Biochemical Activities

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

aeruginosa Pseudomonas

Dark green

Neither

aeruginosa (with vaspar)

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

Observation (existence of clear zone around the area of inoculation, +/-) +

Starch Hydrolysis Absent Present

subtilis

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

Observation (physical state of chilled medium, solid/liquid) Solid Liquid Solid

Gelatin Hydrolysis Absent Present Absent

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

Observation

(alcohol-aqueous Indole Production

separation and color of alcohol layer) The alcohol was separated on the top

Escherichia coli

Present

from the aqueous layer upon standing. The color of the alcohol layer is redorange. The alcohol was separated on the top

Enterobacter aerogenes

Present

from the aqueous layer upon standing. The color of the alcohol layer is redorange.

Table 5: Hydrogen Sulfide Production Test and Indole Production Test on Escherichia coli and Proteus vulgaris Bacteria

Observation Hydrogen sulfide addition After addition of production Kovacs Kovacs reagent

Before of

reagent (motility, (color of the overlay black precipitate) Escherichia coli

Colonies

were

formed

away

from

the

of Kovacs reagent) Yellow-brown

Absent

Red

Absent

stab

area. No black Proteus

precipitate. Colonies were

vulgaris

formed from

away 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 Staphylococcus aureus A. faecalis

surface froth) Bubbles formed vigorously

Present

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 pdimethylaminobenzaldehyde 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 Pseudomonas aeruginosa with vaspar

Medium containing Escherichia coli

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

(a) Bacillus subtilis

(b) Escherichia coli

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

(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 _______________________________________________________________________ _ 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)

Staphylococcus aureus

E. faecalis

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