Bacteriology Mycology Laboratory Manual SPRING - 2022

PTHB 503 Lab Manual, Spring-2022
Department of Pathobiology
LABORATORY
MANUAL
BACTERIOLOGY AND MYCOLOGY
Term 2 – Spring, 2022

1
Table of Contents
Laboratory safety rules and aseptic techniques 3

Submission of clinical specimens, examination: Guidelines 5
Lab-1: Basic bacteriological techniques 7
Gram stain procedure 9
Lab-2: Bacterial isolation and identification 11
Procedure for streak plate 12
Lab-3: Antibiotic sensitivity test and quantitative culture 15
Lab-4: Interpretation of susceptibility testing, urine culture 19
Lab-5: Mycology-I 23
Lab-6: Mycology-II 25
Lab-7 & 8: Gram-negative rods or coccobacilli 28
Lab-9: Gram-positive cocci 36
Lab-10: Aerobic Gram-positive rods or coccobacilli 42
Lab-11: PCR in bacterial diagnosis 47
Bacterial disease conditions and common pathogens 53
Laboratory review slides 54
Faculty & Staff

Dr. Andy Alhassan
Dr. Sharianne Suepaul
Dr. Victor Amadi
Ms. Erica & Ms. Roxanne
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Laboratory Safety Rules and Aseptic Techniques

FYI: The laboratory safety rules, and aseptic techniques listed here should be
considered as a starting point for safe laboratory practice. For Spring 2022 Term,
we will have In-person hands-on laboratory sessions. For students taking the course
online, the hands-on lab session will be converted to online format.
1. Do not eat or drink in the laboratory at any time; and do not bring
food/drinks into the laboratory, particularly to your bench. This will
be strictly enforced, and you will lose 1-2 points if you do not
comply.
2. Wear a laboratory coat at all times to protect clothes from
contamination. If your lab coat becomes contaminated, notify an
instructor at once. Your coat will be sterilized for your protection. If
you do not bring a lab coat, you will be asked to leave the lab until
you get a lab coat, and you will lose 1-2 points if you do not comply.
3. Covered shoes are required for your protection. If you have the wrong
shoes, you will be asked to leave the lab until you are wearing proper
shoes, and you will lose 1-2 points if you do not comply.
4. All cultures are potential pathogens. Use aseptic technique with all
bacteria and DO NOT contaminate yourself, your notebooks, lab
bench or microscope. Keep long hair tied back and loose clothing
away from the Bunsen burner. If you are not properly attired or have
loose hair, you will be asked to leave the lab until you are compliant,
and you will lose 1-2 points if you do not comply.
5. Do not throw away cultures down the sink or leave in the waste
basket. Dispose of all cultures, glass slides, pipettes etc. in the
designated containers in the lab. These items will be autoclaved
before disposal.
6. DO NOT put anything (pens, pencils, fingers) in or near your mouth
or eyes while working in the laboratory. If contaminated material gets
into your eyes or mouth, rinse immediately with running water and
notify the instructors.
7. DO NOT pipette by mouth!
8. Keep only the materials needed for that day’s laboratory on your
bench. Keep coats, books and other items away from work bench.
3
Laboratory Safety Rules and Aseptic Techniques (Cont.)
9. Wash your hands when you leave the laboratory, even if you are
leaving for a short time. Hand washing should become automatic.
10. Disinfect your bench top after working. Clean up your area before
you leave. DO NOT leave cultures or test tubes on the bench unless
directed to do so by the instructors. Under no circumstances are
cultures to be removed from the laboratory.
11. If a culture is dropped or spilled, notify your lab partners and the
instructor immediately. Make sure no one steps in the contaminated
area. Cover the spill with paper towels soaked in disinfectant.
12. Light your Bunsen burner and work in the vicinity of the flame to
take advantage of the upward draft of air around the burner. DO
NOT work in a draft. Avoid coughing or sneezing when handling
your cultures.
13. Hold open containers at an angle to avoid the chance of airborne
contamination. You do not need to flame the lip of sterile test tubes
but DO NOT leave open for any longer than necessary to perform
the inoculation or test.
14. When inoculating Petri dishes, open them only as long as you need to
perform the inoculation or test. When opening an agar plate culture
of bacteria, open the plate slowly to avoid creating an aerosol. DO
NOT put your nose directly into a culture to smell it.
15. Heat inoculating needles and loops to glowing red in the flame before
and after contact with microorganisms, including making smears for
Gram stain. To avoid creating aerosols, always cool your loop before
touching it to agar or broth cultures.
NOTE:
PLEASE HANDLE MICROSCOPES CAREFULLY.
THE OIL-IMMERSION OBJECTIVE LENS MUST BE

CLEANED WITH LENS PAPER BEFORE YOU LEAVE
THE LAB. DO NOT USE OIL ON OTHER OBJECTIVES.
4
SUBMISSION OF CLINICAL SPECIMENS AND EXAMINATION:

GUIDELINES
There are many stages at which laboratory diagnosis of an infection can be invalidated;
the first is in the selection and submission of specimens to the laboratory and is the main
step over which the veterinarian has any control.
The following are guidelines in the selection and submission of specimens:
1. Take sample before antibiotic treatment is started.
2. Take sample using aseptic techniques. Use sterile instruments. Skin disinfectant
is important when collecting blood or aspirates.
3. Take samples from the site of disease where possible, e.g. mare endometritis
swab- uterus; pneumonia – transtracheal aspirate (not nasal swab).
4. Try to avoid contamination by normal flora present on mucous membranes,

especially of intestinal tract, lower urogenital tract, upper respiratory tract – this
will present a major source of confusion in the interpretation of findings. Use
“guarded” swabs.
5. Collect specimens using sterile swabs or containers; where swabs are not
processed within a 1-4 hours they must contain transport medium. Drying and
slow-freezing kills most microbes.
6. If an aerobic specimen is not processed within 1-4 hours, store the specimen at
4°C for no longer than 24-36 hours. If necessary, submit samples by courier.
7. Anaerobic specimens may be held at room temperature.
8. Submit materials cleanly so as not to endanger laboratory staff. Label dangerous

material and ship in accordance to shipping rules and regulations (e.g., permits,
double-bagging, ice pack, special requirements etc.).
9. Give laboratory a full history of animal, including disease suspected, treatment

given etc.
5
SPECIMENS WITH SPECIAL REQUIREMENTS:

1. TISSUES AND ORGANS
Place individual tissues in plastic bags or leak-proof plastic jars. Submit at 4°C
where possible, on ice or dry-ice, by enclosing in insulated containers.
2. FECES
1-2 grams of feces is preferred. Rectal swabs are acceptable.
3. RINGWORM
Clean area well before collecting sample (more details later). Take scrapings of
plucked hairs from edge of the lesions and submit in a closed envelope, sealed small
plastic bag or specimen container.
4. SERUM SAMPLE
Do not use anticoagulant. Pour off serum as soon as clot has retracted. Centrifuge and
remove any blood remaining in serum to prevent hemolysis. Freeze if necessary to
keep for more than a few days.
5. ANAEROBIC SAMPLES (e.g. deep wound with foul-smelling, dark exudate)

Use anaerobic containers.
6. BLOOD CULTURE
For culture of blood (suspected septicemia/bacteremia), you should use special blood
culture bottles, (commercially available) and follow the instructions before submitting
to a diagnostic laboratory.
EXAMINATION OF CLINICAL MATERIAL:

1. Clinical material is examined grossly in the laboratory for evidence of infection, e.g.
pus, sulfur granules, cloudiness in fluids, smell (Yeasts, anaerobes, Pseudomonas).
2. A direct smear, mostly with some type of stain (e.g., Gram, Wright’s, wet mount,
fluorescent antibody) is almost always done on clinical material. This can be a very
useful procedure.
3. Direct smears may not be useful on all tissues collected post mortem.
4. Look for bacteria both outside and inside inflammatory cells.
CULTURE OF CLINICAL SPECIMENS:

The aim of plate culture is to ensure that bacteria present grow as isolated colonies which
can be used for identification or antibiotic sensitivity testing. The number of bacteria
present is crudely quantified as small, moderate or large; where bacteria cause disease they
are present in large numbers and where proper techniques are used they will be recovered
in large numbers.
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LABORATORY - 1
BASIC BACTERIOLOGICAL TECHNIQUES
Purpose
The purpose of this laboratory exercise is to gain skill in basic techniques that are
required in diagnostic microbiology.
Staining
Bacteria and other microorganisms are usually transparent, which makes the study of
their morphology difficult. Staining allow visualization of structural features not apparent
in unstained bacteria. There are several commonly used types of staining in
microbiology: simple stains, differential stains, and negative stains. Simple stain is
application of a single dye or stain to a bacterial suspension. This allows you to visualize
the bacterial morphology since unstained bacteria are difficult to visualize without
staining. Bacterial morphology, i.e. size, shape and arrangement of bacteria, is the first
key in identification of the genus of a bacterial isolate. Differential staining requires
application of more than one stain to differentiate one type of bacteria from another. The
two most important differential stains in diagnostic microbiology are the Gram stain for
most bacteria, and the acid-fast stain for mycobacteria. Negative staining uses India ink
or other material that will not penetrate the bacterial cell. The dye stains, the background
and the bacterial cell stands out in relief as a bright shape against a dark background.
Materials
• Broth cultures or cultures on agar media (such as: Escherichia coli, Pseudomonas
aeruginosa, Corynebacterium renale and Staphylococcus aureus).
• Glass slides
• Inoculating loops
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• Gram stain reagents:

Crystal violet – primary stain
Gram’s iodine – mordant
3:1 95% ethanol/acetone – decolorizer
Safranin – counterstain
• Other items:
Marker
Water
Blotting paper
Bunsen burner
Microscope
Immersion oil
Preparation of a fixed smear
1. If working with a broth culture, place one loopful directly onto the slide, and
spread it to the size of a dime. You can place more than one smear on a slide by
using your marker to draw a circle for each smear on bottom of the slide.
2. If working with culture on agar medium, place a loopful of sterile distilled water
onto a glass slide. Emulsify a small amount of bacterial culture from the slant in
the water, and spread to the size of a dime. For best results the film must be only
slightly cloudy and should be homogeneous.
3. Allow the slide to air dry. DO NOT wave the slide in the air to hasten drying.
This will cause the formation of infectious aerosols.
4. Heat fix by passing the slide, smear side up, through the flame of a Bunsen burner
two or three times. DO NOT overheat, as this causes distortion of the bacterial
morphology. If you touch the slide with the back of your hand, it should not burn
your hand, but just be warm.
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GRAM STAIN PROCEDURE
1. Flood the fixed slide with crystal violet and let stand for 1 minute.
2. Rinse gently with tap water (use forceps or a clothespin to hold the slide).
3. Flood the smear with Gram’s iodine and let stand for one minute.
4. Rinse gently with tap water.
5. Decolorize with acetone/alcohol. This is the most critical step in the staining
procedure. Do not over-decolorize. When decolorizer is applied on an angled slide,
the color will flow off the slide until the solution runs clear. You can also flood the
slide and let it stand (as with the staining solutions). This step should not exceed 15
seconds.
6. Immediately rinse with tap water.
7. Flood the smear with safranin and let stand for one minute (OK up to 3 min.)
8. Gently rinse with tap water.
9. Blot with bibulous paper gently. Do not break the slide during drying
10. Examine under oil immersion (use only oil immersion lens). Never determine the
morphology or staining reaction of bacteria with any objective other than oil
immersion. The diaphragm should be open for the light to pass through, and the
condenser should be all the way up.
11. Record your results.
Mistakes commonly made with Gram stain:
1. Over heat fixing sample (changes staining characteristics).

2. Over decolorizing.
3. Stain sediment identified as positive cocci. The smear may be too thick.
4. Gram-positive cells when aged or damaged may appear pink. Interpretation of these
as Gram negative can lead to a wrong diagnosis.
5. Because tissues and body fluids stain pink, Gram-negative organisms may not be seen
easily.
9
KOH TEST PROCEDURE

This test is used to distinguish Gram-positive from Gram-negative bacteria grown on agar
media (e.g. blood agar).
1. Place a drop of 3% KOH on a clean slide.

2. Take a loopful of the culture and thoroughly mix with KOH.
3. Lift the loop at intervals to see if a gel is forming.
4. Gram-negative bacteria form a viscous gel in a minute or so.
5. Gram-positive bacteria do not (except some Bacillus species).
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LABORATORY - 2
BACTERIAL ISOLATION AND IDENTIFICATION
1. STREAK PLATE METHOD FOR PURE CULTURE
The first step in identification of bacteria is to establish a pure culture of the suspected
etiologic agent. The importance of pure cultures cannot be overemphasized in diagnostic
microbiology. They are essential for the accurate determination of colony characteristics,
biochemical properties, morphology, staining reaction, immunologic reaction and
susceptibility to antimicrobial agents. A pure culture is a microbial population that was
derived from a single bacterial cell. In general, pure cultures are best obtained on solid
media, usually with streak plates. This method is the most practical and most useful for
obtaining discrete colonies and pure cultures. The streak plates method uses the entire
surface of an agar plate so that isolated colonies grow that are representative of the
different organisms present in the clinical sample. Single colonies may then be further
isolated in pure culture by streaking for isolation onto a second agar plate. Identification
by biochemical tests should never be attempted until the microorganism is in pure
culture.
Materials
• Clinical sample. In lab, a mixed broth culture of Staphylococcus aureus and

Escherichia coli is prepared simulating a clinical sample
• 5% sheep blood agar or nutrient agar plates
• Inoculating loops
• Bunsen burner
• Marker
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PROCEDURE FOR STREAK PLATE
1. Check to make sure that your plates are free from condensation. Label the BOTTOM
of your plate with your name, date and source of your sample. Write along the
periphery of the plate, so as not to obscure your view of the resulting colonies.
2. Flame your loop and allow it to cool. Pick up a loopful or two of inoculum from the
broth.
3. Place inoculum at the periphery of the plate as shown in the Figure 1 (page 14).
4. Using the loop, spread the inoculum over the top quadrant of the plate
5. Flame the loop and cool.
6. Overlap the primary streak three or four times at different sites and then streak the
second quadrant of the plate.
8. Overlap the secondary streak three or four times at different sites as before and streak
the third quadrant.
10. Make the fourth streak in a similar manner, being careful to avoid the primary streak.
11. Invert your plates and incubate in the 37°C incubator overnight.
12. Examine plates after 18-24 hours of incubation and record results.
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FIGURE 1. STREAKING METHOD

A) B) C)
D)
FIGURE 2. COLONY MORPHOLOGY
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2. DEMONSTRATION OF ANAEROBIC AND MICROAEROPHILIC CULTURE

EQUIPMENT
Anaerobic bacteria (e.g. Clostridium, Fusobacterium) require absence of all oxygen.
These conditions are met by removal of oxygen by catalytic reaction with hydrogen to
form water, for example, using GasPak Anaerobic System.
Microaerophilic bacteria such as Campylobacter require a reduced oxygen environment

(5% O2, 85% N2, 10% CO2). This can be achieved using commercial products such as
GasPak/Campy Pouch.
3. COMMERCIAL BLOOD-CULTURE BOTTLES DESIGNED FOR

CULTURING BACTERIA FROM BLOOD
These are used when bacteremia is suspected. Strict aseptic precautions should be taken
when collecting blood. After inoculation of blood into the bottle, signal/indicator is
placed and the system is incubated. Gas production forces fluid into the indicator
chamber indicating positive culture. Some organisms may grow in the medium without a
positive signal.
4. COMMERCIAL BACTERIAL IDENTIFICATION SYSTEMS

There are several identification systems available commercially for the identification of
bacteria.
For example, API-20E (Analytical Profile Index) is good for identification of members
of Enterobacteriaceae. It is a plastic strip with several cupules containing biochemical
reagents. A bacterial suspension is inoculated into each cupule, and reactions are read
after incubation. A profile number is obtained based on the results, and the bacterium is
identified on the basis of this number.
Additionally, there are automated identification systems such as “MICROSCAN” for

bacterial identification, and antibiotic susceptibility testing.
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LABORATORY - 3
ANTIBIOTIC SENSITIVITY TEST AND QUANTITATIVE CULTURE
ANTIBIOTIC SUSCEPTIBILITY TESTING
Purpose
Pathogenic bacteria recovered on primary cultures should be tested for their susceptibility
to a variety of antimicrobial agents. Exceptions are Corynebacterium, Erysipelothrix and
Bacillus, which have predictable susceptibility to penicillin. Susceptibility tests are used
as a guide for evaluating which antibiotics can be used for the treatment of the infection.
Susceptibility tests are performed in the clinical laboratory after isolation of a pure
culture from the specimen.
There are several types of assays for determining the susceptibility of a bacterium to an
antibiotic. These include: 1) Broth dilution method, 2) Agar dilution method, 3) Disk
diffusion assay (Kirby-Bauer method) and 4) E-test. Each has its own advantages and
disadvantages. However, regardless of the specific test performed, a result indicating
sensitivity of a pathogen to a particular drug does not guarantee that the drug will be
effective in treating the disease. On the other hand, a result indicating resistance of a
pathogen to a drug strongly suggests that the drug will not be effective in treating, the
disease.
See procedure used for antimicrobial susceptibility test: the disk diffusion test (also
called Kirby-Bauer test) below.
BROTH DILUTION METHOD: In the broth dilution method, a standard inoculum of

bacteria is added to tubes or wells containing broth with different concentrations of the
antibiotic in question. After appropriate incubation the tubes/wells are read for growth
absence of growth.
15
AGAR DILUTION METHOD: In the agar dilution method, agar plates are used instead
of broth. For fastidious bacteria, blood can be added in the agar.
DISK DIFFUSION ASSAY (KIRBY-BAUER METHOD): In the disk diffusion test,

paper disks impregnated with known amounts of antibiotics are placed on the surface of a
Mueller-Hinton agar plate seeded with the bacterial culture. The bacterium is grown to
a standardized turbidity and swabbed onto the plate. The disks are then placed on the
surface of the plate, using a disk dispenser.
After overnight incubation at 37°C, the area around each disk where bacterial growth is
inhibited is measured. Zone sizes (diameters to the nearest mm) are converted to
sensitive, intermediate or resistant categories with respect to each antibiotic using an
interpretive table. This is a qualitative test that tells you that a pathogen is either sensitive
to the drug or resistant to the drug. The interpretive tables are designed to take into
account the minimal inhibitory concentration (MIC) of the organism, the blood levels
obtained with normal dosage of the antibiotic and the distribution of zone sizes among
species with known responsiveness to the antibiotic.
E-TEST: The Epsilometer test (E-test) is similar to Kirby-Bauer in that the bacterium is
grown to standardized turbidity and swabbed onto a plate. Then, an E-test strip with a
labeled gradient of antibiotic concentrations (high to low) is placed on the surface of the
plate. This causes an elliptical or teardrop-shaped growth of inhibition. Where the zone
of inhibition touches the E-test strip is the MIC for the organism.
MIC: The MIC (Minimal Inhibitory Concentration) is defined as the lowest

concentration of the drug at which the bacterium tested does not show visible growth.
For critical cases, MIC may be determined using the broth/agar dilution methods or the
E-test.
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PROCEDURE FOR DISK DIFFUSION ASSAY
Materials
• Mueller-Hinton plates
• Standardized (MacFarland standard 0.5-1.0 [1.5-3.0 × 108 cells]) broth cultures/

suspensions of Pseudomonas aeruginosa and Staphylococcus aureus
• Sterile swabs
• Antibiotic disks – one dispenser per lab: Example- gentamicin, enrofloxacin,

penicillin, ampicillin, tetracycline, cephalothin
Method
1. Compare the density of the culture with that of the density standard if necessary.
2. Saturate a sterile swab with inoculum expel excess fluid by pressing swab on side
of the tube and inoculate a confluent lawn of bacteria on the surface of the
Mueller-Hinton agar plate.
3. Allow to dry (maximum 3-5 minutes).
4. Using the dispenser, place antibiotic disks onto the surface of the plate. Press the
dispenser gently, and firmly.
5. Invert the plates and incubate overnight at 37°C
6. Examine plates after 18-24 hours of incubation and record results
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QUANTITATIVE CULTURE OF URINE FROM DOGS
Bacterial urinary tract infection (UTI) is the most common infectious disease of
dogs. It is less common in cats. The clinical signs and laboratory features of UTI are
nonspecific, except for bacteriuria. For ultimate confirmation of UTI, urine culture is
required. There are several species of bacteria that can cause UTI in dogs. Approximately
75% of UTI in dogs are caused by a single species of pathogen. It is important to collect
samples carefully and transport the containers in ice. Urine is cultured quantitatively.
Low numbers and multiple types of bacteria may indicate contamination.
Materials:
• Urine sample containing an unknown number of bacteria
• A calibrated loop to hold 0.001 ml (1 µl)
• Blood agar and MacConkey agar plates
Procedure:
1. With the calibrated loop remove a sample of urine and inoculate a MacConkey agar
plate by making one straight line down the center of the plate.
2. With the same loop cross streak the entire plate at right angles to the original streak,
beginning at the point of origin of the original streak.
3. Label the plate, place in the incubator basket and incubate at 37°C for 18 to 24 hours.
18
LABORATORY - 4
INTERPRETATION OF SUSCEPTIBILITY TESTING, URINE CULTURE
1. DISK DIFFUSION ASSAY
1. Measure the zones of inhibition diameter (in millimeters) using a ruler and record
your results.
2. Interpret the results by comparing zone sizes to those in Table 1.
TABLE 1. EXAMPLE OF INTERPRETIVE TABLE FOR DISK DIFFUSION

ASSAY
Inhibition zone diameter
Antibiotic Disk content (to the nearest mm)
Moderately
Resistant susceptible Sensitive
(R) (I) (S)
Gentamicin (GM 10) 10 g ≤12 13-14 ≥15
Enrofloxacin (ENO 5) 5 g ≤15 16-20 ≥21
Penicillin (P 10) 10 units

Staphylococcus ≤28 - ≥29
Ampicillin (AM 10) 10 g

Gram-negative enterics ≤11 12-13 ≥14
Staphylococcus ≤28 - ≥29
Tetracycline (TE 30) 30 g ≤14 15-18 ≥19
Cephalothin (CF 30) 30 g ≤14 15-17 ≥18
S = susceptible
I = moderately susceptible
R = resistant
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2. DEMONSTRATION TEST: E-TEST (AB BIODISK, SWEDEN)
This is a quantitative test, unlike the Kirby-Bauer disk diffusion method.
This test uses a thin plastic strip coated with a predefined exponential gradient of
antibiotic.
The inoculum and plates are prepared as described in “Disk diffusion method” and the
plastic strip is placed on the agar surface. After overnight incubation, the MIC is read
directly from the scale printed on the strip at the point of intersection between the
bacterial growth zone and the strip.
3. INTERPRETATION OF QUANTITATIVE URINE CULTURE IN DOGS
URINARY TRACT INFECTIONS (UTI)
UTI refers to urethritis, cystitis, ureteritis, prostatitis, or pyelonephritis. UTIs are most
frequent in dogs.
The following bacteria are most commonly involved:

Escherichia coli
Proteus mirabilis
Proteus vulgaris
Staphylococcus pseudintermedius
Staphylococcus aureus
Streptococcus
Pseudomonas aeruginosa
Klebsiella pneumoniae
(Leptospira, and L-forms/cell wall-deficient bacteria may sometimes be involved)
URINE COLLECTION
There are multiple methods to collect urine. The collection method has a direct
impact on how many bacteria (measured in colony-forming units per ml [CFU/ml])
are required to consider the sample infected.
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1. VOIDED SAMPLES (collected by free catch) will always have contaminants.
2. CATHETERIZATION SAMPLES (collected by catheter) are often contaminated

and may introduce infection.
3. CYSTOCENTESIS SAMPLES (collected by cystocentesis/bladder tap) often

have no bacterial contamination. This is considered a safe procedure.
Urine should be stored at 4°C until culture is performed.
TABLE 2. EXAMPLE OF INTERPRETATIVE TABLE FOR URINE CULTURE

RESULTS (DOGS)
Results not
Collection Results indicative of Equivocal results indicative of
method infection (CFU/ml) (CFU/ml) infection (CFU/ml)
Cystocentesis >1,000 100-1000 <100
Catheterization >10,000 1000-10,000 <1,000
Voided >100,000 10,000-100,000 <10,000
CFU = Colony-forming units; NOTE: the units are CFU/ml (NOT µl)
In the case of cats, >1,000 CFU in catheterized urine is indication of infection.
Example interpretation for Voided urine

1. Count the colonies on any plate containing 100 colonies or fewer.
2. Multiply the colony number by 1000 and compare the number to the row for Voided
in Table 3.
3. 10 colonies on the plate = 10,000 CFU/ml = insignificant.
10-100 colonies = 10,000 to 100,000 CFU/ml = suspicious.
100 (or more) colonies = 100,000 CFU/ml = indicative of urinary tract infection.
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ANTIBIOTIC TREATMENT OF UTI’S
Antibiotic use in UTI’s not only depends on the sensitivity of the pathogen to a given
antibiotic, but also on the ability of the antibiotic to get into the urine in high enough
concentrations to appropriately treat the UTI.
WHAT CONCENTRATION OF ANTIBIOTIC IS NEEDED TO SUCCESSFULLY

TREAT AN INFECTION?
TABLE 3. SENSITIVITY OF COMMON URINARY PATHOGENS

Achievable
concentration MIC values (µg/ml) of
in urine 4 common urinary pathogens
Antibiotic (µg/ml) E. coli Proteus Streptococcus S. aureus
Ampicillin 250+ 16 200 2 0.04
Cephalothin 300 4 4 16 0.1-5
Tetracycline 300 5 R 0.5 200
TMS 1000 8 8 R 4-16
R = Resistant to antibiotic.
**Pay attention to the achievable concentration of each antibiotic in the urine, and
compare these concentrations to the MIC values for the given pathogens.**
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LABORATORY - 5
MYCOLOGY-I
1. EXAMINATION FOR YEASTS USING GRAM STAINING
Draw diagrams and record cell shape, size, color, and weather budding cells are seen.
You should be able to differentiate Candida from Malassezia. Candida may look like
cocci under lower magnifications, but under oil immersion objective, the yeast cells will
look visibly much larger in size than bacteria (cocci).
How does Malassezia differ from Candida in morphology?
Malassezia Candida
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2. CULTURE AND GRAM STAINING OF TRANSPORT SWAB
Remember which of your media are considered general and which are more
specialized; this will impact which is streaked first.
TRANSPORT SWAB CULTURE
1. Culture on blood agar first: Apply the swab on one quarter of the blood agar by
rotating the swab. Then use the loop and streak to get individual colonies by
flaming and cooling the loop after each set of streaks.
2. Culture on Sabauroud agar: Perform the same method as used for blood agar.
After completed, tape the plate around the rim, and incubate for a week at room
temperature.
3. Gram stain: Press and rotate the swab in the center of the slide over an area of a
Dime. Do not use water. Let the smear dry, and then heat fix, and stain with
Gram’s. Examine under oil immersion for bacteria and for peanut/footprint-
shaped Malassezia (ear swab). Also look for other yeasts such as Candida
(oval/rugby or football-shaped/round/budding). Several yeast cells and/or bacteria
in one oil-immersion field may indicate infection, and culture can help confirm.
3. KOH MOUNT OF HAIR SAMPLE
Materials: Hair sample or skin scraping containing hair, forceps, clean glass slide, 10%
KOH, cover slip, microscope.
Using forceps, pick up a few hair segments from sample (~1-3, the fewer the better),
place them gently on a glass slide, try to place them in such a way that they do not clump.
You can tease the hair with the forceps gently to place them slightly away from each
other. Put a drop of 10% KOH, on the sample, try to place the drop in such a way that it
does not run over, but stays on position in a circular pattern. If it does run over slightly,
it’s okay. Cover the drop with cover slip, placing the cover slip sideways first, on the
slide, making a mount, with few to no air bubbles, if possible. Allow 10-20 minutes to
pass, so that the KOH reacts with the hair shaft. Observe the slide using 40× (NO OIL).
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LABORATORY - 6
MYCOLOGY-II
1. DEMONSTRATION SLIDES
Examine the microscopic morphology of the following fungal pathogens and draw
pictures.
Most specimens are mounted in Lactophenol cotton blue (LPCB) mounting medium.
LCPB is used to examine fungi already growing on laboratory medium, but it may be
used to view hyphae and spores directly in clinical material. The blue color provides a
contrasting effect which enhances the hyphae and spores, making them easier to detect.
DIAGRAMS AND NOTES

Use the space below to draw the microscopic appearance of fungi such as
macroconidia of dermatophytes, and other fungi of veterinary significance
demonstrated.
NOTE: IF A DEMO SLIDE IS NOT PRESENT, LEAVE THE CORRESPONDING CIRCLE
BLANK, OR USE IT FOR ANOTHER DRAWING.
1. Aspergillus: 2. Penicillium
(Note septate mycelium, and sunflower-like This is mostly a contaminant

arrangement of conidia)
25
3. Microsporum canis 4. M. gypseum
(Note spindle shaped macroconidia) (Note boat shaped macroconidia)
5. Trichophyton mentagrophytes 6. Trichophyton verrucosum
(Note cigar-shaped macroconidia) (Macroconidia are extremely rare. Note

spores called chlamydospores in chain)
26
7. Zygomycetes (Mucor) 8. Cross section of placenta infected with

zygomycete
Note sporangia (spores enclosed) Note pieces of aseptate hyphae
9. Other fungi (Cryptococcus) 10. Other fungi (Blastomyces)
Note thick capsules Note yeast cells in tissue (pink/red in color)
27
LABORATORY – 7 & 8
GRAM-NEGATIVE RODS OR COCCOBACILLI
Members of the family Enterobacteriaceae are the most frequently isolated Gram-
negative bacteria in aerobically-incubated cultures. They grow on MacConkey agar and
are oxidase negative. These include: Escherichia coli, Klebsiella pneumoniae, Salmonella
spp., Proteus spp., Yersinia spp. and Shigella spp.
Other aerobic Gram-negative rods/coccobacilli include: Pseudomonas aeruginosa,

Pasteurella multocida, Mannheimia haemolytica, Actinobacillus spp., Bordetella
bronchiseptica, Brucella spp., Moraxella bovis and Haemophilus spp.
Campylobacteria species are also Gram negative. They are curvy in shape and are
microaerophilic and thermophilic.
Anaerobic Gram-negative bacteria include Fusobacterium necrophorum, Dichelobacter,

and Bacteroides species.
KEY CHARACTERISTICS OF COMMONLY-ISOLATED

GRAM-NEGATIVE PATHOGENS
1. Escherichia coli: Lactose-fermenting pink colonies with entire margin, flat and
circular colonies. Moist and shiny surface of colonies.
2. Salmonella spp.: Colorless, moist and shiny colonies with entire margin, flat and
circular.
3. Pseudomonas aeruginosa: Colorless to light greenish colonies.
4. Pasteurella multocida: No growth on MacConkey agar. It is not a member of
enterobacteriaceae, hence it gets inhibited, and does not grow when inoculated on
MacConkey agar.
5. Klebsiella pneumoniae: Large mucoid pinkish colonies undulate margins. Colonies
are raised and irregular.
6. Proteus mirabilis: Colorless colonies (swarming is not seen on MacConkey agar).
28
TABLE 1. CHARACTERISTICS OF GRAM-NEGATIVE PATHOGENS
Bacterium Blood agar MacConkey Disease and host(s)

Affects multiple organs and hosts,
Escherichia
Hemolysis ± LF (pink) neonatal diarrhea (many species),
coli
pyometra (dogs)
Salmonella
Hemolysis − NLF (colorless) Diarrhea, septicemia, abortion
Typhimurium
Klebsiella Hemolysis − Otitis (dogs), UTI, Bovine
LF (pink)
pneumoniae Mucoid mastitis, canine pyometra
Proteus Hemolysis – Canine otitis, UTI, other localized
NLF (colorless)
mirabilis Swarming conditions, canine pyometra
UTI, wounds, burns and
Pseudomonas NLF (colorless) respiratory diseases
Hemolysis ±
aeruginosa Green pigment ± (many species),
otitis and pyometria (dogs)
Hemolysis – Wounds (dogs and cats),
Pasteurella Colonies pyometra (dogs);
No growth
multocida may respiratory disease (cattle, pigs)
coalesce septicemia (birds)
Bordetella NLF (colorless to
Hemolysis ± Respiratory: dogs, cats, pigs
bronchiseptica light brown)
LF = Lactose fermenting; NLF = Non-lactose fermenting.
SELECTIVE MEDIA USED FOR CULTURE AND IDENTIFICATION INCLUDE

PLATING MEDIA AND TUBE MEDIA
PLATING MEDIA
Plating media are often both selective (dyes are added to inhibit Gram-positive bacteria)
and differential (carbohydrates and a pH indicator detect fermentation).
MACCONKEY AGAR: MacConkey agar contains crystal violet and bile salts to
inhibit Gram-positive bacteria, lactose and a pH indicator (neutral red) as the important
ingredients. Lactose-fermenting bacteria produce red/pink colonies. Non-lactose
fermenting bacteria produce colorless colonies.
29
TUBE MEDIA
A large variety of tubed media are available to identify organisms recovered from fecal,
urine, or other specimens on plating media. Some of these media are used to detect
motility, urease activity, utilization of a molecule as a sole energy source, cleavage or
alteration of an amino acid, fermentation of sugar etc.
TRIPLE SUGAR IRON AGAR (TSI): TSI agar is used to determine whether or not an
organism ferments lactose, sucrose, or glucose with the formation of acid and gas, and
also determines its ability to produce hydrogen sulfide. Reliable results can be obtained
only if this medium is inoculated with a pure culture. Thus, care must be taken to select
well isolated colonies from primary plating media. The medium contains 1% lactose, 1%
sucrose, 0.1% dextrose, iron salts, and phenol red as a pH indicator. At low pH, phenol
red has a yellow color and at high pH a red color.
Organisms are inoculated into the medium by stabbing the butt and streaking the slant for
aerobic growth. The amounts of lactose and sucrose in the medium are so great that
bacteria, which utilize either compound, produce tremendous amounts of acid and the
entire medium turns yellow in color. Bacteria that utilize only glucose produce small
quantities of acid because of the low concentration of this compound in the medium. The
small amount of acid produced is oxidized to CO2 and H2O on the aerobic surface of the
slant, which retains its red color. Thus the coliforms and certain Proteus spp. which
ferment either lactose or sucrose, produce a yellow butt and yellow slant with gas.
Salmonella produces a yellow butt and red slant; with or without gas. H2S production
turns the iron salt in the medium black. Salmonella and Proteus frequently produce H2S.
Sugar fermentation tubes – Individual sugars in fermentation tubes are sometimes useful
to confirm the results of the TSI medium, especially when H2S has been produced in
large quantities.
30
TABLE 2. TSI INTERPRETATION

Bacterium Slant Butt H2S production
Escherichia coli Yellow Yellow Negative
Salmonella Typhimurium Red Yellow Positive
Pseudomonas aeruginosa Red Red Negative
Klebsiella pneumoniae Yellow Yellow Negative
Proteus mirabilis Red Yellow Positive
Although Pasteurella multocida has a similar TSI profile to E. coli and Klebsiella, will be
differentiated by an absence of growth on MacConkey agar. For differentiation between
E. coli and Klebsiella, the mucoid nature of Klebsiella and the urease test will be helpful
as explained in the differentiating chart at the end of this handout. Proteus swarms on
blood agar, Salmonella does not.
UREASE TEST: Those organisms which produce urease enzyme break down the urea in
the test medium, to release ammonia, which increases the pH, to make it alkaline, causing
a color change of phenol red dye incorporated in the medium. This test helps to
differentiate bacteria which produce urease enzyme to those who don’t.
Procedure: Streak the surface of a urea agar slant with an isolated colony of the
organism to be tested.
Leave the cap of the tube on loosely and incubate the tube for up to 24 hours at 37°C.
Interpretation :
Positive result: When organisms utilize urea, ammonia is formed which makes the
reaction of the media alkaline, producing a pink-red color (due to the change in the
phenol red indicator). Positive organisms include Proteus and Klebsiella.
Negative result: Agar slant and butt of the tube remain light orange. Negative organisms
including E. coli and Salmonella.
31
SIM MEDIUM: This medium is useful to determine if H2S gas is produced (the S), if
indole (the I) is produced from tryptophan, and if the organism is motile (the M) in this
semi-solid agar. H2S is detected by the formation of a black metallic sulfide. Indole is
detected by the formation of a red color after the addition of Kovac’s reagent. Motility is
revealed by a generalized turbidity as opposed to growth only along the inoculation line
produced by non motile bacteria.
Indole test: Some bacteria have the ability to break down tryptophan for nutritional
needs using the enzyme tryptophanase (e.g. E. coli). When tryptophan (present in the
SIM medium) is broken down, indole is released, which in turn is detected through the
use of Kovac’s reagent. The Kovac’s reagent, which is yellow, reacts with indole and
produces a red color on the surface of the test tube.
Procedure:
1. Inoculate a SIM tube with the organism to be tested.
2. Incubate for 24-48 hours at 37°C.
3. Add Kovac’s reagent to the top of the tube, pink/red color formation indicates indole
formation.
4. Black precipitate in the medium indicates hydrogen sulfide formation.
5. Growth away from the stab line indicates motility.
OXIDASE TEST: This test determines the presence or absence of cytochrome

C oxidase enzyme in test bacteria. Organisms producing the enzyme are oxidase positive
and turn the test strip purple (e.g. Pseudomonas aeruginosa), those who do not have the
enzyme do not show any color change on the test strip, within the time limits of the test.
All Enterobacteriaceae are oxidase negative.
Procedure: Open pouch, remove the slide, place on table. Using a plastic loop, pick an
isolated colony from culture to be tested, smear directly into the reaction area, in a
circular manner with 3-4 mm diameter zone. (Do not pick medium; this may give false
positive reaction).
32
Interpretation: Appearance of a dark purple color within 20 seconds indicates a positive

result. Disregard color development after 20 seconds. No color within 20 seconds is
negative test result.
SIMMONS CITRATE TEST: This tests the ability of a bacterium to use citrate as the
sole carbon source. The agar contains bromo-thymol blue as a test reagent.
Procedure: Inoculate solid agar slant of SIM citrate and incubate at 37°C and note color
change to blue (positive).
Positive: Klebsiella, Salmonella, Proteus mirabilis
Negative: E. coli
GROWTH OF CAMPYLOBACTER
This diarrheal pathogen is grown on “Campy” medium or Modified Preston Agar. It
contains charcoal, ferrous sulfate, and sodium pyruvate to enhance growth. Sodium
deosxycholate and cefoperazone are added as selective agents. Smear will show Gram-
negative, small, slender, curvy bacteria. For untrained eyes, it takes some time to see
them.
Fecal samples are directly inoculated onto the medium, and incubated microaerophically
using a gas generating pouch in a special jar. Incubation is between 37°C and 43°C.
Cefoperazone, a 3rd generation cephalosporin inhibits enteric bacteria such as E. coli,
Proteus, and even Pseudomonas. C. jejuni is resistant to cephalosporins.
DEMONSTRATION SLIDES:
1. Observe the cultures on blood and MacConkey agar, and various

biochemical reactions on the DEMO table of common Gram-negative
bacterial pathogens.
2. Observe the Gram stain of Campylobacter jejuni (curved small slender rods)
and culture plate (Preston medium with charcoal and cefoperazone)
33
TABLE 3. OTHER GRAM-NEGATIVE OR CELL WALL-LESS BACTERIA OF

SIGNIFICANCE
Organism Disease(s) Media Comments
Campylobacter Diarrhea (puppies, Preston charcoal Curvy rods,
jejuni calves, hamsters) agar with microaerophilic,
cefoperazone thermophilic
Moraxella bovis IBKC Blood agar Coccobacilli
(Pink eye) (no growth on Hemolysis +
MacConkey) Oxidase +
Brucella spp. Genital disease, Blood agar or Coccobacilli, slow-
abortion (dogs, cattle) special media growing, need CO2
Mycoplasma spp. Various: eyes, Special media with No cell wall,
genital, respiratory, 20% serum difficult to
joints visualize with
Gram stain
Minute colonies,
slow growing, CO2
needed
Rickettsia, Various conditions in Will not grow in lab Diagnose by IFA,
Chlamydia, many species: RMSF, media; Need host ELISA, PCR;
Chlamydophila, PHF, psittacosis, cells Culture can take
Ehrlichia, CME, salmon- 1-2 months
Anaplasma, poisoning disease etc.
Neorickettsia,
Wolbachia
34
FIGURE 1. SIMPLIFIED FLOWCHART FOR IDENTIFICATION OF GRAM-NEGATIVE RODS. Most Gram-positive

organisms cannot grow on MacConkey agar. Lactose fermenters have pink or red colonies. Non-lactose fermenters are colorless.
35
LABORATORY - 9
GRAM-POSITIVE COCCI
STAPHYLOCOCCI
The staphylococci are Gram-positive cocci in grape-like clusters seen in many clinical
specimens or when grown on solid media. In body fluids or in broth cultures single cells,
pairs and even short chains of cells may be seen with these clusters. Staphylococci are
catalase positive. All staphylococci produce the enzyme catalase, which catalyzes the
conversion of hydrogen peroxide to water and oxygen. Streptococci are catalase
negative.
There are several species within the genus Staphylococcus of which Staphylococcus
aureus and Staphylococcus pseudintermedius are important pathogens and
Staphylococcus epidermidis, which is found on skin and mucosa, may be rarely
pathogenic. The species of staphylococci are distinguished by hemolytic reactions,
pigment formation, sugar fermentation reactions, antibiotic resistance, etc. S. aureus and
S. pseudintermedius usually produce double-zone hemolysis on blood agar. S.
epidermidis is non-hemolytic.
COAGULASE PRODUCTION (the ability to cause a fibrin clot to form in citrated

plasma) is considered the classic index of pathogenicity among the staphylococci. S.
aureus and S. pseudintermedius are coagulase – positive, S. epidermidis is coagulase –
negative. Cats are prone to infection with coagulase-negative staphylococci (several
species, S. sciuri, S. simulans etc.).
STREPTOCOCCI
Introduction: The streptococci are Gram- positive cocci which usually forms chains.
They are catalase negative which distinguishes them from the staphylococci. On blood
agar, streptococci may produce hemolysis of the red blood cells.
36
Those, which belong to the alpha-hemolytic type, produce a greenish coloration

surrounding the colony due to the reduction of hemoglobin in the intact but discolored
red blood cells. Others belonging to the beta-hemolytic type produce a sharply defined
clear area surrounding the colony in which the red blood cells are lysed. The third group,
the gamma type, produces no change in the red blood cells. These characteristics can
best be determined by holding the plate up to the light and looking through it. Bacteria
other than streptococci may also produce these changes.
Alpha-hemolytic Streptococci:
They may be non-pathogenic or pathogenic. Examples include S. dysgalactiae and S.

uberis, both mastitis pathogens, and S. suis, a swine pathogen. However, some strains of
these bacteria can show other type of hemolysis as well.
Beta-hemolytic Streptococci:
Lancefield Group A and Groups B and C streptococci are the most frequent pathogens of
humans and animals among the hemolytic streptococci. Group A streptococci can be
identified serologically using latex beads coated with antibody to the Group A cell wall
carbohydrate. A similar test can be used to identify Streptococcus agalactiae, (Group B)
an important animal pathogen whose hemolytic activity is often of the beta type but is
variable. Beta hemolytic streptococci pathogenic to animals include S. equi, S. equisimilis
and S. canis.
37
Observe results of coagulase tube test and purple agar test for staphylococci
TABLE 1. COAGULASE AND PURPLE AGAR TESTS

Species Coagulase Purple Agar
Staphylococcus aureus + + (yellow colonies)
Staphylococcus + − (no change)
pseudintermedius
Staphylococcus − +
epidermidis
DIAGNOSTIC TESTS:
1. CATALASE TEST:
Principle: To detect the presence of the enzyme catalase. This enzyme catalyses the
breakdown of hydrogen peroxide (H2O2) with the release of free oxygen, which is
detected by the presence of bubbles. This test differentiates bacteria which produce
catalase, to those who do not. Staphylococci are catalase positive, while streptococci
are catalase negative.
Procedure: Place a drop of 3% hydrogen peroxide (H2O2), on a clean glass slide.
Using a sterile wire loop, pick up a test colony of bacteria, and dip it on to the drop of
reagent previously placed on the glass slide.
Interpretation: Immediate bubbling = Positive; Absence of bubbles = Negative.
Precautions in interpretation:
• It is important not to contaminate the bacterial colony under test with blood agar.
Red blood cells contain catalase and their presence will give a false postitve
result. So ensure to pick up only bacterial growth from plate, not agar particles.
• Old cultures may lose their catalase activity, possible resulting in false negatives.
2. COAGULASE TEST (to be done for Staphylococcus species only):

Principle: the Coagulase test is a method for differentiating between pathogenic and
non-pathogenic strains of Staphylococcus. Bacteria that produce coagulase enzyme
38
use it as a defense mechanism by clotting the areas of plasma around them, thereby
enabling themselves to resist phagocytosis by the host's immune system.
Slide test: Place a drop of plasma on a slide. Try suspending growth from your
culture plate in the plasma. Clotting and clumping is a positive reaction.
Tube test: Mix 0.5 ml of rabbit plasma in a tube with a loopful of bacteria and
incubate at 37°C for 4 hours. (You do not need to do the tube test; please observe
positive and negative reactions on Demo table).
Interpretation: A positive test is denoted by a solid clot formation in the test tube.
Positive = S. aureus and S. pseudintermedius. Negative (absence of clot) = S.
epidermidis, and several other species.
3. Purple Agar (for Staphylococcus): Purple agar base with the addition of 1% maltose
is a useful medium to differentiate the pathogenic Staphylococci, particularly with
Coagulase positive isolates from dogs that might be either S. aureus or S.
pseudintermedius. The test works in the following way: S. aureus rapidly ferments
maltose, and the acidic metabolic products cause the pH indicator bromocresol
purple, to change the medium, and colonies to yellow. S. epidermidis is positive too,
S. pseudintermedius gives a weak or delayed reaction. While S. hyicus does not
ferment maltose, but attacks the peptone in the medium, producing an alkaline
reaction (a deeper purple) around the colonies. (You need not do this test. Please
observe results on Demo table).
4. Latex particle agglutination test (to differentiate Staphylococcus aureus from

Staphylococcus pseudintermedius or other spp.): In this test, latex particles coated
with species-specific antibodies are added to culture. Clumping (agglutination) of the
beads indicates a positive reaction (Conduct a slide agglutination test if reagents
are available).
5. Sugar fermentation tests (to differentiate streptococci): Although multiple sugars

can be employed for fermentation tests in bacteria (e.g. lactose in MacConkey and
maltose in purple agar), usually a select few are used for differentiation of different
streptococci. The test works in the following manner: Bacteria are added to test
39
medium containing only a single sugar as an energy source. If the streptococcus can
ferment it, the reaction will cause an acidic change in the test medium causing a color
change due to the indicator added. The test requires 24 hours incubation. (Example
sugar fermentations for other bacteria have been shown and will be
demonstrated in later labs).
6. CAMP Test: This test is used for the presumptive identification of Streptococcus
agalactiae. This species is able to complete the partial lysis of red cells produced by
the beta hemolysin of a Staphylococcus species. A hemolytic staphylococcus is
streaked across a blood agar plate; and isolates of streptococci to be tested are
streaked at right angles to the Staph streak.
After incubation at 37°C for 18-24 hours, a positive CAMP reaction is characterized
by an arrowhead zone of clear hemolysis in the partial zone of hemolysis produced
by the staphylococcus (see Figure 1).
Among bovine mastitis pathogens S. agalactiae, S. dysgalactiae, and S. uberis, only S.

agalactiae gives a positive reaction (This test is on the DEMO table; you are not
required to do it).
FIGURE 1: CAMP TEST RENDITION
40
VETERINARY-RELEVANT STAPHYLOCOCCI AND STREPTOCOCCI
TABLE 2: CHARACTERISTICS OF STAPHYLOCOCCI

Staphylococcus Purple
species Disease Hemolysis Catalase Coagulase agar
Bovine:
mastitis and
wound
infections
+
Double-
aureus Dogs and + + +
zone
cats: otitis
externa,
pyoderma,
UTI,
pyometra
+
Double-
zone −
Dogs: same as
pseudintermedius (narrow + + (weak +
S. aureus
inner zone, can occur)
broad outer
zone)
epidermidis and Cats: Skin
related species infections, − + − +
otitis, UTI
TABLE 3: CHARACTERISTICS OF STREPTOCOCCI

Streptococcus
species Disease Hemolysis Catalase CAMP test
agalactiae
Bovine: mastitis +/− (β/α/γ) − +
dysgalactiae
Bovine: mastitis +/− (α/γ) − −
Horses: strangles,
equi (subsp. equi,
genital infection, + (β) − Not done
zooepidemicus)
respiratory infection
Dogs and cats: neonatal
canis septicemia, suppurative + (β) − Not done
conditions
41
LABORATORY - 10
AEROBIC GRAM-POSITIVE RODS OR COCCOBACILLI
Many of the organisms in this classification can be differentiated on the basis of colony
and cell morphology. It should be noted that several of these genera are easily
decolorized, especially when using cultures which are greater than 24 hours old.
BACILLUS: These are some of the largest Gram+ rods, form spores, and produce
catalase. All species of Bacillus are motile except for B. anthracis and a few strains of B.
cereus. Bacillus (other than B. anthracis) and Lactobacillus species are normal flora and
are often recovered as contaminants from clinical samples and are differentiated by
catalase production. Only B. anthracis is of major pathogenic significance.
CORYNEBACTERIA-LIKE SPECIES: Corynebacterium renale, Corynebacterium

pseudotuberculosis, Rhodococcus (formerly Corynebacterium) equi and Trueperella
(formerly Arcanobacterium, Actinomyces, Corynebacterium) pyogenes are small,
nonspore-forming, Gram-positive coccobacilli showing a characteristic arrangement of
short, pleomorphic rods, sometimes referred to as “Chinese letters”.
CORYNEBACTERIA and RHODOCOCCUS EQUI are catalase-positive and can be

speciated based on carbohydrate usage.
TRUEPERELLA (ARCANOBACTERIUM) PYOGENES is catalase-negative with

small white colonies and a very clear but very narrow zone of -hemolysis.
Corynebacterium (Eubacterium) suis is an occasional cause of pyelonephritis in pigs
and is a strict anaerobe.
LISTERIA AND ERYSIPELOTHRIX: These bacteria are similar in morphology but can
be distinguished by catalase production, motility and hemolysis.
LISTERIA: L. monocytogenes is the only species of veterinary importance. It usually
shows a thin zone of -hemolysis, and a positive CAMP reaction with Rhodococcus spp.
ERYSIPELOTHRIX: E. rhusiopathiae is one species associated with disease and
initially shows no or -hemolysis, which will progress to -hemolysis after 48 hours.
42
DEMONSTRATION SLIDES: Examine blood agar cultures of Trueperella

(Arcanobacterium) pyogenes, Corynebacterium renale, Rhodococcus equi,
Corynebacterium pseudotuberculosis, Erysipelothrix rhusiopathiae and Bacillus
cereus on DEMO table. Note colony morphology, including hemolysis.
TABLE 1. CHARACTERISTICS OF GRAM-POSTITIVE RODS

Organism Usual host Colony features Hemolysis Catalase
Trueperella
Ruminants 48 hr- pinpoint + −
pyogenes
Corynebacterium 24 hr- waxy,
Cattle − +
renale yellowish
48 hr- mucoid, slight
Rhodococcus equi Equines − +
pink
Corynebacterium
Sheep, goats Small, dry, chalky + +
pseudotuberculosis
Erysipelothrix Incomplete
Pig Pinpoint −
rhusiopathiae +
All species;
Flat, large, grey with
Bacillus spp. commensal or + or − +
irregular edges
contaminant
DEMONSTRATION SLIDES: Gram-positive or acid-fast bacteria

1. Clostridium tetani: Note characteristic terminal spore
2. Nocardia in milk (Gram stain): Branching with “beady” staining
3. Dermatophilus skin lesion smear: Showing “railroad track” appearance
4. Mycobacterium paratuberculosis in rectal mucosa: Acid-fast pink, short rods
CLOSTRIDIUM PERFRINGENS
This anaerobic bacterium is responsible for a variety of disease conditions in animals.

These include enterotoxemia in farm animals, necrotic enteritis in chickens, diarrhea in
dogs, and myositis/myonecrosis in horses.
A Gram smear will show thick, large Gram-positive rods. Old cells may stain pink.
43
If cultured (anaerobically incubated blood agar) there is double-zone hemolysis, which is

unique for this anaerobic bacterium.
If you see a large number of clostridial organisms and spores in a fecal smear from a dog
with diarrhea, you may suspect Clostridium as a cause. The diagnosis can be confirmed
only by demonstration of C. perfringens toxin in the feces. There are commercial kits
available for this purpose.
NOCARDIA ASTEROIDES QUESTION: A smear from a case of bovine mastitis is

displayed. Note the beady staining Gram-positive branching filaments on pink
background (host cells etc).
Will you treat this case with penicillin (YES/NO)?
What other drugs could you recommend for treatment?
Examine the FLOWCHART (Figure 1, page 45) provided to determine which

differential test to perform in what order for each organism.
44
FIGURE 1. SIMPLIFIED FLOWCHART FOR IDENTIFICATION OF GRAM-POSITIVE AEROBIC BACTERIA
45
TABLE 2. OTHER GRAM-POSITIVE/ACID-FAST BACTERIA OF

SIGNIFICANCE
Organism Disease(s) Media Comments
Neurological, wound, Blood agar or Large rods,
Clostridium spp.
intestinal disease special media anaerobic, spores
Acid-fast,
Tuberculosis, Special egg
Mycobacterium spp. Needs > 2 weeks
Johne’s disease media
for culture
Small
rods/coccobacilli
Listeria Encephalitis in
Blood agar Small, hemolytic
monocytogenes ruminants, abortion
colonies. Multiples
at 4°C
Gram-positive
Canine nocardiosis,
Nocardia asteroides Blood agar branching beady
bovine mastitis
filaments
46
LABORATORY - 11
PCR IN BACTERIAL DIAGNOSIS
Purpose
PCR is a method utilized to amplify a target sequence exponentially. In this way, even
very small quantities of bacterial DNA can be detected and identified. The purpose of this
laboratory exercise is to provide veterinary students with basic information on
polymerase chain reaction (PCR), as well as its uses in diagnosing bacterial infections.
Uses for PCR in Bacteriology include:

1. Determining the presence/absence of a bacterial target sequence.
2. Performing subsequent sequencing of a bacterial target sequence.
3. Creating recombinant proteins.
4. Creating DNA probes.
BASIC SETUP OF PCR:
1. Preincubation: DNA is melted into single linear strands primers are dissociated and
hot-start DNA polymerases are activated.
2. Denaturation: DNA is melted and primers are dissociated.
3. Annealing: Primers adhere to DNA. This step results in the creation of primer-DNA
hybrids.
4. Extension: DNA polymerase creates new DNA at the primer sites. Denaturation,
annealing and extension steps are repeated to amplify the DNA fragments.
5. Final extension: The final extension of any remaining single DNA strands is made.
6. Hold: After PCR, the product is kept at 4°C for better preserving the newly-made
DNA.
47
PCR components:
DNA sample: Template to which the primers will anneal and DNA polymerase will
create copies of the target sequence. Samples from which DNA can be extracted include,
but are not limited to blood, biopsy tissue, fine-needle aspirate, bacterial culture,
transport swabs, vectors (ticks, fleas etc.) and even environmental samples (soil,
water etc.)
Positive control: DNA containing your target DNA sequence. This is used to ensure the
PCR reaction worked.
Negative control: Sample not containing your target DNA sequence. This is used to
ensure there is no contamination in your samples.
PCR buffer + MgCl2: Creates a stable environment in which PCR can most effectively
occur.
dNTPs: Provides bases for use by DNA polymerase to create DNA.
Primers: Used to anneal to DNA to create copies of the target sequence.
DNA polymerase: Creates copies of target DNA.
Thermocycler: The machine used to raise and lower the temperature of the samples
throughout the PCR cycles.
48
REMEMBER THAT IN PCR, YOU ALWAYS PROCEED
CLEANEST (NEGATIVE CONTROL) TO DIRTIEST (POSITIVE CONTROL)
PCR procedure:
1. Retrieve your PCR components from −20°C and allow them to thaw. Keep DNA
polymerase on ice as much as possible to prevent enzyme degradation.
2. Label enough PCR tubes for your sample, negative and positive controls and one
more tube for creating the master mix.
3. Add enough PCR buffer, MgCl2, dNTPs, each primer and DNA polymerase to the
master mix tube to accommodate all PCR reactions (2 controls + samples).
4. Add the master mix to each PCR reaction tube.
5. Add the negative control to its PCR reaction tube.
6. Add the DNA sample to the appropriately-labeled tubes.
7. Add the positive control to the appropriately-labeled tube.
8. Place the PCR tubes in the thermocycler and start the program.
Example thermocycler conditions:
Preincubation at 94°C: 5 minutes
Denaturation at 94°C: 1 minute
Annealing at 60°C: 1 minute 30 cycles
Extension at 72°C: 1 minute
Final extension at 72°C: 7 minutes
Hold at 4°C: Infinity
49
PCR:
Target DNA
Forward primer
3′ 5′
5′ 3′
Reverse primer
MANY DNA COPIES
50
STEPS FOR VIEWING PCR RESULTS:
1. Cast an agarose gel.

2. Load samples, including positive and negative controls and a DNA ladder onto the
gel.
3. Perform electrophoresis to separate DNA by size.
4. Read the gel under UV light to detect DNA bands.
5. Compare bands to positive control and to DNA ladder.
6. Record results.
Components used for PCR results:

Agarose: The gel is a matrix through which DNA passes. When an electrical current is
passed through the gel, DNA moves in the agarose from the negative pole to the positive
pole and is separated according to size; the longer DNA fragments take longer to migrate
than the shorter fragments.
Electrophoresis machine: This device creates an electrical current. DNA, being

negatively charged, migrates from negative to positive.
TAE buffer: Tris-Acetate-EDTA buffer. This buffer is used in the electrophoresis

machine to conduct current through the gel. It is also used to prepare the agarose gel.
DNA ladder: This provides standards for different-sized DNA fragments, which can be
compared to sample bands to obtain an approximate size for sample DNA fragments.
Loading dye: This is added to the DNA samples and DNA ladder to allow visualization
of sample when loading and migration of the sample during electrophoresis.
51
Components used for PCR results (Continued):

Nucleotide dye: This is used to visualize the DNA bands after electrophoresis is
performed.
Examples of dyes are as follows:
Ethidium bromide: Binds to the major groove of DNA and fluoresces under
ultraviolet light.
Sybr Green: Binds to double-stranded DNA and fluoresces under blue light or
ultraviolet light.
DEMONSTRATIONS:
1. Observe the demonstration for PCR. Become aware of the components

required and note why PCR might be useful in the study of bacteria.
2. Observe the demonstration of gel electrophoresis. Observe how a positive

sample is identified and what controls are used to ensure PCR was
successful.
SOME QUESTIONS TO THINK ABOUT:
1. How would you interpret a PCR result in which there was:
A band of the correct size?
No band?
A band of an incorrect size?
2. When could PCR on bacteria be important in your anticipated veterinary field(s)?
52
BACTERIAL DISEASE CONDITIONS AND COMMON PATHOGENS
Disease Bovine Canine Equine Feline Porcine Sheep/goats
Diarrhea E. coli Salmonella Salmonella Salmonella E. coli Clostridia

Salmonella C. jejuni A. equuli Brachyspira
Respiratory Pasteurella/ Bordetella R. equi Bordetella Actino- Pasteurella/

Mannheimia Ps. aeruginosa Str. bacillus Mannheimia
Mycoplasma zooepidem Pasteurella Mycoplasma
H. somni Strep suis A. pyogenes
Mycoplasma
Genital Campy fetus Brucella Klebsiella Leptospira C. jejuni

(incl Leptospira canis Str. zooepi Listeria
abortions) H. somni Leptospira Salmonella
Listeria Chl. abortus
Urinary Coryne E. coli (not E. coli E. coli (not
tract infect. renale Proteus common) Proteus Actinobaculum common)
E. coli Klebsiella Enterococci suis
Ps. aerugin Klebsiella Coryne renale
S. aureus, Pasteurella
pseudintermedius ....
Wounds/ Arcanobact S. intermed Strep equi Pasteurella Streps Coryne
abscesses pyogenes, Strep, Clostridium Staph,strep A. pyogenes pseudotuber
staph, strep, Pseudomon Pseudomon anaerobes Pseudomon
Pseudomonas Nocardia
Infection Cl. chauvoei Staph, clostridia Clostridium (not (not common) Clostridium
of Muscles spp. common) spp.
Eye Moraxella Staph, Pseudomon Chlamydia (not common) Moraxella
infections bovis Pseudomon Mycoplasma Neisseria
Ear H. somni Staph, strep (not (same as for (not common) (not
infections Proteus common) dogs) common)
Pseudomon
Septicemia E. coli Strep canis E. coli (same as for Salmonella E. E. coli
Salmonella Klebsiella Salmonella dogs) coli
(puppies) A. equuli A. suis, S. suis
Mastitis S. aureus (not common) (not (not (E. coli (same as for
Streptococci common) common) Klebsiella) bovine)
Coliforms
53
LABORATORY REVIEW SLIDES
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Bacteriology Mycology Laboratory Manual SPRING - 2022

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PTHB 503 Lab Manual, Spring-2022

BACTERIOLOGY AND MYCOLOGY

Term 2 – Spring, 2022

Laboratory safety rules and aseptic techniques 3

Faculty & Staff

Laboratory Safety Rules and Aseptic Techniques

Laboratory Safety Rules and Aseptic Techniques (Cont.)

PLEASE HANDLE MICROSCOPES CAREFULLY.

THE OIL-IMMERSION OBJECTIVE LENS MUST BE

SUBMISSION OF CLINICAL SPECIMENS AND EXAMINATION:

The following are guidelines in the selection and submission of specimens:

1. Take sample before antibiotic treatment is started.

4. Try to avoid contamination by normal flora present on mucous membranes,

7. Anaerobic specimens may be held at room temperature.

8. Submit materials cleanly so as not to endanger laboratory staff. Label dangerous

9. Give laboratory a full history of animal, including disease suspected, treatment

SPECIMENS WITH SPECIAL REQUIREMENTS:

5. ANAEROBIC SAMPLES (e.g. deep wound with foul-smelling, dark exudate)

EXAMINATION OF CLINICAL MATERIAL:

4. Look for bacteria both outside and inside inflammatory cells.

CULTURE OF CLINICAL SPECIMENS:

BASIC BACTERIOLOGICAL TECHNIQUES

• Gram stain reagents:

Preparation of a fixed smear

GRAM STAIN PROCEDURE

Mistakes commonly made with Gram stain:

1. Over heat fixing sample (changes staining characteristics).

KOH TEST PROCEDURE

1. Place a drop of 3% KOH on a clean slide.

BACTERIAL ISOLATION AND IDENTIFICATION

1. STREAK PLATE METHOD FOR PURE CULTURE

• Clinical sample. In lab, a mixed broth culture of Staphylococcus aureus and

PROCEDURE FOR STREAK PLATE

5. Flame the loop and cool.

7. Flame the loop and cool.

9. Flame the loop and cool.

FIGURE 1. STREAKING METHOD

FIGURE 2. COLONY MORPHOLOGY

2. DEMONSTRATION OF ANAEROBIC AND MICROAEROPHILIC CULTURE

Microaerophilic bacteria such as Campylobacter require a reduced oxygen environment

3. COMMERCIAL BLOOD-CULTURE BOTTLES DESIGNED FOR

4. COMMERCIAL BACTERIAL IDENTIFICATION SYSTEMS

Additionally, there are automated identification systems such as “MICROSCAN” for

ANTIBIOTIC SENSITIVITY TEST AND QUANTITATIVE CULTURE

ANTIBIOTIC SUSCEPTIBILITY TESTING

BROTH DILUTION METHOD: In the broth dilution method, a standard inoculum of

DISK DIFFUSION ASSAY (KIRBY-BAUER METHOD): In the disk diffusion test,

MIC: The MIC (Minimal Inhibitory Concentration) is defined as the lowest

PROCEDURE FOR DISK DIFFUSION ASSAY

• Standardized (MacFarland standard 0.5-1.0 [1.5-3.0 × 108 cells]) broth cultures/

• Antibiotic disks – one dispenser per lab: Example- gentamicin, enrofloxacin,

3. Allow to dry (maximum 3-5 minutes).

5. Invert the plates and incubate overnight at 37°C

6. Examine plates after 18-24 hours of incubation and record results

QUANTITATIVE CULTURE OF URINE FROM DOGS

• Urine sample containing an unknown number of bacteria

• A calibrated loop to hold 0.001 ml (1 µl)

• Blood agar and MacConkey agar plates

INTERPRETATION OF SUSCEPTIBILITY TESTING, URINE CULTURE

1. DISK DIFFUSION ASSAY

2. Interpret the results by comparing zone sizes to those in Table 1.

TABLE 1. EXAMPLE OF INTERPRETIVE TABLE FOR DISK DIFFUSION

Gentamicin (GM 10) 10 g ≤12 13-14 ≥15