1. The image here shows three different genera of bacteria growing on EMB agar.

They are
numbered 1 to 3. Which of the following conclusions is accurate?

a. Organism 1 is Gram-positive and
is unable to ferment glucose.
b. Organism 2 is Gram-negative
and can ferment sucrose.
c. Organism 3 is Gram-negative
and can ferment lactose*.
d. All three organisms are Gram-
negative and can ferment
sucrose.
e. None of the above conclusions
are correct.
2. The product shown in the image here is used to identify

a. Gram-positive cocci.
b. Gram-negative cocci.
c. Gram-positive bacilli .
d. Gram-negative bacilli. *
e. Gram-positive spirochetes.
3. Staphylococcus aureus , a major pathogen, is usually
a. alpha hemolytic.
b. beta hemolytic. *
c. gamma hemolytic.
d. delta hemolytic.
e. incapable of hemolysis.
4. These gelatin tubes have each been inoculated with a pure culture of different bacteria.
The tubes were incubated for 48 hours and have been refrigerated for 30 minutes. The
arrow in this image indicates an organism that is

a. capable of degrading gelatin. *
b. incapable of degrading gelatin.
c. capable of clotting plasma.
d. incapable of clotting plasma .
e. capable of degrading hydrogen
peroxide.
5. These tryptone broths were each inoculated with a different bacteria. After incubation
Kovac's reagent was added to both tubes. Bacteria that produce the result shown at the
arrow in this image are

a. Voges-Proskauer positive.
b. Voges-Proskauer negative.
c. indole positive. *
d. indole negative.
e. methyl red positive.
6. The bacteria that produced the result shown at the arrow in this image

a. produce very acid products from
glucose.
b. produce somewhat alkaline
products from glucose.
c. produce indole when it
degrading trptophan.
d. do not produce indole when
degrading trptophan.
e. are able to utilize citrate as their
sole carbon and energy source .*
7. The media shown in this image is mannitol salt agar. The organism that the arrow is
pointing to is

a. able to ferment mannitol and
probably is Staphylococcus
epidermidis.
b. able to ferment mannitol and
probably is Staphylococcus
aureus.
c. unable to ferment mannitol and
probably is Staphylococcus
epidermidis .*
d. unable to ferment mannitol and
probably is Staphylococcus
aureus.
e. able to ferment glucose and
probably is Staphylococcus
aureus.
8. This image shows the results of bacteria inoculated into motility media. The organism in
tube 2

a. is motile and possesses flagella.
*
b. is nonmotile and lacks flagella.
c. is motile and lacks flagella.
d. is nonmotile and possesses
flagella.
9. Methyl red test results for different organisms are shown in this image. Which
conclusion is correct?

a. Tube 1 indicates an acidic pH as
a result of fermentation of
lactose.
b. Tube 2 indicates an acidic pH as
a result of fermentation of
lactose.
c. Tube 1 indicates an acidic pH as
a result of fermentation of
glucose.
d. Tube 2 indicates an acidic pH as
a result of fermentation of
glucose. *
e. None of the above are correct
conclusions for these results.
10. Pathogens that are usually beta-hemolytic include
a. Staphylococcus aureus.
b. Streptococcus pyogenes (Group A Strep).
c. Streptococcus pneumoniae.
d. a and b only *
e. a, b and c

Catalase test
Posted by: famsbc on: July 30, 2009
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Oxygen is sometimes toxic.
Small amounts of superoxide free radicals are formed during the normal respiration of organisms
that use oxygen as the final electron acceptor.
Obligate anaerobes from some oxygen free radicals that are toxic to the cell. Hence, if bacteria
wants to grow in oxygen environment, enzymes like catalase and superoxidase dismutase must
be present for neutralization of the toxic form of oxygen(oxygen radical)

During normal aerobic respiration, hydrogen ions are produced and have to be removed by
bacterial cell. The electron transport system (ETS) in cellular respiration (a part of glycolysis)
involves these H+ ions and combines them with oxygen to form water. Water is harmless.
Energy is given off and stored in the form of Adenosine Triphosphate.
What is toxic is Hydrogen Peroxide that is formed by the cytochromes in ETS. Water being
harmless is not required to be removed by the bacteria. So, what is harmful to bacteria cell that
requires it to be removed instantly??
answer: H2O2.
Functions of catalase
Protects bacteria from toxic hydrogen peroxide (H
2
O
2
) accumulation, which can occur during
aerobic metabolism. If hydrogen peroxide accumulates, it becomes toxic to the organism.
Since Catalase breaks H
2
O
2
down into water and O
2
, the presence of oxygen can be characterized
by bubbles which indicates a (+) result.
What bacteria could mostly likely be detected?
most aerobic organism make catalase.
http://student.ccbcmd.edu/courses/bio141/labmanua/lab8/catstaph.html
Most aerobic organsims will display (+) results.
e.g. Staphyloccocus aureus.
Some anaerobic organisms will display (-) results, indicating that they do not produce catalase to
prevent oxygen accumulation. Why?
Because since oxygen is totally not used for survival of these organisms, they do not have the
ability to produce catalase.
e.g. Clostridium, Lactobacillus, Streptococcus
Carbohydrate Oxidation-Fermentation
Posted by: famsbc on: July 28, 2009
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Fermentation can occur in the presence of oxygen or absence of oxygen.
If bacteria utilises carbohydrates for nutrients, there may be 2 end products, a gas and acid. The
substrate formed from the metabolism of carbohydrate is either glucose or lactose.
Even if bacteria releases enzymes that enable to use carbohydrates through fermentation and
oxidation, gas may or may not be produced.
FERMENTATION is noted by acid production which can be observed by a colour change in
Durham tubes aka carbohydrate fermentation tube.
Phenol Red indicator is red in neutral or alkaline solution
If acid is present(+), phenol red changes from red –> yellow
If there is a small “space” at the top of the small tube, it means that gas is trapped in the small
inverted tube inside the bigger tube. Therefore, gas is produced from the breakdown of
carbohydrates.

Alfred.E.Brown. (2007). Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.
Look at the 1st picture above, why is it red if gas is produced and it just means fermentation has
occured?
Due to prolonged incubation periods(more than 24h), bacteria will begin to grow
oxidatively(oxygen dependent) on the peptone contents of the fermentation medium after using
up the carbohydrate contents, causing the neutralization of phenol red indicator and turing it red
due to NH3 production.

The only organism that has the ability to break down carbohydrate into glucose and lactose is
E.coli and it also has the ability to produce gas during fermentation.
For P. aerguinosa, it displayed negative results for all, which means it can’t break down
carbohydrate into glucose & lactose.
Another test which is the MRVP test is able to differentiate between organisms that produce
large amounts of acid and organisms that only produce neutral content(acetoin)
M-Methyl Red. VP- Voges -Proskauer. Methyl Red is different from Phenol Red that we
mention earlier. It has a pH of 4.4-6. Hence, it changes colour at this range.
For MR test, if organic acid is produced here, the (+) result is just a no change in colour of
Methyl Red. RED remains.
if neutral acetoin is produced here, the (-) result is a change in colour from RED to YELLOW
because at pH 7, the indicator will change its colour.

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.
To test for the presence of acetoin, we use VP test where potassium hydroxide and α naphthol
are added.
The upper of the medium will turn red. (+) If the medium turns light brown, it is a (-) result.
Please note that production of acetoin is also affected by the duration of incubation, hence, false
negative results may be observed.
We can use Koser’s citrate or Simmon’s citrate to test for the ability of bacteria to ferment
citrate. When citric acid or sodium citrate is in solution, it loses a proton or sodium ion to form
a citrate ion. Bacteria with citrate lyase can break down citrate to form pyruvate. Pyruvate can
be further reduced in fermentation.
Purpose of performing the citrate test
 Tests for the ability of bacteria to convert citrate (an intermediate of the Kreb’s cycle)
into oxaloacetate (another intermediate of the Kreb’s cycle)
Contents of Simmon citrate include
 sodium citrate as the carbon source
 monoammonium phosphate as the nitrogen source
 and bromthymol blue indicator that changes to blue when medium turns alkaline, which
means (+) result.
Why alkaline means citrate is utilised by the bacteria?
When bacteria uses citrate(carbon) and ammonium(nitrogen), medium turns alkaline as ammonia
is produced from ammonium.

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.
Citrobacter, Enterobacter will display (+) result while E. coli & Klebsiella will display (-) result.

After knowing that some bacteria utilises citrate, we can also find out if other products are
formed during metabolism. For example, Hydrogen Sulphide.

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.
Oxidative-Fermentative Metabolism
Posted by: famsbc on: July 27, 2009
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How do we determine if type of metabolism is fermentative or oxidative?

we will test with an glucose OF oxidative- fermentative agar. If agar turns yellow, acid is
produced. If agar turns green, no acid is produced. Motility of the bacteria can also be
determined by the presence of turbidity (cloudiness)
OXIDATIVE metabolism may or may not cause an acid to be produced for aerobic conditions.
No acid will be produced for anaerobic conditions.
FERMENTATIVE metabolism will cause an acid to be produced for aerboic and anaerobic
conditions.

Alfred.E.Brown. (2007). Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

Nitrogen metabolism – urea Hydrolysis
Posted by: famsbc on: July 27, 2009
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UREASE breaks down urea into ammonia
(NH3) & carbon dioxide (CO2).

Alfred.E.Brown. (2007). Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.
Properties of urea medium
Since there’s phenol red pH indicator, pH indicator changes from yellow to bright pink if NH3 is
produced.
At more than pH 6.8, the colour change represents a (+) result
Proteus vulagris is one bacteria that produces urease to break down urea.
Tryptophan Degradation
Posted by: famsbc on: July 25, 2009
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Tryptophan is degraded by tryptophanase into indole, ammonia, pyruvic acid. Pyruvic acid is
then involved in metabolic pathway so that ATP energy for bacterial cell is generated. While
other products like NH3 and Pyruvate is metabolised, indole is not. Hence, it stays in the
medium.
Upon addition of Kovac’s reagent, deep red ring at the top of the agar/ broth is formed when
Kovac’s reagent reacts with indole. This is the (+) result.
E.coli is one of the organism that can display (+) result since exoenzyme tryptophanase is
produced.

Alfred.E.Brown. (2007). Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.
Properties of Tryptic Soy Broth/Agar
-tryptone is one of the contents of TSB/ TSA and is derived from casein.(recall protein from
protein hydrolysis)
Lipolysis
Posted by: famsbc on: July 25, 2009
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Lipolysis is carried out by LIPASES.
Triglycerides(big molecules derived from lipids) are broken down into fatty acids & glycerol
If pH of medium, used for testing whether lipase is present to break down lipids, is lowered. It
indicates acidic product (fatty acid) is formed.
Trigylceride used here is tributyrin that can be found in medium Spirit Blue Agar.
There are 2 indicators for (+) result.
1. dark blue precipitate(tributyrin is completely broken down into fatty acids) OR
2. oil droplets (when tributyrin is not completely broken down into fatty acids)
Organism responsible for exhibiting (+) result is Staphyloccocus aureus.

Alfred.E.Brown. (2007).Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.
Properties of Spirit Blue Agar
Contents- tributyrin (simple animal triglyceride)
Tributyrin acts as a substrate for exoenzyme lipase.
Protein Catabolism (Proteolysis)
Posted by: famsbc on: July 25, 2009
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Proteolysis is carried out by PROTEASE.
In the following posts we will mention
- bacteria’s ability to hydrolyse casein, gelatin
- urea hydrolysis by detecting presence of urease(refer to post on nitrogen metabolism- urea
hydrolysis)
-hydrogen sulphide production

Casein(a protein) is broken down by protease into peptones and amino acids.
During the degradation process, polypeptide bonds are broken.
Once the bonds are broken, amino acids are produced. A clear zone surrounding streak line of
agar indicates a (+) result.
Organism that gives (+) result is Bacillus subtilis, Clostridium spieces. (-) results are indicative
that organismdoes not cause a clear zone. an example of organism displaying (-) result is
Escherichia coli.
Alfred.E.Brown. (2007). Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.
Properties of skim milk agar
 Skim milk agar contents- casein, lactose and other nutrients, which support growth of
lactobacilli.
 Gives the white colour to milk.
As seen from the above, we can infer if bacteria present in milk is able to break down the casein
in milk, milk may have abnormal particles that are not white in colour.
Application of this biochemical this is in food testing.
Another protein commonly found in food products is gelatin.
It is broken down by gelatinase into smaller polypeptides, peptones and amino acids that can
cross the cell membrane and be utilised by the organism.
Property of Gelatin agar
INTERESTING to note: when gelatin is broken down via hydrolysis, it cannot solidify anymore,
the areas of solid gelatin media where the organsim grows, will turn into liquid. Even if you
refrigerate this medium, the media will remains liquid.


Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.
Hence, a (+) result is indicated by the liquid state of gelatin. Bacillus subtilis is able to produce
proteolytic exoenzyme gelatinase to give the (+) result.
Why is gelatin not used widely as a selective media for isolating bacteria? (hint: what is gelatin
agar unique property?)
Most bacteria do not contain enzymes that liquefy gelatin. Hence, it is not uselfy for isolating
microbes for bacterial identification.
Decomposition of amino acid “cysteine” is detected by the formation of ferrous sulphide when
Hydrogen Sulphide is release.
Why? Some bacteria have the ability to give off H2S from sulphur containing amino acids
after proteins are broken down into amino acids by enzymes.
When H2S is produced, sulfide ion reacts with the metal salt to product a black precipitate(+)
result.


Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.
LOOK AT TUBE 3. the black ppt indicates that Hydrogen Sulphide was produced. (+). Bacteria
that produces (+) are Citrobacter, Salmonella.


Title: Motility test
Description (from left to right)
Uninoculated control
Pseudomonas fluorescens (motile; obligate aerobe)
Staphylococcus epidermidis (nonmotile; facultative anaerobe)
Escherichia coli (motile; facultative anaerobe)