Name: _____________________________

                                                                                               Section: _____________ 

MCB101  Introductory Microbiology Lab                                    
Exam – 2                                                                      Fall 2007
For each question choose the one best answer.

Multiple Choice      (25 Questions, 4 points each)

1) Which one of the following statements about carbohydrate fermentation tests is false?

A.  Bacteria that obtain energy by breaking down amino acids excrete basic wastes.
B.  Bacteria that grow by fermenting a sugar usually excrete some acidic waste product.
C.  In MCB 101, the carbohydrate fermentation broth we use contains a Durham tube.  
D.  In MCB 101, the carbohydrate fermentation broth we use contains the pH indicator
     dye brom­thymol blue.
E.  If the bacterium cannot use the sugar in the carbohydrate fermentation broth, 
they
     can’t grow in that medium.

2)  What substance(s) in lactose fermentation broth can act as a carbon source?

A.  The only source for carbon in lactose fermentation broth is lactose.
B.  There are two carbon sources in lactose fermentation broth, lactose and glucose.
C.  There are two carbon sources in lactose fermentation broth, lactose and amino 
acids.
D.  There are three carbon sources in this broth, lactose, ammonium sulfate and vitamins.
E.  The carbon source in lactose fermentation broth is carbon dioxide.

3)  Which one of the following statements best describes the appearance of a reliable
      positive result for a carbohydrate fermentation test.

A.  The medium has turned red.
B.  The medium is cloudy and yellow.
C.  The medium is clear and does not change color.
D.  The medium is cloudy and greenish­blue.
E.  No bubble appears in the Durham tube.

4)  In any bacterial identification scheme, which one of the following procedures must 
     be done before doing biochemical tests?

A.  Prepare a Gram stain of the bacteria.
B.  Obtain a pure culture of the bacteria.
C.  Incubate a plate of L­agar at 37 degrees C.
D.  Examine the bacteria with a microscope.
E.  Grow the bacteria on PEA agar.
5)  A positive result for the catalase test is the appearance of bubbles of oxygen when a 
reagent is placed on the bacterial smear.  What is the reagent used in the catalase test? 
 
A.  methyl red                      B.  hydrogen sulfide                  C.  Kovac’s reagent              
D.  hydrogen peroxide            E.  acidified iron chloride   

6)  Kligler’s iron agar is used to test for the presence of the enzyme cysteine 
desulfhydrase, which catalyzes the breakdown of cysteine.  What product of this reaction 
reacts with the iron that is in the agar, to produce the black precipitate that is the sign of a
positive test?

A.  ammonia                    B.  pyruvate                    C.  hydrogen sulfide
D.  various acids              E.  carbon dioxide

7)  One of the biochemical tests we used to identify bacteria checks for the synthesis of 
the enzyme phenylalanine deaminase.  This enzyme causes phenylalanine to break down 
to form phenylpyruvate + _______________.

A.  ammonia                    B.  pyruvate                    C.  hydrogen sulfide
D.  various acids              E.  carbon dioxide

8)  When the amino acid lysine is broken down by lysine decarboxylase, 
        ______________ is formed and the medium turns ______________.

A.  cadaverine, purple                             B.  putresine, yellow 
C.  citrate, green                                      D.  ammonia,  blue 
E.  2­3 butanediol, does not change
9)  The Methyl Red test is used to check for the production of:

A.  tryptophanase.            B.  acetoin and 2,3­butanediol from glucose.
C.  citric acid.                   D.  large quantities of acid from fermentation of glucose.
E.  starch.

10)  Indole is made from:

A.  a sugar, lactose.                           B.  an amino acid, phenylalanine.
C.  an amino acid, tryptophan.          D.  an amino acid, lysine.
D.  a Krebs cycle intermediate, citrate.

11)  The Vogues Proskaur test uses the same type of medium as the __________ test.

A.  indole                                             B.  methyl red
C.  lysine decarboxylase                     D.  phenylalanine deaminase
E.  amylase

12)  The Voges­Proskauer test shows if a bacterium can produce _____________.

A. pyruvate from cysteine                  B. a mix of organic acids from glucose
C. cadaverine                                      D. phenylpyruvate from phenylalanine
E. 2,3­butanediol as a fermentation waste product

13) Bacteria in the genus Staphylococcus are Gram positive spheres or cocci.
Bacteria in the genus Streptococcus are Gram positive cocci too.
Which one of the following biochemical tests is the best way to tell the
difference between Streptococcus and Staphylococcus?
A. phenylalanine deaminase test B. methyl red test
C. hydrogen sulfide production D. catalase test
E. indole test

14) You set up a ten-tube Minimum Inhibitory Concentration test where the

concentration of the antibiotic in each tube is a 1/2 dilution of the previous tube.
If the concentration of the antibiotic in tube 1 is 3200 micrograms per ml.
What is the concentration of the antibiotic in tube 6?

A.  3200 g/ml                  B.  1600 g/ml                      C.  600 g/ml
D.  100 g/ml                    E.  6 g/ml

15) Serial Dilution: The Final Dilution Factor

In a three step serial dilution:
the first dilution is 1/10,
the second dilution is 1/5,
the third dilution is 1/4.
What is the total dilution factor of this three step serial dilution procedure?
A. 0.05 B. 0.005 C. 1/19 D. 19 E. 200

16)  If a viable plate count of a 0.1 ml sample yields 210 colonies
      and the dilution factor was 104, 
      what was the concentration of cells in the original sample? 

A.  2.1 x 104  cells/ml                 B.  2.1 x 106  cells/ml                C.  2.1 x 107  cells/ml
D.  210 x 104  cells/ml                E.  210,000 cells/ml 

17)  A bacterial culture is started with 60,000 cells and is allowed to grow for 30
        generations.   Determine the final number of cells in the culture.

A.   1.80 x 105                      B.   6.44 x 1013                      C.   6.00 x 1034
D.   1.20 x 1035                     E.   6.44 x 1036
Questions 18 and 19 refer to the information given in this table.

18)  You are attempting to identify an unknown enteric bacterium that is one of the 12 
species shown in the biochemical test key that is given above.  Your unknown bacterium 
is positive for the Voges­Proskauer test and positive for lactose fermentation.  Which one
of the following tests would allow you to unambiguously determine the identity of the 
unknown?

A.  arabinose fermentation              B.  indole test                     C.  urease test
D.  dulcitol fermentation                 E. esculin hydrolysis 

19)  You are attempting to identify an unknown enteric bacterium that is one of the 12 
species shown in the biochemical test key that is given above.  Your unknown bacterium 
is positive for dulcitol fermentation, negative for raffinose fermentation and positive for 
Simmon’s citrate.  
Which one of the following would you expect to be true?

A. Your unknown is positive for the indole test.
B.  Your unknown produces a black precipitate in Kligler’s iron agar.
C.  Your unknown is Citrobacter freundii.
D. Your unknown is positive for lactose fermentation.
E.  Your unknown can create a clear zone around the area of growth on egg yolk agar.  
20)  What is the generation time for the bacteria depicted in this growth curve?

A.  30 minutes        B.  60 minutes       C.  90 minutes          D.  5.5 hours        E.  7 hours 

21)  For the growth curve depicted below, the bacterial culture called ________ grows 
       the fastest while the bacterial culture called ________ shows the greatest number
       of doublings during the experiment.

A. P, P                   B. P, S                    C. S, S                      D. S, P
22)  Which one of the following bacteria is the best fit to this description?
         It is purple in the Gram stain.  The cells are shaped like little round balls.
         The cells are arranged in chains.  It is negative for the catalase test.
The normal habitat for this bacterium is the mammalian large intestine.

A. Bacillus subtilis B. Enterococcus faecium C. Escherichia coli

D. Pseudomonas aeruginosa E. Staphylococcus aureus

23) Which one of the following statements about the Kirby-Bauer disc diffusion test
for antibiotic susceptibility is true?

A. If the bacterium is resistant to an antibiotic there is a big clear zone around that disc.
B. If there is any clear zone at all around a given antidiotic disc, even a very small one,
the bacterium is considered to be resistant to that antibiotic.
C. The concentration of the antibiotic on a disc has no effect on the size of any clear
zone that may be seen around that disc.
D. If there is a big clear zone around a given antibiotic disc, that drug might be clinically
useful in treating an infection caused by the bacteria being tested.
E. If the bacterium is susceptible to any of the antibiotics on the discs it won’t grow on
the agar plate at all

24) You perform a MIC test using a new antibiotic, Hexacycline, against an old nemesis,
Bordetella pertussis the bacterium that causes whooping cough.
From the data shown, determine the MIC for Hexacycline against this bacteria.

Antibiotic Concentration (g/ml): 1000 500 250 125 62.5 31.3 15.6 7.8
Growth?: - - - - + + + +

A. 500 g/ml B. 250 g/ml C. 125 g/ml D. 62.5 g/ml E. 31.3 g/ml

25) Which one of the following statement about bacterial growth in broth culture is false?

A. After inoculating the broth, there may be a delay before you begin to see growth.
B. Calculations of the growth rate require data from the exponential phase.
C. A shorter generation time equals a faster growth rate.
D. The generation time is the time it takes for the bacterial population to double.
E. The bacterial population is at its’ maximum density during the middle of the log
phase.
MCB101  Microbiology         Exam – 2         Answer Key  

1) __ E ___        8) ___ A ___         15) ___ E ___        22) ___ B __

2) __ C ___        9) ___ D ___         16) ___ C ___        23) ___ D __

3) __ B ___       10) ___ C ___         17) ___ B ___       24) ___ C __

4) __ B ___       11) ___ B ___         18) ___ C ___       25) ___ E __

5) __ D ___       12) ___ E ___         19) ___ B ___  

6) __ C ___       13) ___ D ___         20) ___ C ___  

7) __ A ___       14) ___ D ___         21) ___ B ___  

The Collection of Clean Milk Samples (illustrated in Figure 1)

1. Wear gloves.
2. Remove (forestrip) three or four streams of milk from the quarter being sampled
to minimize chances of sample contamination from bacteria in the teat end.
3. Brush any dirt, debris, or bedding particles from the udder and teats. Predip with
an effective teat dip (for example, 0.5% iodine or 4% hypochlorite) leaving the predip
on the teat for at least 20 to 30 seconds before removal.
4. Dry each teat thoroughly, and remove the predip using a single, dry paper or cloth
towel per cow with particular emphasis on the teat end.
5. Double check to ensure that the teats and udder are clean and dry.
6. For 15 to 20 seconds, carefully and vigorously scrub the teat end and orifice with
a cotton or cloth gauze pad moistened (but not dripping wet) with 70 to 80% ethyl or
isopropyl alcohol. Use a separate swab for each teat being sampled, even within the
same cow. Continue to clean the teat end until the swab is completely clean and
 white. In order to prevent recontamination of teat ends, clean the teats on the far side
of the udder first followed by the teats on the near side of the udder.
7. Open the collection vial immediately before the sample is taken. Do not let the
teat end touch the container or let skin debris or dirt enter the container. Do not put
the cap on the floor. Keep the cap upside down, and do not touch the inside of the cap
so that no debris contaminates the inside of the cap. Hold the collection vial at a 45°
angle to keep debris (hair, manure, dirt) from accidentally falling into the collection
vial. Turn the teat toward the collection vial, striving for direct streams of milk into
the vial. The teat should never touch the collection vial or cap. Sample as rapidly as
possible, starting with the teats on the near side of the udder followed by the teats on
the far side of the udder.
8. You only need to collect 3 to 5 ml of milk (a few streams). Do not fill the
collection vial. Attempting to fill the collection vial increases the likelihood of
contamination. In addition, if a full collection vial is frozen, it may burst.
Immediately place the cap on the container and seal so it is airtight.

9. Label the sample vials using a waterproof marker that will not come off during
transport to the laboratory. Be sure to identify both the cow and quarter from which
the sample was obtained. Designate each quarter sampled as RF (right front), RR
(right rear), LF (left front), or LR (left rear).

10. Immediately place collection vial on ice and keep refrigerated or on ice until
delivered to the lab. Best results are obtained if samples are chilled or placed on ice
during transport to the laboratory. When samples cannot be delivered to the
laboratory within 24 hours, they should be frozen.

Citrate
Principle:
When an organic acid such as citrate (remember Krebs cycle) is used as a carbon and
energy source, alkaline carbonates and bicarbonates are produced ultimately. In addition,
ammonium hydroxide is produced when the ammonium salts in the medium are used as
the sole nitrogen source.
Utilization of exogenous citrate requires the presence of citrate transport proteins
(permeases). Upon uptake by the cell, citrate is cleaved by citrate lyase to oxaloacetate
and acetate. The oxaloacetate is then metabolized to pyruvate and CO2.
Citrate = oxaloacetate + acetate
oxalacetate = pyruvate + CO2
 Citrate Utilization Test
Further metabolic breakdown is dependent upon the pH of the medium.
A. Under alkaline conditions, pyruvate is metabolized to acetate and formate.
pyruvate = acetate + formate
B. At pH 7.0 and below, lactate and acetoin are also produced.
pyruvate = acetate + lactate + CO2
pyruvate = acetoin + CO2
The carbon dioxide that is released will subsequently react with water and the sodium ion
in the medium to produce sodium carbonate, an alkaline compound that will raise the
pH. In addition, ammonium hydroxide is produced when the ammonium salts in the
medium are used as the sole nitrogen source.
Growth usually results in the bromothymol blue indicator, turning from green to
blue. The bromothymol blue pH indicator is a deep forest green at neutral pH. With an
increase in medium pH to above 7.6, bromothymol blue changes to blue
Ureaese test
Medium used for urease test: Any urea medium, agar or broth (It can be a sole medium
or part of panel like motility indole urease (MIU) test)
Indicator used in urease test: Phenol red
Color change:
 Original: orange yellow color
 Final color (in positive test): Bright pink
Urease test principle
Urea is a diamide of carbonic acid. It is hydrolyzed with the release of ammonia and
carbon dioxide.
Many organisms especially those that infect the urinary tract, have an urease enzyme
which is able to split urea in the presence of water to release ammonia and carbon
dioxide. The ammonia combines with carbon dioxide and water to form ammonium
carbonate which turns the medium alkaline, turning the indicator phenol red from its
original orange yellow color to bright pink.

Procedure for urease test

 1. The broth medium is inoculated with a loopful of a pure culture of the test
organism; the surface of the agar slant is streaked with the test organism.
2. Leave the cap on loosely and incubate the test tube at 35 °C in ambient air for 18
to 24 hours; unless specified for longer incubation.
Result and Interpretation
Organisms that hydrolyze urea rapidly (e.g. Proteus spp) may produce positive reactions
within 1 or 2 hours; less active species (e.g. Klebsiella spp) may require 3 or more days.
In routine diagnostic laboratories the urease test result is read within 24 hours.
 If organism produces urease enzyme, the color of the slant changes from light
orange to magenta.
 If organism do not produce urease the agar slant and butt remain light orange
(medium retains original color).
Urease Test Results
Diagnostic utility of Urease test
Urease test is used for the presumptive evidence of the presence of Helicobacter pylori in
tissue biopsy material. This is done by placing a portion of crushed tissue biopsy material
directly into urease broth. A positive urease test is considered presence of Helicobacter
pylori. Commercially available urease agar kits are also available.
₋ Rapid Urease test is can be used to differentiate between the yeasts, Candida
albicans and Cryptococcus neoformans. A presumptive identification of C.
neoformans may be based on rapid urease production, whereas Candida
albicans do not.
₋ Urea breath test: A common noninvasive test to detect Helicobacter pylorialso
based on urease activity. This is highly sensitive and specific test.

Principle of Urea Breath Test:

Patient ingests radioactively labeled (13C or 14C) Urea. If infection is present, the urease
produced by Helicobacter pylori hydrolyzes the urea to form ammonia and labeled
bicarbonate that is exhaled as CO2. The labeled CO2 is detected either by a scintillation
counter or a special spectrometer.
Urease test is useful test:
This test can be used as part of the identification of several genera and species
of Enterobacteriaceae including Proteus and Klebsiella. It is also useful to
identify Cryptococcus species, Brucella, Helicobacter pylori.
Name of urease positive organisms (Bacteria)
1. Proteus spp
2. Cryptococcus spp
3. Corynebacterium spp
4. Helicobacter pylori
5. Brucella spp

Thử nghiệm kháng huyết thanh

+ Kiểm tra sự tự dính kết của các chủng;
Cho một giọt dung dịch nước muối sinh l. lên một phiến kính đ. rửa sạch. Dàn đều
khuẩn lạc cần thử nghiệm trong giọt dung dịch sao cho thu được một thể huyền
phù
đục đồng nhất. Lắc nhẹ phiến kính trong 30-60 giây.
Quan sát kết quả trên nền tối, tốt nhất nên dùng kính lúp.
Nếu các vi khuẩn vón lại thành các đơn vị ít nhiều có kết dính th. chủng này được
coi là tự kết dính và không cần phải thử tiếp v. việc phát hiện kháng nguyên không
thể thực hiện được.
+ Kiểm tra kháng nguyên O:
Sử dụng một khuẩn lạc thuần khiết không tự ngưng kết, tiến hành như phương
pháp kiểm tra sự tự kết dính ở trên, bằng cách thay một giọt kháng huyết thanh O
thay cho dung dịch nước muối.
Nếu xuất hiện dính kết, phản ứng được coi là dương tính. Sử dụng tuần tự một
kháng huyết thanh đa giá và đơn giá.
+ Kiểm tra kháng nguyên Vi:
Tiến hành như phương pháp kiểm tra sự tự kết dính bằng cách thay một giọt huyết
thanh Vi thay cho dung dịch nước muối.
Nếu xuất hiện dính kết, phản ứng coi là dương tính.
+ Kiểm tra kháng nguyên H:
Cấy từ 1 khuẩn lạc không tự dính kết thuần khiết lên thạch dinh dưỡng thể nữa đặc
(phần dính kèm), ủ ấm 18-24 giờ, 371oC.
Dùng giống này để kiểm tra kháng nguyên H, tiến hành như phương pháp kiểm tra
sự tự dính kết, nhưng sử dụng một giọt kháng huyết thanh H thay cho dung dịch
nước muối sinh lý. Nếu xuất hiện dính kết, phản ứng coi là dương tính.
LƯU Ý
Có thể sử dụng kháng huyết thanh đa giá OMA, OMB để phát hiện sự có mặt của
các kháng nguyên Salmonella. Trên 98% các chủng Salmonella cho phản ứng
ngưng kết với một trong hai kháng huyết thanh OMA, OMB. Do vậy khi thấy có
sự dính kết
với một trong hai kháng huyết thanh đa giá trên có thể kết luận là Salmonella.

Buffer (1%) Peptone water (BPW): a pre-enrichment medium designed to help

recovery of sub-lethally damaged bacteria that have been injured by processes of food
preservation before transfer to a selective medium cells.
➢ Proteose peptone: provides essential growing nutrient for microorganism.
➢ Sodium chloride: provide the osmotic balance condition for bacteria.
➢ Disodium phosphate and Monopotassium phosphate: buffering agents preventing
change
in pH during microbial growth which can produce acid.

Rappaport – Vassiliadis Soybean (RVS) broth: An enrichment medium designed to

stimulate the growth of Salmonella by creating an ideal condition for it.
➢ Papaic digest of soyabean meal: provides essential growth nutrients and enhances the
growth of Salmonella.
➢ Sodium chloride: provide the osmotic balance condition for bacteria.
➢ Potassium dihydrogen phosphate and Dipotassium hydrogen phosphate: buffering
agents
preventing change in pH during microbial growth which can produce acid.
➢ MgCl2.6H2O: increase the osmotic pressure.
➢ Malachite green: inhibits growth of most gram-positive bacteria, while selectively
enriches growth of Salmonella.

Xylose lysine desoxycholate (XLD) agar: is selective medium which is recommended for
the purpose of the enumeration and isolation of Salmonella spp. and Salmonella Typhi. 
XLD agar promote the growth of Shigella (white colonies) significantly in both 2
samples (pork and chicken) and Salmonellae bacteria (black colonies),
➢ Yeast extract: nitrogen and vitamins source
➢ L-lysine: differentiates the Salmonella and the non-pathogens.
➢ Lactose, sucrose and xylose: provide sources of fermentable carbohydrates.
➢ Sodium chloride: provide the osmotic balance condition for bacteria.
➢ Sodium dexycholate: inhibits growth of Gram-positive organisms.
➢ Sodium Thiosulfate and Ferric Ammonium Citrate: an H2S indicator system for the
visualization for the production of hydrogen sulphide.
➢ Phenol red: pH indicator.

Bismuth sulfite (BS) agar: is selective medium which is recommended for the purpose
of the isolation of Salmonella. Bismuth Sulfite Indicator and Brilliant Green are
complementary, inhibiting Gram-positive bacteria and coliforms, allowing Salmonella
spp. to grow.
➢ Peptic digest of animal tissue and beef extract: serve essential nutrients of growing
factor.
➢ Dextrose: carbon source.
➢ Disodium phosphate: provide osmotic equilibrium for Salmonella.
➢ Ferrous sulphate: detects hydrogen sulphide production.
➢ Bismuth sulphite: indicator along with brilliant green inhibit the intestinal Gram-
positive and Gram-negative bacteria.

Nutrient agar: basic culture media used for maintaining microorganisms, cultivating
fastidious organisms by enriching with serum or blood and are also used for purity
checking prior to biochemical or serological testing
➢ Peptic digest of animal tissue, beef extract and yeast extract: nitrogen compounds,
carbon,
vitamins and also some trace ingredients necessary for the growth of bacteria.
➢ Sodium chloride: maintains a salt concentration in the medium.

Triple sugar iron (TSI) agar: test for ability sugar fermentation, H2S and CO2
production of microorganism. for identification of members of Enterobacteriaceae
especially Salmonella species:
1. If lactose (or sucrose) is fermented, a large amount of acid is produced,
which turns the phenol red indicator yellow both in butt and in the slant. Some organisms
generate gases, which produces bubbles/cracks on the medium.
2. If lactose is not fermented but the small amount of glucose is, the oxygen deficient butt
will be yellow (remember that butt comparatively have more glucose compared to slant
i.e. more media more glucose),but on the slant the acid (less acid as media in slant is very
less) will be oxidized to carbondioxide and water by the organism and the slant will be
red (alkaline or neutral pH).
3. If neither lactose/sucrose nor glucose is fermented, both the but and the slant will be
red. The slant can become a deeper red-purple (more alkaline) as a result of production of
ammonia from the oxidative deamination of amino acids (remember peoptone is a major
constitutents of TSI Agar) .
4. if H2S is produced, the black color of ferrous sulfide is seen
➢ Peptic digest of animal tissue, beef extract and yeast extract: nitrogen compounds,
carbon,
vitamins and also some trace ingredients necessary for the growth of bacteria.
➢ Lactose, sucrose, and dextrose: fermentable carbohydrates checking fermenting
ability.
➢ Sodium chloride: provide the osmotic balance condition for bacteria.
➢ Ferrous sulphate and sodium Thiosulphate: H2S indicator
➢ Phenol red: pH indicator

Brain Heart Infusion (BHI) broth: highly nutritive medium, used to prepare the inocula
for antimicrobial susceptibility testing. Brain Heart Infusion (BHI) broth: used for
propagation of fastidious pathogenic cocci and other organism associated with blood
culture applied pathological investigation
➢ Calf brain, Beef heart infusion form, and Proteose peptone: Sources of essential
nutrients.
➢ Dextrose: Carbon source.
➢ Disodium phosphate: buffering agents preventing change in pH during microbial
growth
which can produce acid.
➢ Sodium chloride: provide the osmotic balance condition for bacteria.

Baird Parker (BP) agar: used for purpose of isolation and enumeration of
Staphylococci in food and other material since it allows a good differentiation of
coagulase positive strains. Principle that staphylococci are able to reduce tellurite to
tellurium and
to detect lecithinase from egg lecithin. Staphylococcus aureus reduces tellurite to form
grey-black shiny colonies and then produces clear zones around the colonies by
proteolytic action. This clear zone with typical grey-black colony is diagnostic
for Staphylococcus aureus
➢ Casein enzymeic hydrolysate, Meat extract B, Yeast extract : Nutrients sources.
➢ Glycine: enhances growth of Staphylococcus.
➢ Lithium chloride: inhibits other microflora that contaminated except S.aureus.
➢ Sodium pyruvate: acts as resuscitating agent, protects injured cells and supports the
recovery, stimulate S. aureus’s growth.
Egg yolk tellurite emulsion diagnostic agent; makes the medium yellow and opaque;
Staphylococcus aureus reduces tellurite to form grey-black shiny colonies
Egg yolk tellurite emulsion: served as identification of coagulase- positive
Staphylococci. Egg
yolk: differentiates Staphylococcus from others capable of growing in the base agar and
must be mixed well with the Baird Parker agar before inoculation.
Triptose sulphite cycloserin agar (TSC): with the addition of selective supplement and
enrichment is used for the presumptive identification and enumeration of Clostridium
perfringens from food.
Coagulase plasma: used for diagnosing Staphylococcus aureus in coagulase test.

Tryptose sulfite cycloserine (TSC) Agar: mixed with egg yolk emulsion, Enrichment
medium. Egg yolk emulsion promote the growth of Clostridium perfringens. D-
cycloserine inhibits bacteria flora.
➢ Meat peptone, Soya peptone and Yeast extract: sources of nutrients and vitamins for
the
development and growth of Clostridia.
➢ Sodium disulfite and Ammonium ferric citrate: Indicator for sulfite reduction, indicate
by black coloured colonies.

Lactose-gelatin medium (LGM): test for ability lactose fermentation and liquefaction of
microbe.
➢ Meat peptone and Yeast extract: sources of nutrients and vitamins for the development
and growth of Clostridia.
➢ Lactose: Carbon source.
➢ Phenol red: pH indicator.
➢ Disodium hydrogen phosphate: buffering agent.

Nitrate-motility medium (NMM): A test medium for presumptive C. perfringens that

identify the motility of microorganisms and calculate the nitrate reduction.
➢ Meat extract, Gelatin peptone and Galactose: Provide essential nutrients for growth.
➢ Potassium nitrate: Substrate for nitrate reduction, result of nitrate reduction checked
by
indole test with Kovac’s reagent.
➢ Disodium hydrogen phosphate: buffering agent.

Saline Peptone Water (SPW): a pre-enrichment medium designed to help recovery of

sublethally damaged bacteria that have been injured by processes of food preservation
before
transfer to a selective medium cells.

Lactobacillus MRS Agar: for the isolation, enumeration and cultivation of

Lactobacillus species.
➢ Proteose peptone, Beef extract: Nitrogen sources.
➢ Yeast extract: Vitamin B-complex source.
➢ Dextrose: fermentable carbonhydrate and energy source.
➢ Polysorbate 80: supply fatty acids for the metabolism of Lactobacilli.
➢ Ammonium citrate, Sodium acetate: inhibit the growth of Streptococci, molds and
other
microorganisms.
➢ Magnesium sulfate, Manganese sulfate: provide essential ions for multiplication of
Lactobacilli.
➢ Dipotassium phosphate: buffering agent.
Lactobacilli MRS medium is based on the formulation of deMan, Rogosa and Sharpe
with slight modification. It support luxuriant growth of allLactobacilli from dairy
products, foods, feces and other sources. Polysorbate fourscore supplies fatso acids
required for the metabolism of MRS agar at dilution
Lactobacilli. Na acetate rayon and ammonium citrate inhibit Streptococci,
moulds and many other microorganisms. Magnesium sulphate and Mn sulphate provide
requisite ion for multiplication of lactobacilli. MRS agar has cream to light yellow
semblance and Lactobacilli appear as large, white colonies in rice-shaped, embedded in
or on MRS agar. Growth can be sub-cultured onto appropriate media for use in additional
function .

Alkaline Saline Peptone water (ASPW): Pre-enrichment and enrichment broth used for
homogenizing sample.
➢ Peptone: provides carbonaceous, nitrogeneous and essential nutrients to the
organisms.
➢ Sodium chloride: Provide the osmotic balance condition for bacteria, also has an
inhibitory action on the accompanying microflora.

Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar: selective medium for isolating
Vibrio spp. After incubation, we see various coming into court of Vibrio species like
V.parahaemolyticus (blueing to super acid centered colony ), V.vulnificus (green
colonies), V. cholerae (yellow green or colourless) on TCBS agar plates.
- For one accurate plates in figure 1, we can see that the colonies coloring is yellow,
then they should be V.cholerae.
- Gramme - staining : The morphology of Vibrio was observed under x100 lens
microscope. The result is pinko color → gram-negative, curvature pole shape →
Vibrio species.
➢ Proteose peptone and yeast extract: provide nitrogenous compounds, vitamin B
complex
and other essential growth nutrients.
➢ Oxgall and sodium citrate: inhibit gram-positive bacteria and coliforms.
➢ Sodium thiosulphate ferric citrate: H2S indicator.
➢ Sucrose: fermentable carbohydrate, which colonies have yellow color.
➢ Sodium chloride: Provide the osmotic balance condition for bacteria, also has an
inhibitory action on the accompanying microflora.
➢ Bromo thymol Blue and Thymol Blue: pH indicator.

Saline Nutrient agar (SNA): selective and purify the culture of Vibrio spp.
➢ Peptone: provide and Meat extract: provide nitrogen compounds, growth factors and
vitamins.
➢ Sodium chloride: Provide the osmotic balance condition for bacteria, also has an
inhibitory action on the accompanying microflora.

Saline Triple Sugar Iron (TSI) agar: test for ability to sugar fermentation, H2S
production.
➢ Peptic digest of animal tissue and Beef extract: provide nitrogenous compounds,
sulphur, trace elements and vitamin B complex.
➢ Sodium chloride: Provide the osmotic balance condition for bacteria.
➢ Lactose, sucrose and glucose: fermentable carbohydrates.
➢ Sodium thiosulphate and ferric ions: H2S indicator.
➢ Phenol red: pH indicator.
➢ Bismulth sulphite indicator: inhibiting Gram-positive bacteria and coliforms.
➢ Briliant green: inhibiting Gram-positive bacteria and coliforms.

Sulphide Indole Motility (SIM) agar: test for motility ability of

microorganism.
➢ Beef extract: provide nutrients and Peptic digest of animal tissue:
provide nitrogenous
compounds, sulphur, trace elements and vitamin B complex.
➢ Peptonized iron and Sodium thiosulphate: H2S indicator.

Polyvalent O antiserum is used to psychometric test the isolate

before testing with Monovalent antisera to identify O antigen (s).
Polyvalent Sextuplet antiserum is used to detect Vi antigen(s), which is
added after the reaction with Polyvalent O antiserum is negative.
Salmonella species are serotyped according to their O (somatic) antigens, Vi (capsular) antigens, and H
(flagellar) antigens. The antigenic formula of Salmonella serotypes are listed in the Kauffman-White scheme
and are expressed as follows: O antigens; Vi when present; H antigens phase 1; H antigens phase 2 (when
present).

SELECTIVE AND DIFFERENTIAL MEDIA
Selective and differential media are used to isolate or identify particular organisms.
Selective media allow certain types of organisms to grow, and inhibit the growth of other
organisms. The selectivity is accomplished in several ways. For example, organisms that
can utilize a given sugar are easily screened by making that sugar the only carbon source
in the medium. On the other hand, selective inhibition of some types of microorganisms
can be achieved by adding dyes, antibiotics, salts or specific inhibitors which affect the
metabolism or enzyme systems of the organisms. For example, media containing
potassium tellurite, sodium azide or thallium acetate (at concentrations of 0.1 - 0.5 g/l)
will inhibit the growth of Gram-negative bacteria. Media supplemented with penicillin
(5-50 units/ml) or crystal violet (2 mg/l) will inhibit the growth of Gram-positive
bacteria. Tellurite agar, therefore, is used to select for Gram-positive organisms, and
nutrient agar supplemented with penicillin can be used to select for Gramnegative
organisms.
Differential media are used to differentiate closely related organisms or groups of
organisms. Owing to the presence of certain dyes or chemicals in the media, the
organisms will produce characteristic changes or growth patterns that are used for
identification or differentiation. A variety of selective and differential media are used in
medical, diagnostic and water pollution laboratories, and in food and dairy laboratories.
Three of the more common selective and differential media are described below and will
be used in the laboratory exercise.

MANNITOL SALT AGAR (MSA) Mannitol salt agar is a selective medium used for the
isolation of pathogenic staphylococci. The medium contains mannitol, a phenol red
indicator, and 7.5% sodium chloride. The high salt concentration inhibits the growth of
most bacteria other than staphylococci. On MSA, pathogenic Staphylococcus aureus
produces small colonies surrounded by yellow zones. The reason for this change in color
is that S. aureus ferments the mannitol, producing an acid, which, in turn, changes the
indicator from red to yellow. The growth of other types of bacteria is generally inhibited.
EOSIN METHYLENE BLUE AGAR (EMB agar) Eosin methylene blue agar is a
differential medium used for the detection and isolation of Gram-negative intestinal
pathogens. A combination of eosin and methylene blue is used as an indicator and allows
differentiation between organisms that ferment lactose and those that do not. Saccharose
is also included in the medium because certain members of the Enterobacteria or coliform
group ferment saccharose more readily than they ferment lactose. In addition, methylene
blue acts as an inhibitor to Gram-positive organisms. Colonies of E. coli normally have a
dark center and a greenish metallic sheen, whereas the pinkish colonies of Enterobacter
aerogenes are usually mucoid and much larger than colonies of E. coli. Other organisms,
such as Salmonella (one of the causative agents of food poisoning), do not ferment
lactose or saccharose and produce colonies that are noncolored.
MacCONKEY'S AGAR
MacConkey's agar is a differential plating medium used in the detection and isolation of
all types of dysentery, typhoid and paratyphoid organisms. It is generally used for
differentiating strains of Salmonella typhosa from members of the coliform group;
however, the medium supports the growth of all Salmonella and Shigella strains and
gives good differentiation between these enteric pathogens and the coliform group. When
grown on MacConkeyís medium, colonies of coliform bacteria are brick-red in color and
are surrounded by a zone of precipitated bile. These reactions are due to the acid
produced by the fermentation of lactose. The acid end-products act on bile salts, and
neutral red is absorbed by the precipitated salts. Dysentery, typhoid and paratyphoid
bacilli do not ferment lactose but give an alkaline reaction when grown on the medium.
Colonies of these organisms are noncolored and transparent. The growth of Grampositive
organisms is inhibited because of the crystal violet and bile salts in the medium.
Triple Sugar Iron Agar (TSIA) Test
The Triple Sugar Iron agar (TSIA) test is designed to differentiate among the different
groups or genera of the Enterobacteriaceae, which are all gram negative bacilli capable of
fermenting glucose with the production of acid, and to distinguish them from other gram
negative intestinal bacilli. The differentiation is based on fermentation of glucose and
lactose or sucrose and hydrogen sulfide (H 2S) production. TSIA medium contains 10
parts of lactose: 10 parts of sucrose: 1 part of glucose and peptone. Phenol red and
ferrous sulfate serve as indicators of acidification and H2S formation, respectively. The
acid base indicator phenol red incorporated for detecting carbohydrate fermentation is
indicated by the change in color of the carbohydrate medium from orange red to yellow
in the presence of acids. In case of oxidative decarboxylation of peptone, alkaline
products are built and the pH rises. This is indicated by the change in colour of the
medium from orange red to deep red. Sodium thiosulfate and ferrous ammonium sulfate
present in the medium detects the production of hydrogen sulfide and is indicated by the
black color in the butt of the tube.

Glucose is utilized first by a fermentative organism and the entire medium becomes
acidic (yellow) in 8 to 12 hours. Butt remains acidic even after 18 to 24 hours incubation
period because of the presence of organic acids resulting from the fermentation of
glucose under anaerobic conditions in the butt of the tube. The slant, however, reverts to
the alkaline (red) state because of oxidation of the fermentation products under aerobic
conditions on the slant. This change is a result of the formation of CO2 and H2O and the
oxidation of peptones in the medium to alkaline amines. When, in addition to glucose,
lactose and/or sucrose are fermented, the large amount of fermentation products formed
on the slant neutralizes the alkaline amines and renders the slant acidic (yellow), provided
the reaction is read in 18 to 24 hours. If the slant and butt become alkaline, glucose has
not been fermented. Organisms showing this reaction are defined as non-fermenters, and
derive their nutrients from the peptones present in the medium. The formation of CO2
and hydrogen gas (H2) is indicated by the presence of bubbles or cracks in the agar or by
separation of the agar from the sides or bottom of the tube. The production of H2S
(sodium thiosulfate reduced to H2S) requires an acidic environment, and reaction with
the ferric ammonium citrate produces a blackening of the agar butt in the tube.

VP Test

Voges Proskauer test, commonly known as VP test is used to determine the ability of
some organisms to produce neutral end products (e.g., 2, 3-butanediol or acetoin) from
glucose fermentation. All members of the Enterobacteriaceae can convert glucose to
pyruvic acid by the Embden-Meyerhof pathway, but bacteria can further metabolize
pyruvic acid by two different pathways. Organisms metabolizing pyruvic acid by the
mixed acid pathway will produce more acid end products, such as lactic acid and acetic
acid, and maintain an acidic environment. If the organism produces large amount of
organic acids that includes formic acid, acetic acid, lactic acid, and succinic acid from
glucose fermentation, the broth medium will remain red after the addition of methyl red,
a pH indicator. However, MR-negative organisms further metabolize the initial
fermentation products by decarboxylation to produce neutral acetyl methylcarbinol
(acetoin), which results in decreased acidity in the medium and raises the pH towards
neutrality (pH 6.0 or above). MR test along with VP test is performed simultaneously
because they are physiologically related and are performed on MRVP broth.
Bacteria fermenting sugars via the butanediol pathway produce acetoin (i.e., acetyl
methyl carbinol or 3-hydroxybutanone) as an intermediate which can be further reduced
to 2,3-butanediol.
2 pyruvate = acetoin + 2CO 2
acetoin + NADH + H + = 2,3-butanediol + NAD +
In the presence of KOH the intermediate acetoin is oxidized to diacetyl, a reaction which
is catalyzed by alpha naphthol. Diacetyl reacts with the guanidine group associated with
molecules contributed by peptone in the medium, to form a pinkish-red-colored product.
The alpha naphthol in the Barritt’s modification of the VP test serves as a color
intensifier.
Positive: Crimson to ruby pink (red) color
Negative: No change in colouration
Limitations of VP Test
The Voges-Proskauer test may be used in the identification of gram-negative bacteria.
Additional biochemical testing using pure culture is recommended for complete
identification.
Prolonged incubation (greater than 72 hours at 35ºC) of certain VP-positive strains
of Enterobacteriaceaemay result in false-negative reactions due to the breakdown of
acetylmethyl carbiol.
The order of the addition of reagents is extremely important. Reversal of the order may
result in weak positive reactions or false-negative reactions.
MR-VP testing should be used in conjunction with other confirmatory tests to
differentiate organisms among the Enterobacteriaceae.
Quality control of VP Test
VP positive: Enterobacter aerogenes (ATCC13048)
VP negative: Escherichia coli (ATCC25922)

Principle of Oxidase Test

The oxidase test is designed for specifically detecting the presence of the
terminal enzyme system in aerobic respiration called cytochrome C oxidase or
cytochrome a3. Cytochrome C oxidase is the terminal or last H2 electron
acceptor in aerobic respiratory mechanism which is composed of a number of
enzymes which alternatively oxidize and reduce each other by donating or
accepting electrons derived from H2.

The ability of an organism to produce the cytochrome C oxidase can be

determined by using the reagent tetramethyl-p-phenylenediamine
 dihydrochloride impregnated in filter disk. The reagent serves as an artificial
substrate donating electrons and thereby becoming oxidized to a deep purple
compound in the presence of the enzyme oxidase and free O2. Development of
pink, then maroon and finally dark purple colouration after rubbing the organism
in the oxidase disc containing the reagent indicates positive reaction. The
positive reaction involve the conversion of colourless, reduced tetramethyl-p-
phenylenediamine to oxidized form into deep purple colour in presence of
Cytochrome C oxidase. No colour change is indicative of the negative test result.

Oxidase Test

Objective of Oxidase Test

To determine the ability of the organism to produce the cytochrome oxidase enzyme.

Principle of Oxidase Test

The oxidase test is designed for specifically detecting the presence of the terminal enzyme system
in aerobic respiration called cytochrome C oxidase or cytochrome a3. Cytochrome C oxidase is
the terminal or last H2 electron acceptor in aerobic respiratory mechanism which is composed of
a number of enzymes which alternatively oxidize and reduce each other by donating or accepting
electrons derived from H2.

The ability of an organism to produce the cytochrome C oxidase can be determined by using the
reagent tetramethyl-p-phenylenediamine dihydrochloride impregnated in filter disk. The reagent
serves as an artificial substrate donating electrons and thereby becoming oxidized to a deep
purple compound in the presence of the enzyme oxidase and free O2. Development of pink, then
maroon and finally dark purple colouration after rubbing the organism in the oxidase disc
containing the reagent indicates positive reaction. The positive reaction involve the conversion of
colourless, reduced tetramethyl-p-phenylenediamine to oxidized form into deep purple colour in
presence of Cytochrome C oxidase. No colour change is indicative of the negative test result.

Positive test: Development of deep purple color within 10 seconds

Negative test: Absence of color

Limitations of Oxidase Test

1. The reagents used in the oxidase test have been shown to autooxidize, and hence false
positive result may be obtained.
2. Nickel, steel, and other wire loops may give false-positive results, so it is important to
use only platinum or inert transfer loops, such as sterile wood sticks commonly used in teaching
laboratories.
3. Bacteria grown on media containing high concentrations of glucose show inhibited
oxidase activity, so it is recommended to test colonies grown on media without excess sugar, such
as nutrient agar.
 Quality Control of Oxidase Test
Positive: Pseudomonas aeruginosa ATCC27853
Negative: Escherichia coli ATCC 25922
Limitations of Methyl Red (MR) Test
 The MR test should not be read before 48 hours, because some organisms will not
have produced enough products from the fermentation of glucose.
 MR-negative organisms may also not have had sufficient time to convert those
products and will appear MR positive.
 If the methyl red test results are inconclusive (orange) after 48 hours, continue
incubation of the broth for an additional three days and retest the broth culture.
 MR-VP testing should be used in conjunction with other confirmatory tests to
differentiate organisms among the Enterobacteriaceae.
Quality Control of Methyl Red (MR) Test
MR positive: Escherichia coli (ATCC25922)
MR negative: Enterobacter aerogenes (ATCC13048)

Limitations of Hydrogen Sulfide (H2S) Production Test

1. H2S production may be inhibited on TSI for organisms that utilize sucrose and
suppress the enzyme mechanism that results in production of H2S.
2. SIM is more sensitive in the detection of H2S than either TSI or KIA, because
of its semisolid nature, its lack of interfering carbohydrates, and the use of
peptonized iron as an indicator.
3. Lead acetate paper is 10 times more sensitive than other media.
4. Lead acetate is toxic to bacteria and may inhibit the growth of some bacteria. Do
not allow the media to touch the strip.

Quality Control of Hydrogen Sulfide (H2S) Production Test

Positive control: Proteus vulgaris

Negative control: Escherichia coli

Limitation of Indole Test

 Ehrlich’s method may also be used to differentiate organisms under anaerobic
conditions.

Quality Control of Indole Test

Kovac’s Method
Positive: Escherichia coli (ATCC25922)
Negative: Klebsiella pneumoniae (ATCC13883)
Ehrlich’s Method
Positive: Haemophilus influenzae (ATCC49766)
Negative: Haemophilus parainfluenzae (ATCC76901)
Ehrlich’s Method (Anaerobic)
Positive: Porphyromonas asaccharolytica (ATCC25260)
Negative: Bacteroides fragilis (ATCC25285)
Positive: Deep pink coloration, Light Orange, Magneta

Negative: No Color change, Yellowish orange coloration

Limitations for urease

Some organisms rapidly split urea (Proteus spp, H. pylori), while others react slowly.
Urea Agar should not be used to determine the quantitative rate of urease activity, as
organisms vary in their capability and rate of hydrolysis.
Prolonged incubation may cause alkaline reaction in the medium and give false positive
result.

Quality Control
Positive: Proteus vulgaris (ATCC13315)

Weak positive: Klebsiella pneumoniae (ATCC13883)

Negative: Escherichia coli (ATCC25922)

Result Interpretation of Citrate Utilization Test

Positive: Growth on the medium, with or without a change in the color of the indicator.
Growth typically results in the bromthymol blue indicator turning from green to blue.
Negative: Absence of growth.

Limitations of Citrate Utilization Test

Some organisms are capable of growth on citrate and do not produce a color change.
Growth is considered a positive citrate utilization test, even in the absence of a color
change.

Quality Control

Positive: Enterobacter aerogenes (ATCC13048)—growth, blue color

Negative: Escherichia coli (ATCC25922)—little to no growth, no color change

Result Interpretation of Catalase Test

Positive: Copious bubbles are produced.
Negative: No or few bubbles are produced.
Limitations of Catalase Test
Some organisms (enterococci) produce a peroxidase that slowly catalyzes the breakdown
of H2O2, and the test may appear weakly positive. This reaction is not a truly positive
test. False positives may occur if the sample is contaminated with blood agar.
Quality Control
Positive: Staphylococcus aureus (ATCC25923)
Negative: Streptococcus pyogenes (ATCC19615)
Biofilms are a collective of one or more types of microorganisms that can grow
on many different surfaces. Microorganisms that form biofilms
include bacteria, fungi and protists. Biofilms are densely packed communities of
microbial cells that grow on living or inert surfaces and surround themselves with
secreted polymers.