UDBB2114 Microbiology

Experiment 5: Biochemical Activities of Microorganisms

Student: Harpreet Singh
Student ID: 1206650
Course: Bachelor of Biotechnology
Practical class : P4
Lecture name: Dr.Lisa Ong Gaik Ai

I.

Result :
A. Carbohydrates Fermentation Test on Escherichia coli and Pseudomonas
aeruginosa

From this figure, we noticed that

both tube containing E.coli with
and without vaspar show yellow
colour through thus it is
fermentative culture however for
two tubes containing Pseudomonas
aeruginosa with or without vaspar
show the upperside is yellow
colour but at the bottom, it is
greenish blue in colour thus it is
oxidative culture.

B. Starch Hydrolysis Test on Escherichia coli and Bacillus subtilis

Before grams iodine test

This figure indicate the culture dish

inoculated with Escherichia coli
and Bacillus subtilis before starch
hydrolysis test

After grams iodine test

From this figure, we noticed that
the culture dish containing
Escherichia coli doesnt show clear
zone thus starch hydrolysis is
absent. However, culture dish
containing Bacillus subtilis show
clear zone around the area of
innoculation thus starch hydrolysis
is present.

C. Gelatin Hydrolysis Test on Escherichia coli, Bacillus subtilis, and Proteus

vulgaris

From the figure, we noticed that

tubes containing Proteus vulgaris
and Bacillus subtilis turns into
liquid thus gelatin hydrolysis is
present however for tube of E.coli
remain solid thus gelatin hydrolysis
is absent.

D. Utilization of Amino Acids

1. Indole Production Test on Escherichia coli and Enterobacter aerogenes.
From this figure, we noticed that
tube containing E.coli show alcohol
is separated on the top from the
aqueous layer and the colour of the
alcohol layer is purple thus indole
production is present however tube
of Enterobacter aerogenes show no
difference thus it there is no indole
production.

2. Hydrogen Sulfide Production Test on Escherichia coli and Proteus vulgaris

From this figure, we noticed that
tube containing E.coli show the
colour of overlay of Kovacs
solution is purple in colour and
colonies were formed away from
the stab area however no black
precipitate is formed. Tube
containing Proteus vulgaris show
the colour of overlay of Kovacs
solution is purple in colour and
colonies were formed away from
the stab area. Black precipitate is
also formed thus there is
production of hydrogen sulfide.

E. Catalase Activity Test on Staphylococcus aureus and Enterobacter aerogenes

From this figure, we noticed that

tube containing Staphylococcus
aureus show Bubbles formed
vigorously thus catalase activity is
present. Tube of Enterobacter
aerogenes show bubbles is formed
but lesser compare to S.aureus,
catalase activity is present.

II.

Discussion :
From the experiment in carbohydrates fermentation, both tubes containing E.coli with
or without vaspar each show fermentative culture as their culture medium remain yellow
in colour. The function of vaspar is to block the oxygen supply into the medium. This

shows that with or without oxygen, fermentation activity wont be affected. In both tube
containing Pseudomonas aeruginosa with or without vaspar each show oxidative culture
as the upper region of the tubes remain yellow but at the bottom part of OxidationFermentation medium, greenish blue colour can be seen. This shows the oxidative culture
require oxygen however the tube of P.aeruginosa containing vaspar also show oxidation
due to thin and insufficient layer of vaspar.
In starch hydrolysis experiment, we uses Bacillus subtilis and Escherichia coli as a
specimen. B.subtilis showed postitive result and there is clear zone around the area of
inoculation. Grams iodine solution is used to test the starch hydrolysis. 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 thus it gives a positive result and since all the
starch is completely remove from the area around the inoculum,it gives a clear zone.
In Gelatin Hydrolysis test, Bacillus subtilis and Proteus vulgaris display a positive
results. Both tubes containing the microbes have turn the medium into liquid. 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 and P.vulgaris, which can keep the
medium remain liquid even at temperature of 37oC. E.coli shows negative result because
of its inability to produce gelatinase.
In Indole production test, tube containing Escherichia coli show alcohol is
separated on the top from the aqueous layer and the colour of the alcohol layer is purple.
This showed the ability of producing indole from tryptophan. E. coli have the enzyme
tryptophanase that can degrade the amino acid tryptophan into indole, pyruvicacid and

ammonia. The indole produced by both the bacteria can therefore binds with pdimethylaminobenzaldehyde in Kovacs reagent to produce quinoidal red-violet
compound. Enterobacter aerogenes should show the same positive results however no
changes occur, this may be due to contamination.
In Hydrogen Sulfide Production test,both E. coli and P. vulgaris are motile bacteria
since colonies were formed away from the area of stab. However, only P. vulgaris
showed positive result for hydrogen sulfide production, formation of blackening on the
area of stab can be observe. Sulfide-Indole-Motility (SIM) medium is used here because
it contain sulfates, high level of tryptophan and also 0.5% agar content. Sulfates is
necessary to determine sulfide production. High amount of tryptophan is important for
indole production and lastly 0.5% agar content to allow the bacterial motility. Sulfates
(H2S) from the microbe will combined with Iron(II) cation (FE2+) present in media to
form FeS ,a black precipitate.Upon the addition of Kovacs reagent, medium with P.
vulgaris and E. coli showed purple color on the overlay of the Kovacs reagent. This
means that both microbes showed positive result on indole production.
In catalase activity test, both Staphylococcus aureus and Enterobacter aerogene
showed positive results and are capable of producing Catalase. Hydrogen peroxide is an
extremely toxic superoxide and its accumulation will bring harm if not degraded. S.
aureus and Enterobacter aerogenes are capable to 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 E.aerogenes are nonobligate anaerobes.

III.

Conclusion :
We manage to identify and determine the characteristic of microbes through test on their
biochemical activities.
1. Escherichia coli are fermenter and not capable to hydrolyze starch or gelatin.
It able to produce indole from tryptophan but unable to produce hydrogen
sulfide from sulfur-containing amino acid.
2. Pseudomonas aeruginosa are oxidizer.
3. Bacillus subtilis able to hydrolyze starch and gelatin.
4. Proteus vulgaris able to hydrolyze gelatin, produce hydrogen sulfide from
sulfur-containing amino acids, and showed positive result for indole
production.
5. Both Staphylococcus aureus and Enterobacter aerogenes able to degrade

IV.

hydrogen peroxide.
References :
1. 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.
2. Cappuccino, J. G. & Sherman, N. (2008). Microbiology. A Laboratory Manual, 8th
ed.,San Francisco, CA: Pearson Benjamin Cummings.
3. Willey, M. J., Sherwood, M.L., & Woovlvertoon, J.C., (2008) Microbiology. 7th
Ed. Singapore : McGraw-Hill.
4. Parker, J., Wilson, G., Outland, L. and Wilson, M. 2014. Hydrogen Sulfide
Production Test. [online] Available at:
http://www.vumicro.com/vumie/help/VUMICRO/Hydrogen_Sulfide_Production_
Test.htm [Accessed: 1 Apr 2014].