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

vaspar)

Yellow at the upper portion of the

tube; dark green in lower portion of

the tube

Pseudomonas aeruginosa Dark green Neither

Pseudomonas aeruginosa

(with vaspar)

Dark green

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

Bacteria Observation (existence of clear zone

around the area of inoculation, +/-)

Starch Hydrolysis

Escherichia

coli

- Absent

Bacillus subtilis + Present

Table 3: Gelatin Hydrolysis Test on Escherichia coli, Bacillus subtilis, and Proteus

vulgaris

Bacteria Observation (physical state of chilled

medium, solid/liquid)

Gelatin Hydrolysis

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

separation and color of alcohol layer)

Indole Production

Escherichia coli The alcohol was separated on the top

from the aqueous layer upon standing.

The color of the alcohol layer is red-

orange.

Present

Enterobacter aerogenes The alcohol was separated on the top

from the aqueous layer upon standing.

The color of the alcohol layer is red-

orange.

Present

Table 5: Hydrogen Sulfide Production Test and Indole Production Test on Escherichia

coli and Proteus vulgaris

Bacteria Observation Hydrogen sulfide

productionBefore addition

of Kovacs

reagent (motility,

black precipitate)

After addition of

Kovacs reagent

(color of the overlay

of Kovacs reagent)

Escherichia

coli

Colonies were

formed away

from the stab

area. No black

precipitate.

Yellow-brown Absent

Proteus

vulgaris

Colonies were

formed away

from the stab

area. No black

precipitate.

Red Absent

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

Bacteria Observation (formation of

bubbles and formation of

surface froth)

Catalase activity

Staphylococcus aureus Bubbles formed vigorously Present

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:

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Picture 1: Carbohydrate Fermentation Test on Escherichia coli and Pseudomonas aeruginosa (Table 1)

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

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Medium containing Pseudomonas aeruginosa with vaspar

Medium containing Pseudomonas aeruginosa

Medium containing Escherichia coli with vaspar

Medium containing Escherichia coli

(a) Bacillus subtilis (b) Escherichia coli

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(a) Bacillus subtilis (b) Escherichia coli

(c) Proteus vulgaris

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

on Escherichia coli, Bacillus subtilis, and Proteus

vulgaris (Table 3)

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

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

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

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Staphylococcus aureus

E. faecalis