Advanced Micriobiology Lab Exercises - January

Advanced Microbiology
Lab Exercises
Manalo, Karen Paula V.

Microscopy
• Beginnings: Robert Hoooke and
Anton van Leuuwenhoek
• Light compound microscope: visible
light to magnify and resolve objects
Bacterial cells not easily seen
1. Extremely small
2. Most appear colorless against
bright background
The Compound Microscope
• Basic tool of microbiology lab
• Contains a series of lenses allowing a
specimen to be magnified up to a
thousand times (1000x)
• Have mechanical adjustments and
supportive features  broad range of
possibilities in viewing microorganisms
• Combines principles of optical and
illumination system 
magnification in a bright field
• Light projected toward an object 
passes through object  collected
by objective lens  formation of
magnified image
• Light projected toward an object 
passes through object  collected
by objective lens  formation of
magnified image object for a
second lens, ocular lens 
magnifies image further
• Resolving power – determines size of the
smallest object can be clearly seen under
specified conditions
• Working distance – proximity of the slide
to the bottom of the objective lens
• Refractive index – light-bending ability of
glass, oil, air-media where light must pass
during image formation
• Lens paper = to clean the lenses and
stage area
• Ocular lens = eyepiece
• Objective lenses
• Stage
• Slide holder (mechanical stage)
• Condenser
• Diaphragm
• Coarse adjustment
• Fine adjustment
• Total magnification = ocular
magnification x objective magnification
• Diaphragm lever adjustment = to achieve
optimal lighting
• Field = singular circular area
• Parfocal = an object in view under one
objective will still be in view under other
lenses
• Oil immersion lens
• Immersion oil = has the same
refractive index or light bending ability
as glass; oil keeps light in a straight
line as it passes from the glass slide to
the oil and then to the glass of the
objective lens
Microscopic Troubleshooting
• Problem: Object observed clearly
under LPO but lost when moved to
HPO.
• Solution: Place the object in the center
field before changing objectives.
Microscopic Troubleshooting
• Problem: Object observed clearly
under LPO but lost when moved to
HPO.
• Solution: Place the object in the center
field before changing objectives.
Microscope
Q and A
Q: Explain the problem encountered when
magnifying an object with the oil immersion lens
and indicate how immersion oil helps solve the
problem.
A: The immersion oil allows more light to be

directed to the objective lens (OIO) for a clearer
image.
The Compound Microscope Q
and A
Q: Why do you use only the fine adjustment knob when
viewing specimens with the high power (40x) objective and
with the oil-immersion (100x) objective?
A: The fine adjustment know is used only when viewing
specimens with the high and oil-immersion objectives as
there is a short working distance between these objectives
and the glass slide where the specimen is. It is likely that
the glass slide will be hit and then broken when the coarse
adjustment knob will also be adjusted.
and A
Q: Describe the important steps that should be taken to
care for the microscope during and after the use in the
laboratory.
A: It is to be noted that the microscope is properly and
securely placed on a stable desk or table. It is important to
make sure that the microscope is clean particularly the
objective lenses after use. The LPO should be arrange din
the middle and the stage should be as close to the
objective lenses.
and A
Q: What does the adjective parfocal mean when applied to
a microscope? How is it a valuable asset in the use of the
microscope?
A: According to Pommerville (2018), parfocal means that an
object being observed under one objective is also viewed
under other lenses. Also, Wilson (n.d.) also said that
parfocal is the capability of a microscope to maintain its
focus despite changing the objectives being used. This is
supported by Schorr (n.d.) this is advantageous.
and A
Q: Explain the resolving power and working distance as they apply to the
microscope. Determine the resolving power of your microscope, using the oil
immersion objective. Can a spherical bacterium measuring 3m in diameter be
seen with the oil immersion lens? Explain.
A: Pommerville (2018), described the resolving power as the aspect of microscopy

that allows for the a clear distinction of the smallest specimen size observed under
the microscope. It is important to determine the resolving power of the microscope
so as to know down to which sample or specimen can still be distinguished or
clearly observed upon observation. Pommerville (2018), also described the working
distance as the distance between the objective lens and the glass slide on the
stage. It is important to be aware of the working distance so as to not hit or break
the glass slide while working with a particular objective lens.
References for Microscopy
HOW MOULDS CAN BE ISOLATED. How fungi are constructed. [accessed 2018 Nov 9].
http://website.nbm-mnb.ca/mycologywebpages/Moulds/Isolation.html
Microscope Immersion Oil Explained. Microscope History. [accessed 2018 Nov 9].
https://www.microscopeworld.com/t-using_microscope_immersion_oil.aspx
Pommerville JC. Fundamentals of microbiology. Burlington, MA: Jones & Bartlett

Learning; 2018.
Schorr R. Why Is It Desirable That Microscope Objectives Be Parfocal? Sciencing. 2018

Jul 17 [accessed 2018 Nov 6]. https://sciencing.com/why-is-it-desirable-that-
microscope-objectives-be-parfocal-12742456.html
Streak Plate Method. Determination of Planck's Constant (Theory) : Modern Physics

Virtual Lab : Physical Sciences : Amrita Vishwa Vidyapeetham Virtual Lab. [accessed
2018 Nov 8]. http://vlab.amrita.edu/?sub=3&brch=73&sim=213&cnt=1
Zwenger S. General Microbiology Laboratory Manual Biology 490.

Culture Techniques

Isolation Methods
• pipette is an instrument often used to transfer
aliquots of culture, to prepare serial dilutions of
microorganisms, and to dispense chemical reagents
• Inoculating needles and loops are used to aseptically
transfer microorganisms from broth, slant, or agar
cultures to other media
– Both may consist of handles, a shaft, and a turret,
which holds a nickel- chromium or platinum wire
– Before using either, the end of the wire must be
sterilized
Isolation Methods
Isolation Methods
• Microorganisms are transferred from one culture medium
to another by subculturing using specific procedures and
aseptic technique
• Asepsis - free from sepsis [a toxic condition resulting from
the presence of microorganisms.]
• pure or stock culture - growth of a single species of
microorganism
• maintenance of pure stock cultures over a long period by
refrigeration or desiccation
• lyophilization (freeze-drying) - best way to preserve many
stock cultures for long periods is through
Aseptic Technique for Bacterial Removal
and Subculturing
Transferring Techniques
Spread-Plate Technique
• an easy, direct way of achieving an individual bacterial

species or a pure culture
• a small volume of dilute bacterial mixture containing
100 to 200 cells or less is transferred to the center of
an agar plate and is spread evenly over the surface
with a sterile, L-shaped glass rod
• colony is a large number of bacterial cells on solid
medium, which is visible to the naked eye as a discrete
entity; derived from one cell and therefore represents
a clone of a pure culture
Spread-Plate Technique
The Streak-Plate Technique
• Where isolated, pure colonies can also be obtained

• the bacterial mixture is transferred to the edge of an
agar plate with an inoculating loop and then streaked
out over the surface in one of several patterns
• At some point on the streaks, individual cells will be
removed from the loop as it glides along the agar
surface and will give rise to separate colonies
• key principle of this method is that by streaking, a
dilution gradient is established on the surface of the
plate as cells are deposited on the agar surface
The Streak-Plate Technique
Preparation of a Streak Plate
Pour-Plate Technique
• will yield isolated colonies and has been extensively
used with bacteria and fungi
• original sample is diluted several times to reduce the
microbial population sufficiently to obtain separate
colonies upon plating
• small volumes of several diluted samples are added to
sterile petri plates and mixed with liquid tryptic soy
agar that has been cooled to about 48° to 50°C
• To prepare pure cultures, colonies growing on the
surface or subsurface can be inoculated into fresh
medium
The Pour-Plate Technique
Quantitative Plating Procedure
Microbial culture media
• survival and growth of microorganisms depend on
available nutrients and a favorable growth
environment
• nutrient preparations that are used for culturing
microorganisms
• Three physical forms are used: liquid, or broth,
media; semisolid media; and solid media
• Semisolid media can also be used in fermentation
studies, in determining bacterial motility, and in
promoting anaerobic growth
• Solid media, such as nutrient agar or blood agar, are
used
– (1) for the surface growth of microorganisms in
order to observe colony appearance
– (2) for pure culture isolations
– (3) for storage of cultures
– (4) to observe specific biochemical reactions
• liquefied state, solid media can be poured into either
a test tube or petri plate (dish)
• agar slant - the medium in the test tube is allowed to
harden in a slanted position
• agar deep tube - tube is allowed to harden in an
upright position
• agar plate - agar is poured into a petri plate
• Agar pours (the same as Agar deeps) containing
about 15 to 16 ml of media are often used to prepare
agar plates
CULTURE TECHNIQUES
Two different types of media
• Chemically defined or synthetic, media are
composed of known amounts of pure chemicals
– often used in culturing autotrophic
microorganisms such as algae or nonfastidious
heterotrophs
• complex, or nonsynthetic, media - routine
bacteriology laboratory exercises
– composed of complex materials that are rich in
vitamins and nutrients
Pouring agar
plates
Sterilization of Media and Equipment
• Sterilization is the process of rendering a medium or
material free of all forms of life
• Autoclaving - items are sterilized by exposure to
steam at 121°C and 15 lbs of pressure for 15 minutes
or longer, depending on the nature of the item
• dry-heat sterilized for dry glassware such as pipettes
and petri plates; Steam tends to etch glassware and
also leaves it damp
Staining Techniques

STAINING TECHNIQUES
• Simple
• Differential
• Special (endospore, capsule)
BACTERIAL STAINING
• One of the procedures used to observe
bacteria is to stain them with dyes.
Stains
• Colored chemical
• Imparts color to bacterial cells or
structures
Stains: Simple and Differential
• Simple staining
–Provide contrast needed to carry out
cell measurements and observations
• Differential staining
–Use two contrasting colored stains to
separate bacteria into one of two
basic groups
–To visualize bacterial structures such
as endospores and capsules
Stains: Simple
• Provide contrast needed to carry
out cell measurements and
observations
• uses one dye
• can be positive or negative staining
Stains: Positive and Negative
• Positive simple staining -
uses basic dyes
Stains: Negative
• indirect, or background staining
• uses acidic dyes as nigrosin, India ink, or
eosin
• spreading out the mixture on a slide to
form a film
Stains: Negative
• spreading out the mixture on a slide to form a film
• stains will not penetrate and stain the bacterial cells
due to repulsion between the negative charge of the
stains and the negatively charged bacterial wall
• stains either produce a deposit around the bacteria
or produce a dark background so that the bacteria
appear as unstained cells with a clear area around
them
Stains: Differential
• uses two or more dyes that
will distinguish bacteria
from each other because
their cell walls have
different reactions on dyes
• distinguish between types of bacteria
• Gram stain - most useful and widely
employed differential stain in
bacteriology
• divides bacteria into two groups— gram
negative and gram positive
Types and Uses for Staining
TYPE OF STAINING PURPOSE
Simple (uses one stain) Measure cell size
Determine cell shape (bacillus,
coccus, spiral)
Determine cell arrangements
Differential (uses two contrasting Measure cell size and determine
stains) cell shape and arrangements
Separate bacteria into groups
(Gram positive and negative)
Visualize cell structures
(endospores, capsules
Q and A
1. Why do you have to heat fix the smear prior to
staining?
Heat fixing should be done before staining for the
bacterial smear to securely adhere to the glass
slide and immediately absorb stains. It also lets the
smear to dry (vlab.amrita.edu, 2001). Collins.edu
(n.d.) also stated that heat fixing destroys the
bacteria and allow them to be “more permeable to
stains.”
Q and A
2. Draw the specimen observed under positive simple
staining
Q and A
3. Explain the principle of staining.
Staining is a supplemental method in microscopy to improve visualization of

microscopic images. Staining makes use of stains and dyes to emphasize
structure and parts of biological samples such as cells and tissues. The most
common staining technique in microbiology is the Gram stain created by Hans
Christian Joachim Gram in the 1800s. It is a kind of differential staining, which
from the word itself, differentiates bacteria into two categories namely the
gram-positive and the gram-negative. The technique makes use of the
capability of microorganisms to maintain the color of the stains applied in the
reaction. Gram-positive bacteria are normally purple when the primary stain is
applied and then is decolorized by alcohol. It will then become pink after
counterstaining. Gram-positive bacteria, on the other hand, will not lose the
purple color even after decolorizing by alcohol (vlab.amrita.edu, 2001).
Q and A
4. Draw and describe the appearance of the representative
bacteria under gram staining.
Specimen: Vibrio cholerae

Shape: curved or comma shape
Arrangement: singles
Gram negative
Q and A
bacteria under gram staining.
Specimen: Escherichia coli

Shape: rod-shaped
Arrangement: single-cell
individually in large clumps
Gram negative
Q and A
bacteria under gram staining
Specimen: Streptococcus sp
Shape: spheroidal
Arrangement: pairs or
chains
Gram positive
Q and A
5. Give the importance of the different dyes and reagents
used in gram staining and their expected result.
Dyes/Reagents Role Expected result
Crystal Violet Simple or primary stain G+ Cells that maintain the primary stain
which colors cell wall of will be blue or purple indicating gram
many bacteria. positive bacteria.
G- Cells appear as blue or purple as well.
Gram Iodine Functions as a mordant G+
which reacts with the G-
crystal violet to form large Both gram positive and gram negative
crystals that are not easily cells will have the same color.
removed from the cell.
Q and A
Ethanol The ethanol or alcohol functions as G+ Alcohol dehydrates gram positive
a decolorizer. It does not affect cell bacteria shrinking the cell wall pores.
walls made of peptidoglycan but The crystal violet-iodine complex
affects those composed of lipid then firmly attaches to the cell wall
components. Such cell walls will with different layers staining cells
have large holes as lipids will be purple.
dissolved by acetone and alcohol. G- Gram negative bacteria become
The large holes will then allow the colorless as alcohol or acetone
crystal violet-iodine complex to be dissolves the lipid outer layer of the
removed from the cell thereby bacteria. It also exposes the
making the cell colorless. peptidoglycan layer.
Q and A
Safranin Functions as a counterstain which G+ Gram positive cells will
dyes the colorless cells. appear blue or purple as
they retain the primary
stain.
G- Gram negative cells will
appear pink or red due to
the counterstain.
Q and A
6. Why is it necessary to use a young culture (24-hr old
culture) in gram staining?
A young culture (18-24 hours)
should be used in gram staining
because there are bacteria that can
alter their Gram reactions as they
age (Chan, 1997).
Q and A
7. What would be the color of gram positive and negative
bacteria if:
crystal violet was G+ Gram positive bacteria appear blue as methylene blue is
replaced with as effective as crystal violet (Bentz, Wilber, Fitzgerald,
methylene blue? Muldavin and Peterson, 2018).
G- Gram negative bacteria appear blue as methylene blue is
as effective as crystal violet (Bentz, Wilber, Fitzgerald,
Muldavin and Peterson, 2018).
Q and A
bacteria if:
mordant was G+
skipped? G-
The mordant strengthens the attachment of the primary
stain (crystal violet or methylene blue) with bacteria. If the
mordant is skipped, the cells will be easily discolored by
alcohol for both gram positive and gram negative
(Washington University School of Medicine, n.d.).
Q and A
bacteria if:
decolorizing step G+ Cells will still appear purple or blue.
was omitted? G- Cells will still appear purple or blue
since they will not be decolorized.
counterstain was G+ Gram positive cells will appear blue
not used? or purple as they retain the primary
stain.
G- Cells will appear colorless.
Q and A
8. Why did you flood the smear with malachite green and
with continuous steaming?
Malachite Green is the main stain while Safranin is the
counterstain. They are both parts of the standard
endospore stain. Steaming the smear while flooding it
with Malachite green to allow the stain to be attached to
the spore. The endospore of bacteria protects the cells
and does not allow stains to be absorbed, thus heating
or steaming was done (Portland Community Colleges,
2006).
Q and A
9. Draw the specimen observed under endospore staining.
Q and A
ENDOSPORE
• resistant structure capable of surviving for long
periods in an unfavorable environment and then
giving rise to a new bacterial cell
• spherical to elliptical in shape and may be either
smaller or larger than the parent bacterial cell
• position within the cell is characteristic and may be
central, subterminal, or terminal
Q and A
ENDOSPORE
• do not stain easily, but, once stained, they strongly resist
decolorization
• stained with malachite green
• Heat is used to provide stain penetration
• The rest of the cell is then decolorized and counterstained
a light red with safranin
Q and A
10. Draw the specimen observed under positive simple
staining.
Q and A
11. Explain the principle of capsule staining?
CAPSULE
• Large polysaccharide capsule and give rise to large mucoid

colonies
• Slimy layer surrounding bacteria
• composition, as well as its thickness, varies with
individual bacterial species
• Polysaccharides, polypeptides, and glycoproteins have all
been found
Q and A
CAPSULE
• one cannot always determine if a capsule is present by
simple staining procedures, such as using negative
staining
• nonionic and the primary stain cannot adhere
Q and A
CAPSULE
• Copper sulfate is the decolorizing agent. It removes excess
primary stain as well as color from the capsule
• copper sulfate acts as a counterstain by being absorbed
into the capsule and turning it a light blue or pink
• smears should not be heat-fixed since shrinkage is likely
to occur and create a clear zone around the bacterium,
which can be mistaken for a capsule
References for Bacterial Staining
Bentz K, Wilber P, Fitzgerald H, Muldavin D, Andrea A. Differential Staining Technique: Gram Stain and Streak Isolation. 2018.
https://www.cnm.edu/programs-of-study/math-science...lab.../unit-5-summer.docx
Chan SL. What Does My Bacteria Look Like? Summer Research Program for Science Teachers. 1997 [accessed 2018 Nov 1].
http://www.scienceteacherprogram.org/biology/chan97.html
Endospore Stain. [accessed 2018 Nov 1]. http://spot.pcc.edu/~jvolpe/b/bi234/lab/differentialTests/endospore_stain.htm
Gram Stain Technique. Amrita Vishwa Vidyapeetham Virtual Lab. 2011 [accessed 2018 Nov 1]. vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=2
Harley JP, Prescott LM, Harley JP. Instructors manual to accompany Laboratory exercises in microbiology, fifth edition. Boston: McGraw-Hill; 2002.
Lepp P. General Microbiology Laboratory Manual. 2010. Retrieved 1 November 2018 from
http://yourspace.minotstateu.edu/paul.lepp/BIOL%20592/Introductory%20Microbiology%20Laboratory%20Manual.pdf?fbclid=IwAR2kz_NIi108Blss7j
0-CufTgKauL5_B4OALArtZ-bmTxk67UgE1C1cOyi4
Microbiology Laboratory Syllabus. Portland Community College. 2006. [accessed 2018 Nov 1].
http://spot.pcc.edu/~jvolpe/b/bi234/lab/differentialTests/endospore_stain.htm
STAINING BACTERIA AND USE OF THE MICROSCOPE. Washington University School of Medicine. [accessed 2018 Nov 1].
http://medicine.wustl.edu/ysp
Zwenger S. General Microbiology Laboratory Manual. [accessed 2018 Nov 1]. http://themodern.farm/studies/Microbiology-Laboratory-Manual.pdf
ENZYMATIC ACTIVITIES
IN BACTERIA

Enzymes
• Most important chemical mediators
• Organic substances
• Catalyze or promote the uptake and use
of raw materials  synthesis of cellular
components or for energy
Enzymes
• Breakdown of unneeded substances
• Breakdown of metabolic side products
eliminated from the cell and returned to
the environment
• Promote changes only in very specific
substances or substrates
Enzymes of Bacteria
• Highly varied
• With different metabolic processes
• Identified in terms of the type of change
produced in the substrate
Fermentation
• energy-producing biochemical reactions in
which organic molecules serve both as
electron acceptors and donors
• given carbohydrate may be fermented to a
number of different end products depending
upon the microorganism involved
Outline of Major Fermentation Pathways. Microorganisms produce various waste products when they ferment
glucose. The by-products released (shaded boxes) are often characteristic of the microorganisms and can be used as
Fermentation
• end products (alcohols, acids, gases, or other
organic molecules) are characteristic of the
particular microorganisms
• Example: bacteria grown in a liquid culture medium
with carbohydrate glucose  they may produce
organic acids (by-products)
• acids are released into the medium and lower its pH
• pH indicators: phenol red or bromcresol purple 
acid production  original color to yellow
Fermentation
• Durham tube: Gases produced during the
fermentation process can be detected by using a
small, inverted tube
• During autoclaving  air expelled from the Durham
tubes  become filled with the medium
• gas is produced, the liquid medium inside the
Durham tube will be displaced, entrapping the gas
in the form of a bubble
Fermentation
(a) Possible carbohydrate fermentation patterns of microorganisms, with phenol red as

indicator.
Fermentation
(b) The tube on the left is the control. The next tube shows alcohol fermentation. Notice the gas
bubble at the top. The third tube from the left shows no carbohydrate fermentation (negative).
The tube on the right shows acid and gas production.
Fermentation of Carbohydrates:
Sugars (Methods)
• 4 sets of tubes each composed of BMglu,
BMfru, BMgal, BMsuc, BMlac and
Bmman prepared
• Tubes inoculated with unknown bacteria
• Incubated at 37˚C for 48 hrs.
• Gas production and color changed
observed
Sugars
Table 5.1. Fermentation reactions of the test bacteria
Bacteria Sugars
Glucose Fructos Lactose Sucrose Mannito Galactos

(1) e (5) (3) l (4) e
S. aureus A A A A A A
M. luteus N N N N N N
P. N N N N A N
aeruginosa
Unknown A - N A A -
Sugars
Q: E. coli ferments glucose but not sucrose although
sucrose is a disaccharide of glucose and fructose.
Explain why this bacterium uses glucose and not
sucrose.
A: E. coli does not have a sucrose operon responsible
for sucrose fermentation. However, many studies have
already developed E. coli strains that can ferment
sucrose (Lee et al., 2010).
Sugars
• E. coli can use lactose as their sole source of carbon.
• An essential enzyme in the metabolism of this sugar
is R-galactosidase.
• R-galactosidase hydrolyzes lactose to galactose and
glucose
• Instead of lactose, the natural substrate of this
enzyme, an artificial substrate ONPG (o-nitro-phenyl-
ONPG is colorless but upon hydrolysis yields
onitrophenol, which is yellow in an alkaline solution.
Methyl Red and Voges Proskauer Test
• Three tubes labeled with MRVP broth
• Tubes inoculated with the unknown bacteria then
incubate at 37˚C for 48 hrs.
• Five drops of Methyl red added after 48 hrs into the
first MRVP tube.
• Ten drops of 40% KOH and 15 drops of alpha-naphthol
reagent were added into the second MRVP tube.
• Color changes observed.
Methyl Red
• pH indicator; detects a pH change to the acid range as a
result of acidic end products such as lactic, acetic, and
formic acids
• test is of value in distinguishing between E. coli (a
mixed acid fermenter) and E. aerogenes (a butanediol
fermenter)
• pH of 4: methyl red indicator turns red—a positive
methyl red test
• pH of 6, the indicator turns yellow—a negative methyl
red test
Methyl Red
Methyl Red Test. (a) Escherichia coli, MR+. (b) Enterobacter aerogenes, M
Voges Proskauer Test
• identifies bacteria that ferment glucose, leading to 2,3-
butanediol accumulation in the medium
• addition of 40% KOH and a 5% solution of alpha-naphthol in
absolute ethanol (Barritt’s reagent) will detect the presence of
acetoin—a precursor in the synthesis of 2,3-butanediol
• the presence of the reagents and acetoin, a cherry-red color
develops
• Development of a red color in the culture medium 15 minutes
following the addition of Barritt’s reagent represents a
positive VP test
• absence of a red color is a negative VP test
Voges-Proskauer Test
Voges-Proskauer Test. (a) Enterobacter aerogenes, VP+. (b) Escherichia c

Methyl Red and Voges Proskauer Test
Fill in Table 5.2. Place a (+) for positive result and a (-) for negative result.
Table 5.2. Methyl Red and Voges Proskauer Test of the Bacteria
Test Bacteria Methyl Red Voges-Proskauer
E. aerogenes - +
E. coli + -
Unknown
+ -
Methyl Red and Voges-Proskauer Test
Would it be possible to have an MR+ and
VP+? Provide an explanation.
Whether or not an organism be both MR and VP positive
depends on the fact that the Methyl Red test is primarily
used to identify gram-negative rods. The process of what
is tested for in the Voges-Proskauer, or VP test, could also
show up in gram-negative rods. As a result, although it is
not very common—the answer is yes— a gram-negative
organism could potentially show positive results in both
the MR and the VP tests.
Respiration of Carbohydrates:
Oxidase Test (Methods)
• Unknown bacteria were inoculated in an NA
plate then incubated at 37˚C for 24 hrs.
• After 24 hrs, 2 drops of oxidase reagent
were added.
• Color change observed.
Oxidase Test
• distinguishes between groups of bacteria
based on cytochrome oxidase activity
• play an important role in the operation of
the electron transport system during aerobic
respiration
Oxidase Test
• Cytochrome oxidase (aa3 type) uses O2 as an electron
acceptor during the oxidation of reduced cytochrome c to
form water and oxidized cytochrome c
• ability of bacteria to produce cytochrome oxidase can be
determined by the addition of the oxidase test reagent or
test strip (tetramethyl-p-phenylenedi- amine
dihydrochloride or an Oxidase Disk, p-amino-
dimethylaniline) to colonies that have grown on a plate
medium
Oxidase Test
• light pink oxidase test reagent serves as an artificial
substrate, donating electrons to cytochrome oxidase and
in the process becoming oxidized to a purple and then
dark purple compound in the presence of free O2 and the
oxidase
• presence of this dark purple coloration represents a
positive test
• No color change or a light pink coloration on the colonies
indicates the absence of oxidase and is a negative test
Catalase Test (Methods)
• A clean glass slide was divided into two sections with a marking
pen.
• With a sterilized and cooled inoculating loop, a small amount of
the unknown culture was obtained from the nutrient agar slant
and smeared directly onto the left-hand side of the slide. The
smear was about the size of a pea.
• The loop was sterilized again and a small amount of unknown
culture was smeared on the right-hand side of the slide.
• A drop of hydrogen peroxide was placed over each smear. The
appearance of gas bubbles was observed.
Oxidase Test
Table 5.3. Catalase and oxidase Test of the Bacteria
Test Bacteria Catalase Oxidase
S. epidermidis + -
S. aureus + -
P. aeruginosa + +
E. coli + -
Unknown
Activity of Urease (Methods)
• A tube of urea agar was inoculated with
unknown bacteria.
• The tubes were incubated at 35°C for
24 hrs.
• Changes were observed after 24 hours.
Urease Test
• to determine the ability of bacteria to degrade
urea by means of the enzyme urease
• urease – enzyme that attacks the nitrogen and

carbon bond in amide compounds such as urea,
forming the end products ammonia, CO2, and
water
Urease Test
• detected by growing bacteria in a medium containing urea
and using a pH indicator such as phenol red
• When urea is hydrolyzed, ammonia accumulates in the

medium and makes it alkaline. This increase in pH causes
the indicator to change from orange-red to deep pink or
purplish red and is a positive test for urea hydrolysis.
• Failure of a deep pink color to develop is a negative test

Urease Test
Urea Hydrolysis. (a) Uninoculated control. (b) Weakly positive reaction (delayed positive). (c) Very rapid
positive reaction. (d) Negative reaction.
Urease Test
Citrate Utilization Test (Methods)
1. Tubes were labeled of SCA.
2. Unknown bacteria were inoculated
on tubes and then incubated at 37˚C
for 24 hrs.
3. Color changes were observed.
Citrate Utilization Test
• ability of bacteria  use citrate as a sole
carbon source for their energy needs
• ability depends on the presence of a citrate
permease that facilitates transport of citrate
into the bacterium
• citrate  pyruvic acid and CO2
• Simmons citrate agar slants
• sodium citrate as the carbon source
• NH4+ as a nitrogen source
• pH indicator bromothymol blue
• Done on slants since O2 is necessary for citrate
utilization.
• When bacteria oxidize citrate, they remove it from
the medium and liberate CO2.
• CO2 + sodium (supplied by sodium citrate) + water 
sodium carbonate—an alkaline product.
• raises the pH  pH indicator - blue color : positive
• Absence of a color change is a negative citrate test
• Citrate-negative cultures will also show no growth in the

medium.
Gelatin Hydrolysis
• When boiled in water, the connective tissue
collagen (which is stringy, insoluble, and
indigestible) changes into gelatin, a soluble mixture
of polypeptides
• Certain bacteria are able to hydrolyze gelatin by
secreting a proteolytic enzyme called gelatinase
Gelatin Hydrolysis
• The resulting amino acids can then be used as
nutrients by the bacteria
• used to assess the pathogenicity of certain bacteria
• production of gelatinase can often be correlated
with the ability of a bacterium to break down tissue
collagen and spread throughout the body of a host
Gelatin Hydrolysis
• Gelatin liquefaction (the formation of a liquid) can be
tested for by stabbing nutrient gelatin deep tubes.
• Following incubation, the cultures are placed in a

refrigerator or ice bath at 4°C until the bottom resolidify.
Gelatin Hydrolysis
• If gelatin has been hydrolyzed, the medium will remain
liquid after refrigeration.
• If gelatin has not been hydrolyzed, the medium will
resolidify during the time it is in the refrigerator
• Nutrient gelatin may require up to a 14-day incubation
period for positive results.
Gelatin Hydrolysis
Fill in Table 5.4. Place a (+) for positive result and
a (-) for negative result.
Table 5.4. Utilization of urea, gelatin and citrate
Test Bacteria Urea Gelatin Citrate
S. epidermidis + + -
S. aureus + + +
P. aeruginosa +/- + +
E. coli - - -
Unknown +
Utilization of Amino Acids
1. Tryptophan
2. S-containing Amino Acid
3. Lysine
4. Phenylalanine
Utilization of Amino Acids:
Tryptophan (Methods)
• Label 3 tubes of SIM.
• Inoculate the tubes with E. coli, E. aerogenes, and
the unknown bacteria. Incubate at 37˚C for 48 hrs.
• After 24 hrs, add 10 drops of Kovac’s reagent.
• Observe for color change.
Tryptophan
• tryptophan - found in nearly all proteins
• Bacteria with tryptophanase can hydrolyze
tryptophan to its metabolic products, namely,
indole, pyruvic acid, and ammonia
• The bacteria use the pyruvic acid and ammonia to
satisfy nutritional needs
Tryptophan
• Indole is not used and accumulates in the
medium.
• The presence of indole can be detected by the
addition of Kovacs’ reagent.
• Kovacs’ reagent reacts with the indole, producing
a bright red compound on the surface of the
medium
Tryptophan
• Bacteria producing a red layer following addition
of Kovacs’ reagent are indole positive
• the absence of a red color indicates tryptophan
was not hydrolyzed, and the bacteria are indole
negative
Tryptophan
S-containing Amino Acid (Methods)
• Observe for the presence of black line of
streak on the SIM tube.
• Sulphide Indole Motility medium
S-containing Amino Acid
• Many proteins are rich in sulfur-containing amino acids such as cysteine.
• When these proteins are hydrolyzed by some bacteria, the amino acids are
released and taken up as nutrients.
• Cysteine, in the presence of cysteine desulfurase, loses its sulfur atom
through the addition of hydrogen from water to form hydrogen sulfide gas
S-containing Amino Acid (Methods)
• Gaseous hydrogen sulfide may also be produced by the reduction of
inorganic sulfur-containing compounds such as thiosulfate (S2O32–),
sulfate (SO42–), or sulfite (SO32–)
• When certain bacteria take up sodium thiosulfate, they can reduce it to
sulfite using the enzyme thiosulfate reductase, with the release of
hydrogen sulfide gas
• the SIM medium (named after J. S. Simmons in 1926)
contains peptones and sodium thiosulfate as substrates,
and ferrous ammonium sulfate, Fe(NH4)SO4, as the H2S
indicator
• Cysteine is a component of the peptones used in SIM
medium
• Sufficient agar is present to make the medium semisolid
• Once H2S is produced, it combines with the ferrous ammonium
sulfate, forming an insoluble, black ferrous sulfide precipitate
that can be seen along the line of the stab inoculation
• If the organism is also motile, the entire tube may turn black.
This black line or tube indicates a positive H2S reaction
• absence of a black precipitate indicates a negative reaction
Lysine (Methods)
• The tubes were inoculated with the unknown
bacteria then layered with mineral oil and
incubated at 37˚C for 24 hrs.
• Color change observed.
Lysine
• Decarboxylation is the removal of a carboxyl group from an organic
molecule
• Bacteria growing in liquid media decarboxylate amino acids most
actively when conditions are anaerobic and slightly acidic.
Decarboxylation of amino acids, such as lysine and ornithine,
results in the production of an amine and CO2
Lysine
• Bacteria  enzymes lysine decarboxylase and ornithine
decarboxylase  decarboxylate lysine and ornithine  amines as
precursors utilized
• bacteria carry  fermentation  acidic waste products making

the medium acidic and inhospitable
• decarboxylases - activated by a low pH

• acid groups removed from amino acids  alkaline amines  which
raise the pH  hospitable
Lysine
• Decarboxylation of lysine or ornithine can be detected by culturing
bacteria in a medium containing the desired amino acid, glucose,
and a pH indicator (bromcresol purple)
• Before incubation, sterile mineral oil is layered onto the broth to

prevent oxygen from reaching the
• acids produced by the bacteria from the fermentation of glucose 

lower the pH  pH indicator: from purple to yellow
Lysine
• acid pH activates the enzyme that causes decarboxylation
of lysine or ornithine to amines and the subsequent
neutralization of the medium
• results in another color change from yellow back to
purple
• Bacteria that decarboxylate lysine turn the medium
purple
• Bacteria that produce H2S appear as black colonies
Lysine
Phenylalanine (Methods)
• Three tubes were labeled PA.
• Tubes were inoculated with the unknown
bacteria and then incubated at 37˚C for 48
hrs.
• After 24 hrs, 10 drops of Ferric chloride were
added to each tube.
• Color changed observed.
Phenylalanine
• phenylalanine deaminase test to differentiate between
various enteric bacteria
• ability of certain bacteria to oxidatively degrade
phenylalanine is of taxonomic importance
• P. vulgaris produces the enzyme phenylalanine deaminase
• E. coli does not
• phenylalanine deamination can be used to differentiate
the genera Morganella, Proteus, and Providencia (+) from
the Enterobacteriaceae (–)
Phenylalanine
• Phenylalanine
deaminase catalyzes
the removal of the
amino group (NH3+)
from phenylalanine
Phenylalanine
• The resulting products include organic acids, water,
and ammonia.
• Certain enteric bacteria (e.g., Proteus, Morganella,
and Providencia) can use the organic acids in
biosynthesis reactions.
• Deamination detoxifies inhibitory amines
Phenylalanine
• phenylalanine deaminase test
• can be used to differentiate among enteric bacteria such as E.
coli and P. vulgaris. P. vulgaris produces the enzyme
phenylalanine deaminase, which deaminates phenylalanine,
producing phenylpyruvic acid
• When ferric chloride is added to the medium, it reacts with
phenylpyruvic acid, forming a green compound
• E. coli does not produce the enzyme, it cannot deaminate
phenylalanine. When ferric chloride is added to an E. coli culture,
there is no color change.
Phenylalanine
Table 5.5. Utilization of amino acids
Test Bacteria Tryptophan Cysteine Lysine Phenylalanine
S. epidermidis + + + -
S. aureus - + - -
P. aeruginosa + + - -
E. coli + + + -
Unknown -
• Once H2S is produced, it combines with the ferrous ammonium
sulfate, forming an insoluble, black ferrous sulfide precipitate
that can be seen along the line of the stab inoculation
• If the organism is also motile, the entire tube may turn black.
This black line or tube indicates a positive H2S reaction
• absence of a black precipitate indicates a negative reaction
Unknown Bacteria and Conclusion
Methyl Red Test. (a) Escherichia coli, MR+. (b) Enterobacter aerogenes
Table
Table 5.1.5.4. Utilizationreactions
Fermentation of urea,ofgelatin
the test
bacteria
and citrate
• E. coli Bacteria SugarsGelatin
Test Bacteria Urea Citrate
• Fermentation Gluco Fruct Lacto Sucro Mann Galact
• Glucose – A se(1) ose se (5) se (3) itol ose
• Lactose – N S. epidermidis + +
(4)
-
S. aureus A A A A A A
• Sucrose – A S.M.
aureus +N
luteus N N N + N N+
• Mannitol – A
P.P.aeruginosa N N +/-N N + A N+
• MR (+), VP ( - ) aeruginos
• Citrate (+) E.
a coli - - -
Unknown A - N A A -
• Phenylalanine (-) Unknown +
Voges-Proskauer Test. (a) Enterobacter aerogenes, VP+. (b)
Escherichia coli, VP–.
Table 5.5. Utilization of amino acids
Test Bacteria Tryptophan Cysteine Lysine Phenylalanine
S. epidermidis + + + -
S. aureus - + - -
P. aeruginosa + + - -
E. coli + + + -
Unknown -
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Manalo, Karen Paula V.

A: The immersion oil allows more light to be

A: Pommerville (2018), described the resolving power as the aspect of microscopy

Pommerville JC. Fundamentals of microbiology. Burlington, MA: Jones & Bartlett

Schorr R. Why Is It Desirable That Microscope Objectives Be Parfocal? Sciencing. 2018

Streak Plate Method. Determination of Planck's Constant (Theory) : Modern Physics

Zwenger S. General Microbiology Laboratory Manual Biology 490.

Manalo, Karen Paula V.

• an easy, direct way of achieving an individual bacterial

• Where isolated, pure colonies can also be obtained

Manalo, Karen Paula V.

Staining is a supplemental method in microscopy to improve visualization of

Specimen: Vibrio cholerae

Specimen: Escherichia coli

• Large polysaccharide capsule and give rise to large mucoid

Endospore Stain. [accessed 2018 Nov 1]. http://spot.pcc.edu/~jvolpe/b/bi234/lab/differentialTests/endospore_stain.htm

Manalo, Karen Paula V.

(a) Possible carbohydrate fermentation patterns of microorganisms, with phenol red as

Glucose Fructos Lactose Sucrose Mannito Galactos

Voges-Proskauer Test. (a) Enterobacter aerogenes, VP+. (b) Escherichia c

Test Bacteria Methyl Red Voges-Proskauer

Test Bacteria Catalase Oxidase

• urease – enzyme that attacks the nitrogen and

• When urea is hydrolyzed, ammonia accumulates in the

• Failure of a deep pink color to develop is a negative test

• raises the pH  pH indicator - blue color : positive

• Absence of a color change is a negative citrate test

• Citrate-negative cultures will also show no growth in the

• Following incubation, the cultures are placed in a

Test Bacteria Urea Gelatin Citrate

• bacteria carry  fermentation  acidic waste products making

• decarboxylases - activated by a low pH

• Before incubation, sterile mineral oil is layered onto the broth to

• acids produced by the bacteria from the fermentation of glucose 

Lab #7. Quizlet. [accessed 2018 Dec 14]. https://quizlet.com/145711211/lab-7-flash-cards/

Microbiology Tests. Antranikorg. [accessed 2018 Nov 23]. https://antranik.org/microbiology-tests

Micrococcus. MRI History. [accessed 2018 Nov 23]. http://web2.uwindsor.ca/courses/biology/fackrell/Microbes/17275.htm

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