Biochemical Tests

Tests To Know
Case Study Tests
Indole
Citrate
Urea hydrolysis
Bile solubility
Oxidase
Methyl Red/Voges Proskauer
KIA & TSI
Motility
Catalase
Coagulase

Indole Test
Principle test: This test is performed to help differentiate species of the family
Enterobacteriaceae. It tests for the bacteria species ability to produce indole, e.g.
E. coli, P. vulgaris, M. morganii. Bacteria use an enzyme, tryptophanase to break
down the amino acid, tryptophan, which makes by-products, of which, indole is
one.
Media and Reagents Used: Tryptone broth contains tryptophan. Kovacs
reagent {4 (p)-dimethylaminobenzaldehyde}.
How to Perform Test: Inoculate Tryptone or Peptone water broth with inoculating
loop.
Reading Results: Kovacs reagent reacts with indole and creates a red color at
the top part of the test tube.

Citrate utilization test

Property it tests for: This test is used to help differentiate species of the family
Enterobacteriaceae. It is selective for bacteria that has the ability to consume
citrate as its sole source of carbon and ammonium as sole nitrogen source, e.g. K.
pneumoniae.
Media and Reagents Used: Simmons Citrate Agar contains sodium citrate
(carbon source), ammonium ion (nitrogen source), & pH indicatorbromthymol
blue.
How to Perform Test: Inoculate slope with inoculating loop.
Reading Results:
A +ve result is blue (meaning the bacteria metabolised citrate and produced
an acid end product) and
A ve result remains green.

Urea Hydrolysis
This test is done to determine a bacterias ability to hydrolyze urea to make
ammonia using the enzyme urease. It used to differentiate enterobacteria. Proteus
strain are strong urease producers. Y. enterocolitica also shows urease activity.
Principle: The test organism is cultured in a medium which contains urea and the
indicator phenol red. When the strain is urease-producing, the enzyme will break
down the urea (by hydrolysis) to give ammonia and carbon dioxide. With the
release of ammonia, the medium becomes alkaline as shown by a change in colour
of the indicator to pink-red.
Media and Reagents Used: Urea broth or agar contain a yeast extract,
monopotassium phosphate, disodium phosphate, urea, and phenol red indicator.
Reading Results:
Pink colour.+ve urease
No pink colour-ve urease

Bile solubility test

This test helps to differentiate S. pneumonae, which is soluble in bile and bile salt,
from other alpha-haemolytic streptococci (Viridans streptococci) which are
insoluble.
Principle: A heavy inoculum of the test organism is emulsified in physiological
saline and the bile salt sodium deoxycholate is added. This dissolves S.
pneumonae as shown by clearing of the turbidity within 10-15 mins
Media and Reagents Used:
Sodium deoxycholate 100 g/l (10% w/v).
Physiological saline (Nacl, 8.5 g/l)

 Reading Results:
Clearing of turbidity.....Probably S. pneumonae.
No clearing of turbidityOrganism is probably not S. pneumonae.

Oxidase test
The oxidase test is used to assist in the identification of Pseudomonas, Neisseria,
Brucella, and Pasteurella species, all of which produce the enzyme cytochrome
oxidase.
Principle: Oxidase positive organisms are detected by the use of oxidase reagent
(N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride). Oxidase reagent is
colorless in its reduced state and dark purple in its oxidized state.
Media and Reagents Used:
Freshly prepared oxidase reagent or oxidase disc.
Important: Acidity inhibit oxidase enzyme activity, therefore the oxidase test
must not be performed on colonies that produce fermentation on carbohydratecontaining media ?.
Reading Results: (Within 10 seconds)
Blue-purple colour..+ve oxidase test.
No blue-purple colour.-ve oxidase test

Methyl Red/Voges Proskauer (MR/VP)

Properties they test for: Both tests are used to help differentiate species of the
family Enterobacteriaceae.
MRtests for acid end products from glucose fermentation.
VPtests for acetoin production from glucose fermentation.
How to Perform Tests: Inoculate 2 glucose broths with inoculating loop. After 48
hours of incubation, add a few drops of MR to one tube, and VP reagents to the
other tube.
Media and Reagents Used:
Glucose Broth
Methyl Red indicator for acid
Voges Proskauer reagentsA: 5% Alpha-Naphthol, & ethanol, B: Potassium Hydroxide,
& Deionized Water.
Reading Results:
MR a + result is red (indicating pH below 6) and a result is yellow
(indicating no acid production)
VPA + result is red after VP reagents are added (indicating the presence of
acetoin) and a result is no color change.

Kliger's iron agar (KIA) and triple sugar iron agar

(TSI)
Are widely used in the identification of gram negative bacteria particularly the
Enterobacteriaceae. The media are poured as slants and are inoculated with a stab
to the butt followed by a streak of the slant surface. The bacteria therefore are
exposed to both an anaerobic environment (butt) and an aerobic one (slant).
Phenol red is present as an indicator.

Motility Test
The motility test is not a biochemical test since we are not looking at metabolic
properties of the bacteria. Rather, this test can be used to check for the ability of
bacteria to migrate away from a line of inoculation .
Property it tests for: This test is done to help differentiate species of bacteria
that are motile.
Media and Reagents Used: Motility media contains tryptose, sodium chloride,
agar, and a color indicator. Or use cavity slide in hanging drop method.
Reading Results: If bacteria is motile, there will be growth going out away from
the stab line, and test is positive. If bacteria is not motile. A colored indicator can
be used to make the results easier to see.

Catalase test
This test is used to differentiate those bacteria that produce the enzyme catalase,
such as staphylococci, from non-catalase producing bacteria such as streptococci.
Principle: Catalase acts as a catalyst in the breakdown of hydrogen peroxide.
Bubbles of oxygen are released if the organism is catalase producer.
Reagents Used: Hydrogen peroxide 3% H2O2.
Results
Active bubbling+ve catalase.
No bubbles..-ve. Catalase.
Caution:
The culture is not be more than 24 hours old.?
The test is not performed from blood agar.?
Performing the test on a slid is not recommended because of the risk of
contamination from active bubbling.

Coagulase

 This test is used to identify S. aureus which produces the enzyme coagulase.
Principle: Coagulase causes plasma to clot by converting fibrinogen to fibrin. Two
types of coagulase are produced by most strains of S. aureus:
1- Free coagulase which converts fibrinogen to fibrin by activating a coagulase
reacting factor present in plasma. Free coagulase is detected by clotting in the
tube test.
2- Bound coagulase (Clumping factor) which convert fibrinogen directly to fibrin. It
detected by rapid slide test.
Coagulase Results
Reading Results:
If the organism is has coagulase it will clump the plasma.
If the organism does not have coagulase it will not clump the plasma.