboratory use only.
Contents • Do not use beyond stated expiry date.
The box contains 10 vials. Each vial is sufficient to sup-
plement 250 mL of Tryptose Sulfite Cycloserine Agar Applicable media
Base (TSC Agar) Ref. 01-278. Ref. 01-278 Tryptose Sulfite Cycloserine Agar (TSC
Agar)
Vial contents
Necessary amount for 250 mL of medium.
D-Cycloserine ..........................................100 mg
Distilled water ..............................................5 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solu-
tion to 250 mL of sterile agar base cooled to 50°C. If
it is desired, add 20 mL of Egg Yolk Sterile Emulsion
(Ref. 06-016). Homogenize and distribute the complete
medium into the plates.
Note: Don’t heat the media once the supplements has
been added.

222
 Ferric Ammonium Citrate for Bacteriology
Ref. 06-112CASE
Contents
The box contains 10 vials. Each vial is sufficient to sup-
plement 500 mL of Listeria Enrichment Broth acc. Fraser
Ref. 02-496 in order to prepare 500 mL of Fraser broth.
Vial contents
Necessary amount for 500 mL of complete medium.
Ferric ammonium citrate ......................... 250 mg
Distilled water ............................................. 5 mL
Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500 mL of sterile broth base cooled to 50°C.
Note: Don’t heat the media once the supplement has
been added.
Precautions
• This product should be for laboratory use only.
• Do not use beyond stated expiry date.
Applicable media
Ref. 02-496 Listeria Enrichment Broth Base
acc. to Fraser
Listeria Selective Supplement for Enrichment
acc. FDA/IDF
T Directions
Ref. 06-107CASE R-61-25-68
S-36/37-45-53
Contents
The box contains 10 vials. Each vial is sufficient to sup-
plement 500 mL of Listeria Enrichment Broth Base acc.
Lovett Ref. 02-498 in order to prepare 500 mL of Listeria
enrichment broth according FDA and IDF/FIL.
Vial contents
Necessary amount for 500 mL of complete medium.
Nalidixic acid, sodium salt ...................... 20,0 mg
Cycloheximide ........................................ 25,0 mg
Acriflavine ................................................. 7,5 mg
Distilled water ........................................... 5,0 mL
Ref. 06-113CASE
Contents
The box contains 10 vials. Each vial is sufficient to supple-
ment 500 ml of Lactose Sulfite Broth Base Ref. 02-519 in
order to prepare 500 ml of Lactose Sulfite Broth.
Vial contents
Necessary amount for 500 ml of complete medium.
Ferric ammonium citrate ......................... 312 mg
Distilled water ............................................. 5 mL
Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution to
500 ml of sterile broth base cooled to 50°C.
Note: Don’t heat the media once the supplement has been
added.
Precautions
• This product should be for laboratory use only.
• Do not use beyond stated expiry date.
Applicable media
Ref. 02-519 Lactose Sulfite Broth Base
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500 mL of sterile broth base cooled to 50°C.
Note: Don’t heat the media once the supplement has
been added.
Precautions
• This product should be for laboratory use only.
• Do not use beyond stated expiry date.
Applicable media
Ref. 02-498 Listeria Enrichment Broth Base acc. Lovett.
223
 Listeria Selective Supplement for Primary
Enrichment (UVM I)
T Precautions
Ref. 06-106CASE • This product should be for laboratory use only.
R-61-25-68
S-36/37-45-53
• Do not use beyond stated expiry date.
Contents
The box contains 10 vials. Each vial is sufficient to
supplement 500 mL of Listeria Enrichment Broth Base Applicable media
(UVM) Ref. 02-472 in order to prepare 500 mL of Liste- Ref. 02-472 Listeria Enrichment Broth Base (UVM)
ria primary enrichment medium.

Vial contents
Necessary amount for 500 mL of complete medium.
Nalidixic acid, sodium salt ......................... 10 mg
Acriflavine .................................................... 6 mg
Distilled water .............................................. 5 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500 mL of sterile broth base cooled to 50°C.
Note: Don’t heat the media once the supplement has
been added.

Listeria Selective Supplement for Secondary Enrichment

(UVM II / Fraser)

Ref. 06-111CASE T Precautions

R-61-25-68
Contents S-36/37-45-53
The box contains 10 vials. Each vial is sufficient to
supplement 500 mL of Listeria Enrichment Broth Base
(UVM) Ref. 02-472 in order to prepare 500 mL of Liste-
ria secondary enrichment medium (UVM II formulation);
or to supplement 500 mL of Listeria Enrichment Broth
acc. Fraser Ref. 02-496 in order to prepare 500 mL of
Fraser Broth.
• This product should be for laboratory use only.
• Do not use beyond stated expiry date.
Applicable media
Ref. 02-472 Listeria Enrichment Broth Base (UVM)
Ref. 02-496 Listeria Enrichment Broth Base acc. to
Fraser

Vial contents
Necessary amount for 500 mL of complete medium.
Nalidixic acid, sodium salt ..................... 10,0 mg
Acriflavine ..............................................12,5 mg
Distilled water ..........................................5,0 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500 mL of sterile broth base cooled to 50°C.
Note: Don’t heat the media once the supplement has
been added.

224
 MUG Supplement
Ref. 06-102CASE
Contents
The box contains 10 vials. Each vial is sufficient to sup-
plement 500 mL of Coliforms Agar or Broth.
Vial contents
Necessary amount for 500 mL of complete medium.
MUG (4-methilumbeliferil-ß-D-glucuronide) .....50 mg
Distilled water ..............................................5 mL
Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500 mL of agar or broth cooled to 50°C.
MUG supplement may be added to almost all the media
that allow the growth of Escherichia coli for its identifica-
tion. However, results will be more reliable in all those
media that are selective for coliforms. Attached is a list of
the most currently used. In our Culture Media Handbook
you will find a table indicating the aspect of Escherichia
coli in each medium.
Note: Don’t heat the media once the supplement has
been added.
Nalidixic Acid Selective Supplement
Ref. 06-124CASE
Contents
The box contains 10 vials. Each vial is sufficient to sup-
plement 500 mL of CN Selective Agar for Pseudomonas
(Ref. 01-609).
Vial Contents
Necessary amount for 500 mL of complete medium:
Nalidixic acid sodium salt ........................ 7,5 mg
Distilled water .......................................... 5,0 mL
Precautions
• This product should be for laboratory use only.
• Do not use beyond stated expiry date.
Applicable media
Ref. 01-047 CLED Agar (Brolacin Agar)
Ref. 01-118 MacConkey Agar (Eur. Phar. Medium H)
Ref. 01-164 Violet Red Bile Agar (VRB Agar)
Ref. 01-220 Violet Red Bile Dextrose Lactose Agar
(VRBDL Agar)
Ref. 01-484 E.coli Direct Agar (ECD Agar)
Ref. 02-041 Brilliant Green Bile 2% Broth
Ref. 02-060 EC Broth
Ref. 02-105 Lactose Broth (Eur. Phar. Broth Medium D)
Ref. 02-108 Tryptose Lauryl Sulfate Broth
Ref. 02-118 MacConkey Broth
Ref. 02-120 MacConkey Modified Broth
Ref. 02-611 MacConkey G Broth (Eur. Phar. Medium G)
Directions
Mix the liquid with the powder by pressing downd on the
cap. Shake to dissolve and asseptically add the solution
to 500 mL of sterile agar base cooled to 50ºC.
Note: Don’t heat the media once the supplement has
been added.
Precautions
• This product should be for laboratory use only.
• Do not use beyond stated expiry date.
Applicable media
Ref. 01-609 CN Selective Agar Base for Pseudomonas.
225
 Oxford Agar Selective Supplement
T
Ref. 06-109CASE R-25-52/53-61/68
S-36/37-45-53-61 Precautions
Contents • This product should be for laboratory use only.
The box contains 10 vials. Each vial is sufficient to sup- •Do not use beyond stated expiry date.
plement 500 mL of Oxford Agar Base Ref. 01-471 in
order to prepare 500 mL of Listeria selective agar (Oxford
formulation). Applicable media
Ref. 01-471 Oxford Agar Base
Vial contents
Necessary amount for 500 mL of complete medium.
Acriflavine ................................................... 2,5 mg
Fosfomicyn ................................................. 5,0 mg
Sodium cefotaxim ....................................... 1,0 mg
Colystin ..................................................... 10,0 mg
Cycloheximide ........................................ 200,0 mg
Distilled water ............................................. 5,0 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution to
500 mL of sterile agar base cooled to 50°C.
Note: Don’t heat the media once the supplement has
been added.

Oxytetracycline Selective Supplement

Ref. 06-115CASE
Precautions
Contents • This product should be for laboratory use only.
The box contains 10 vials. Each vial is sufficient to sup- • Do not use beyond stated expiry date.
plement 500 mL of Sabouraud with Oxytetracycline Agar
Base Ref. 01-275. Applicable media
Ref. 01-275 Saboraud with Oxytetracycline Agar (OG-
Vial contents YEA)
Necessary amount for 500 mL of medium.
Oxytetracycline HCl .................................. 50 mg
Distilled water ............................................. 5 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500 mL of sterile agar base cooled to 50°C. Homoge-
nize and distribute the complete medium into the plates.
Note: Don’t heat the media once the supplement has
been added.

226
 Palcam Agar Selective Supplement

Ref. 06-110CASE Precautions

• This product should be for laboratory use only.
Contents • Do not use beyond stated expiry date.
The box contains 10 vials. Each vial is sufficient to
supplement 500 mL of Palcam Agar Base Ref. 01-470
in order to prepare 500 mL of Listeria Selective Agar Applicable media
(Palcam formulation). Ref. 01-470 Palcam Agar Base

Vial contents
Necessary amount for 500 mL of complete medium.
Acriflavine ............................................... 2,5 mg
Polymixin B sulphate .............................. 5,0 mg
Sodium ceftazidime ............................. 10,0 mg
Distilled water ......................................... 5,0 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 500 mL of sterile agar base cooled to 50°C.
Note: Don’t heat the media once the supplement has
been added.

Polymixin B Sulfate Selective Supplement

Ref. 06-021CASE Precautions

• This product should be for laboratory use only.
Contents • Do not use beyond stated expiry date.
The box contains 10 vials. Each vial is sufficient to
supplement 500 mL of Bacillus cereus Agar Base Ref. Applicable media
01-262. Ref. 01-262 Bacillus cereus Agar
Ref. 01-487 Bacillus cereus Selective Agar
Vial contents
Necessary amount for 500 mL of medium.
Polymixin B sulphate ................................ 50 mg
Distilled water ............................................. 5 mL

Directions
Mix the liquid with the powder by pressing down on the
cap Shake to dissolve and aseptically add the solution to
450 mL of sterile agar base cooled to 50°C.
Add also 50 mL of sterile Egg Yolk Emulsion. Homoge-
nize and distribute the complete medium into the plates.
Note: Don’t heat the media once the supplements have
been added.

227
 Rosolic Acid Selective Supplement
F
Ref. 06-085CASE Precautions
R-11
S-7-16
• This product should be for laboratory use only.
Contents • Do not use beyond stated expiry date.
The box contains 10 vials. Each vial is sufficient to sup-
plement 500 mL of Fecal Coliforms Agar or Broth (m-FC)
Ref.1-287 or 2-287 in order to prepare 500 mL of m-FC Applicable media
complete medium. Ref. 01-287 Fecal Coliforms Agar (FC Agar)
Ref. 02-287 Fecal Coliforms Broth (FC Broth)
Vial contents
Necessary amount for 500 mL of complete medium.
Rosolic Acid ............................................. 50 mg
Ethanol ...................................................... 5 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake till total dissolution and aseptically add the
solution to 500 mL of agar or broth cooled to 50°C.
Use medium newly made.
Note: Don’t heat the media once the supplement has
been added.

228
 SC Selective Supplement

Ref. 06-012CASE T N Precautions

R-25-32-42/43-51/53
Contents S-7-22-24-37-45-61
The box contains 10 vials. Each vial is sufficient to sup-
plement 500 mL of Blood Columbia Agar Base Ref. 01-
034 or Blood Agar Base Ref. 01-352 in order to prepare
500 mL of Clostridium ssp. selective agar.
• This product should be for laboratory use only.
• Do not use beyond stated expiry date.
Applicable Media
Ref. 01-034 Blood Columbia Agar Base
Ref. 01-352 Blood Agar Base

Vial contents
Necessary amount for 500 mL of complete medium.
Sodium Azide .......................................120,0 mg
Neomycine sulfate ..................................90,0 mg
Distilled water ...........................................5,0 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 475 mL of sterile agar base cooled to 50°C. Add 25
mL of defibrinated blood . Homogenize and distribute
into the plates.
Note: Don’t heat the media once the supplements have
been added.

Disodium disulfite (meta-bisulfite) for Bacteriology

Ref. 06-114CASE

Contents Precautions
The box contains 10 vials. Each vial is sufficient to supple- • Reagent for laboratory use only.
ment 500 ml of Lactose Sulfite Broth Base Ref. 02-519 in • Do not use beyond stated expiry date.
order to prepare 500 ml of Lactose Sulfite Broth.
Applicable Media
Ref. 02-519 Supplement for Lactose Sulfite Broth
Vial contents
Necessary amount for 500 mL of complete medium.
Disodium sulfite ....................................375,0 mg
Distilled water ...........................................5,0 mL

Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution to
500 ml of sterile broth base cooled to 50°C.
Note: Don’t heat the media once the supplement has
been added.

229
Reagents
 Barrit’s Reagent
Ref. 06-027 F
R-11-36
Presentation S-7-16-26
100 mL dropper flask
Specification
Reagent for the Voges-Proskauer test in enterobacte-
riaceae.
Description
All the enterobacteria ferment dextrose, but some spe-
cies like Klebsiella, Enterobacter, etc..., do it following
the 2-3-butanediol path and other species like E.coli,
Salmonella, etc..., do it by the mix acid path.
Voges Proskauer test shows the production of 2-3-
butanediol and acetoine, that are only produced in big
amounts in the 2-3-butanediol path. The basis of the test
is that these compounds, in alkaline medium and with
air, bear an oxidation and become diacethyl, which at the
same time reacts with guanidine producing very visible
coloured compounds.
O’Meara, in 1931, observed that adding creatine to the
alkaline solution (O’Meara’s Reagent, Ref. 06-006) aids
diacethyl reaction with guanidine, and then it was easier
to detect the red coloured compounds.
Later, in 1936, Barrit demonstrated that the addition
of an alcoholic solution of alpha-naphtol 5% (Barrit’s
Reagent, Ref. 06-027) increased very much sensibility,
and it was possible to obtain positive reaction even when
the final concentration of diacethyl was very low. It is
important to add the Barrit’s Reagent before the alkaline
solution.
Technique
Microorganism to be assayed is inoculated in MRVP
Broth (Ref. 02-207) and is incubated at 30°C for a period
between 3 and 5 days maximum.
Just before read, add Barrit’s Reagent (Ref. 06-027)
until all the medium gets a milky look. Following, add
O’Meara’s Reagent (Ref. 06-006) until the milky look
disappears and then shake chiefly. Relative volumes of
each reagent depend on the initial volume of inoculated
medium.
When test is positive a violet pinked colour appears be-
fore 5 minutes, starting from top. When test is negative
there is no change ol colour.
There is a quicker way to perform the Voges-Proskauer
test, with very little volumes of medium and massive
inocules. This way allows very short incubations (18-20
hours) and the read may be accelerated by heating up
the culture almost to boiling after adding the reagents.
However, this method increases the possibility of getting
wrong results.
References
BARRY, A.L., K.L. FEENEY (1967) Two Quick Methods
for the Voges Proskauer Test. Appl. Microbiol. 15:1138-
1141.
BARRIT, M.M. (1936) The intensification of The Voges
Proskauer Reaction by the Addition of alpha-Naphtol. J.
Pathol. Bacteriol. 42:441-453.
BLAZEVIC, D.J. and EDERER, G.M. (1975) Principles
of Biochemical Tests in Diagnostics Microbiology. John
Wiley Sons. N.Y.
O’MEARA, R.A.Q. (1931) A simple, Delicated and Rapid
Method of Detecting the Formation of Acetylmethyl-car-
binol by Bacteria Fermenting Carbohydrate. J. Pathol.
Bacteriol. 34:401-406.
McFADDIN, J.E. (2000) Biochemical tests for identifica-
tion of medical bacteria. 3rd. Ed. Cippincott William &
Wilkins. Philadelphia.
233
 Crystal Violet Dye Solution for Gram Staining
Ref. 06-029 Xn
R-40-52/53
Presentation S-36/37-61
100 mL dropper flask
1 L dropper flask
Description
This solution has been prepared according to the speci-
fications by Hucker for the Gram staining, and it is very
stable though, when it is too old, it may require filtration
immediately before the use. Elderliness does not affect
the staining properties but may force to make decoulor-
ing times longer.
Technique
Fix the smear following the habitual method and let it
cool or dry.
Cover the extension with Crystal Violet Dye Solution
(Ref. 06-029) and let it act for 1 minute
Wash the exceed of colouriser. The best way is to put
the preparation in a precipitate glass with fluent water.
Do not wash excessively. This step may be critical for
the rest of the test.
Cover the preparation with Lugol Solution (Ref. 06-030)
and drain immediately. Cover again with new solution
and let it act for 1 minute.
Wash softly again with water. To put the preparation in
water fluent for 5 seconds will be enough.
Decolourate, pouring the Gram Decoluriser (Ref. 06-
031), drop to drop, over the slanted microscopical slide
until total decolourising. Anycase, this step may not be
longer than 60 seconds.
Wash with water to stop the decolouring action.
234
Cover the preparation with Safranine Dye Solution (Ref.
06-032) and let it act for 1 minute.
Wash gently to remove the excess of colouriser, putting
the preparation in fluent water for 1-2 seconds.
Dry and observe under microscope in homogeneous
inmersion.
Microorganisms that get coloured by the first colouriser,
Crystal Violet, become dark blue coloured and it is said
that they take the gram, and they are call grampositive
(G+). Those microorganisms that just get coloured by
the contrast colouriser become red and they are called
gramnegative (G-).
Most of eukariote cells, except yeasts, are coloured as
gramnegative and thus the staining is not very significa-
tive. In spite of, it is one of the first levels in the system-
atic identification of prokariote: between the bacteria, all
the coci, except Neisseria and Veillonella, are gramposi-
tive, and all the sporogen bacilli and some part of the
other bacilli are grampositive too. Spiriles, vibria, rikett-
sia, clamidia and most bacilli are gramnegative.
Should actinomycete presence is suspected or microor-
ganisms are not well coloured as grampositives, it is ad-
visable to use Crystal Violet with Anilin for Actinomycete.
References
BARTHOLOMEW, J.W. (1962) Variables Influencing Re-
sults, and the Precise Definition of Steps in Gram Stain-
ing as a Means of Standardizing the Results Obtained.
Stain Technol. 37:139-155.
PAIK, G. (1980) Reagents, Stain and Miscellaneous
Procedures, in Manual of Clinical Microbiolgy by Lenette,
Balows, Hausler and Truant (eds.). ASM, Washington.
CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed.
William & Wilkins. Baltimore.
 Decolouriser for Gram Staining

Ref. 06-031 F Xi Dry and observe under microscope in homogeneous

R-11-36-66-67
inmersion.
Presentation S-7-9-16-26

100 mL dropper flask
1 L dropper flask
Description
Gram’s decolouriser is a mixture of alcohol and acetone
especially adapted to act softly and quickly over base
colourings. It use to be enough 15 or 20 drops to achive
an total decolourising of a correctly coloured smear.
Technique
Fix the smear following the habitual method and let it
cool.
Cover the extension with Crystal Violet Dye Solution
(Ref. 06-029) and let it act for 1 minute
Wash the exceed of colouriser. The best way is to put
the preparation in a precipitate glass with fluent water.
Do not wash excessively. This step may be critical for
the rest of the test.
Microorganisms that get coloured by the first colouriser,
Crystal Violet, become dark blue coloured and it is said
that they take the gram, and they are call grampositive
(G+). Those microorganisms that just get coloured by
the contrast colouriser become red and they are called
gramnegative (G-).
Most of eukariote cells, except yeasts, are coloured as
gramnegative and thus the staining is not very significa-
tive. In spite of, it is one of the first levels in the system-
atic identification of prokariote: between the bacteria, all
the coci, except Neisseria and Veillonella, are gramposi-
tive, and all the sporogen bacilli and some part of the
other bacilli are grampositive too. Spiriles, vibria, rikett-
sia, clamidia and most bacilli are gramnegative.
Should actinomycete presence is suspected or microor-
ganisms are not well coloured as grampositives, it is ad-
visable to use Crystal Violet with Anilin for Actinomycete.

Cover the preparation with Lugol Solution (Ref. 06-030)

and drain immediately. Cover again with new solution
References
and let it act for 1 minute.
BARTHOLOMEW, J.W. (1962) Variables Influencing Re-
Wash softly again with water. To put the preparation in
sults, and the Precise Definition of Steps in Gram Stain-
water fluent for 5 seconds will be enough.
ing as a Means of Standardizing the Results Obtained.
Stain Technol. 37:139-155.
Decolourate, pouring the Gram Decoluriser (Ref. 06-
PAIK, G. (1980) Reagents, Stain and Miscellaneous
031), drop to drop, over the slanted microscopical slide
Procedures, in Manual of Clinical Microbiolgy by Lenette,
until total decolourising. Anycase, this step may not be
Balows, Hausler and Truant (eds.). ASM, Washington.
longer than 60 seconds.
CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed.
Wash with water to stop the decolouring action.
William & Wilkins. Baltimore.
Cover the preparation with Safranine Dye Solution (Ref.
06-032) and let it act for 1 minute.
Wash gently to remove the excess of colouriser, putting
the preparation in fluent water for 1-2 seconds.

235
 Kovac’s Reagent
Ref. 06-018 C Since indole is one of the most volatile compounds of
R-10-20-34
Presentation S-26-36/37/39-45
100 mL flask
1L in flask
Description
Many microorganisms can produce indole (=benzopyr-
role) from Tryptophane thanks to a Tryptophanase, in
a process favored by oxygen and inhibited by glucose.
We therefore recommend that media used in this test
contain no glucose, present a high Tryptophane content
and are incubated aerobically.
The indole production property constitutes a classical
test for the differentiation of Escherichia and Entero-
bacter, integrated in the IMViC. It is also widely used for
the differentiation of other non enteric microorganisms.
The indole test can be conducted by various means, still
the biochemical basis of the reaction remain the same.
When a Pyrrol is mixed with a heated alcoholic p-dimeth-
ylaminobenzaldehyde solution, a peculiar cherry-red
coloring develops (Rosindole). If the reagent’s solution
is prepared with concentrated hydrochloric acid it will not
be necessary for it to progress in hot, as is the case with
our reagents.
The reagent which was initially used was that of Ehrlich-
Böhme (Ref. 06-024) with a previous extraction and con-
centration in Xylene, Ether, Chloroform or Toluol. Later
on Kovacs modified the original reagent by replacing
the ethanol with amyl-alcohol, so that a previous extrac-
tion was no longer necessary. In 1956 Gadenbusch and
Gabriel proved that the Kovacs’s reagent was much
stable if instead of Amyl alcohol, Butilic or Isoamyl were
used.
Nevertheless, the indole test reaction with paradiami-
nobenzaldehyde is not very specific since at least 17
compounds close to Indole are known to react similarly.
Although other reagents such as Oxalic acid and Hy-
droxilamine HCI have been proposed, their use has not
been widespread.
236
the group, some authors choose to conduct the test
using strips impregnated in reagent. Others suggest
placing the reagent directly on the cap’s swabs, avoiding
all contact with the culture medium so that the reaction
will occur with just the indole’s steam. Isenberg and
Suddenheim demonstrated that if a previous extraction
with Toluene was performed, only indole and Alpha-
Methylindole (=indoleacetic acid) were detected. Such
is the most common practice nowadays when using the
Ehrlich-Böhme reagent.
Technique
When conducting the indole production test on vari-
ous groups of bacteria, an appropriate reagent for each
group must be considered. Kovacs’s reagent (Ref.
06-018) is recommended for enterobacteria while the
Ehrlich reagent is for non-fermentating and anaerobes in
general.
The directions to follow during the assay are:
Inoculate the pure culture to be verified in a high Tryp-
tophane content medium, as for example the Indole-Ni-
trite Fluid Medium (Ref. 03-101), the SIM Medium (Ref.
03-176) or a non-glucose tryptone broth. Incubate at
35°C for 48 hours. Incubation time can be reduced to 4
hours if a massive inoculum in solid medium is done fol-
lowed by seeding of a small volume (0,5 mL) of culture
medium.
In both cases, examination after incubation in the follow-
ing way:
a) Kovacs’s Reagent (Ref. 06-018, for enterobacteria)
Add 0,5 mL of reagent to the broth’s surface, shaking
lightly to help extraction. If a cherry-red color develops in
less than a minute it will be considered a POSITIVE RE-
ACTION. No change in the original coloring constitutes a
negative reaction.
b) Ehrlich-Böhme Test
Add 1 mL of Xylene or Toluene to the broth and shake
energically to help extraction. Allow to stand for 2 min-
utes until both layers separate. Then slide 0.5 mL of the
reagent carefully down the sides of the tube, making
sure there is no agitation. Should a dark red colored ring
appear in the interface, it will be considered a POSITIVE
REACTION.
Storage
Reagents must be stored refrigerated and avoiding
direct light.
References
BÖHME, A. (1905) Die Anwendung der Ehrlichschen In-
dolreaktion für bakteriologische zwecke. Zentralbl. Bakt.
Parasit. Abt 1, Jena 40:129-133
 Kovac’s Reagent

KOVACS, N. (1928) Eine vereinfachte Methode zum

Nachweis der Indolbihdung duch bakterien. Z. Immuni- Kovac’s reaction on: Ref. 02-277 Tryptone Phosphate
tats. Forsch. Exp. Ther. 55:311-315 Water, Ref. 02-460 Tryptose Lauryl Sulfate Mannitol
GADEBUSCH, H.H. and GABRIELS, S. (1956) Modified Tryptophan Broth, Ref. 02-494 Buffered Peptone Water
Stable Kovacs’s Reagent for the detection of Indol. Am. Eur. Phar., Ref. 03-156 Tryptone Water.
J. Clin. Pathol. 26:1373-1375
ISENGERG, H.D. and SUNDHEIM, L.H. (1958) «Indole
Reactions in Bacteria» J. Bact. 75:682-690
CENTER FOR DISEASE CONTROL (1968)»Identifica-
tion of Unusual Pathogenic Bacteria» Atlanta G.
VIRGINIA POLYTECHNICAL INSTITUTE (1972) Anaer-
obe Laboratory Manual Blaksburg,Va.
EDWARDS, P.R. and EWING, W.H. (1972) «Identifica-
tion of Enterobacteriaceae» 3rd Ed. Burgess
Pub. Co. Minneapolis
McFADDIN, J.F. (2000) Biochemical tests for identifica-
tion of medical bacteria. 3rd. Ed. Lippincott William &
Wilkins. Philadelphia.
ISO 9308-2 Standard (1990) Water Quality - Detection
of coliforms thermotolerant coliforms and presumptive
E.coli - MPN method.

Left: control; center: Salmonella typhimurium ATCC

14028 (negative reaction); right: Escherichia coli ATCC
25922 (positive reaction).

Lactophenol Blue

Ref. 06-037 C efficacy in the staining of moulds and plants material has
R-21/22-34-41
been demonstrated.
Presentation S-26-36/37/39-45

100 mL flask Technique

1 L flask
Specification
Reagent for staining of fungi in fresh and fixed prepara-
tions.
Description
Lactophenol Blue is an excellent colouriser for fresh
preparation of fungi, since it has, in a single solution, the
propierties of a mordant, a fixer and a colouriser.
In fungi preparations for microscopical examination is
not usual to use water neither aquose colouriser solu-
tions, since most of the moulds expel water and remain
trapped in air microbubbles. This reason makes Lac-
tophenol Blue an idoneous medium for the examination,
becase it moistens the structures at the same time that it
acts like a fixer and soft mordant.
In other hand, its nature makes the preparations useful
longer because evaporation is smaller. This effect can be
enhanced if the preparations are sealed with vaspar or
nails varnish.
The Cotton Blue, China Blue or Soluble Anilin Blue is
probably, an impurified and complex colouriser but, its
Put a bit of mould to be assayed in a clean microscopical
slide and pour one or two drops of Lactophenol Blue.
Disperse the material with two needles, mixing it with the
colouriser.
Add a couple of drops of water and homogenize all
before putting the overglass.
Heat slightly the preparation over a flave until it will be
almost boiling. In that precise moment, press to remove
all the excess of liquid and seal the borders with vaspar
or nails varnish.
Preparation is ready for the microscopical examination.
References
HARRIGAN, W.F. and McCANCE, M.E. (1976) Labora-
tory Methods in Food and Dairy Microbiology. Academic
Press. London.
CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed.
William and Wilkins. Baltimore
LARONE, D.H. (2002) Medically important fungi. ASM
Press. Washington, DC.

237
 Lugol Solution for Gram Staining

Ref. 06-030
Presentation
100 mL dropper flask
1 L dropper flask
Description
Iodine solution has been prepared according to the
specifications by Burke, therefore it is more stable than
the classical Lugol formulation, and it does not affect
the colouring. The solution may be stored for months at
room temperature, but if a characteristic amber colour is
observed, it must be discarded.
Technique
Fix the smear following the habitual method and let it
cool.
Cover the extension with Crystal Violet Dye Solution
(Ref. 06-029) and let it act for 1 minute
Wash the exceed of colouriser. The best way is to put
the preparation in a precipitate glass with fluent water.
Do not wash excessively. This step may be critical for
the rest of the test.
Dry and observe under microscope in homogeneous
inmersion.
Microorganisms that get coloured by the first colouriser,
Crystal Violet, become dark blue coloured and it is said
that they take the gram, and they are call grampositive
(G+). Those microorganisms that just get coloured by
the contrast colouriser become red and they are called
gramnegative (G-).
Most of eukariote cells, except yeasts, are coloured as
gramnegative and thus the staining is not very significa-
tive. In spite of, it is one of the first levels in the system-
atic identification of prokariote: between the bacteria, all
the coci, except Neisseria and Veillonella, are gramposi-
tive, and all the sporogen bacilli and some part of the
other bacilli are grampositive too. Spiriles, vibria, rikett-
sia, clamidia and most bacilli are gramnegative.
Should actinomycete presence is suspected or microor-
ganisms are not well coloured as grampositives, it is ad-
visable to use Crystal Violet with Anilin for Actinomycete.

Cover the preparation with Lugol Solution (Ref. 06-030)

References
and drain immediately. Cover again with new solution
BARTHOLOMEW, J.W. (1962) Variables Influencing Re-
and let it act for 1 minute.
sults, and the Precise Definition of Steps in Gram Stain-
Wash softly again with water. To put the preparation in
ing as a Means of Standardizing the Results Obtained.
water fluent for 5 seconds will be enough.
Stain Technol. 37:139-155.
PAIK, G. (1980) Reagents, Stain and Miscellaneous
Decolourate, pouring the Gram Decoluriser (Ref. 06-
Procedures, in Manual of Clinical Microbiolgy by Lenette,
031), drop to drop, over the slanted microscopical slide
Balows, Hausler and Truant (eds.). ASM, Washington.
until total decolourising. Anycase, this step may not be
CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed.
longer than 60 seconds.
William and Wilkins. Baltimore.
Wash with water to stop the decolouring action.

Cover the preparation with Safranine Dye Solution (Ref.

06-032) and let it act for 1 minute.
Wash gently to remove the excess of colouriser, putting
the preparation in fluent water for 1-2 seconds.

238
 Malachite Green
Ref. 06-038
Presentation
100 mL flask
1 L flask
Description
Malachite Green for Spores has been prepared accord-
ing to the formulation by Barholomew and Mittwer, in
1950, which was a modification over the procedure by
Schaeffer and Fulton, in 1933, that was based in the
original one by Wirtz.
Essencially, the modification is the supression of the step
where the preparation was heated up. To achieve this
without affecting the test, they prepared a more concen-
trated colouriser and let it act longer, and afterwards they
did the contrast with a softer colouriser, Safranine Dye
Solution (Ref. 06-032).
This stain is a saturated and stabilized aquose solution
of malachite green. To use it with the Schaeffer tech-
nique it has to be diluted at 50% to avoid the formation
of precipitates.
Technique
Prepare a smear of microorganism, in the habitual way,
and let it dry. Fix strongly by passing the microscopical
slide over a flame about 20 times.
Before doing the staining, let cool the microscopical
slide. Cover all the smear with Malachite Green for
Spores Stain and let it act for 10 minutes.
Wash with water to remove the excess of colouriser.
Contrast by covering the smear with Safranine Dye Solu-
tion (Ref. 06-032) and letting it act for 15-30 seconds.
Wash again, dry and perform the microscopical exami-
nation in homogeneous inmersion.
Cellular bodies appear red or pinked coloured, whereas
spores are green.
Should the Schaeffer’s technique is wanted to be used
diluting the colouriser, once the smear is covered with
Malachite Green, do not let it act for 10 minutes and
instead of this, bring it to the boiling 3 consecutive times.
Then follow the described technique.
References
BARTHOLOMEW, J.W., MITTWER, T. (1950) A Simpli-
fied Bacterial Spore Stain. Stain Technol. 24:153-156.
SCHAEFFER, A.B., M. FULTON (1933) A Simplified
Method of Staining Endospores. Science, 77, 194.
CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed.
William and Wilkins. Baltimore.
239
 Methyl Red
Ref. 06-007 F Technique
R-11
Presentation S-7-16
100 mL flask
1 L flask
Specification
Indicator solution for the fermentation test in enterobac-
teria.
Description
Clark and Lubs, in 1915, described the Methyl Red test
to distinguish between the E. coli group and Entero-
bacter.
All the enteric bacteria ferment dextrose, some do it
following the 2-3-butanediol path, like Klebsiella, En-
terobacter, etc..., and other follow the mix acid path,
like E.coli, Salmonella, etc...In this last case, there is an
accumulation of acid products produced by the sugar
unfoldment. This causes a decrease of pH, reaching a
value up to 4 after the incubation. Adding the methyl red
alcoholic solution the change of the indicator may be de-
tected: it remains yellow over pH 5,1 and red below pH
4,4. If the change is positive it means that the dextrose
fermentation followed the mix acid path, since in the 2-
3-butanediol path, after 3 days of incubation, predomi-
nate the neutral or alkaline products.
Too early reads may produce false positive results.
240
Inoculate a tube of MRVP Broth (Ref. 02-207) and
incubate at 30°C for 3-5 days. Take 5 mL of culture and
transfer them to a clean tube and then add 5 or 6 drops
of indicator.
Positive reaction is shown by the presence of a red
colour, whereas the negative reaction produce a yellow
or orange colour.
Most cases, a 48 hours incubation at 37°C is enough,
but if the results are doubtous, the assay must be re-
peated incubating at 30°C for 5 days.
There is a quicker way to perform the test: suspend a
loop of bacterial growth from a solid medium in 0,5 mL
of Azide Dextrose Broth acc. to Rothe (Ref. 02-027) and
incubate at 37°C for 18 hours. Add a couple of drops of
indicator and read the results as in the last case.
References
BARRY, A.L., K.L. BERNSOHN, A.P. ADAMS, L.D.
THRUPP (1970) Improved 18-hour methyl red test. Appl.
Microbiol. 20:886-870.
CLARK, W.M. and LUBBS, H.A. (1915) The Differentia-
tion of Bacteria of the Colon-Aerogenes Family by the
use of indicators. J. Infect. Dis. 17:161-173.
EDWARDS, P.R., W.H. EWING (1972) Identification of
Enterobacteriaceae. 3rd. Ed. Burgess Pub Co. Minne-
apolis.
BLAZEVIC, D.J. and EDERER, G.M. (1975) Principles
of Biochemical Tests in Diagnostics Microbiology. John
Wiley Sons. N.Y.
McFADDIN, J.F. (2000) Biochemical tests for identifica-
tion of medical bacteria. 3rd. Ed. Lippincott William and
Wilkins. Philadelphia.
 Nitrates Reduction Reagents

Nitrates A Solution The scheme for the global process is the following:
C
Ref. 06-003
R-10-35
S-23.2-51-26-36/37/39-45
Presentation
100 mL dropper flask

Nitrates B Solution
C
Ref. 06-004
R-10-35 Generally, the Griess-Ilosvay’s reagents detect the
S-23.2-51-26-36/37/39-45
Presentation presence of nitrites with bacterial origin in a medium that
100 mL dropper flask initially has no nitrites (Indole Nitrite Fluid Medium, Ref.
03-101 and Nitrate Broth, Ref. 02-138).
The scheme for the complete reaction is the following:
Specification
Griess-Ilosvay’s Reagents for the verification of the
nitrates reduction through nitrites detection.

Description
To use them, mix equal parts of the solutions A and B.
Once they are mixed, the reagents are stable just for a
few hours. Alone they may be stored for several months
at room temperature. Nitrates B solution may produce a
slight cristalization that does not affect its efficacy. This
process is accelerated with refrigeration, therefore it is
recommended not to store them in the refrigerator.

Nitrates reduction in bacteria is performed through sev- References

eral ways and it obeys different procedures.
Nitrates Assimilation involves a reduction to ammonia
in several steps where nitrite may be detected. The am-
monia that is produced is finally incorporated to the cel-
lular material. However, in the Deassimilation process,
nitrite is used as the final receiver of electrons, and thus,
more than an assimilation process it is an energetic re-
action of respiration without oxygen, and this fact allows
the facultative growth of many aerobic in anaerobiosis.
In this case, it is usual the presence of nitrite acumula-
tions, which may be toxical for the microorganism. In
other cases, nitrate may reduce itself to gas states and it
is expeled as free nitrogen bubles. This process is called
Denitrification, since it makes the active ion (NO3) an
inert gas (N2).
WALLACE, G.I., S.L. NEAVE (1927) The nitrite test as
applied to bacterial cultures. J.Bact. 14:377-384.
BLAZEVIC, D.J., G.M. EDERER (1975) Principles of
Biochemical Tests in Diagnostics Microbiology. John
Wiley Sons. NY.
FORBES, B.A., D.F. SAMM, A.S. WEISSFELD (1998)
Bailey & Scott’s Diagnostics Microbiology. 10th. Ed.
Mosby. St. Louis.
GRIESS, P. (1879) Liebereinige Azoverbindungen. Ber.
Deutsch. Chem. Geselkch. 12:426-427.
McFADDIN, J.F. (2000) Biochemical Tests for identifica-
tion of medical bacteria. 3rd. Ed. Lippincott William and
Wilkins. Philadelphia.

241
 O’Meara’s Reagent
Ref. 06-006 C Technique
R-22-35
Presentation S-26-36/37/39-45
100 mL dropper flask
Specification
Reagent for the Voges-Proskauer test in enterobacte-
riaceae.
Description
All the enterobacteria ferment dextrose, but some spe-
cies like Klebsiella, Enterobacter, etc..., do it following
the 2-3-butanediol path and other species like E.coli,
Salmonella..., do it by the mix acid path.
Voges Proskauer test shows the production of 2-3-
butanediol and acetoine, that are only produced in big
amounts in the 2-3-butanediol path. The basis of the test
is that these compounds, in alkaline medium and with
air, bear an oxidation and become diacethyl, which at the
same time reacts with guanidine producing very visible
coloured compounds.
O’Meara, in 1931, observed that adding creatine to the
alkaline solution (O’Meara’s Reagent, Ref. 06-006) aids
diacethyl reaction with guanidine, and then it was easier
to detect the red coloured compounds.
Later, in 1936, Barrit demonstrated that the addition
of an alcoholic solution of alpha-naphtol 5% (Barrit’s
Reagent, Ref. 06-027) increased very much sensibility,
and it was possible to obtain positive reaction even when
the final concentration of diacethyl was very low. It is
important to add the Barrit’s Reagent before the alkaline
solution.
242
Microorganism to be assayed is inoculated in MRVP
Broth (Ref. 02-207) and is incubated at 30°C for a period
between 3 and 5 days maximum.
Just before read, add Barrit’s Reagent (Ref. 06-027)
until all the medium gets a milky look. Following, add
O’Meara’s Reagent (Ref. 06-006) until the milky look
disappears and then shake chiefly. Relative volumes of
each reagent depend on the initial volume of inoculated
medium.
When test is positive a violet pinked colour appears be-
fore 5 minutes, starting from top. When test is negative
there is no change ol colour.
There is a quicker way to perform the Voges-Proskauer
test, with very little volumes of medium and massive
inocules. This way allows very short incubations (18-20
hours) and the read may be accelerated by heating up
the culture almost to boiling after adding the reagents.
However, this method increases the possibility of getting
wrong results.
References
BARRY, A.L., K.L. FEENEY (1967) Two Quick Methods
for the Voges Proskauer Test. Appl. Microbiol. 15:1138-
1141.
BARRIT, M.M. (1936) The intensification of The Voges
Proskauer Reaction by the Addition of alpha-Naphtol. J.
Path. Bact. 42:441-453.
BLAZEVIC, D.J. and EDERER, G.M. (1975) Principles
of Biochemical Tests in Diagnostics Microbiology. John
Wiley Sons. N.Y.
O’MEARA, R.A.Q. (1931) A simple, Delicated and Rapid
Method of Detecting the Formation of Acetylmethyl-car-
binol by Bacteria Fermenting Carbohydrate. J. Pathol.
Bact. 34:401-406.
McFADDIN, J.F. (2000) Biochemical tests for identifica-
tion of medical bacteria. 3rd. Ed. Lippincott William and
Wilkins. Philadelphia.
 Oxidase Reagent
Ref. 06-057
Presentation
5 g flask
Specification
Reagent for the detection and verification of bacterial
citochrome-oxidase.
Description
The chemical formulation for the Oxidase Reagent is
N,N-dimethyl-P-phenyldiamine-2-HCl, and it is also
designated as 4-amino-N,N-dimethylaniline-2-HCl. It is
advisable to store it in powder at 4°C since it has a very
short life time when is dissolved.
Directions
Prepare an aquose solution of Oxidase Reagent 1% im-
mediately before the use. It is recommended to prepare
just the amount that is going to be used, since once
diluted it will work just for a week, even if it is kept at
4°C and avoiding direct light. Althought self oxidation is
restrained with the addition of ascorbic acid 0,01%, if the
liquid is dark it must not be used. The normal colour for
the solution is transparent or slightly pinked.
Technique
There are several techniques to determinate citrocrome-
oxidase in the different genus. The more standardized
are the following:
a) Soak a filtration paper disc with the reagent and put it
over a clean Petri plate. Take a colony and spread
it over the paper. This step must be performed with
a Platinum-Iridium loop (Ref. 5-006) or a Pasteur pi-
pette. The use of metallic objects (nicrom loop, etc...)
may produce wrong positive results.
b) Flood the colony with reagent directly in the plate. Fol-
lowing this way, colonies are not able to subculture,
but the test does not interfere with the Gram staining
and the colony may be observed at the microscope.
Positive reaction is shown by the presence of a pink
colouring, that becomes dark red and finally black after
10 minutes.
The basis of the test is the following:
Citochromes are hemoproteins that act as oxidant enz-
imes in the transportant chains of oxidative phosphorila-
tion electrons. Generally, citochromes are found just in
the aerobic bacteria, whereas the strict anaerobic does
not present them.
Citochrome-oxidase found even in the enterobacte-
riaceae is composed by a3 citochrome. However, the
substance that the Oxidase Reagent is able to reduce is
c citochrome, thus the test just determinates the pres-
ence of c citochrome and therefore test is positive only
for the bacteria that have c citochrome in their respira-
tory chains.
Reagent is very unstable to oxygen, and light acts as a
catalyst in the self oxidation and so the reagent may be
stored in amber flask. When the reaction in the colony is
very slow, it has to be considered negative or very weak,
since the lately appaerance of colour is more attributable
to the spontaneous oxidation than to the truly presence
of c citochrome.
Generally, except a few cases, oxidase production is
linked to flagelation in the following way: Polar flagela-
tion is related with oxidase positive; peritrichous flagela-
tion is related with oxidase negative.
The oxidation of c citochrome for the positive reaction
of oxidase is the following and it may be observed that
molecullar oxygen is absolutely necessary:
References
GABY, W.L., C. MARTLEY (1957) Practical laboratory
test for the identification of Pseudomonas aeruginosa. J.
Bact. 74:356-358.
BLAZEVIC, D.J., G.M. EDERER (1975) Principles of
biochemical test in diagnostic Microbiology. John Wiley
Sons. NY.
FORBES, B.A., D.F. SAHM, A.S. WEISSFELD (1998)
Bailey & Scott’s Diagnostic Microbiology. 10th. Ed.
Mosby. St. Louis.
ISO 9308-2 Standard (1990) Water Quality - Detection
of coliforms, thermotolerant coliforms and presumptive
E.coli - MPN method.
243
 Safranin Dye Solution for Gram Staining

Ref. 06-032 Dry and observe under microscope in homogeneous

Presentation
100 mL dropper flask
1 L dropper flask
Description
The contrast colouriser is composed by the classic
Safranine solution 0,25%. It is demonstrated that this
solution is more effective than the fuchsine. This con-
trast colouriser is employed also in many other staining
methods.
Technique
Fix the smear following the habitual method and let it
cool.
Cover the extension with Crystal Violet Dye Solution
(Ref. 06-029) and let it act for 1 minute
Wash the exceed of colouriser. The best way is to put
the preparation in a precipitate glass with fluent water.
Do not wash excessively. This step may be critical for
the rest of the test.
inmersion.
Microorganisms that get coloured by the first colouriser,
Crystal Violet, become dark blue coloured and it is said
that they take the gram, and they are call grampositive
(G+). Those microorganisms that just get coloured by
the contrast colouriser become red and they are called
gramnegative (G-).
Most of eukariote cells, except yeasts, are coloured as
gramnegative and thus the staining is not very significa-
tive. In spite of, it is one of the first levels in the system-
atic identification of prokariote: between the bacteria, all
the coci, except Neisseria and Veillonella, are gramposi-
tive, and all the sporogen bacilli and some part of the
other bacilli are grampositive too. Spiriles, vibria, rikett-
sia, clamidia and most bacilli are gramnegative.
Should actinomycete presence is suspected or microor-
ganisms are not well coloured as grampositives, it is ad-
visable to use Crystal Violet with Anilin for Actinomycete.

Cover the preparation with Lugol Solution (Ref. 06-030)

References
and drain immediately. Cover again with new solution
BARTHOLOMEW, J.W. (1962) Variables Influencing Re-
and let it act for 1 minute.
sults, and the Precise Definition of Steps in Gram Stain-
Wash softly again with water. To put the preparation in
ing as a Means of Standardizing the Results Obtained.
water fluent for 5 seconds will be enough.
Stain Technol. 37:139-155.
PAIK, G. (1980) Reagents, Stain and Miscellaneous
Decolourate, pouring the Gram Decoluriser (Ref. 06-
Procedures, in Manual of Clinical Microbiolgy by Lenette,
031), drop to drop, over the slanted microscopical slide
Balows, Hausler and Truant (eds.). ASM, Washington.
until total decolourising. Anycase, this step may not be
CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed.
longer than 60 seconds.
William and Wilkins. Baltimore.
Wash with water to stop the decolouring action.

Cover the preparation with Safranine Dye Solution (Ref.

06-032) and let it act for 1 minute.
Wash gently to remove the excess of colouriser, putting
the preparation in fluent water for 1-2 seconds.

244