[Medical Microbiology] [2022]

HCMC INTERNATIONAL UNIVERSITY

SCHOOL OF BIOTECHNOLOGY

Medical Microbiology Practical Course

Laboratory Manual

Prepared by: Assoc Prof Dr Nguyen Thi Thu Hoai

Semester:……….Academic year:………………..
Student’s name:…………………………………..
Student’s ID:……………....Class:….……………
Demonstrator’s name: ……………

CONTENT:
Lab 1: Introduction to the practical course: safety issue, chemical and reagent preparation
and bacterial isolation and culture.
Lab 2: Identification of some important bacterial pathogens by using biochemical methods.
Lab 3&4: Antimicrobial susceptibility testing (AST): disc diffusion and micro-dilution
method.
Lab 5&6: Detection of staphylococcal enterotoxins by using multiplex PCR.

1
 [Medical Microbiology] [2022]

LABORATORY RULES
1. Always wear protective clothing: lab coat, closed shoes.
2. Do not eat, drink or smoke in the laboratory.
3. Regard the bench as a contaminated area. Do not place any object on the laboratory
bench that could later transfer to your mouth. Do not sit on the bench.
4. Practice good aseptic techniques and take care when disposing of class materials.
5. Care must be taken when handling samples to avoid any infection incident.
6. Do not discard any demonstration slides.
7. Dispose used lancets, broken glass in the “SHARPS” discard containers.
8. At the end of your class, tidy your bench.
9. While working, concentrate, avoid talking and consult the instructor immediately when
having questions or feeling uncertain in doing or using something.
10. Before you leave the laboratory, wash your hands thoroughly with soap and water.

2
 [Medical Microbiology] [2022]

Lab 1:
Introduction to the practical course: safety issue, chemical and reagent
preparation and bacterial isolation and culture

1. Objectives
- Know the rules, safety regulations, locations and functions of equipments.
- Know the general content of the practical course and evaluation.
- Be assigned in team with strangers to be able to work with new colleagues.
- Be able to work safely with pathogenic microorganisms.
- Perform nasal culture to isolate Staphylococcus aureus and Haemophilus influenza.
- Be able to distinguish culturing, differentiate and selective media.

2. Introduction
While working with pathogens and potential pathogens, bio-safe laboratories are requires
A biosafety level is the level of the biocontainment precautions required to isolate dangerous
biological agents in an enclosed facility. The levels of containment range from the lowest
biosafety level 1 (BSL-1) to the highest at level 4 (BSL-4).
BSL-1: This level is suitable for work involving well-characterized agents not known to
consistently cause disease in healthy adult humans, and of minimal potential hazard to
laboratory personnel and the environment (CDC, 1997)
BSL-2: This level is similar to Biosafety Level 1 and is suitable for work involving agents of
moderate potential hazard to personnel and the environment. It includes various bacteria and
viruses that cause only mild disease to humans or are difficult to contract via aerosol in a lab
setting, such as C. difficile, most Chlamydiae, hepatitis A, B, and C, orthopoxviruses (other
than smallpox), influenza A, Lyme disease, Salmonella, mumps, measles, scrapie, MRSA,
and VRSA.
BSL-3: This level is applicable to clinical, diagnostic, teaching, research, or production
facilities in which work is done with indigenous or exotic agents which may cause serious or
potentially lethal disease after inhalation. It includes various bacteria, parasites and viruses
that can cause severe to fatal disease in humans but for which treatments exist, such as
Yersinia pestis (causative agent of plague), Francisella tularensis, Leishmania donovani,
Mycobacterium tuberculosis, Chlamydia psittaci, West Nile virus, Venezuelan equine
encephalitis virus, Eastern equine encephalitis virus, SARS coronavirus, Coxiella burnetii,
Rift Valley fever virus, Rickettsia rickettsii, several species of Brucella, rabies virus, and
yellow fever virus.
BSL-4: This level is required for work with dangerous and exotic agents that pose a high
individual risk of aerosol-transmitted laboratory infections, agents which cause severe to fatal
disease in humans for which vaccines or other treatments are not available, such as Bolivian
and Argentine hemorrhagic fevers, Marburg virus, Ebola virus, Lassa virus, Crimean-Congo
hemorrhagic fever, and various other hemorrhagic diseases.
3. Procedure
3.1 Required Materials and Instruments
Lab requirement: BSL-2
Bacteria strains: Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa;
Staphylococcus aureus; Salmonella typhi
Reagents and Labwares:
- Ready Petri dishes of MacConkey Agar, Brain Heart Infusion (BHI) Agar, Blood agar/
Chocolate agar (slowly heat blood agar to 56OC), Thiosulfate-citrate-bile salts-sucrose agar
(TCBS) agar, Manitol Salt Agar (MSA), Cetrimide agar (CTAB): 10 dishes/ each type.

3
 [Medical Microbiology] [2022]

- BHI broth, glycerol

- Mueller Hinton broth
- Duran bottles
Instruments: Autoclave, Incubator, hood (laminar box), microscopes

3.2 Experimental Procedure

General laboratory techniques
- Prepare: 200 ml MHB and autoclave for lab 5&6
- Prepare: 20 ml BHI/glycerol 20% and autoclave for sample storage

Culture bacteria on Differentiate/ Selective Media:

- Culture defined bacterial strains: Vibrio cholerae, Escherichia coli, Pseudomonas

aeruginosa; Staphylococcus aureus; Salmonella typhi on MSA, Blood agar,
McConkey agar, TCBS and CTAB agar to observe and compare their features
- Inoculate the agar plate with a pure culture by streaking over the entire surface of the
slant / agar plate (zig-zag to cover surface) in case of streaking on slant then stabbing
deep into the butt if necessary.
- Practice to inoculate 2-4 strains on one agar plate.

Incubate at 37ºC for 24 hours.

Isolation of opportunistic pathogenic bacteria from nasal cavity

- Use autoclaved cotton buds to take sample from the anterior nare, streak on: MSA,
Chocolate agar, Blood agar and Mc Conkey agar.

4
 [Medical Microbiology] [2022]

Lab 2:
Identification of some important bacterial pathogens
by using biochemical methods
1. Objectives
- Be able to differentiate several important pathogens by simple culturing techniques,
biochemical tests and Gram staining.

2. Introduction
Generally, pathogens are identified via phenotypic (biochemical properties), serotypic
(immunological properties) or genotypic (DNA sequences) methods.
In this practice, several basic and traditional phenotypic methods are introduced.

3. Procedure
3.1 Required Materials and Instruments
Bacteria strains:
- Defined strains Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa;
Staphylococcus aureus; Salmonella typhi on MSA, Blood agar, McConkey agar,
TCBS agar
- Undefined strains: Nasal samples on MSA, Chocolate agar, Blood agar and Mc
Conkey agar
Reagents:
- Coagulase plasma eg. Coagulase plasma, rabbits with EDTA-BD sciences), H2O2
- Gram staining kit
- Gram-negative identification kit (NamKhoa Biotek)
Instruments: Incubator, hood (laminar box), microscopes

3.2 Experimental Procedure

A. Classical biochemical tests:

Catalase (using H2O2 as the detecting reagent):

- Hand on some drops of H2O2 on the agar plate and observe.
If the test organism produces catalase, bubbles will be formed

Coagulase (Using Coagulase plasma as detecting reagent: eg. Coagulase plasma, rabbits with
EDTA-BD sciences). This test is used to differentiate Staphylococcus aureus (positive) from
coagulase negative Staphylococci. S. aureus produces two forms of coagulase: bound and
free. Bound coagulase or clumping factor is bound to the bacterial cell wall and reacts
directly with fibrinogen. When a bacterial suspension is mixed with plasma, this enzyme
causes alteration in fibrinogen of the plasma to precipitate on the staphylococcal cells,
causing the cells to clump. Free coagulase is produced extra-cellularly by the bacteria that
causes the formation of a clot when S. aureus colonies are incubated with plasma.
This test could be performed as slide test for coagulase screening or tube test for
confirmation of coagulase presence:
- Add a generous loopful of the organism to be tested to a tube of citrated rabbit plasma.
Thoroughly homogenize the inoculum with the loop and incubate the tube at 37o C for one to
four hours. Examine the tube at 30 minute to hourly intervals for the first couple of hours for
the presence of a clot by tipping the tube gently on its side.
- A test that shows any degree of clotting within 24 hours is considered coagulase positive.
Reincubate the tube overnight to see if the clot subsequently lyses. In strains that produce

5
 [Medical Microbiology] [2022]

fibrinolysin, the clot will be slowly digested. This illustrates the importance of reading the
coagulase results within 24 hours. Thereafter, the lack of clotting could be a false negative
reaction with a coagulase-positive strain.
(+) --- Delayed reaction
d --- 11- 89% of strains are positive.

B. Gram staining:

1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent.
Please note that the quality of the smear (too heavy or too light cell concentration) will affect
the Gram Stain results. Young cell culture should be used for obtaining standard
morphology. Heat fixation is performed by moving the slide into a flame.
2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
3. Flood slide with the mordant: Gram's iodine (Lugol solution). Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
5. Flood slide with decolorizing agent. Wait 15 seconds or add drop by drop to slide until
decolorizing agent running from the slide runs clear (see Comments and Tips section).
6. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
7. Wash slide in a gentile and indirect stream of tap water until no color appears in the
effluent and then blot dry with absorbent paper.
8. Observe the results of the staining procedure under oil immersion using a Brightfield
microscope. At the completion of the Gram Stain, gram-negative bacteria will stain pink/red
and gram-positive bacteria will stain blue/purple. (The iris of the microscope condenser
should be opened as wide as possible. With a closed condenser colours can hardly be
discriminated.). In a smear that has been stained using the Gram Stain protocol, the shape,
arrangement and Gram reaction of a bacterial culture will be revealed.

C. Identify bacteria by using commercial kit

Gram- negative identification kit (NamKhoa Biotek) is used to identify different Gram-
negative bacterial strains: IDS 14 GNR- Identification system for Gram- Negative Rod-
shaped non- fastidious Bacteria including Enterobacteriaceae and non- Enterobacteriaceae.

IDS 14 GNR includes:

- OXI test: Oxidase paper disc
- 1 plastic plate containing 12 wells with 12 biochemical paper discs for 12 biochemical
tests.

Table 2.1: The 14 biochemical tests in Nam Khoa IDS 14 GNR Kit

Reaction Well Label BIOCHEMICAL TEST Additive Reagent

1 OXI Oxidase
2 1 GLU Glucose fermentation Parafilm solution must be added
3 2 NIT Nitrate reduction Nitrite reactive paper disc- added 5- minutes
before reading the result in the next day
4 3 ONPG ONPG hydrolysis
5 4 URE Urease production Parafilm solution must be added
6 5 PAD Phenylalanine FeCl3 10% added 5- minutes before reading
Deaminase production the result in the next day
7 6 CIT Citrate usage

6
 [Medical Microbiology] [2022]

8 7 ESC Esculin Hydrolysis

9 8 H2S H2S production
10 9 IND Indole production Kovacs solution, added 5- minutes before
reading IND result in the next day
11 10 VP Voges- Proskauer KOH, alpha-naphthol added 5- minutes before
reading the result in the next day
12 11 MLO Malonate usage
13 12 LDC Lysine decarboxylase Parafilm solution must be added
14 MOB Motility
Note: reactions on 12 wells:
1
: glucose fermenters because they can metabolize glucose anaerobically. Glucose fermenter
will release acid product thus change the pH indicator, Bromocresol purple from deep purple
to yellow (anerobic condition is required).
2
: This test is used to determine the ability of the organism to reduce nitrate to nitrites or fee
nitrogen gas. The reduction of nitrate to nitrite is detected by adding sulphanilic acid and
alpha‐naphthylamine. The sulphanilic acid and nitrite reacts to form a diazonium salt which
then reacts with alpha‐naphthylamine to produce a red, water soluble azo‐dye.
3
: ortho-Nitrophenyl-β-galactoside (ONPG) is a colorimetric and spectrophotometric
substrate for detection of β-galactosidase activity. This compound is normally colorless.
However, if β-galactosidase is present, it hydrolyzes the ONPG molecule into galactose and
ortho-nitrophenol. The latter compound has a yellow color.
4
: To determine the ability of the organism to split urea forming 2 molecules of ammonia
(plus CO2) by the action of the enzyme Urease with resulting alkalinity. Urease test media
contain 2% urea and phenol red as a pH indicator. An increase in pH due to the production of
ammonia results in a color change from yellow (pH 6.8) to bright pink (pH 8.2). (anerobic
condition is required).
5
: PAD removes the amine group from the amino acid phenylalanine and releases the amine
group as free ammonia. As a result of this reaction, phenylpyruvic acid is also produced.
Phenylpyruvic acid will then react with the ferric chloride and turn dark green.
6
: the test is based on the ability of an organism to use citrate as its only sole source of
carbon and ammonia as its only source of nitrogen. Bacteria are inoculated on a medium
containing sodium citrate and a pH indicator such as bromothymol blue. The medium also
contains inorganic ammonium salts, which are utilized as sole source of nitrogen. Use of
citrate involves the enzyme citritase, which breaks down citrate to oxaloacetate and acetate.
Oxaloacetate is further broken down to pyruvate and carbon dioxide (CO2). Production of
sodium bicarbonate (NaHCO3) as well as ammonia (NH3) from the use of sodium citrate and
ammonium salts results in alkaline pH. This results in a change of the medium’s color from
green to blue. In acidic condition, the pH indicator has yellow color.
7
: When an organism hydrolyzes the glycoside esculin to form esculetin and dextrose, the
esculetin reacts with the ferric citrate to produce a dark brown or black phenolic iron
complex.
8
: This test determines whether the microbe reduces sulfur-containing compounds to sulfides
during the process of metabolism. If sulfide is produced, it combines with iron compounds to
produce FeS, a black precipitate.
9
: Indole test is performed to determine the ability of the organism to split tryptophan
molecule (tryptophanase) into Indole, Pyruvic acid and Ammonia. Indole can react with with
p-Dimethylaminobenzaldehyde (Kovac’s reagent) at an acid pH in alcohol to produce a red-
violet compound.

7
 [Medical Microbiology] [2022]

10
: VP is a test used to detect acetoin in a bacterial broth culture. If glucose is being broken
down to acetylmethylcarbinol (also called acetoin), it will react with alpha-naphthol and
potassium hydroxide (KOH) to form a red color.
11
: The purpose is to see if the microbe can use the compound malonate as its sole source of
carbon and energy for growth. If a microbe can use malonate for carbon and energy, it will
grow on malonate broth which contains mineral salts, sodium malonate for carbon, and
ammonium phosphate for its nitrogen source. Organisms which simultaneously utilize
malonate and ammonium sulfate produce sodium hydroxide leading to a rise in pH of the
medium, and a pH indicator changes color. The pH indicator is bromothymol blue, which is
green at neutral pH, yellow at acidic pH <6.0 and turns blue at alkaline (basic) pH >7.6.
12:
LDC (Lysin Decarboxylase) bottle to test for LDC production. The medium used is
lysine decarboxylase broth. The medium is a nutrient broth to which 0.5% lysine is added. An
important component of the medium is a modest amount of glucose, necessary for the process
to proceed. The microbe must first use the glucose present to cause the pH to drop. The pH
indicator bromocresol purple is purple at neutral or alkaline/basic pH but turns yellow at pH
<5.2. Once the medium has been acidified, the enzyme lysine decarboxylase is activated.
When lysine is used, the pH of the medium rises and the indicator changes color. Change
back to purple/ violet from yellow indicates a positive test for lysine decarboxylase.
(Anerobic condition is required). Note that some obligate aerobes (Pseudomonas, Vibrio, etc)
cannot grow thus turbidity of the medium should also check together with color change. If
there is no bacterial growth, the result should be noted as negative even though color of the
bottle is violet.
- Oxidase Test: Each Oxidase reagent tube contains 0.5 ml N, N, N`, N`- tetramethyl-p-
phenylenediamine dihydrochloride 1% (1% TMPD, or 1% Kovács oxidase reagent). In the
presence of cytochrome oxidase, this reagent, reduced TMPD- riched in electron- colorless
will be converted to oxidised- TMPD- blue/ purple.
- MOB (Motility Bottle) to test for motility.
Motility test medium with triphenyltetrazolium chloride (TTC) provides an easy method for
determining motility. TTC in its oxidized form is colorless. As bacteria grow in the presence
of TTC, the dye is absorbed into the bacterial cells where it is reduced to the insoluble red-
colored pigment formazan. Growth is indicated by the presence of the red color, and as
motility occurs, small to very large regions of color can be observed around the area of
inoculation.

Procedure: Remember to perform the experiment in laminar flow box and using sterile
materials to avoid contamination and wrong identification result
- Prepare physiological solution: NaCl 0.85% for preparing bacterial suspension
(desired OD600nm is about 0.08- 0.1, i.e approx. 10^8 CFU- 10^9 CFU/ mL). The
bacteria suspension must be prepared by using physiological solution not by using
medium as this can cause false positive result.
- OXI test: add a drop of bacterial suspension on Oxidase paper disc. If the test
organism produces cytochrome oxidase (sometime called indophenol oxidase), the
oxidase reagent will turn blue or purple within 15 seconds.
- Add 100 µL of the bacterial suspension on each well of the 10- well plate. In well 1,
4, 12, 50 µL (1 drop) of parafilm should be further added to provide anaerobic
condition.
→ Incubate at 37oC overnight and read result on the next day.
- For the well 2, 5, 9, 10: before reading result, add 1 drop (or a disc) of testing
reagent: nitrite reactive paper, FeCl3 10%, Kovacs solution, KOH plus alpha-
naphthol, into the well respectively (Table 2.1) and read the result within 5 minutes.

8
 [Medical Microbiology] [2022]

- MOB test: Use needle upright to inoculate the bacteria into the bottle, reach 2/3 of the
bottle. Keep the needle straight.

Result Interpretation:
Table 2.2 Result Interpretation Table for Nam Khoa IDS 14 GNR

Test Well Label Result: Positive/ Negative SCORE FINAL

(+/-) SCORE
1 Disc OXI Blue or purple/ white 1/0 A=1+2+3
2 1 GLU Yellow/ Purple 2/0
3 2 NIT Red or pink/ No color change 4/0
4 3 ONPG Yellow/ Colorless 1/0 B= 4+5+6
5 4 URE Red/ Orange or Yellow 2/0
6 5 PAD Green/ Colorless or Yellow 4/0
7 6 CIT Marine blue/ Green or Pale yellow 1/0 C= 7+8+9
8 7 ESC Black/ Colorless or Yellow 2/0
9 8 H2S Black/ Colorless or Yellow 4/0
10 9 IND Red or pink on the surface/ Colorless or 1/0 D= 10+11+12
Yellow
11 10 VP Red/ Colorless or Yellow 2/0
12 11 MLO Blue or marine blue/ Green or Yellow 4/0
13 12 LDC Violet/ Yellow 1/0 E= 13+14
14 Bottle MOB Red color spreads out of/ only at streak line 2/0
The obtained final score (A B C D E) will be used to match with the Reference Identification
Table provided in the Kit Manual.
For example: 6 1 0 1 3→ E. coli;
3 0 2 4 2 → Pseudomonas vesicularis
4. Results and Discussion
Observe results and categorize the pathogens.

5. Review Exercises
1. You would consult Bergey's Manual if you were (circle only one correct answer)
A. repairing your dune bergey
B. classify a bacteria isolate
C. identifying a bacteria isolate
D. determining whether bacteria species A is more closely related to species B or species C
E. all of the above
F. none of the above
2. Two pure cultures of bacteria differ only in terms of the stream (i.e., running water) in which they resided
prior to their domestication. The bacteria in those cultures are considered to be separate __________. (chose
best answer)
A. strains
B. isolates
C. species
D. serovars
E. biovars
F. all of the above
G. none of the above
3. Two pure cultures of bacteria are created by the splitting of a single broth culture in two and then growing the
resulting broth cultures for 24 hours. The bacteria in those cultures are considered to be different __________.
(chose best answer)
A. strains
B. isolates

9
 [Medical Microbiology] [2022]

C. species
D. serovars
E. biovars
F. all of the above
G. none of the above
4. Two bacteria differ by only a single genetic change, one which has no noticeable effect on the bacteria.
Nevertheless, one is considered to be a mutant of the other. These two bacteria are considered to be different
__________. (chose best answer)
A. strains
B. isolates
C. species
D. serovars
E. biovars
F. all of the above
G. none of the above
5. Name a circumstance in which consultation of Bergey'sManual might be helpful.
6. Given that all of the following comparisons are between two stocks of bacteria of the same species, fill in
blanks with the appropriate letter (i.e., best answer):
A. strains
B. isolates
C. serovars
D. biovars
E. none of the above
i. Different antibody reactivity: __________
ii. Different cell shape: __________
iii. Differentiable, but not as living cells (no phenotypic differences): __________
iv. Obtained from different locations, otherwise identical: __________
v. Differentiable, but both descended from single isolate: __________
vi. Not differentiable, both descended from single isolate: __________
7. A microorganism purified from a wild, heterogeneous mixture of microorganisms is identified as being a
member of a particular species, but is morphologically distinct from other members of that species. How might
you describe such an organism? As ________. (CIRCLE ALL APPLICABLE ANSWERS)
A. an isolate.
B. a serovar.
C. a morphovar.
D. a biovar.
E. a strain.
F. a new species.
G. a type strain.
8. Describe a reason you might resort to protein analysis to distinguish two similar strains.
9. Agglutination tests, ELISAs, and Western blotting all detect ___________ binding to samples, and thus are
considered serological techniques.
10. Usually one of the first strains of a species studied, the __________ is often more fully characterized than
other strains.
11. Two pure cultures of virus are obtained from two different patients. By all possible criteria, biological,
morphological, serological, and genetic, the two viruses are deemed identical. Consequently, we classify the two
viruses as being two examples of the same strain of virus. Given this information, we infer that the virus
infecting each of the two patients had a common source, either one patient infected the other, or both were
infected by a common third party. Since the two pure cultures of virus were obtained from different patients,
despite the otherwise identity of the two viruses (extremely high degree of organismal similarity), we would still
classify the two viruses as distinct and separate __________.

10
 [Medical Microbiology] [2022]

Lab 3&4:
Antimicrobial Susceptibility Testings
1. Objectives
To evaluate the antibiotic susceptibility of S. aureus and E. coli via disk- diffusion method
and determine minimum inhibitory concentration (MIC) values of the tested antibiotics
towards the microorganisms using micro-dilution method.

2. Introduction
Antibiotic sensitivity is the susceptibility of bacteria to antibiotics. Antibiotic susceptibility
testing (AST) is usually carried out to determine which antibiotic will be most successful in
treating a bacterial infection in vivo. Ideal antibiotic therapy is based on determination of the
aetiological agent and its relevant antibiotic sensitivity. However, empiric treatment is often
started before laboratory microbiological reports are available when treatment should not be
delayed due to the seriousness of the disease.
Measuring antibiotic sensitivity of pathogens is not only important in clinic settings but also
in pathogen research. Some antibiotics actually kill the bacteria (bactericidal), whereas others
merely prevent the bacteria from multiplying (bacteriostatic) so that the host's immune
system can overcome them. Some pathogens are still sensitive to many antibiotics, some are
resistant to various drug types. The mechanisms underlying their drug resistance are varied
and are of research interest since decades.

3. Procedure
3.1 Required Materials and Instruments
Bacteria strains: Streptococcus, Pseudomonas, Staphylococcus, Lactobacillus, cultured on
LB/TSB dishes
Reagents: Ampicillin, Kanamycin, Gentamycin, Media (MHA, MHB)
Instruments: Hood, incubator, spectrometer

3.2 Experimental Procedure

A. Disc diffusion testing:
- Bacterial solution in MH medium was prepared from colonies, OD 620 nm 0.08- 0.1 (0.5
McFarland). We can use directly this solution or dilute 1:10, 1:100, poured on MHA plate (5
ml, for example, attention to pour evenly on the plate, and then pipette the left volume out for
drying-up sooner, let the plate dry for another few minutes.
- Antibiotic solution preparation: the corresponding amount of antibiotic on the disc
should range from 10- 1000 µg depending on type of antibiotics.
- Procedure: lay the paper disc on the plate with light press (can use the tip 1-ml to pick
the disc to the plate)→ pipette 10µl of antibiotic solution which corresponds to the desire
amount on it. Make sure to write down notes on the plate. Incubate overnight.
- Scan and measure the diameter of the inhibition zone:
With the ruler, measure the zones of inhibition in millimeters from the underside of
the plate. Measure the entire diameter of the zone, including the disc.

11
 [Medical Microbiology] [2022]

Figure 1. Illustrative data of an antibiotic sensitivity disk test with

Meropenem on E. aerogenes: 10 microliter of antibiotics at different
concentrations equal to the amount of 2, 4, 8, 12, 16, 14 mg was loaded
on the paper disc.

B. Micro-dilution method
- Take 1 to 4 colonies and dilute them by shaking in 3 mL sterile cation-adjusted MHB
medium.
- Check that optical density at 620 nm is 0.08 to 0.1 or 0.5 McFarland which
corresponds to 1 - 2 x10^8 CFU/mL (Blank is 1 mL sterile cation adjusted MHB
medium).
- The antibiotic concentration of the first well should range from 16- 512 µg/ mL
depending on type of antibiotics.
Calculations of volume of bacterial suspension needed for 11 well plates
- Put 400 µL of bacterial suspension adjusted between 0.08 and 0.1 in 36.6 mL sterile
cation adjusted MHB medium (dilution 100 fold) that corresponded to 1-2 x10^6
CFU/mL.
On the 96- well sterile plate:
- Pipett 100 µl MHB for all wells 1-11.
- 1st well, 100µl of 4-fold antibiotic solution→ perform serial dilution for next wells,
using of 100µl mixed from the previous, discard the 100 µl of the last mixed (well
Nr10).
- Pipett 100 µl of bacteria suspension for all wells 1-11.
- Pipett 200 µl of MHB for well 12.
- Plate will be incubated at 37C and observed with mirror in the next day (16-20 h),
opacity is qualified by comparing with negative control.
Abt 1 2 3 4 5 6 7 8 9 10 11 12

A 1 :2^1 :2^2 :2^3 :2^4 :2^5 :2^6 :2^7 :2^8 :2^9

B
C
D

F
G
H

12
 [Medical Microbiology] [2022]

Notice: In case, 96-well plates are not available, glass tubes can be used but the volumes must be
increased 10x, i.e the total volume instead of 200 ul, it is 2 ml.
4. Results and Discussion
Record: Diameter of the inhibition zone; MIC value.
Compare the two results in evaluating the antibiotic sensitivity of a certain bacterial strain.

5. Review Exercises
- What could happen if bacterial culture for antibiotic susceptibility test was not pure
culture?
- If that was the case, explain the achieved result when: a) Disc diffusion; b) micro-
dilution method was used.
- Why is it recommended to use MHA in antibiotic sensitivity testing?

13
 [Medical Microbiology] [2022]

Lab 5&6:
Detection of staphylococcal enterotoxins by using multiplex PCR
1. Objectives
- Able to extract and quantify DNA from a bacterial sample
- Able to use multiplex PCR method to detect multiple staphylococcal enterotoxins. The
outcome is to determine the virulence of a certain Staphylococcus aureus strain.

2. Introduction
Enterotoxins are chromosomally encoded exotoxins targeting intestine. They are produced
and secreted from several bacterial organisms such as E. coli. Salmonella spp, Staphylococcus spp,
Vibrio spp, Shigella spp and Clostridium spp.
Enterotoxins are often heat-stable, and are of low molecular weight and water-soluble. They
are frequently cytotoxic and kill cells by altering the apical membrane permeability of the mucosal
(epithelial) cells of the intestinal wall. They are mostly pore-forming toxins (mostly chloride pores),
secreted by bacteria, that assemble to form pores in cell membranes. This causes the cells to die.
Staphylococcus aureus (S. aureus) is a Gram positive bacterium that is carried by about one
third of the general population and is responsible for common and serious diseases. These diseases
include food poisoning and toxic shock syndrome, which are caused by exotoxins produced by S.
aureus. Of the more than 20 Staphylococcal enterotoxins, SEA and SEB are the best characterized
and are also regarded as superantigens because of their ability to bind to class II MHC molecules on
antigen presenting cells and stimulate large populations of T cells that share variable regions on the b
chain of the T cell receptor.
In this practice, students will detect staphylococcal toxin encoding genes including eta, etb
(exfoliative toxin a and b); tst (toxic shock syndrome toxin, TSST-1). Additionally, mecA, whose
product is a 78-kDa protein called penicillin binding protein 2a, which is responsible for intrinsic
resistance to antibiotics is also detected. Positive control for the experiment is femA which is a gene
encoding a factor essential for methicillin resistance and is universally present in all S. aureus isolates.

3. Procedure
3.1 Required Materials and Instruments
- Bacterial strains: Staphylococcus aureus nasal isolates and standard Staphylococcus
aureus strain.
- Reagents: Quick-gDNA™ MiniPrep (Zymoresearch), primers, PCR master mix,
agarose, Ethidium bromide (or GelRed, CyberGreen, Runsafe).
- Instruments: PCR machine (Thermocycler), agarose gel electrophoresis apparatus,
bioimaging apparatus.

3.2 Experimental Procedure

Day 1:
- Extract DNA from bacterial pellets by using Quick-gDNA™ MiniPrep
(Zymoresearch) and conventional phenol-chloroform method.
Protocol for Quick-gDNA™ MiniPrep
Step 1: Add 400 µl of Genomic Lysis Buffer to 100 µl of bacterial culture or suspension
(4:1). Mix completely by vortexing 4-6 seconds, then let stand 5-10 minutes at room
temperature.
Note: Add 200 µl Genomic Lysis Buffer to all samples <50 µl. For samples larger than 50
µl, add a proportional amount (4:1) of Genomic Lysis Buffer (e.g., Add 800 µl Genomic Lysis
Buffer to 200 µl blood).
Step 2: Transfer the mixture to a Zymo-Spin™ Column in a Collection Tube. Centrifuge at
10,000 x g for one minute. Discard the Collection Tube with the flow through.

14
 [Medical Microbiology] [2022]

Step 3: Transfer the Zymo-Spin™ Column to a new Collection Tube. Add 200 µl of DNA
Pre-Wash Buffer to the spin column. Centrifuge at 10,000 x g for one minute.
Step 4: Add 500 µl of g-DNA Wash Buffer to the spin column. Centrifuge at 10,000 x g for
one minute.
Step 5: Transfer the spin column to a clean micro-centrifuge tube. Add ≥50 µl (eg. 100 µl )
DNA Elution Buffer or water to the spin column. Incubate 2-5 minutes at room temperature
and then centrifuge at top speed for 30 seconds to elute the DNA. The eluted DNA can be
used immediately for molecular based applications or stored ≤-20ºC for future use.
Conventional phenol-chloroform method:
Step 1: Centrifuge 1ml of overnight culture at 13,000 rpm in 5 min to harvest cell pellet
Step 2: Add 500 µl cell lysis buffer to the cell pellet and mix thoroughly.
Step 3: Add 500 µl ice-cold Phenol-Chloroform (1:1) (v/v) then centrifuge at 13,000 rpm in 2
min to collect the supernatant.
Step 4: Add 300 µl absolute EtOH, incubate 30’-1h at -20C, then centrifuge at 12,000 rpm,
15 min to collect the precipitant
Step 5: Add 300 µl EtOH 70% to wash to precipitant and then centrifuge at 12,000 rpm, 10
min to collect the cleaned precipitant.
Let the precipitant dry and resuspend in ≥ 50 µl (eg. 100 µl TE buffer or ddH2O).
Quantify DNA concentration by using The Synergy™HT - Bio-Tek.
Note: This machine is a spectrometer which will measure the absorbance of the sample at the
wavelength 260 nm. In general, A260 value of 0.1 corresponds to 5 µg/mL. The overall
sensitivity of the method is approximately 50-250 ng/mL of DNA. In addition, absorbance at
the wavelength 280 nm is also measured to check for the quality of the sample because
proteins (a common contaminant) absorb light at 280nm. For DNA, a pure sample will have
an A260/A280 value of 1.8.
For RNA, a pure sample will have an A260/A280 value of 2.0. Furthermore, absorbance at
wavelength 230 nm should also be measured to check for contaminant with carbohydrates,
peptides, phenols, and aromatic compounds. A pure sample should have an A260/A230 ratio
of 2.2.
DNA concentration calculation: http://www.endmemo.com/bio/OD260.php
•1 OD260 Unit = 50ug/ml for double stranded DNA.
•1 OD260 Unit = 40ug/ml for single stranded RNA.
•1 OD260 Unit = 33ug/ml for single stranded DNA (ssDNA).
•1 OD260 Unit = 20ug/ml for single stranded oligo (ssOligo).
Day 2:
Perform multiplex PCR with a set of primer mixes. Reactions were prepared with master
mixes of components from the TopTaq Master Mix kit (Qiagen) with slight modifications to
the given instructions.
Multiplex primer set contained 200 mM deoxynucleoside triphosphates; 5 ul of reaction
buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl); 2.0 mM MgCl2; 50 pmol for eta and 20
pmol each for etb, tst, mecA, and femA; 2.5 U of Taq DNA polymerase, and 10 to 1,000 ng
of template DNA. The volume of this mix was adjusted to 50 µl with sterile water.

STOCK PRIMER SOLUTION: 100 µM

Table 3.1 Primers used in the practical course (Mehrotra et al. 2000)
Genes Forward (5’-3’) Reverse (5’-3’) Location within Expected
gene amplified
length (bp)
Eta GCAGGTGTTGATT AGATGTCCCTATT 775-794//848–867 93
TAGCATT TTTGCTG
Etb ACAAGCAAAAGA GTTTTTGGCTGCTT 509–528//715– 226

15
 [Medical Microbiology] [2022]

ATACAGCG CTCTTG 734

Tst ACCCCTGTTCCCT TTTTCAGTATTTGT 88–107//394–113 326
TATCATC AACGCC
MecA ACTGCTATCCACC CTGGTGAAGTTGT 1182– 163
CTCAAAC AATCTGG 1201//1325–1344
FemA AAAAAAGCACAT GATAAAGAAGAA 1444– 132
AACAAGCG ACCAGCAG 1463//1556–1575
Prepared solution for eta primers: 1.25 uM;
Prepared solution for etb, tst, mecA, femA is 0.5 uM;
DNA amplification was carried out in a thermocycler with the following thermal cycling
profile: an initial denaturation at 94°C for 5 min was followed by 35 cycles of amplification
(denaturation at 94°C for 2 min, annealing at 57°C for 2 min, and extension at 72°C for 1
min), ending with a final extension at 72°C for 7 min.
3.3 Perform gel electrophoresis
Prepare agarose gel: Instead of using Ethidium Bromide, we now use SYBR Safe Stain or
Gel-Red reagent at the concentration of 1: 10,000 in the agarose 2 % solution in 1X SB
buffer (see below).
- Place the agarose gel in the buffer tank containing 1X SB buffer serving as running
buffer.
- Mix PCR product with loading buffer and load 5-10 µl sample/ well.
- Load 2-5 µl of DNA ladder.
- Set up 50 V, 60 minutes for optimal result (however, this duration can be adjusted).
Loading buffer:
6X Loading buffer: 6 ml of 100% glycerol; 0.6 ml of 1M Tris HCl, pH 8.0; 120 µl of 0.5 M
EDTA, pH 8.0; 6 mg Bromophenol Blue. Add 3.28 ml of dH2O.

ELECTROPHORESIS BUFFERS:
10 X SB (Sodium Borate) buffer: 4 g NaOH; pH to 8.0 with approx. 24 g H3BO3
(boric acid powder); bring to 1 L with dH2O.
NOTE: This stock may precipitate over time, requiring heat to re-dissolve crystallized salts
before dilution.
SB buffer is made up of sodium borate, usually 1–10 mM at pH 8.0. It has a lower
conductivity, produces sharper bands, and can be run at higher speeds than can gels made
from TBE buffer (Tris Borate EDTA) or TAE buffer (Tris Acetate EDTA) (5–35 V/cm
as compared to 5–10 V/cm). At a given voltage, heat will be generated and thus the gel will
be heated. However, SB buffer has lower conductivity than TBE and TAE, and thus the gel
temperature is much lower than with TBE or TAE buffers. Therefore, the voltage can be
increased to speed up electrophoresis so that a gel run takes only a fraction of the usual time.
LB buffer containing lithium borate is similar to sodium borate and has all of its
advantages, but permits use of even higher voltages due to the lower conductivity of lithium
ions as compared to sodium ions. However, lithium borate is somewhat more expensive so
that we do not use in our practical lab.
3.4 Record image result with the VisionCapture (Quantum P4) under UV light.

4. Results and Discussion

Record data and discuss on the presence/ absence of PCR products

5. Review Exercises
1. Explain the use of positive and negative controls in each PCR run.
2. Is there any relationship between intensity of PCR bands and expression of the virulence
gene?

16
 [Medical Microbiology] [2022]

APPENDIX
A. DIFFERENTIATE MEDIA AND BIOCHEMICAL TESTS
A.1 Available commercial kits: API strips (Slideshow)
A.2 Differentiate Media
Blood agar: A nutrient medium augmented with the addition of 5% sheep blood routinely is used.
This test provides information on what hemolytic enzymes a bacterium possesses. By providing a culture
medium enriched with red blood cells, it is possible to determine whether a bacterium can destroy the cells and
whether it can digest the hemoglobin inside.
Bacteria possessing hemolysins will be able to discolor or clear blood agar in the vicinity of their colonies.

Figure A.1: Hemolyses of Streptococcus spp. (left) α-hemolysis (S. mitis); (middle) β-hemolysis (S. pyogenes);
(right) γ-hemolysis (= non-hemolytic, S. salivarius) (source: Wikipedia).
When alpha hemolysis (α-hemolysis) is present, the agar under the colony is dark and greenish. Streptococcus
pneumoniae and a group of oral streptococci (Streptococcus viridans or viridans streptococci) display alpha
hemolysis. This is sometimes called green hemolysis because of the color change in the agar. Other synonymous
terms are incomplete hemolysis and partial hemolysis. Alpha hemolysis is caused by hydrogen peroxide
produced by the bacterium, oxidizing hemoglobin to green methemoglobin.
Beta hemolysis (β-hemolysis), sometimes called complete hemolysis, is a complete lysis of red cells in the
media around and under the colonies: the area appears lightened (yellow) and transparent. Streptolysin, an
exotoxin, is the enzyme produced by the bacteria which causes the complete lysis of red blood cells. There are
two types of streptolysin: Streptolysin O (SLO) and streptolysin S (SLS). Streptolysin O is an oxygen-sensitive
cytotoxin, secreted by most Group A streptococcus (GAS), and interacts with cholesterol in the membrane of
eukaryotic cells (mainly red and white blood cells, macrophages, and platelets), and usually results in β-
hemolysis under the surface of blood agar. Streptolysin S is an oxygen-stable cytotoxin also produced by most
GAS strains which results in clearing on the surface of blood agar. SLS affects immune cells, including
polymorphonuclear leukocytes and lymphocytes, and is thought to prevent the host immune system from
clearing infection. Streptococcus pyogenes, or Group A beta-hemolytic Strep (GAS), displays beta hemolysis.
Some weakly beta-hemolytic species cause intense beta hemolysis when grown together with a strain
of Staphylococcus. This is called the CAMP test1. Streptococcus agalactiae displays this property. Clostridium
perfringens can be identified presumptively with this test. Listeria monocytogenes is also positive on sheep's
blood agar.
If an organism does not induce hemolysis, it is said to display gamma hemolysis (γ-hemolysis): the agar under
and around the colony is unchanged (this is also called non-hemolytic). Enterococcus faecalis (formerly called
Group D Strep) displays gamma hemolysis.

Mc Conkey agar
History
MacConkey agar was the first solid differential media to be formulated. It was developed at the turn of the 20th
century by Alfred Theodore MacConkey, M.D, then Assistant Bacteriologist to the Royal Commission on
Sewage Disposal, in the Thompson-Yates Laboratories of Liverpool University, England. The goal was to
formulate a medium that would select for the growth of gram-negative microorganisms and inhibit the growth of
gram-positive microorganisms. Dr. MacConkey first developed a bile salt medium containing glycocholate,
lactose and litmus, to be incubated at 22°C (MacConkey, 1900). This formula was soon altered by the
replacement of glycocholate with taurocholate and the incubation temperature was raised to 42°C (MacConkey,
1901). MacConkey later changed the recipe again by substituting neutral red for litmus (MacConkey, 1905),

17
 [Medical Microbiology] [2022]

following the suggestion that neutral red be used as an indicator in bile salt lactose medium (Grunbaum and
Hume, 1902). The final media formulation was designed to support growth of Shigella and is the one that is
most commonly used today.
Purpose
MacConkey agar is used for the isolation of gram-negative enteric bacteria and the differentiation of lactose
fermenting from lactose non-fermenting gram-negative bacteria. It has also become common to use the media to
differentiate bacteria by their abilities to ferment sugars other than lactose. In these cases lactose is replaced in
the medium by another sugar. These modified media are used to differentiate gram-negative bacteria or to
distinguish between phenotypes with mutations that confer varying abilities to ferment particular sugars.
Theory
MacConkey agar is a selective and differential media used for the isolation and differentiation of non-fastidious
gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas. The
inclusion of crystal violet and bile salts in the media prevent the growth of gram-positive bacteria and fastidious
gram-negative bacteria, such as Neisseria and Pasteurella. The tolerance of gram-negative enteric bacteria to
bile is partly a result of the relatively bile-resistant outer membrane, which hides the bile-sensitive cytoplasmic
membrane (Nikaido, 1996). Other species specific bile-resistance mechanisms have also been identified
(Provenzano, et al. 2000; Thanassi et al. 1997).
Gram-negative bacteria growing on the media are differentiated by their ability to ferment the sugar lactose.
Bacteria that ferment lactose cause the pH of the media to drop and the resultant change in pH is detected by
neutral red, which is red in color at pH's below 6.8. As the pH drops, neutral red is absorbed by the bacteria,
which appear as bright pink to red colonies on the agar.
The color of the medium surrounding Gram negative bacteria may also change. Strongly lactose fermenting
bacteria produce sufficient acid to cause precipitation of the bile salts, resulting in a pink halo in the medium
surrounding individual colonies or areas of confluent growth. Bacteria with weaker lactose fermentation
growing on MacConkey agar will still appear pink to red but will not be surrounded by a pink halo in the
surrounding medium.
Gram-negative bacteria that grow on MacConkey agar but do not ferment lactose appear colorless on the
medium and the agar surrounding the bacteria remains relatively transparent.
Lactose can be replaced in the medium by other sugars and the abilities of gram-negative bacteria to ferment
these replacement sugars is detectable in the same way as is lactose fermentation (for example Farmer and
Davis, 1985).
RECIPE
Peptone (Difco) or Gelysate (BBL) 17.0 g
Proteose peptone (Difco) or Polypeptone (BBL) 3.0 g
Lactose 10.0 g
NaCl 5.0 g
Crystal Violet 1.0 mg
Neutral Red 30.0 mg
Bile Salts 1.5 g
Agar 13.5 g
Distilled Water Add to make 1 Liter
Adjust pH to 7.1 +/-0.2. Boil to dissolve agar. Sterilize at 121° C for 15 minutes. (Holt and Krieg, 1994, Remel
2005)
PROTOCOL
Streak a plate of MacConkey's agar with the desired pure culture or mixed culture. If using a mixed culture use a
streak plate or spread plate to achieve colony isolation. Good colony separation will ensure the best
differentiation of lactose fermenting and non-fermenting colonies of bacteria.
Streak plate of Escherichia coli and Serratia marcescens on MacConkey agar. Both microorganisms grow on
this selective media because they are gram-negative non-fastidious rods. Growth of E. coli, which ferments
lactose, appears red/pink on the agar. Growth of S. marcescsens, which does not ferment lactose, appears
colorless and translucent.

COMMENTS AND TIPS

This section is to evolve as feedback on the protocol is discussed at ASMCUE. Please contact the project
manager for further information.

18
 [Medical Microbiology] [2022]

Figure A.2: Different types of bacterial growth on Mc Conkey agar. E. coli produces pink to red colonies
often with a reddish bile precipitate surrounding colonies on MacConkey's agar. E. aerogenes produces pink to
red mucoid colonies on MacConkey's agar. Gram-negative bacteria Proteus vulgaris and Salmonella
typhimurium grow on MacConkey's agar, but do not ferment lactose (media appears yellow to light pink in color
& colonies are colorless; swarming of Proteus is inhibited. Gram positive such as S. aureus is unable to grow on
Mc Conkey agar (source: Wikipedia).
(Mary E. Allen 2005)
TCBS agar
TCBS Agar is prepared according to the formula developed by Kobayashi, et al. It is highly selective for the
isolation of V. cholerae and V. parahaemolyticus , in addition to other Vibrio spp. TCBS has a very high pH
(8.5-9.5) which suppresses growth of intestinal flora other than Vibrio spp. The medium consists of plant and
animal proteins, a mixture of bile salts, one percent sodium chloride, sodium thiosulfate, ferric citrate, sucrose,
and yeast extract. The bile salts inhibit growth of Gram-positive microorganisms; one percent sodium chloride is
incorporated into the medium to provide optimum growth and metabolic activity of halophilic Vibrio spp.;
sodium thiosulfate provides a source of sulfur and also acts in combination with ferric citrate to detect the
production of hydrogen sulfide; sucrose serves as the fermentable carbohydrate that, with the help of
bromothymol blue and thymol blue indicators, allows for the differentiation of those Vibrio spp. which utilize
sucrose.
TCBS is not autoclaved because: This media contains Ox bile (Oxgall), a derivative of bile salts which inhibit
gram-positive bacteria might be sensitive while autoclaving. This agar needs to be boiled and not autoclaved to
avoid the caramelization (browning) of sucrose. Autoclaving may precipitate and/or alter the chemistry of the
media.
Ox bile is manufactured from large quantities of fresh bile by rapid evaporation of the water content. Bile is
composed of fatty acids, bile acids, inorganic salts, sulfates, bile pigments, cholesterol, mucin, lecithin,
glycuronicacids, porphyrins, and urea. The use of Oxbile insures a regular supply of bile, and uniformity
impossible to obtain with fresh materials.
Ox bile is used in bacteriological media formulations for gastro-intestinal tract organisms, specifically for
differentiation of bile tolerant microorganisms. A 10% solution of dehydrated bile is equivalent to a fresh bile
solution. It is usually incorporated into media e.g., Bile Esculin Agar and Brilliant Green Bile Agar, used for the
determination of enteric pathogens. Oxbile is also found in Littman Agar, a selective fungal medium.
V. cholerae and its biotype Eltor ferment sucrose, which results in a pH shift and production of yellow-brown
colonies. According to Fishbein, et al., V. parahaemolyticus will produce light bluish colonies. Certain strains
of Proteus and enterococci may grow and produce small, yellow colonies that are easily distinguished.
(Source: From Hardydiagnostics)
MSA agar
Mannitol Salt Agar contains peptones and beef extract, which supply nitrogen, vitamins, minerals and amino
acids essential for growth. The 7.5% concentration of sodium chloride results in the partial or complete
inhibition of bacterial organisms other than staphylococci. Sodium chloride also supplies essential electrolytes
for transport and osmotic balance. Mannitol is the fermentable carbohydrate, fermentation of which leads to
acid production, detected by phenol red indicator, aids in the differentiation of staphylococcal species.
Coagulase positive staphylococci (e.g., Staphylococcus aureus) produce yellow colonies and a surrounding
yellow medium while coagulase negative staphylococci produce red colonies and no color change of the phenol
red indicator. Micrococci can also grow yielding large white or orange colonies. Gram negatives are usually
inhibited or only show trace growth.

19
 [Medical Microbiology] [2022]

Addition of 5% v/v Egg Yolk Emulsion enables the detection of lipase activity of staphylococci along with
mannitol fermentation. The salt clears the egg yolk emulsion and lipase production is detected as yellow opaque
zone around the colonies.
(Source: From https://microbiologyinfo.com)
Cetrimide agar
Cetrimide agar is a type of agar used for the selective isolation of the Gram-negative bacterium, Pseudomonas
aeruginosa. As the name suggests, it contains cetrimide, which is the selective agent against alternate microbial
flora.Cetrimide also enhances the production of Pseudomonas pigments such as pyocyanin and pyoverdine,
which show a characteristic blue-green and yellow-green colour, respectively.
Cetrimide agar is widely used in the examination of cosmetics, pharmaceuticals and clinical specimens to test
for the presence of Pseudomonas aeruginosa.
A.3. Biochemical tests
Catalase (using H2O2 as the detecting reagent):
Microorganisms able to live in oxygenated environments produce enzymes which neutralize toxic forms of
oxygen. One such enzyme is catalase, which breaks hydrogen peroxide into water and molecular oxygen.
Organisms which produce catalase will bubble when placed into hydrogen peroxide. The catalase test is
primarily used to distinguish among Gram-positive cocci: Member of the genus Staphylococcus are catalase-
positive, and members of the genera Streptococcus and Enterococcus are catalase-negative.
Limitation of the test:
• Hydrogen peroxide is unstable and should undergo a control check daily prior to use.
• Growth for catalase testing must be taken from an 18-24 hour culture. Organisms lose their catalase
activity with age, resulting in a false-negative reaction.
• Catalase activity is a function of aerobic process. Organisms incubated anaerobically must be exposed
to atmospheric oxygen for a minimum of 30 minutes before a catalase test is performed. Failure to
complete this step may produce false-negative results.
• A positive catalase reaction with anaerobic organisms may be delayed for up to a minute after addition
of the reagent.
• A weak catalase or pseudocatalase reaction may be produced by some strains of Aerococcus species
and Enterococcus species.
Hints and precaution:
• Dispose the hydrogen peroxide slides in the appropriate container filled with disinfectant.
• Nichrome wire should be used when testing the organism. Platinum wires may cause a false-positive
reaction.
• When using a slant for other purposes in the same laboratory period, it is possible to save material by
adding H2O2 to the slant after finishing with it.
• Extreme care must be taken if a colony is taken from a blood agar plate. Erythrocytes contain catalase,
and a transfer of only a few blood cells can give a false-positive reaction.
• Always use fresh hydrogen peroxide, since it is unstable.
• Do not add organism to reagent, particularly if iron-containing inoculating loops are used. Iron
containing loops will cause false positive test results if exposed to hydrogen peroxide.

Table A.1: Catalase production property of some bacteria (source: Wikipedia).

20
 [Medical Microbiology] [2022]

Figure A.3: Detection of Catalase production. Staphylococcus and Micrococcus. Visible bubbles on the right
indicate a Catalase-positive organism (e.g. Staphylococcus and Micrococcus). The test on the left is Catalase-
negative (e.g. Streptococcus and Enterococcus). (source: Wikipedia).

Coagulase (Using Coagulase plasma as detecting reagent: eg. Coagulase plasma, rabbits with
EDTA-BD sciences);
The coagulase test is used to differentiate the potentially pathogenic species Staphylococcus aureus from the
usually non-pathogenic species Staphylococcus epidermidis. The presence of coagulase results in the formation
of a clot in a tube of citrated platelet-rich plasma (~ >150 x 106 platelets/cc plasma). The citrate is an
anticoagulant that is added to avoid autoclotting.
Application
1. The coagulase test is used to distinguish between pathogenic and nonpathogenic members of the genus
Staphylococcus. All pathogenic strains of S. aureus are coagulase positive whereas the nonpathogenic species
(S. epidermidis) are coagulase negative.
2. While slide coagulase test is useful in screening, tube coagulase test is useful in confirmation of
coagulase test.
3. Not all S.aureus strains produce coagulase; such rare strains are identified by thermonuclease test.
Limitation:
1. The slide test should be read very quickly, as false positives can occur.
2. Auto agglutination may occur.
3. Use water instead of saline for mixing.
4. The slide test should not be performed with organisms taken from high-salt media such as Mannitol
Salt Agar, as the salt content can create false positives.
5. Over mixing may cause the clot to break down.
6. The tube test is more reliable than the slide test.
7. We generally don’t use the coagulase test when identifying unknowns.
8. Samples must be observed for clotting within 24 hours. This is because some strains that produce
coagulase also produce an enzyme called fibrinolysin, which can dissolve the clot. Therefore, the absence of a
clot after 24 hours is no guarantee that a clot never formed. The formation of a clot by 12 hours and the

21
 [Medical Microbiology] [2022]

subsequent disappearance of the clot by 24 hours could produce a so-called false negative if the test were only
observed at the 24-hour time.

Figure A.4: The enzyme Coagulase is present in S. aureus but not in S. epidermidis. Agglutination of rabbit
plasma on the right (or above tube) is a positive result for Coagulase. The smooth emulsion on the left (or below
tube) is negative for Coagulase. (Source: Wikipedia).

Oxidase test (Oxidase reagent Dropper-BD sciences)

Figure A.5. This is a mixed culture of oxidase-negative Escherichia coli and

oxidase-positive Vibrio cholerae showing how the direct oxidase test
differentiates between the two organisms. Kovács oxidase reagent was added
directly to the plate.

A.4. Gram staining

History
The Gram stain was first used in 1884 by Hans Christian Gram (Gram,1884). Gram was searching for a method
that would allow visualization of cocci in tissue sections of lungs of those who had died of pneumonia. Already
available was a staining method designed by Robert Koch for visualizing turbercle bacilli. Gram devised his
method that used Crystal Violet (Gentian Violet) as the primary stain, an iodine solution as a mordant followed
by treatment with ethanol as a decolorizer. This staining procedure left the nuclei of eukaryotic cells in tissue
samples unstained while the cocci found in the lungs of those who had succumbed to pneumonia were stained
blue/violet. Gram found that his stain worked for visualizing a series of bacteria associated with disease such as
the “cocci of suppurative arthritis following scarlet fever”. He found however that Typhoid bacilli were easily
decolorized after the treatment with crystal violet and iodine, when ethanol was added. We now know that those
organisms that stained blue/violet with Gram’s stain are gram-positive bacteria and include Streptococcus
pneumoniae (found in the lungs of those with pneumonia) and Streptococcus pyogenes (from patients with
Scarlet fever) while those that were decolorized are gram-negative bacteria such as the Salmonella Typhi that is
associated with Typhoid fever.
You may read the original publication of the staining procedure in the translated article "The Differential
Staining of Schizomycetes in tissue sections and in dried preparations".
Purpose
The Gram stain is fundamental to the phenotypic characterization of bacteria. The staining procedure
differentiates organisms of the domain Bacteria according to cell wall structure. Gram-positive cells have a thick
peptidoglycan layer and stain blue to purple. Gram-negative cells have a thin peptidoglycan layer and stain red
to pink.
Theory
The Gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining
procedure. Through a series of staining and decolorization steps, organisms in the Domain Bacteria are
differentiated according to cell wall composition. Gram-positive bacteria have cell walls that contain thick
layers of peptidoglycan (90% of cell wall). These stain purple. Gram-negative bacteria have walls with thin
layers of peptidoglycan (10% of wall), and high lipid content. These stain pink. This staining procedure is not
used for Archeae or Eukaryotes as both lack peptidoglycan. The performance of the Gram Stain on any sample
requires four basic steps that include applying a primary stain (crystal violet) to a heat-fixed smear, followed by
the addition of a mordant (Gram’s Iodine), rapid decolorization with alcohol, acetone, or a mixture of alcohol

22
 [Medical Microbiology] [2022]

and acetone and lastly, counterstaining with safranin.

Details of the chemical mechanism of the Gram stain were determined in 1983 (Davies et al.,1983 and
Beveridge and Davies, 1983). In aqueous solutions crystal violet dissociates into CV + and Cl – ions that
penetrate through the wall and membrane of both gram-positive and gram-negative cells. The CV+ interacts with
negatively charged components of bacterial cells, staining the cells purple. When added, iodine (I- or I3-)
interacts with CV+ to form large CVI complexes within the cytoplasm and outer layers of the cell. The
decolorizing agent, (95% ethanol or an ethanol 95% and acetone 95% solution (1:1), interacts with the lipids of
the membranes of both gram-positive and gram-negative Bacteria. The outer membrane of the gram-negative
cell is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of
peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of gram-
positive cells (Dmitriev, 2004).With ethanol treatment, gram-negative cell walls become leaky and allow the
large CV-I complexes to be washed from the cell. The highly cross-linked and multi-layered peptidoglycan of
the gram-positive cell is dehydrated by the addition of ethanol. The multi-layered nature of the peptidoglycan
along with the dehydration from the ethanol treatment traps the large CV-I complexes within the cell. After
decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple
color and is only revealed when the counterstain, the positively charged dye safranin, is added. At the
completion of the Gram stain the gram-positive cell is purple and the gram-negative cell is pink to red.
Some bacteria, after staining with the Gram Stain yeild a pattern called gram-variable where a mix of pink and
purple cells are seen. The genera Actinomyces, Arthrobacter, Corynebacterium, Mycobacterium, and
Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in gram-
negative staining of these gram-positive cells. In cultures of Bacillus, Butyrivibrio, and Clostridium a decrease
in peptidoglycan thickness during growth coincides with an in increasing number cells that stain gram-negative
(Beveridge, 1990). In addition, in all bacteria stained using the Gram stain, the age of the culture may influence
the results of the stain.
Some bacteria do not stain as expected with the Gram stain. For example, members of the genus Acinetobacter
are gram-negative cocci that are resistant to the decolorization step of the Gram stain. Acinetobacter spp. often
appear gram-positive after a well prepared Gram stain (Visca et al. 2001). For Mycobacterium spp., the waxy
nature of the coat renders the bacteria not readily stainable with dyes used in the Gram stain, though the bacteria
are considered to be gram positive (Saviola and Bishai, 2000). Gardnella has an unusual gram-positive cell wall
structure that causes bacteria of this genus to stain gram-negative or gram-variable (Sadhu et al 1989).
Misinterpretation of the Gram stain has led to misdiagnosis or delayed diagnosis of infectious disease (Visca et
al., 2001, Noviello et al., 2004 )
RECIPE
(Gephardt et al., 1981)This is Hucker’s modification of the Gram Stain method. Gram originally used Gentian
Violet as the primary stain in the Gram stain. Crystal violet is generally used today. In Hucker’s method
ammonium oxalate is added to prevent precipitation of the dye (McClelland, 2001) and uses an alcoholic
solution of the counterstain. Burke’s modification of the Gram Stain adds sodium bicarbonate to the crystal
violet solution. Sodium bicarbonate prevents the acidification of the solution as iodine oxidizes (McClelland,
2001) and uses an aqueous solution of Safranin for the counterstain (Gephardt et al., 1981).
The reagents listed below can be made or purchased commercially from biological supply houses.

1. Primary Stain: Crystal Violet Staining Reagent.

Solution A for crystal violet staining reagent
Crystal violet (certified 90% dye content), 2g
Ethanol, 95% (vol/vol), 20 ml
Solution B for crystal violet staining reagent
Ammonium oxalate, 0.8 g
Distilled water, 80 ml
Mix A and B to obtain crystal violet staining reagent. Store for 24 h and filter through paper prior to use.
2. Mordant: Gram's Iodine
Iodine, 1.0 g
Potassium iodide, 2.0 g
Distilled water, 300 ml
Grind the iodine and potassium iodide in a mortar and add water slowly with continuous grinding until the
iodine is dissolved. Store in amber bottles.
3. Decolorizing Agent
Ethanol, 95% (vol/vol)
*Alternate Decolorizing Agent
Some professionals prefer an acetone decolorizer while others use a 1:1 acetone and ethanol mixture.
Commercially, a variety of mixtures are available, most using 25 – 50% acetone with the ethanol. A few include

23
 [Medical Microbiology] [2022]

a small quantity of isopropyl alcohol and/or methanol in the formulation.

Acetone, 50 ml
Ethanol (95%), 50 ml
4. Counterstain: Safranin
Stock solution:
2.5g Safranin O
100 ml 95% Ethanol
Working Solution:
10 ml Stock Solution
90 ml Distilled water
OR: Another option
Solutions:
Hucker's crystal violet reagent is made by mixing two solutions:
Solution A: Solution B:
Crystal violet 2.0g Ammoniumoxalate 0.8g
Ethanol (95%) 20.0ml Distilled water 80.0ml
This mixture is stable and can be kept at room temperature for months. An iodine solution and counterstain are
also required:
Stabilised Lugol- PVP complex: Counterstain:
Iodine 1.3g Safranin 0.25g
KI 2.0g Ethanol (95%) 10.0ml
Polyvinylpyrrolidone 1.0g Distilled water 100.0ml
Add distilled water to 100ml

PROTOCOL (Gephardt et al, 1981, Feedback from ASMCUE participants, ASMCUE , 2005)
1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note that the
quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results.
2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
3. Flood slide with the mordant: Gram's iodine. Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
5. Flood slide with decolorizing agent. Wait 15 seconds or add drop by drop to slide until decolorizing agent
running from the slide runs clear (see Comments and Tips section).
6. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry
with absorbent paper.
8. Observe the results of the staining procedure under oil immersion using a Brightfield microscope. At the
completion of the Gram Stain, gram-negative bacteria will stain pink/red and gram-positive bacteria will stain
blue/purple.

COMMENTS AND TIPS

Comments and tips come from discussions at ASM Conference for Undergraduate Educators 2005.
The thickness of the smear used in the Gram stain will affect the result of the stain. The step that is most
crucial in effecting the outcome of the stain is the decolorizing step. Over-decolorizing will lead to an
erroneous result where gram-positive cells may stain pink to red indicating a gram-negative result, and under-
decolorizing will lead to an erroneous result where gram-negative cells may appear blue to purple indicating a
gram-positive result. The degree of decolorizing required is determined by the thickness of the smear (number
of cells on the slide). The Gram stain was discussed in detail at the American Society for Microbiology
Conference for Undergraduate Educators in 2005 when this site was reviewed. The group recommends that cells
be prepared with a thin smear with no areas of clumping or inconsistency. When staining the thin smear a short
decolorizing time should be used. Some individuals will flood the slide for 15 seconds or less with decolorzing
agent, while others recommend adding decolorizing agent drop wise for 5- 15 seconds or until the color of the
decolorizing agent running from the slide no longer shows any color.
It is recommended that young, actively growing cultures be used for gram staining. An intact cell wall is
required for an accurate gram stain. Older cultures may have breaks in the cell wall and often give gram-variable
results where a mixture of pink/red cells are seen among blue/purple cells.
Using a gram stain control is recommended. On the same slide as the test culture include a sample of cells with

24
 [Medical Microbiology] [2022]

a known gram stain reaction to serve as a control for success in the gram stain technique.
Gram-stained bacteria should be viewed with a brightfield microscope at 1000X magnification with oil
immersion. If the smear of cells is crowded it will be difficult to note cell shape and arrangement.
When viewing slides use brightfield microscopy and adjust the brightness sufficiently to reveal the color of the
specimen.
Freshly made staining reagents are recommended. With older staining reagents, filter stains before use.
In the Gram Stain technique, two positively charged dyes are used: crystal violet and safranin. The use of the
designation “gram-positive” should not be confused with the concept of staining cells with a simple stain that
has a positive charge.
KOH string test may be used as a confirmatory test for the Gram Stain (Powers, 1995, Arthi et al., 2003): The
formation of a string (DNA) in 3% KOH indicates that the isolate is a gram-negative organism.
Procedure:
Place a drop of 3% KOH onto a glass slide.
Emulsify in KOH a loopful of the culture from a BA incubated for 18-24 hours.
Continue to mix the suspension for 60 sec and by slowly lifting the loop, observe for the formation of a string.
Interpretation:
Gram-negative cells form a string within 60 seconds.
Gram-positive cells are not affected.
Various formulations of decolorizing agents may be used (acetone, acetone/ethanol, ethanol). Acetone is the
most rapid decolorizer followed by acetone/ethanol and then ethanol. Ethanol is recommended for student use to
prevent over-decolorization of samples (McClelland, 2001)
When the counterstain is added this positively charged dye will replace the crystal violet dye in the gram-
positive bacteria as well as stain the gram-negative bacteria, although the presence of the mordant slows this
process considerably it is important not to overexpose cells to counterstain (McClelland, 2001)

Figure A.6: A representative Gram staining result. Gram-

positive (blue/purple) rods (Left) and Gram-negative
(pink/red) rods (Right).

B. E test for antibiotic sensitivity testing- a modified disc agar diffusion

method.
The Epsilometer test (usually abbreviated Etest) is a laboratory test used by microbiologists to determine
whether or not a specific strain of bacterium or fungus is susceptible to the action of a specific antibiotic. This is
most commonly used in the setting of medicine, where a particular organism has been found to infect a patient,
and the doctor treating the patient is seeking guidance on what concentration of antibiotic is suitable.
The Etest utilises a rectangular strip that has been impregnated with the drug to be studied. A lawn of bacteria is
spread and grown on an agar plate, and the Etest strip is laid on top; the drug diffuses out into the agar,
producing an exponential gradient of the drug to be tested. There is an exponential scale printed on the strip.
After 24 hours of incubation, an elliptical zone of inhibition is produced and the point at which the ellipse meets
the strip gives a reading for the minimum inhibitory concentration (MIC) of the drug.

Figure A.7: E-test of N. gonorrhoeae

25
 [Medical Microbiology] [2022]

C. Different cell lysis buffers (wikipedia)

NP-40 lysis buffer
It may be the most widely used lysis buffer. The solubilizing agent is NP-40, which can be replaced by other
detergents at different concentrations. Since NP-40 is a nonionic detergent, this lysis buffer has a milder effect
than RIPA buffer. It can be used when protein functions are to be retained with minimal disruption.
150 mM NaCl
1.0% Nonidet P-40 (NP-40)
50 mM Tris-Cl
Adjust pH to 7.4
RIPA (RadioImmunoPrecipitation Assay) lysis buffer
RIPA buffer is a commonly used lysis buffer for immunoprecipitation and general protein extraction from cells
and tissues. The buffer can be stored without vanadate at 4 °C for up to 1 year. RIPA buffer releases proteins
from cells as well as disrupts most weak interactions between proteins.
1% (w/w) Nonidet P-40 (NP-40)
1% (w/v) sodium deoxycholate
0.1% (w/v) SDS
0.15 M NaCl
0.01 M sodium phosphate, pH 7.2
2 mM EDTA
50 mM sodium fluoride (NaF)
0.2 mM fresh sodium orthovanadate (Na3VO4.2H2O, it has phosphatase inhibitor function
because it mimics phosphate)
100 U/ml protease inhibitor, such as aprotinin
SDS (sodium dodecyl sulfate) lysis buffer
SDS is ionic denaturing detergent. Hot SDS buffer is often used when the proteins need to be completely
solubilized and denatured.
0.5% (w/v) SDS
0.05 M Tris⋅Cl
Adjust pH to 8.0
Add 1 mM fresh dithiothreitol (DTT)
ACK (Ammonium-Chloride-Potassium) lysing buffer
ACK is used for lysis of red blood cells in biological samples where other cells such as white blood cells are of
greater interest.
150mM ammonium chloride
10mM potassium bicarbonate
0.1mM EDTA
Adjust pH to 7.2-7.4
D. DNA ladders
Figure A.8: DNA ladder. Tech and Innovation Inc., Korea

26
 [Medical Microbiology] [2022]

E. BERGEY’S MANUAL OF DETERMINATIVE BACTERIOLOGY

(attached)

F. KEYs
Lesson Nr 2
1. B, identifying a bacteria isolate
2. A, isolates since they must be the same species and even same strain (since they differ minimally) but
they do differ specifically in where or when they were isolated.
3. G, none of the above. Ideally, at least, there should be no difference between the bacteria making up
the two cultures.
4. A, strains. The lack of phenotypic differences is consistent with they not being serovars or biovars,
both kinds of strains.
5. You are attempting to identify an unknown bacteria; you are interested in knowing the characteristics
of an identified microorganism.
6. A-. c serovars; B-e, biovars; C-a, strains; D-b, isolates; E-a, strains or d, biovar; F-e, none of the above.
7. A, C, E and also, even, D: an isolate, morphovar, biovar (since morphovars are also biovars), and
strain.
8. A- establishment of clonality, B epidemiological reasons, C determination of whether an epidemic is
occurring due to a change in an organism (increase in virulence---establishment of clonality), or some
sort of organism-independent procedural change, D linking of contamination to potential source, etc.
9. antibody
10. type strain
11. isolates

REFERENCES

1. American Society for Microbiology Conference for Undergraduate Educators (ASMCUE). 2005.
[cited August 10, 2005]
2. Arthi K, Appalaraju B, Parvathi S. Vancomycin sensitivity and KOH string test as an alternative to
gram staining of bacteria. Indian J Med Microbiol 2003;21:121-123
3. Anderson, G.K. Beveridge, T. J., and H. C. Clark. 1983. Chemical Mechanism of the gram stain and
synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the
stain. J. Bacteriol. 156 (2):837-845.
4. Beveridge T.J, Davies J.A. (1983) Cellular responses of Bacillus subtilis and Escherichia coli to the
Gram stain. J Bacteriol. 1983 Nov;156(2):846-58.
5. Beveridge, T.J. 1990. Mechanism of Gram Variability in Select Bacteria J. Bacteriol. 172(3): 1609-
1620.
6. Davies, J. A., Anderson, G.K., Beveridge, T. J., and H. C. Clark. 1983. Chemical Mechanism of the
gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine
mordant of the stain. J. Bacteriol. 156 (2):837-845.
7. Dmitriev, B.A., Toukach, F.V., Holst,O., Rietshel,E.T., and Ehlers, S. 2004. Tertiary Structure of
Staphylococcus aureus cell wall murein. J. Bacteriol. 186 (21): 7141-7148.
8. Gephart, P., R.G. E. Murray, R. N.Costilow, E.W., Nester, W.A. Wood, N.R. Krieg, and G. B
Phillips. 1981. Manual of Methods for General Bacteriology, ASM Press, Washington D.C. Gram. C. 1884.
Ueber die isolirte Färbung der Schizomyceten in Schnitt-und Trockenpräparaten. Fortschritte der Medcin,
(2):185-189. Available at: (pdf)
9. Manisha Mehrotra, Gehua Wang, Wendy M. Johnson. Multiplex PCR for Detection of Genes for
Staphylococcus aureus Enterotoxins, Exfoliative Toxins, Toxic Shock Syndrome Toxin 1, and Methicillin
Resistance. J Clin Microbiol. 2000 March; 38(3): 1032–1035.
10. Noviello S, Gallo R, Kelly M, Limberger RJ, DeAngelis K, Cain L, et al. Laboratory-acquired
brucellosis. Emerg Infect Dis [serial on the Internet]. 2004 Oct [cited August 6,2005]. Available from
http://www.cdc.gov/ncidod/EID/vol10no10/04-0076.htm
11. McClelland, R., 2001. Gram’s stain the key to microbiology. Medical Laboratory Observer [serial on
the internet]. April 2001 [cited August 6, 2005] Available from: http://www.mlo-online.com/ce/pdfs/apr01.pdf.

27
 [Medical Microbiology] [2022]

12. Microbe Notes, https://microbenotes.com/

13. Powers, E.M., Efficacy of the Ryu nonstaining KOH technique for rapidly determining gram
reactions of food-borne and waterborne bacteria and yeasts. Appl Environ Microbiol. 1995 October; 61(10):
3756–3758.
14. Saviola, B., and Bishai, W. “The Genus Mycobacteria-Medical” in M. Dworkin et al., eds., The
Prokaryotes: An Evolving Electronic Resource for the Microbiological Community, 3rd edition, release 3.4,
December 1, 2000, Springer-Verlag, New York, http://link.springer-ny.com/link/service/books/10125/.
15. Sadhu, K., P. A. G. Dominigue, A. W. Chow, J. Nellingan, N. Cheng. and J. W. Costerton; 1989.
Gardnerella vaginalis has a Gram-positive cell-wall ultrastructure and lacks classical cell-wall
lipopolysaccharide J. Med. Microbiol., vol. 29, pp. 229–235.
16. Visca, P, Petrucca, A., De Mori, P., Festa, A., Evangelo, B., Antinori, A., and Petrosillo, N., (2001)
Community –Acquired Acinetobacter radioresistens Bacteremia in an HIV-Positive Patients. Emerging
Infectious Disease 7 (6):1032 – 1035. http://www.cdc.gov/ncidod/eid/vol7no6/visca.htm
17. Wikipedia, https://en.wikipedia.org

28