Group 5B

202101254 SIYONGWANA, E.S

202101291 MPHOMELA, T
202101372 BANGANI, N
202101493 MAKOYI, A
202101533 SILWANYANA, S
202101676 MABUYA, K
MIC 211
PRACTICAL 5
May 2023
TITLE
Selected physiological tests used for identification of bacteria.
INRODUCTION

AIM
METHODS AND MATERIALS
Part 1: Indole production
Two test tubes were inoculated with 1% tryptone broth with E. coli and B. subtilis and left
one test tube uninoculated, which is control. The plates were incubated for 48 hours at 37°
Celsius. In next period, added Kovac’s reagent to each tube to detect indole production. Mix
well by rotating between the hands. A change of colour to red will be indicating positive test
for indole production.
Part 2: Gelatin hydrolysis

Part 3: Starch hydrolysis

Firstly, the plates were inverted, and labelled. The starch agar plates were inoculated with
provided culture of B. subtilis and E. coli. The single streak method was used. The plates
were incubated for 48 hours at 37° Celsius. The next period, Gram’s iodine was added to see
if the starch will remain in the agar or will be hydrolysed. If starch has been hydrolysed there
was going to be a clear zone around the bacterial growth.

RESULTS
Part 1
Table1: Represent the Indole production by B. subtilis and E. coli indicated by colour change
after the addition of Kovac reagent.
Culture Colour Indole production
E. coli Yellowish Negative indole
B. subtilis Pink Positive indole
Control Yellow Negative indole

Part 2
Table2: Shows the Gelatin hydrolysis by E. coli and B. subtilis indicated by liquification of
the medium.
Culture Liquification Gelatin hydrolysis
E. coli Solid media No gelatin hydrolysis
B. subtilis Liquid media Gelatin hydrolysis
Control Solid media No gelatin hydrolysis

Part3
Table3: Shows the starch hydrolysis by E. coli and B. subtilis indicated by clear zones after
the addition of Gram’s Iodine.
Culture Clear zone Colour Starch hydrolysis
E. coli Clear zone Decolourized Positive
hydrolysis
B. subtilis No clear zone No decolouration Negative
hydrolysis
DISCUSSION
Some bacteria use the enzyme tryptophanase to convert the amino acid tryptophan into
molecules of indole, pyruvic acid, and ammonia (Pattern et.al.,2013). Since only a few
bacteria contain tryptophanase, the formation of indole from a tryptophan substrate can be
another useful diagnostic tool for the identification of an organism. Indole production is a key
test for the identification of Escherichia coli. By adding Kovac's reagent to the medium after
incubation we can determine if indole was produced. Kovac's reagent will react with the
indole and turn red.

Starch is a polysaccharide which appears as a branched polymer of the simple sugar glucose.
This means that starch is really a series of glucose molecules hooked together to form a long
chain (Power, 2003). Additional glucose molecules then branch off this chain. Some bacteria
can use starch as a source of carbohydrate but to do this, they must first hydrolyse or break
down the starch so it may enter the cell. The bacterium secretes an exoenzyme which
hydrolyses the starch by breaking the bonds between the glucose molecules. This enzyme is
called a diastase. The glucose can then enter the bacterium and be used for metabolism. When
iodine is added to starch, the iodine-starch complex that forms give a characteristic dark
brown or deep purple colour reaction. If the starch has been hydrolysed into glucose
molecules by the diastase exoenzyme, it no longer gives this reaction. Flood the surface of
the Starch agar plate with gram's iodine. If the bacterium produced an exoenzyme that
hydrolysed the starch in the agar, a clear zone will surround the bacterial growth because the
starch is no longer there to react with the iodine. If the bacterium lacks the exoenzyme to
break down the starch, the agar around the growth should turn dark brown or blue/black
colour due to the iodine-starch complex (Chemjong, 2019).

REFERENCES
Chemjong, K., 2019. Study of A-Amylase Enzyme in hibition Activity and Antimicrobial
Activity of Local Tea Camellia Sinensis (L.) Kuntze, Leaves of Buddleja Asiatica Lour, And
Roots of Polygala Arillata Buch.-Ham. Ex D. Don (Doctoral dissertation, Department of
Chemistry).
Patten, C.L., Blakney, A.J. and Coulson, T.J., 2013. Activity, distribution and function of
indole-3-acetic acid biosynthetic pathways in bacteria. Critical reviews in microbiology,
39(4), pp.395-415.
Power, R.F., 2003. Enzymatic conversion of starch to fermentable sugars. The Alcohol
Textbook,, pp.23-32.