SURNAME: KHALIPHA

NAME: NOSIBUSISO

STUDENT NO: 201614708

TASK: ASSIGNMENT 2

COURSE: MIC 211

DUE DATE: 21/10/2020

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Contents
INTRODUCTION.....................................................................................................................................3
Indole production......................................................................................................................................3
Uses of Indole Test..................................................................................................................................4
Gelatin hydrolysis......................................................................................................................................4
Uses of Gelatin Hydrolysis test...............................................................................................................4
Starch hydrolysis.......................................................................................................................................5
Acetylmethylcarbinol production and the methyl red test.....................................................................5
REFERENCE LIST:.................................................................................................................................6

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MIC 211 ASSIGNMENT
TITLE: SELECTED PHYSIOLOGICAL TESTS USED FOR IDENTIFICATION OF
BACTERIA

INTRODUCTION

Traditional methods for identifying bacteria are based on morphological and

physiological basis. Since bacteria are so small, little detail can be deduced from
microscopical examinations of their morphology. Therefore, physiological criteria are
used to identify the genera and species of bacteria (Facklam, 1972).

Indole production

This test demonstrates the ability of certain bacteria to decompose the amino acid
tryptophane to indole, which accumulates in the medium. Indole production test is
important in the identification of Enterobacteria. Most strains of E. coli, P. vulgaris, P.
rettgeri, M. morgani and Providencia species break down the amino acid tryptophan
with the release of indole. This is performed by a chain of a number of different
intracellular enzymes, a system generally referred to as “tryptophanase.” It is used as
part of the IMViC procedures, a test designed to distinguish among members of the
family Enterobacteriaceae (Reva et al, 2001).

A variation on this test using Ehrlich’s reagent (using ethyl alcohol in place of isoamyl
alcohol, developed by Paul Ehrlich) is used when performing the test on non-fermenters
and anaerobes. Tryptophan is an amino acid that can undergo deamination and
hydrolysis by bacteria that express tryptophanase enzyme. Indole is generated by
reductive deamination from tryptophan via the intermediate molecule indole pyruvic
acid. Tryptophanase catalyses the deamination reaction, during which the amine (-NH2)
group of the tryptophan molecule is removed. Final products of the reaction are indole,
pyruvic acid, ammonium (NH4+) and energy. Pyridoxal phosphate is required as a
coenzyme (Reva et al, 2001).

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Uses of Indole Test

To differentiate Proteus mirabilis (indole negative) from all other Proteus species (indole
positive).To differentiate Klebssiella pneumoniae (indole negative) from Klebsiella
oxytoca (indole positive).To differentiate Citrobacter freundii (indole negative) from
Citrobacter koseri (indole positive) (Reva et al, 2001).

Gelatin hydrolysis

Gelatin is a protein derived from the animal protein collagen– component of vertebrate
connective tissue. It has been used as a solidifying agent in food for a long time. Gelatin
hydrolysis test is a great way to highlight proteolysis by bacteria

Principle of Gelatin hydrolysis test Gelatin hydrolysis test is used to detect the ability of
an organism to produce gelatinase (proteolytic enzyme) that liquefy gelatin. Hydrolysis
of gelatin indicates the presence of gelatinases. This process takes place in two
sequential reactions. In the first reaction, gelatinases degrade gelatin to polypeptides.
Then, the polypeptides are further converted into amino acids. The bacterial cells can
then take up these amino acids and use them in their metabolic processes (Morea et al,
1999).

Uses of Gelatin Hydrolysis test

Gelatin hydrolysis test is helpful in identifying and differentiating species of Bacillus,

Clostridium, Proteus, Pseudomonas, and Serratia. It distinguishes the gelatinase-
positive, pathogenic Staphylococcus aureus from the gelatinase-negative, non-
pathogenic S. epidermidis. Gram-positive, spore-forming, rod-shaped, aerobic, or
anaerobic bacteria such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis,
Clostridium perfringens and Clostridium tetani, are also positive for gelatin hydrolysis.
The test can also be used to differentiate genera of gelatinase-producing bacteria such
Serratia and Proteus from other members of the family Enterobacteriaceae (Morea et al,
2001).

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Starch hydrolysis

This test is used to identify bacteria that can hydrolyse starch (amylose and
amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. Often used to
differentiate species from the genera Clostridium and Bacillus. Because of the large size
of amylose and amylopectin molecules, these organisms cannot pass through the
bacterial cell wall. In order to use these starches as a carbon source, bacteria must
secrete a-amylase and oligo-1,6-glucosidase into the extracellular space. These
enzymes break the starch molecules into smaller glucose subunits which can then enter
directly into the glycolytic pathway. In order to interpret the results of the starch
hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to
form a dark brown colour. Thus, hydrolysis of the starch will create a clear zone around
the bacterial growth. Bacillus subtilis is positive for starch hydrolysis (Reva et al, 2001).

Acetylmethylcarbinol production and the methyl red test

These two tests can be performed on the same medium and they are both great
importance in the characterization of gram-positive non-spore forming organisms.
Instead of accumulating mostly acid products from fermentation of glucose, some
bacteria convert an intermediate substance pyruvic acid, to neutral products and carbon
dioxide. The formation of the most easily detected neutral product,
acetylmethylcarbinol(ch2coochohch3) is illustrated in the present experiment. This test is
Vosges-Proskauer test, organisms that do not convert acid end products into neutral
products produce lower pH than those they do. By employing the indicator methyl red
(yellow at pH 6.0, completely red at 4.5) (Facklam, 1972).

REFERENCE LIST:

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1. Facklam, R.R., 1972. Recognition of group D streptococcal species of human
origin by biochemical and physiological tests. Applied microbiology, 23(6), pp.
1131-1139.
2. Morea, M., Baruzzi, F. and Cocencelli, P.S., 1999. Molecular and physiological
characterization and dominant bacterial populations in traditional mozzarella
cheese processing, journal of applied microbiology, 87(4), pp. 5774-5782.
3. Reva, O.N., Sorokulova, I.B. and Smirnov, V.V., 2001. Simplified technique for
identification of the aerobic spore-forming bacteria by phenotype. International
journal of systematic and evolutionary microbiology), 51(4), pp.1361-1371.