MEDICAL MICROBIOLOGY

LAB 2
IDENTIFICATION OF SOME IMPORTANT BACTERIA
PATHOGENS BY USING BIOCHEMICAL METHODS

Classical biochemical tests

Catalase test:
Catalase is common enzyme, which is found in almost living organisms exposed
to oxygen. It protects the cell from oxidative damaging by reactive oxygen
species.
Theoretically, EC, VC, SA, PA, ST are all catalase positive.
Experiment:
Procedure: Hand on some drops of H2O2 on agar plates of ST, PA, VC, EC, SA.
If there is any catalase, it then oxidizes added H2O2 to create bubbles on the
surface of agar plates.
Results: Bubbles appeared on 4 experimented agar plate of PA,VS, ST, EC.
Conclusion: These bacteria pathogens have catalase.

The BHI medium contained

4 species PA, VS, ST, EC
releasing bubbles after
adding drops of H2O2.
Comment: there are maybe some mistakes because 4 species PA, ST, VC, EC
are only in 1 agar plate and not separated to each other.
Coagulase test:
Coagulase is an enzyme produced by S.aureus that converts soluble fibrinogen
in plasma to insoluble fibrin. There are 2 forms of coagulase: bound and free.
Coagulase is used to distinguish between different types of Staphylococcus
isolates.
Coagulase reacts with prothrombin in the blood to form Staphylothrombin,
which enables the enzyme protease to convert fibrinogen, a plasma protein
produced by the liver, to fibrin. This is the result of clotting in blood.
This test can be performed in slide test or tube test.
Procedure:
-Add a generous loopful of the organism to be tested to a tube of citrated rabbit
plasma.
-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.
Results: if there is any clot within 24 hours that means coagulase positive. After
24 hours, the clot will be slowly digested by fibrinolysin.
Oxidase test:
The oxidase test is used to identify bacteria that produce cytochrome C oxidase,
an enzyme of the bacterial electron transport chain.
If oxidase test is positive, it means the tested bacteria are aerobic. In contrast,
the negative result shows anaerobic characteristic.
Procedure:
- The reagent can be N,N,N,N-tetramethyl-p-phenylenediamine (TMPD)
or N,N-dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator.
- Hand on some drops of oxidase reagent on the agar plate and observe.
Results: the positive result gives out blue or purple color within 15 seconds.
Experimental results:
- Oxidase-positive: PA, VC.
- Oxidase-negative: ST, EC, SA.

Gram Staining
1. Vibrio cholerae

The gram stain of Vibrio cholerae bacteria

has pink color and comma shaped. It
indicates that Vibrio cholerae is Gram
Negative bacterium.

2. Staphylococcus aureus

The gram stain of Staphylococcus aureus

bacteria has purple color and cocci shaped.
It indicates that Staphylococcus aureus is
Gram Possitive bacterium.

3. Pseudomonas aeruginosa

The gram stain of Pseudomonas aeruginosa

bacteria has pink color and rod shaped. It
indicates that Pseudomonas aeruginosa is
Gram Negative bacterium.
 4. Salmonella typhi

The gram stain of Salmonella typhi

bacteria has pink color and rod shaped.
It indicates that Salmonella typhi is
Gram Negative bacterium.

5. Escherichia coli

The gram stain of Escherichia

coli bacteria has pink color and rod
shaped. It indicates that Escherichia
coli is Gram Negative bacterium.

Comment : The results of gram staining of all bacterial strains are not very good
as my group expected. It may because of these factors:
the microscope lens are not clean enough to visualize.
the amount and quality of staining reagent or bacterial colonies is taken
too much which makes the sample is difficult to observe.
the technique when staining the samples is not good.
the time during adding reagents is measured incorrectly
Therefore, it is hard to distinguish the morphology of organisms. However, we
can still differentiate between G (+) and G (-) among groups of bacteria based
on colors presence.

Identify bacteria by using commercial kit (NamKhoa kit):

Test Label Actual Results Score Final score

1 OXI White 0 2
2 GLU Yellow 2
3 NIT White 0
4 ONPG Yellow 1 5
5 URE Pale yellow 0
6 PAD Green 4
7 CIT Yellow 1 1
8 ESC Pale yellow 0
9 H2S Yellow 0
10 IND Pale yellow 0 2
11 VP Red 2
12 MLO Yellow 0
13* LDC Yellow 0 2
14 MOB Red color mixed in medium 2

1. OXI :
Result: Color does not change Negative result

2. Glucose Fermentation: 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.
Result: Yellow color appears Positive result

3. Nitrate Reduction: 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 alphanaphthylamine to produce a
red, water soluble azodye.
Result: Yellow color does not change Negative result

4. ONPG Hydrolysis: ortho-Nitrophenyl-_-galactoside (ONPG) is a

colorimetric and spectrophotometric substrate for detection of _-
galactosidase activity. This compound is normally colorless. However, if
galactosidase is present, it hydrolyzes the ONPG molecule into galactose
and ortho-nitrophenol. The latter compound has a yellow color.
Result: Yellow does not change more Positive result
5. Urease Production: 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).
Result: Pink become yellow Negative result

6. Phenyl Alanine Deaminase production: 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.
Result: Green color exist Positive result

7. Citrate Usage: 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 mediums color
from green to blue. In acidic condition, the pH indicator has yellow color.
Result: Green turns to blue color Positive result

8. Esculin Hydrolysis: 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.
Result: Light Pink Color appear not the exact result , so this may
be mistake happen when taken the sample into the seventh well or it
is contamited.

9. H2S Production: 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.
Result: No black precipitate happens Negative result
10. Indole Production: 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 p-
Dimethylaminobenzaldehyde (Kovacs reagent) at an acid pH in alcohol
to produce a redviolet compound
Result: Yellow on the surface of the well Negative result

11.Voges Proskauer: 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 an potassium hydroxide
(KOH) to form a red color.
Result: Red color appear Positive Result

12. Malonase Usage: 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 brom thymol blue, which is green at
neutral pH, yellow at acidic pH <6.0 and turns blue at alkaline (basic)
pH >7.6.
Result: Light red color appear Positive result

13. LDC: Lysine Decarboxylase Test which Decarboxylases are a group of

substrate specific enzymes that are capable of reacting with the
carboxyl (COOH) portion of amino acids, forming alkaline-reacting amines
and byproduct Carbon dioxide. Increased pH of the medium is detected by
color change of the pH indicators bromcresol purple and cresol red.
Bromcresol purple turns purple at an alkaline pH and turns yellow at an
acidic pH.
Each decarboxylase enzyme is specific for an amino acid. Lysine, Ornithine,
and arginine are the three amino acids routinely tested in the identification of
Enterobacteriaceae.
These byproducts are sufficient to raise the pH of the media so that the broth
turns purple.
If the inoculated medium is yellow, or if there is no color change, the
organism is decarboxylase-negative for that amino acid. If the medium turns
purple, the organism is decarboxylase-positive for that amino acid.
 The inoculated medium is yellow that means there is no color
change. The organism is decarboxylase-negative for that amino
acid. However, the bacteria our group used is Salmonella typhi,
which is decarboxylase-positive.

Result: Yellow does not change more Negative result

14. MOB: which used to test the mobility of microorganisms.

The red color is mixed with the yellow color of medium

that mean it is positive for mobility test.

Result: Light red color mixed with yellow color of medium Positive
result

Comment: The final score is 25122. Based on the Reference Identification

Table, the bacteria is but as we have known that the bacterial suspension used
in this test is Salmonella typhi. In our group opinion, the mistake might occur
either in our techniques when performing this test or in the preparation of the
bacterial suspension or due to the comtamination.