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Atlas of Medical Bacteriology

Research · June 2016

DOI: 10.13140/RG.2.1.2130.9048

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 Atlas
of
Medical
Bacteriology

1
 Atlas of Medical Bacteriology
Simplified Guide for Primary Differentiation and Diagnosis
of Pathogenic Bacteria that can be isolated from patients of
different diseases.

University of Babylon
College of Pharmacy
Department of Clinical and laboratory Sciences
Second Class
2014-2015
1st. Edition

2
Preface:
To help students in the comprehension of Practical Part of Medical
Microbiology especially the Bacteriology which is the concept of this
Atlas, this work may introduce some help .
Some bacteria are rare, the others very dangerous or may be obligate
intracellular parasite, all of the mentioned cases need to study these
bacteria by pictures in addition to the theoretical information, therefore I
dedicate this work to everyone can get any benefit from it.

Msc. Teacher Assistant

Halah Dawood Salman
2014-2015

3
 LIST OF CONTENTS
Subject No. Subject Page
List of Contests 4
Gram Positive Bacteria
1-
1.1 Staphylococcus 5
1.2 Streptococcus 10
1.3 Bacillus 15
1.4 Closteridium 20
1.5 Corynebacterium 25
1.6 Listeria 27
1.7 Actinomycetes 29
1.8 Propionebacterium 31
2-Gram Negative Bacteria
2.1 Escherichia coli 33
2.2 Klebsiella 35
2.3 Enterobacter 37
2.4 Proteus 39
2.5 Salmonella 41
2.6 Shigella 43
2.7 Serratia 45
2.8 Pseudomonas 47
2.9 Vibrio 50
2.10 Campylobacter 54
2.11 Helicobacter 57
2.12 Haemophilus 59
2.13 Brucella 62
2.14 Bordetella 63
2.15 Yersinia 65
2.16 Neisseria 68
3-Acid Fast Stain Bacteria
3.1 Mycobacterium tuberculosis 72
3.2 Mycobacterium leprae 75
4- Cell Wall-Defective Bacteria (Mycoplasma) 76
5- Chlamydia 79
List of abbreviations 83
Appendix 84
References 99

4
1-GRAM POSITIVE BACTERIA
1-1-Staphyllococcus spp.
The staphylococci are gram-positive spherical cells, non-sporing, non-motile, usually non-
capsulate, true facultatively anaerobic organisms. (1micrometer in diameter) usually
arranged in grapelike irregular clusters. They grow readily on many types of media and are
active metabolically, fermenting carbohydrates and producing pigments that vary from white
to deep yellow. Colonies on solid media are round, smooth, raised, and glistening S. aureus
usually forms gray to deep golden yellow colonies. S. epidermidis colonies usually are gray
to white on primary isolation; many colonies develop pigment only upon prolonged
incubation. No pigment is produced anaerobically or in broth. Various degrees of hemolysis
are produced by S aureus and occasionally by other species. Staphylococcus saprophyticus
is a frequent cause of cystitis in women, probably related to its occurrence as part of normal
vaginal flora. S. saprophyticus can be distinguished from S. epidermidis and most other
coagulase-negative staphylococci by its natural resistance to novobiocin . [Note: Urinary
coagulase-negative staphylococcus is often presumed to be S. saprophyticus; novobiocin
resistance can be used for confirmation.].

Light Microscope Electron Microscope

5
Scanning electron micrograph (SEM) depicting numerous clumps of methicillin-resistant Staphylococcus aureus
bacteria, commonly referred to by the acronym, MRSA; Magnified 9560x Source: Centers for Disease Control and
Prevention

S. aureus on Nutrient Agar S. epidermidis on Nutrient Agar

6
S. aureus mannitol salt agar S. aureus on Luria Agar (LA) Media

blood agar S.aureus on Trypticase soy agar(TSA)

7
On blood agar:

S.auerus S. epidermidis S. saprophyticus

(ß-hemolysis) (No hemolysis) (No hemolysis)

On Mannitol Salt agar:

S. aureus S. epidermidis S. saprophyticus

Growth with + mannitol Growth with no mannitol
fermentation fermentation

8
 On DNase agar:

S. aureus S. aureus S. epidermidis

White colonies with clear halo around colonies. White colonies with NO
clear halo around colonies

MRSA: Methicillin-Resistant Staphylococcus aureus, MRSA are a major cause of nosocomial and life
threatening infections. Infections with MRSA have been associated with a significantly higher morbidity,
mortality and costs than methicillin-susceptible S. aureus (MSSA). Selection of these organisms has been
greatest in the healthcare setting; however, MRSA have also become more prevalent in the community.

9
1-CHROMagar MRSA 2-Spectra MRSA 3- Brilliance MRSA 2 Agar

1- CHROMagar MRSA: is a selective and differential medium for the qualitative direct detection of nasal
colonization by methicillin-resistant Staphylococcus aureus (MRSA) to aid in the prevention and control of
MRSA infections in healthcare settings.

2-Spectra MRSA medium plated with methicillin-resistant Staphylococcus aureus. The ability of Spectra
MRSA to identify methicillin-resistant S. aureus based on the color of the colony, in which colonies of
methicillin-resistant S. aureus appear denim blue.

3- Newly Improved Chromogenic Medium for Rapid and Reliable MRSA Screening in Just 18 Hours.

1-2 Streptococcus spp.

The streptococci are gram-positive spherical bacteria that characteristically form pairs or chains during
growth. They are widely distributed in nature. Streptococci elaborate a variety of extracellular substances
and enzymes. The streptococci are a large and heterogeneous group of bacteria, and no one system suffices
to classify them. Many species of streptococci, including Streptococcus pyogenes (group A), S agalactiae
(group B), and the enterococci (group D), are characterized by combinations of features, including colony
growth characteristics, hemolysis patterns on blood agar (α-hemolysis, β-hemolysis, or no hemolysis).

Light Microscope Micrograph

10
 Gram stain of S. pneumoniae with WBC

Scanning Electron Microscope Micrograph

Electron Microscope Micrograph

11
 S. pneumoniae colonies on a BAP(Blood Agar Plate)

S. pneumoniae colonies have a flattened and depressed - The small grey, flat colonies surrounded by a
greenishcenter after 24-48 hours of growth on a BAP, zone of alpha-hemolysis are S. pneumoniae and
whereas the viridans streptococci retain a raised center. the white colonies with no hemolytic activity
. are S. epidermidis (black arrow).

12
Streptococcus agalactiae colonies growing on GBS Detect™ showing beta- hemolytic colonies. This
strain is not hemolytic on a regular blood agar plate. Incubated aerobically for 24 hours at 35 deg. C.
( GBS Detect™ is recommended for the isolation and detection of gamma-hemolytic (non-hemolytic)
Group B Streptococcus by inducing beta-hemolysis on sheep blood agar upon subculture from enrich-
ment broth procedures; such as Strep B Carrot Broth™ and LIM Broth).GBS*: Group B Streptococci.

Optochin test for S. pneumoniae using optochin disks. The strain on the left is resistant to optochin
with no zone of inhibition, and therefore is not a pneumococcus. The strain on the right is susceptible
to optochin and is S. pneumoniae.

13
 CAMP-test
GAS*: Group A Streptococci.
GBS*: Group B Streptococci.

14
S. pneumoniae in spinal fluid Streptococcus pneumonia Quellung
FA: Florescent Antibody (capsular swelling) reaction can be use
)digitally colored). to demonstrate the presence of a specific
capsular type of the bacterium.

Encapsulated Streptococcus pneumonia

15
 1-3-Bacillus
Bacillus are gram-positive spore-forming bacilli. These bacilli are ubiquitous, and because
they form spores, they can survive in the environment for many years. Bacillus species are
aerobes. Colonies of B. anthracis are round and have a ―cut glass ‖appearance in transmitted
light. Hemolysis is uncommon with B .anthracis but common with B. cereus and the
saprophytic bacilli. Bacillus anthracis, commonly known as anthrax. These rod-shaped,
bacteria can infect the skin (cutaneous anthrax), causing raised itchy lesions, the lungs
(pulmonary anthrax), which is fatal unless treated quickly, and the digestive system
(gastrointestinal anthrax), causing vomiting of blood and severe diarrhoea. All forms can be
fatal if left untreated

-
-Light Microscope- Bacillus spp. Malachite Green
spore stain

Electron Microscope image Bacillus Anthracis (Anthrax)Scanning electron

micrograph of Bacillus anthracis spores

16
 Image of Bacillus spp. as a result of capsule stain.

Bacillus anthracis spores seen under phase contrast

microscopy. Bacillus anthracis endospores are seen
under phase contrast microscopy as lighter areas, i.e.
"points of light", due to the fact that they are dehydr-
ated, and therefore, more refractile.

17
Bacillus anthracis on chocolate agar Colonies of B. subtilis grown on a culture dish

Bacillus spp. On nutrient agar B.cereus on Luria Agar (LA)

18
 -1- -2-

1- Mucoid colonies of Bacillus anthracis. This culture was probably incubated at an increased CO2
tension (5% CO2) which greatly enhances production of the poly-D-glutamyl capsule and accounts for
the mucoid colony type

2-Bacillus sp. on Mueller-Hinton agar. Cultivation in aerobic atmosphere, 28°C, 48 hours.

Environmental isolate

-1- -2-

1- Lysis of Bacillus anthracis by the lytic phage gamma. The plaque (clear area) in the region of confluent
growth is where the gamma phage was applied. The plaque results from the phage's ability to lyse the
bacterial cells. Since the gamma phage is specific for B. anthracis, and will not lyse B. thuringiensis or B.
cereus.

2- Colonies of Bacillus cereus on the left; colonies of Bacillus anthracis on the right. B. cereus colonies are
larger, more mucoid, and this strain exhibits a slight zone of hemolysis on blood agar.

19
B.cereus: Large, irregular colonies surrounded Bacillus cereus colonies on blood agar
by a wide haemolytic
zone.

-1- -2-

1-Oxoid Brilliance Bacillus cereus Agar: (formerly Chromogenic Bacillus cereus Agar) is a chromogenic
medium for the isolation and differentiation of Bacillus cereus from food samples.

2-A Bacillus subtilis bacterial colony showing signs of stationary growth after 48 hours of incubation at 37
°C (98.6 °F; magnified about 9 times).

20
1-4- Clostridium spp.
The clostridia are large anaerobic, gram-positive, motile rods. Many decompose proteins or
form toxins, and some do both.Their natural habitat is the soil or the intestinal tract of
animals and humans, where they live as saprophytes. In general, the clostridia grow well on
the blood-enriched media or other media used to grow anaerobes. Some clostridia produce
large raised colonies (eg, C. perfringens); others produce smaller colonies form colonies
that spread on the agar surface. Many clostridia produce a zone of hemolysis on
blood agar. C. perfringens characteristically produces a double zone of -hemolysis
around colonies.

C. perfringens Light Microscope

Clostridium tetani
Light Microscope

21
This Gram-stained micrograph of Clostridium botulinum Type-A in thioglycollate broth was incubated for
48hrs at 35°C

A photomicrograph of Clostridium botulinum type A viewed using a Gram stain technique.

Light Microscope

Clostridium difficile bacteria, light micrograph.

22
"Clostridium botulinum". This picture shows the rod-shaped bacteria, under a (SEM) scanning electron
microscope. Courtesy of Science Photo Library.com
Scanning Electron Microscope

C.deficile
Electron Microscope

C.tetani C.tetani -Light Microscope

23
Botulism (Clostridium botulinum). These are Clostridium botulinum Type E colonies displaying an opaque
zone grown on a 48hr egg yolk agar.

-1- -2-

1- Clostridium difficile Cefoxitin Cycloserine Egg-Yolk (CCEY) AGAR.

2-Clostridium perfringens colonies on tryptose sulphite cycloserine agar (TSC agar).

Ultraviolet irradiation shows yellow-green fluorescence of Clostridium difficile in agar medium. The CDC
lists drug-resistant C diff as a pathogen of urgent concern.

24
 -1- -2-

1-Clostridium difficile On blood agar

2-This photograph depicts a colony of Clostridium sp., which had been grown on a 4% blood agar plate
(BAP) over a 48 hour time period.

-1- -2-
1-Clostridium Perfringens on blood agar
2-Clostridium botulinum which produces the botulism toxins growing on egg yolk agar showing the lipase
reaction after 72 hours of incubation. CDC PHIL (1/3/2008).

25
1-5- Corynebacterium spp.
Corynebacteria are non–spore-forming gram-positive bacilli , characteristically, they possess
irregular swellings at one end that give them the ―club-shaped‖ appearance. Irregularly
distributed within the rod (often near the poles) are granules staining deeply with aniline dyes
(metachromatic granules) that give the rod a beaded appearance. A simple stain with methy -
lene blue is often used to stain these granules.
On blood agar, the C. diphtheriae colonies are small, granular, and gray with irregular edges
and may have small zones of hemolysis . On agar containing potassium tellurite, the colonies
are brown to black with a brown-black halo because the tellurite is reduced intracellularly
(staphylococci and streptococci can also produce black colonies). C. diphtheriae and other
corynebacteria grow aerobically on most ordinary laboratory media. On Loeffler serum
medium, corynebacteria grow much more readily than other respiratory organisms,
Corynebacteria tend to pleomorphism in microscopic and colonial morphology.

Light Microscope Scanning Electron Micrograph

-1- -2- -3-

1-Corynebacterium diphtheriae cultivated on Tinsdale agar. Cultivation 72 hours, 37 °C in an aerobic

atmosphere.
2 -Corynebacterium diptheriae on Tinsdale agar.
3- Corynebacterium diphtheriae on Tellurite agar.

26
 Hoyle's tellurite agar Blood agar

1-6- Listeria monocytogenes

L. monocytogenes is a short, gram-positive, non–spore-forming rod . It has a tumbling end-
over-end motility at 22–28°C but not at 37°C; the motility test rapidly differentiates Listeria
from diphtheroids that are members of the normal microbiota of the skin. Listeria grows
well on media such as 5% sheep blood agar on which it exhibits the characteristic small zone
of hemolysis around and under colonies. The organism is a facultative anaerobes. The
motility at room temperature and hemolysin production are primary findings that help
differentiate Listeria from coryneform bacteria.

-Light Microscope -Coloured Transmission Electron Microscope Image

Listeria monocytogenes

27
 -1- -2- -3-
1-Colorex Listeria (ISO):It is a selective medium for the isolation and presumptive identification of
Listeria monocytogenes from clinical and food samples.

2-CHROMagar Listeria : It is a chromogenic media for isolation of specimins allowing direct

differenciation of Listeria monocytogenes by blue colony colour with a white halo. This chromogenic
culture media is based on phospholipases specific of Listeria monocytogenes.

3-Blood Agar: Listeria monocytogenes, removing colonies to see the subtle hemolysis directly beneath the
colonies. (Rebecca Buxton, University of Utah).

-1- -2-

1- Listeria monocytogenes residing within white blood cells.

2- Listeria bacteria. Coloured Transmission Electron Micrograph (TEM) of a human cell seen with a group
of Listeria monocytogenes bacteria. The human cell is coloured green; the rod-shaped bacteria are yellow.

28
 Actin Tail

Bacterium
-1- -2-

1- Listeria moving through cell by actin-based motility, Green = actin filament stain, Red =Listeria stain.
Note the long actin filament tails behind the moving bacteria.

2-Scanning Electron Microscopy (SEM). Adhesion of L. monocytogenes on a cell wall. Experiment

performed at Pasteur Institute.
*Listeria monocytogenes )Facultative, Intracellular Parasite(

1-7-Actinomycetes
Actinomycetes are a group of filamentous, branching, gram-positive organisms that easily
fragment into slender rods . Although they superficially resemble fungi on morphologic
grounds, they are prokaryotes of bacterial size. They are free-living, mostly soil organisms
that are related to Corynebacteria and Mycobacteria, as well as to the Streptomycetes that are
sources of important antibiotics. Actinomycosis is an opportunistic infection . The infection
is probably initiated by accidental introduction of organisms into the underlying soft tissue
during conditions of sufficient anaerobiasis to support their growth. They are related to the
corynebacteria and include multiple genera of clinical significance such as Mycobacteria and
saprophytic organisms such as Streptomyces. Members of the aerobic Actinomycetes can be
categorized on the basis of the acid fast stain. Mycobacteria are truly positive acid fast
organisms; weakly positive genera include Nocardia, Rhodococcus, and a few others of
clinical significance. Streptomyces and Actinomadura, two agents that cause actinomycotic
mycetomas, are acid fast stain negative.

- Actinomyces israelii, and Arachnia propionica:

A. israelii and A. propionica are part of the normal oral and intestinal flora in humans, they
are strict anaerobes. Most strains of A.israelii and the other agents of actinomycosis are
facultative anaerobes that grow best in an atmosphere with increased carbon dioxide. On
enriched medium, such as brain-heart infusion agar, young colonies (24–48 hours) produce
gram-positive substrate filaments that fragment into short chains, diphtheroids, and
29
coccobacilli. After a week, these "spider" colonies develop into white, heaped-up "molar
tooth" colonies. In thioglycolate broth, A. israelii grows below the surface in compact
colonies. Species are identified based on cell wall chemotype and biochemical reactions.

Laboratory identification: The most typical and diagnostic finding in

Actinomycosis is the presence of sulfur granules• in the draining pus. These are small, firm,
usually yellowish particles, which in fact do not contain sulfur. When examined under the
microscope, these appear as microcolonies composed of filaments of the organism embedded
in an amorphous, eosinophilic material thought to be antigen-antibody complexes. The
organism can be grown anaerobically on enriched media such as thioglycollate broth or
blood agar. Growth is slow, often requiring ten to fourteen days for visible colonies.

30
 A B
-Nocardia asteroids in seputum:(A-Acid fast stain) ,(B-Gram,s stain)

1-8- Propionibacterium
On Gram stain, they are highly pleomorphic, showing curved, clubbed, or pointed ends; long
forms with beaded uneven staining; and occasionally coccoid or spherical forms
Propionibacterium species a members of the normal microbiota of the skin, oral cavity,
large intestine, conjunctiva, and external ear canal. Their metabolic products include
propionic acid, from which the genus name derives. P.cnes sometimes contaminates blood or

31
cerebrospinal fluid cultures that are obtained by penetrating the skin. It is therefore important
(but often difficult) to differentiate a contaminated culture from one that is positive and
indicates infection.

Light Microscope Electron Microscope

Electron Microscope

Colonies of Propionibacterium acnes on Blood agar.

32
2-Gram Negative Bacteria:
2-1-E.coli

short gram-negative rods , motile , E.coli are facultative anaerobes and form circular,
convex, smooth, flat, nonviscous colonies with distinct edges. Lactose fermented rapidly,
some strains of E .coli produce hemolysis on blood agar , have metallic sheen on differential
media.

Light Microscope Electron Microscope

-1- -2-
1- MacConkey agar.

2-Hektoen - Escherichia coli. Note: Orange color indicates acid production as a result of lactose
fermentation.

33
 -1- -2- -3-
(1) & (2)- E.coli on MacConkey agar.
3-CLED Agar . A: Proteus vulgaris (blue colour due to pH-shift to >7.6). B: Eschericha coli (colour
greenish-yellow due to acid production caused by lactose fermentation).

Eosin Methylene blue agar(EMB) Trypticase soy agar E.coli on Chrom agar
E.coli-Green metallic sheen colonies

E.coli on Endo agar

34
2-2- Klebsiella
short gram-negative rods , motile and facultative anaerobes .Capsules are large and regular in
Klebsiella species, Klebsiella colonies are large and very mucoid and tend to coalesce with
prolonged incubation and Lactose fermented rapidly . Klebsiella pneumonia are very
viscous and have mucoid growth. The organism grows with difficulty on media containing
egg yolk.

Light Microscope Electron Microscope

Electron Microscope Image

35
MacConkey agar

EMB agar

36
 Blood agar

-1- -2-
1-Cystine -Lactose Electrolyte - Deficient agar (CLED).

2- Eosin Methylene Blue Agar - Klebsiella pneumonia .Note: Mucoid colonies with dark
centers due to capsule production and lactose fermentation respectively.

2-3- Enterobacter
short gram-negative rods , facultative anaerobes, most Enterobacter species give positive test
results for motility, produce mucoid colonies, Enterobacter aerogenes has small capsules.
raised colonies, no metallic sheen; often motile; more viscous growth E nterobacter cloacae
has similar to Enterobacter aerogenes .

37
 Light Microscope Electron Microscope

MacConkey agar EMB XLD

Luria Agar (LA) Blood agar

38
 Chromagar CLED

2-4- PROTEUS
short gram-negative rods , facultative anaerobes , Proteus species move very actively by
means of peritrichous flagella, resulting in ―swarming‖ on solid media unless the swarming
is inhibited by chemicals, such as phenylethyl alcohol or CLED (cystine-lactose-electroly-
tedeficient) medium.; Proteus species are urease positive (smell of ammonia) . The Proteus
ferments lactose very slowly or not at all. Proteus mirabilis is more susceptible to anti-
microbial drugs, including penicillins, than other members of the group.

Light microscope

39
 Coloured TEM of the bacterium Proteus mirabilis

MacConkey agar Chrom agar

Swarming of Proteus mirabilis on an agar plate showing the typical growth rings

40
 -1- -2-
1- CLED Agar inhibits swarming of Proteus. A: Proteus vulgaris (blue colour due to pH-shift to >7.6)
B: Eschericha coli (colour greenish-yellow due to acid production caused by lactose fermentation).

2-proteus mirabilis growing on XLD agar.

2-5- Salmonella
Salmonellae are short gram-negative rods but vary in length, facultative anaerobes most
isolates of Salmonellae are motile with peritrichous flagella that characteristically
ferment glucose and mannose without producing gas, from dextrose they produce acid and
usually gas but they almost never ferment lactose or sucrose. Salmonellae grow readily on
simple media, most salmonellae produce H2S. They are often pathogenic for humans or
animals when ingested. They survive freezing in water for long periods. Salmonellae are
resistant to certain chemicals(eg, brilliant green, sodium tetrathionate, sodium deoxycholate)
that inhibit other enteric bacteria; therefore useful for inclusion in media to isolate
salmonellae from feces.

Light Microscope Electron Microscope

41
MAcConkey agar EMB agar

XLD agar

Hektoen agar
SS agar
42
 Bood agar

Salmonella on bismuth-sulphite agar

2-6- Shigella
Short gram-negative rods , Shigella species are nonmotile, not produce gas from dextrose .
Shigellae are facultative anaerobes but grow best aerobically. Convex, circular, transparent
colonies with intact edges reach a diameter of about 2 mm in 24 hours. Shigellae are usually
do not ferment lactose but do ferment other carbohydrates, producing acid but not gas. They
do not produce H2S. The four Shigella species are closely related to E. coli. Many share
common antigens with one another and with other enteric bacteria (eg, Hafnia alvei and
Plesiomonas shigelloides).

43
Electron Microscope Light Microscope

MAcConkey agar Blood agar

44
 SS agar XLD agar

Hektoen agar ENDO agar

2-7- Serratia
Short gram - negative rods , facultative anaerobes, slowly fermented Lactose Serratia
species produces DNase, lipase, and gelatinase. Serratia marcescens is a common
opportunistic pathogen in hospitalized patients . Serratia (usually nonpigmented)
causes pneumonia, bacteremia, and endocarditis. Only about 10% of isolates form the red
pigment (prodigiosin) that has long characterized S. marcescens.

Light Microscope Electron Microscope

45
 Pigment Production by Serratia marcescens

Serratia marcesens on MacConkey Agar Serratia marcesens on Blood Agar

S. marcesens appear as smooth, round, medium sized

colonies on blood agar. Some strains produce

a pinkish-orange pigment.

46
Serratia marcescens growing on XLD agar Serratia marcescens growing on Nutrient agar

2-8-Pseudomonas
The pseudomonads are gram-negative rods occurs as single bacteria, in pairs, and occasi-
onally in short chains, motile, obligate aerobic, some of which produce water - soluble
pigments. The pseudomonads occur widely in soil, water, plants, and animals. P.aeruginosa
is frequently present in small numbers in the normal intestinal fl ora and on the skin of
humans and is the major pathogen of the group. Other pseudomonads infrequently cause
disease. P. aeruginosa grows readily on many types of culture media, sometimes producing
a sweet or grapelike or corn taco–like odor. Some strains hemolyze blood. P. aeruginosa
forms smooth round colonies with pigments which diffuses into the agar. Other
Pseudomonas species do not produce the same pigments. P. aeruginosa in a culture
can produce multiple colony types.

Electron Microscope
Electron Microscope Light Microscope

47
MacConkey agar EMB- P. aeruginosa (lactose nonfermentor)

XLD agar P.aeroginosa Trypticase soy agar

Blood Agar

48
 Mullar Hinton Agar CHROMagar Pseudomonas

-TSA agar- Pseudomonas isolation agar

Pseudomonas aeruginosa on Trypic Soy Agar (TSA).
2007 Environmental Microbiology Laboratory, Inc.

49
 -1- -2-
1- Pseudomonas aeruginosa on HiFluoro Pseudomonas Agar
2- Pseudomonas Agar: Pseudomonas aeruginosa on HiFluoro Pseudomonas Agar under UV light.

2-9- Vibrio spp.

Vibrios are among the most common bacteria in surface waters worldwide. They are Gram
negative , curved aerobic rods and are motile, possessing a polar flagellum. V. cholerae
serogroups O1 and O139 cause cholera in humans, and other vibrios may cause sepsis or
enteritis. Upon first isolation typical Organisms of V cholerae is a comma-shaped, curved
rod 2–4 μm long . It is actively motile by means of a polar flagellum. On prolonged
cultivation, vibrios may become straight rods that resemble the gram-negative enteric
bacteria. V cholerae produces convex, smooth, round colonies that are opaque and granular
in transmitted light. V cholera and most other vibrios grow well at 37°C on many kinds
of media, including defined media containing mineral salts and asparagine as sources of
carbon and nitrogen. V. cholera grows well on thiosulfate-citrate-bile-sucrose (TCBS)
colonies (sucrose fermented) agar, a media selective for vibrios, on which it produces yellow
that are readily visible against the dark-green background of the agar.

50
 Light Microscope

Vibrio cholerae. Leifson flagella stain (digitally colorized). CDC/Dr. William A. Clark

51
 Electron Microscope

V. cholreae V. parahaem.
TCBS Agar
Thiosulphate Citrate Bile Salts Sucrose Agar

52
 Vibrio cholerae on Columbia Horse Blood Agar

Vibrio cholerae on C.L.E.D.

Vibrio species On MacConkey Vibrio species On Chrom agar

53
2-10- Campylobacter
C. jejuni and Campylobacter coli have emerged as common human pathogens, C jejuni and
the other campylobacters are gram-negative rods with comma, S, or ―gull wing‖ shapes.
They are motile, with a single polar flagellum, and do not form spores. Selective media are
needed, and incubation must be in an atmosphere with reduced O2 (5% O2) with added
CO2(10% CO2). Incubation of primary plates for isolation of C jejuni should be at 42°C.
Several selective media are in widespread use . Skirrow’s medium .The colonies tend to be
colorless or gray. They may be watery and spreading or round and convex, and both colony
types may appear on one agar plate.

Light Microscope

54
Electron Microscope

Electron Microscope

55
 Blood Agar

Campylobacter selective agar

56
2-11- Helicobacter pylori
H pylori is a spiral-shaped gram-negative rod. H pylori has many characteristics in common
with campylobacters. It has multiple flagella at one pole and is actively motile. H. pylori
grows in 3–6 days when incubated at 37°C in a microaerophilic environment, as for C.jejuni.
The media for primary isolation include Skirrow’s medium , chocolate medium, and other
selective media with antibiotics. The colonies are translucent and 1–2 mm in diameter.
H. pylori is a strong producer of urease.

Helicobacter pylori Gram-negative, spiral to pleomorphic, spiral rod prokaryote. It can move by means of tiny flagella at the
end of the cell. Light Microscope

Helicobacter pylori -SEM (Scanning Electron Microscope)

57
 Helicobacter pylori electron micrographs; fastidious microaerophile; typical helical shape shown in EM; causative agent of chronic gastritis,
peptic ulcers and gastric cancer.

Helicobacter pylori (yellow)

coloured SEM (Scanning Electron Microscope)

58
 H. pylori culture on sheep blood agar H. pylori culture on selective HP agar

Helicobacter Pylori Agar Base-HP-Selective Medium

2-12-Haemophillus
small, gram-negative, pleomorphic bacteria that require enriched media, usually containing
blood or its derivatives, for isolation. Haemophilus infl uenzae type b is an important human
pathogen; In specimens from acute infections, Haemophilus influenzae are short (1.5 μm)
coccoid bacilli, sometimes occurring in pairs or short chains. In cultures, the morphology
depends both on the length of incubation and on the medium. At 6–8 hours in rich medium,
the small coccobacillary forms predominate. Later there are longer rods, lysed bacteria, and
very pleomorphic forms. Organisms in young cultures (6–18 hours) on enriched medium
have a definite capsule. On chocolate agar, flat, grayish brown colonies with diameters
of 1–2 mm are present after 24 hours of incubation. IsoVitaleX in media enhances growth.
H influenzae does not grow on sheep blood agar except around colonies of staphylococci
(―satellite phenomenon‖).

59
 Light Microscope
Haemophilus influenzae : are small, pleomorphic, gram-negative bacilli or coccobacilli with random
arrangements.

Haemophilus influenzae

In specimens from patients with pneumonia (Sputum) caused by Haemophilus influenzae, both neutrophils
and bacteria are usually plentiful. However, if examination of the slide is not thorough, the coccobacilli may
be inconspicuous in the background of pink-staining mucus. Other organisms, such as Eikenella corrodans
or Bacteroides species, are also pleomorphic gram-negative coccobacilli; but they rarely cause pneumonia.

60
Haemophilus influenzae Bacteria TEM picture ( Transmission Electron Microscope)
False-colour scanning electron micrograph (SEM) of Haemophilus influenzae (Pfeiffer's bacillus),
a Gram negative, non-motile, non-sporing species of bacilli (rod-shaped bacteria)

Haemophilus influenzae type b bacteria(HIB) TEM of Haemophilus influenzae

TEM ( Transmission Electron Microscope)-
False-colour transmission electron micrograph
(TEM) of Haemophilus influenza.

61
 Haemophilus influenzae (only grows on chocolate)
Must strains of Haemophilus spp does not grow on 5% Sheep Blood Agar, which contains hemin (factor X)
but lacks NAD (factor V).

H. influenzae colonies on a CAP(Chocolate Agar Plate).

2-13- Brucella
The brucellae are obligate parasites of animals and humans and are characteristically located
intracellularly. They are relatively inactive metabolically. The appearance in young cultures
varies from cocci to rods 1.2 μm in length, with short coccobacillary forms predominating.
They are gram negative but often stain irregularly, and they are aerobic, nonmotile, and
nonspore forming. Brucellae have Small, convex, smooth colonies appear on enriched
media in 2–5 days. Brucellae are adapted to an intracellular habitat, and their nutritional
requirements are complex. Some strains have been cultivated on defined media containing
amino acids, vitamins, salts, and glucose..

62
 Light Microscope

A magnified view of Brucella TEM of Brucella abortus bacteria

- Scan Microscopy TEM ( Transmission Electron Microscope)

Brucella colonies on agar

2-14-Bordetella
minute, gram-negative coccobacilli bacteria resembling H influenzae. With toluidine blue
stain, bipolar metachromatic granules can be demonstrated. A capsule is present. Primary
requires enriched media. Bordet-Gengou medium (potato-blood- isolation of B. pertussis
glycerol agar) that contains penicillin G, 0.5 μg/mL, can be used; however, a charcoal-
(Regan Lowe) is preferable because of containing medium supplemented with horse blood
the longer shelf life. The plates are incubated at 35–37°C for 3–7 days aerobically in a moist
environment (eg, a sealed plastic bag).

63
 Light Microscope

Electron Microscope

Blood agar Charcoal agar

64
 Charcoal Blood Agar with cephalexin
*With the addition of blood this medium is used to isolate Bordetella pertussis.

2-15-Yersinia
Short, pleomorphic gram-negative rods that can exhibit bipolar staining. Microaerophilic or
facultatively anaerobic. Most have animals as their natural hosts, but they can produce
serious disease in humans. Y pestis exhibits striking bipolar staining with special stains such
as Wright, Giemsa, Wayson, or methylene blue . It is nonmotile. It grows as a facultative
anaerobe on many bacteriologic media. Growth is more rapid in media containing blood or
tissue fluids and fastest at 30°C. In cultures on blood agar at 37°C, colonies may be very
small at 24 hours. A virulent inoculum, derived from infected tissue, produces gray and
viscous colonies, but after passage in the laboratory, the colonies become irregular and rough.
The organism has little biochemical activity, and this is somewhat variable. Y enterocolitica
and Y. pseudotuberculosis these are nonlactose-fermenting gram-negative rods that are urease
positive and oxidase negative. They grow best at 25°C and are motile at 25°C but nonmotile
at 37°C.

65
Photomicrograph of Gram stain of Yersinia enterocolitica, Yersinia pestis — Gram Stain Gram-negative

the causative agent of yersiniosis.Credit: Centers for bacilli (0.5 to 0.8 by 1 to 3 microns), single or
Disease Control and Prevention short chained. Sometimes bipolar staining

(CDC)(Image Number: 2153) (―closed safety pin‖).

-Light Microscope-

Electron Microscope

66
 Electron Microscope

Cefsulodin Irgasan Novobiocin (CIN) Agar

-A differential and selective medium for the isolation of Yersinia enterocolitica. Fermentation of 
mannitol in the presence of neutral red produces characteristic "bull's-eye" colonies. These are colourless
with a red centre. A zone of precipitated bile may also be present.Crystal violet, sodium desoxycholate,
cefsulodin, Irgasan (triclosan) and novobiocin are inhibitory agents. Typical Y. enterocolitica colonies will
have deep-red centres surrounded by a transparent border giving the appearance of a "bull's-eye". Other
Yersinia species and Enterobacteriaceae grow on the medium 
and may look similar to Y. enterocolitica. 

67
2-16-Neisseria
The typical Neisseria is a gram-negative, nonmotile diplococcus, approximately 0.8 μm in
diameter. Individual cocci are kidney shaped; when the organisms occur in pairs, the flat or
concave sides are adjacent. In 48 hours on enriched media (eg, modified Thayer-Martin,
Martin-Lewis, GC-Lect, and New York City), gonococci and meningococci form convex,
glistening, elevated, mucoid colonies 1–5 mm in diameter. Colonies are transparent or
opaque, nonpigmented, and nonhemolytic . The neisseriae grow best under aerobic
conditions, but some grow in an anaerobic environment. They have complex growth
requirements.

-1- -2-

1-Gram stain of Neisseria gonorrhoeae from blood culture growth.

2-Gram stain of Neisseria gonorrhoeae, the agent of the STD gonorrhea. The bacteria are seen as pairs of
cocci (diplococci) in association with host pmn's(polymorphonuclear leukocytes).CDC .

-1- -2-
1-Light microscopy of Neisseria meningitidis. Gram stain.Gram-negative cocci that typically appear in pairs with the
opposing sides flattened.

2-Gram stain of N. meningitides.

68
 Neisseria meningitidis Neisseria gonorrhoeae
Scanning electron micrograph images Scanning electron micrograph images
of gonococcal-lymphocyte interactions.

False-colour transmission electron micrograph Neisseria gonorrhoeae

(TEM) of Neisseria meningitides. a species of Electron microscope picture
non- motile, non-capsulate, aerobic, Gram-
negative bacteria causing meningitis.

69
Neisseria meningitides colonies on CAP (Chocolate Agar Plate) at 35-37°Cwith ~5%CO2 (or in a candle-
jar).

Colonies of Neisseria meningitidis on Blood Agar Plate (BAP) . Some strains form pearl-like colonies on the
surface of this media. Cultivation 24 hr. in an aerobic atmosphere enriched with 5% CO2, 37 °C.

70
 N. meningitidis N.gonorrhoeae
colonies on a Blood Agar plate colonies on chocolate agar plate

(Chocolate Agar / Martin Lewis with Lincomycin)

Chocolate Agar- Gonococcal Agar For the cultivation and isolation of Neisseria gonorrhoeae.

Neisseria meningitidis colonies on chocolate agar (Thayer-Martin agar)with positive oxidase test as
indicated by black colonies.

71
 GC SELECTIVE AGAR (NEW YORK CITY)

3-Acid-fast‖ bacilli
3-1: Mycobacterium tuberculosis.
The mycobacteria are rod-shaped, aerobic bacteria that do not form spores. Although they do
not stain readily, after being stained, they resist decolorization by acid or alcohol and are
therefore called ―acid-fast‖ bacilli. The Ziehl-Neelsen technique of staining is used for
identification of acid-fast bacteria. Mycobacterium tuberculosis causes tuberculosis and is a
very important pathogen of humans. Mycobacterium leprae causes leprosy. In tissue,
tubercle bacilli are thin, straight rods measuring about 0.4 × 3 μm . The media for primary
culture of mycobacteria should include a nonselective medium and a selective medium:
Semisynthetic agar media—Inspissated egg media and Broth media. (LÖwenstein-
Jensen medium is example for Inspissated egg media , small inocula in specimens from
patients will grow on these media in 3–6 weeks. These media with added antibiotics are used
as selective media) . Mycobacteria are obligate aerobes and derive energy from the oxidation
of many simple carbon compounds. Increased CO2 tension enhances growth. The growth
rate is much slower than that of most bacteria. The doubling time of tubercle bacilli is
about18 hours. Saprophytic forms tend to grow more rapidly, to proliferate well at 22–33°C,
to produce more pigment, and to be less acid fast than pathogenic forms.

72
Mycobacterium tuberculosis. Acid-fast stain. CDC. Mycobacterium tuberculosis revealed with a special
stain specific for Mycobacteria,acid-fast Ziehl-Neelsen
stain; Magnified 1000X. Image courtesy: CDC/Dr.
George P. Kubica (1979).

Mycobacteria
http://www.flickr.com/photos/ajc1/510668360/
Mycobacterium tuberculosis
scanning electron micrograph. Mag 15549X. CDC

73
 Transmission electron micrograph of
Mycobacterium tuberculosis

In vitro culture of Mycobacterium tuberculosis

on an agar slope. James King-Holmes 2011

.
Mycobacterium tuberculosis colonies Mycobacterium tuberculosis colonies
on7H 11 agar on agar. Courtesy Center for
Hopkins University Tuberculosis Research, Johns

74
 Mycobacterium tuberculosis Colonies on Lowenstein-Jensen medium. CDC.

3-2: Mycobacterium leprae

Typical acid-fast bacilli—singly, in parallel bundles, or in globular masses—are regularly
found in scrapings from skin or mucous membranes (particularly the nasal septum) in
lepromatous leprosy. Since discovering of leprosy it has not been cultivated on nonliving
bacteriologic media. Scrapings with a scalpel blade from skin or nasal mucosa or from a
biopsy of earlobe skin are smeared on a slide and stained by the Ziehl-Neelsen technique.
Biopsy of skin or of a thickened nerve gives a typical histologic picture. The bacilli are often
found within the endothelial cells of blood vessels or in mononuclear cells. When bacilli
from human leprosy (ground tissue nasal scrapings) are inoculated into footpads of mice,
local granulomatous lesions develop with limited multiplication of bacilli. Inoculated
armadillos develop extensive lepromatous leprosy.

Photomicrograph depicting an acid fast stain Electron Microscope Micrograph

of Mycobacterium leprae bacteria.

75
4-Cell Wall-Defective Bacteria (Mycoplasma )
- Overview
Mycoplasmas are small, prokaryotic organisms with no peptidoglycan cell walls.
Instead, they are enclosed in a single, trilaminar plasma membrane. They are,
therefore, plastic and pleomorphic, and thus cannot be classified as either cocci or
rods. Because of their extremely small size, mycoplasmas frequently pass through
bacteriologic filters. Mycoplasmas are also the smallest of known free-living, self-
replicating prokaryotic cells.
The many Mycoplasma species are widely distributed in nature, and include
several commensals commonly found in the mouth and genitourinary tract of
humans and other mammals. For these reasons, Mycoplasmas are often recovered
as contaminants or adventitious flora from biologic materials, including clinical
samples. Three Mycoplasma species are definitively associated with human
disease, namely Mycoplasma pneumoniae, which is the cause of a primary
atypical pneumonia, and Mycoplasma hominis and Ureaplasma urealyticum,
which are associated with a variety of genitourinary diseases, such as urethritis,
pelvic inflammatory disease, and intrapartum infections .

-Colony production
Mycoplasmas produce minute colonies on specialized agar after several days of
incubation. These are best visualized under 30 to 100x magnification. The central
portion of the colony penetrates the agar while the periphery spreads over the
adjacent surface, in some cases giving the colony a characteristic fried egg
appearance.

-Mycoplasma Pneumoniae
causes a lower respiratory tract infection (primary atypical pneumonia, so named
because the signs and symptoms are unlike typical lobar pneumonia).

-Laboratory identification
Direct microscopic examination of clinical material for M.pneumoniae is of
limited value. Sputum is scanty and nonpurulent, and the pathogen stains poorly or
not at all using standard bacteriologic stains. Sputum samples or throat swabs can
be cultured on special media; however, isolation of the organism usually requires
eight to fifteen days and, therefore, cannot aid in early treatment decisions.
Serologic tests are the most widely used procedures

-Genital Mycoplasmas
Mycoplasma hominis and Ureaplasma urealyticum are common inhabitants of
the genitourinary tract . They grow more rapidly than M.pneumoniae, and can be

76
distinguished by their carbon utilization patterns; M. hominis degrades arginine,
U. urealyticum hydrolyses urea.

- Other Mycoplasmas
Several other species of Mycoplasma can be recovered from human sources, for
which no pathogenic role is established to date. One such organism, AIDS-
associated Mycoplasma, or M. incognitus, has been isolated in high frequency
from patients with that disease, in which the organism may play a role, possibly as
a secondary invader.

77
78
5-Chlamydiae
I. Overview

Chlamydia is a genus of small bacteria that are obligate intracellular

parasites, depending on the host cell for energy in the forms of adenosine
triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+).
They grow in cytoplasmic vacuoles in a limited number of host cell
types. The genus is divided into three species: Chlamydia trachomatis,
Chlamydia psittaci, and Chlamydia pneumoniae. C.trachomatis
infections cause diseases of the genitourinary tract and the eye, including
many cases of nongonococcal urethritis and ocular infections such as
trachoma. C.psittaci and C.pneumoniae infect various levels of the
respiratory tract. For example, C.psittaci causes psittacosis, and C.
pneumoniae causes atypical pneumonia

- General Features of Chlamydiae

Chlamydiae are small, round-to-ovoid organisms that vary in size during
the different stages of their replicative cycle. The chlamydial DNA
genome is less than 109 daltons in size, making it among the smallest
found in prokaryotic cells. Chlamydiae possess ribosomes and synthesize
their own proteins and, therefore, are sensitive to antibiotics that inhibit
this process, such as tetracyclines and macrolides

- Laboratory identification: Useful stains: Chlamydiae are not stained

using the Gram stain, but can be visualized under light microscopy by
stains that preserve the host cell architecture. Direct immunofluorescence
is also a common and useful procedure. In C.trachomatis only, a matrix
of glycogenlike material accumulates in the inclusions, which can be
shown by staining with iodine. Other species do not give this reaction.

-C. trachomatis causes a range of genitourinary and eye infections

-Chlamydia Psittaci

Psittacosis, or more broadly, ornithosis, denotes a zoonotic disease that is

transmitted to humans by inhalation of dust contaminated with
respiratory secretions or feces of infected birds. The human disease
usually targets the lower respiratory.

79
-Chlamydia Pneumoniae

C.pneumoniae is a respiratory pathogen causing pharyngitis, sometimes

followed by laryngitis, bronchitis, or interstitial pneumonia. It is a
significant cause of community-acquired respiratory infection, occurring
worldwide and without seasonal incidence.

80
81
82
 List of Abbreviations

Abbreviations Meaning
CLED Cystine -Lactose Electrolyte - Deficient
EMB Eosin Methylene Blue
SS agar Salmonella Shigella Agar
Methicillin-Resistant Staphylococcus
MRSA
aureus
GBS Group B Streptococci
XLD Xylose lysine deoxycholate
TSA Tryptic Soy Agar
TEM Transmission Electron Microscope
SEM Scanning Electron Microscope
WBC White Blood Cell
Spp. Species
CDC Center for Disease Control and Prevention
CIN Cefsulodin Irgasan Novobiocin
CAMP test Christie-Atkins, Munch-Petersen test
CAP Chocolate Agar Plate
BAP Blood Agar Plate
GC Gonococcal
STD Sexually Transmitted Disease
CCEY Cefoxitin Cycloserine Egg-Yolk
TSC Tryptose Sulphite Cycloserine
HP Helicobacter Pylori

83
Appendix-1

84
Appendix -2

85
Appendix -3

86
Appendix -4

87
Appendix -5

88
Appendix -6

89
Appendix -7

90
Appendix -8

91
Appendix -9

92
Appendix -10

93
Appendix -11

94
Appendix -12

95
Appendix13

96
Appendix -14

97
Appendix -15

98
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