Universiti Tunku Abdul Rahman (Kampar Campus)

Faculty of Science, Engineering, and Technology

Bachelor of Science (Hons) Biotechnology

Year 2 Semester 1

UESB 2142 Laboratory 2A

(II) The Properties of Matter

Lecturer: Dr. Teh Yok Lan

Student’s Name: Cheah Hong Leong

Student’s ID: 08AIB03788

Experiment No. 6

Title: Biochemical Activities of Microorganisms

Date: August 3, 2009

Title: Biochemical Activities of Microorganisms

Objective:
– To examine the ability of certain microorganisms in carry out a particular biochemical
reaction.
– Examine the chemical activities of microorganisms by carry out biochemical tests on
the culture of the particular microorganism.
Results:
Table 1: Carbohydrate Fermentation Test on Escherichia coli and Pseudomonas
aeruginosa
Bacteria Observation (color of medium) Oxidizer / Fermenter
Escherichia coli Dark green Fermenter
Escherichia coli (with Yellow at the upper portion of the
vaspar) tube; dark green in lower portion of
the tube
Pseudomonas Dark green Neither
aeruginosa
Pseudomonas Dark green
aeruginosa (with vaspar)

Table 2: Starch Hydrolysis Test on Escherichia coli and Bacillus subtilis

Bacteria Observation (existence of clear zone Starch Hydrolysis
around the area of inoculation, +/-)
Escherichia - Absent
coli
Bacillus + Present
subtilis

Table 3: Gelatin Hydrolysis Test on Escherichia coli, Bacillus subtilis, and Proteus
vulgaris
Bacteria Observation (physical state of chilled Gelatin Hydrolysis
medium, solid/liquid)
Escherichia coli Solid Absent
Bacillus subtilis Liquid Present
Proteus vulgaris Solid Absent

Table 4: Indole Production Test on Escherichia coli and Enterobacter aerogenes

Bacteria Observation (alcohol-aqueous Indole Production
 separation and color of alcohol layer)
Escherichia coli The alcohol was separated on the top Present
from the aqueous layer upon standing.
The color of the alcohol layer is red-
orange.
Enterobacter aerogenes The alcohol was separated on the top Present
from the aqueous layer upon standing.
The color of the alcohol layer is red-
orange.

Table 5: Hydrogen Sulfide Production Test and Indole Production Test on Escherichia
coli and Proteus vulgaris
Bacteria Observation Hydrogen sulfide
Before addition After addition of
production
of Kovacs Kovacs reagent
reagent (motility, (color of the overlay
black precipitate) of Kovacs reagent)
Escherichia Colonies were Yellow-brown Absent
coli formed away
from the stab
area. No black
precipitate.
Proteus Colonies were Red Absent
vulgaris formed away
from the stab
area. No black
 precipitate.

Table 6: Test for Catalase Activity on Staphylococcus aureus and E. faecalis

Bacteria Observation (formation of Catalase activity
bubbles and formation of
surface froth)
Staphylococcus Bubbles formed vigorously Present
aureus
A. faecalis Bubbles formed Present

Note: The pictures of tube and plate media for the observation from table 1 to 6 are
shown in attachment.

Discussion:
According to Table 1, only tube with Escherichia coli and vaspar show
fermentative activity. This showed that Escherichia coli are fermenter. However, another
tube with Escherichia coli without vaspar did not turned the medium yellow and
therefore no fermentative activity showed. Theoretically, fermenter should show the
fermentation with or without oxygen. Therefore, the second medium of E. coli should
also show turned yellow. Pseudomonas aeruginosa did not show any effect on both the
tube of OF medium, this indicates that P. aeruginosa are neither oxidizer nor fermenter.
From Table 2, Bacillus subtilis showed positive result on the starch hydrolysis
whereas Escherichia coli did not. Starch hydrolysis requires presence of exoenzyme
amylase to hydrolyze starch into shorter polysaccharides, dextrin, maltose and glucose.
Positive result of starch hydrolysis showed the ability of the microorganism to produce
amylase, and vice versa. Bacillus subtilis are capable of producing exoenzyme amylase.
Table 3 showed that only Bacillus subtilis have the activity of gelatin hydrolysis.
This showed that B. subtilis have the ability to produce proteolytic exoenzyme of
gelatinase, which can hydrolyzes gelatin into amino acids. The degradation of gelatin into
its amino acids gives the ability of liquefaction to B. subtilis, which can keep the medium
remain liquid even at temperature of 4 oC.
 From Table 4, both the Escherichia coli and Enterobacter aerogenes showed the
ability of producing indole from tryptophan. Bothe E. coli and E. aerogenes have the
enzyme tryptophanase that can degrade the amino acid tryptophan into indole, pyruvic
acid and ammonia. The indole produced by both the bacteria can therefore binds with p-
dimethylaminobenzaldehyde in Kovacs reagent to produce quinoidal red-violet
compound.

Table 5 showed that both E. coli and P. vulgaris are motile bacteria since colonies
were formed away from the area of stab. However, both E. coli and P. vulgaris showed
negative result for hydrogen sulfide production, none of the tubes showed formation of
blackening on the area of stab. Upon the addition of Kovacs reagent, only medium with
P. vulgaris showed red color on the overlay of the Kovacs reagent. This means that P.
vulgaris showed positive result on indole production.
Table 6 showed that both Staphylococcus aureus and A. faecalis are both capable of
producing Catalase. Hydrogen peroxide is very an extremely toxic superoxide and its
accumulation will bring fatal if not degraded. S. aureus and A. faecalis are capable of
degrade hydrogen peroxide rapidly by producing Catalase. Degradation of hydrogen
peroxide rapidly also produce large amount of oxygen gas, which is the reason for the
formation of bubbles upon addition of hydrogen peroxide into the medium. Ability of
degrading hydrogen peroxide with production of oxygen gas also indicates that both S.
aureus and A. faecalis are non obligate anaerobes.

Conclusion:
1. Escherichia coli are fermenter, not capable of hydrolyze starch, not capable of
hydrolyze gelatin, production of indole from tryptophan, and not capable of
producing hydrogen sulfide from sulfur-containing amino acid.
2. Pseudomonas aeruginosa are neither oxidizer nor fermenter.
3. Bacillus subtilis are able to hydrolyze starch and gelatin.
4. Proteus vulgaris are not able to hydrolyze gelatin, not able to produce hydrogen
sulfide from sulfur-containing amino acids, but showed positive result for indole
production.
5. Enterobacter aerogenes are able to produce indole from tryptophan.
6. Both Staphylococcus aureus and A. faecalis are able to degrade hydrogen peroxide.

References:

Madigan, M. T., Martinko, J. M., Dunlap, P. V., & Clark, D. P. (2009). Brock Biology of

Microorganisms, 12th ed., San Francisco, CA: Pearson Benjamin Cummings.

Cappuccino, J. G. & Sherman, N. (2008). Microbiology. A Laboratory Manual, 8th ed.,

San Francisco, CA: Pearson Benjamin Cummings.
Attachment:
_______________________________________________________________________
_Medium containing Medium containing
Pseudomonas
Escherichia coli
aeruginosa
with vaspar

Medium
containing
Medium containing Escherichia coli
Pseudomonas
aeruginosa with
vaspar

Picture 1: Carbohydrate Fermentation Test on Escherichia coli and Pseudomonas

aeruginosa (Table 1)
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_

(a) Bacillus subtilis (b) Escherichia coli

Picture 2 (a) and (b): Starch Hydrolysis Test on Escherichia coli and Bacillus subtilis
(Table 2)
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_
 (a) Bacillus subtilis (b) Escherichia coli

Picture 3 (a), (b) and (c): Gelatin Hydrolysis Test

on Escherichia coli, Bacillus subtilis, and
Proteus vulgaris (Table 3)

(c) Proteus vulgaris

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_

Picture 4: Indole Production Test on

Escherichia coli and Enterobacter aerogenes
(Table 4)

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_
 Picture 5: Hydrogen Sulfide Production Test
and Indole Production Test on Escherichia
coli and Proteus vulgaris (Table 5)

Staphylococcus aureus

E. faecalis

Picture 6: Test for Catalase Activity on Staphylococcus aureus and E. faecalis (Table 6)
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_