BIOCHEMICAL TEST TO IDENTIFY
BACTERIA
Prepared by:
Miss Norzawani Jaffar
Bsc(Hons) Biomedical Sciences
Learning Outcomes
• Able to understand the principle and function of biochemical
test;
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Beta-glucuronidase
Bile solubility
Catalase
Citrate utilization
Coagulase
DNA-ase
Indole
Litmus milk decolorization
Lysine decarboxylase
Oxidase
Urease
Introduction
• While several commercial system for
identifying bacteria are available, these are
often difficult to obtain or too expensive to
use in developing countries.
• This subunit includes a range of conventional
biochemical test and tablet identification test
which most district laboratories will be able to
perform.
TEST
PURPOSE
Beta-glucuronidase
To identify E.coli
Bile Solubility
To differentiate S. pneumoniae from other
alpha-haemoytic streptococci
Catalase
Citrate utilization
To differentiate staphylococci from
streptococci
To differentiate enterobacteria
Coagulase
To identify S. aureus
DNA-ase
To help identify S. aureus
Indole
To differentiate Gram negative rods,
particularly E.coli
TEST
PURPOSE
Litmus milk decolorization
To help identify Enterococcus
and some clostrodia
Oxidase
To help identify Neisseria,
Pasteurella, Vibrio,
Pseudomonas
To help identify Proteus,
Morganella, Y. enterocolitica, H.
pylori
To assist in the identification os
Salmonella and shigella
Urease
Lysine decarboxylase
Bile Solubility Test
• To differentiate S. pneumoniae, which is soluble
in bile and bile salt, from other alpha-hemolytic
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. pneumoniae
as shown by a clearing of the turbidity with in 10-15
minutes. Viridans and other streptococci are not
dissolved and therefore there is no clearing of the
turbidity.
• Used to differentiate those bacteria that
produce the enzyme catalase, such as
staphylococci, from non-catalase producing
bacteria such as streptococci.
• PRINCIPLE:
– Catalase act as a catalyst in the breakdown of
hydrogen peroxide to oxygen and water. An
organism is tested for catalase production by
bringing it into contact with hydrogen peroxide.
Bubbles of oxygen are released if the organism is a
catalase producer. The culture should not be more
than 24 hrs old.
Most aerobic organsims will display (+) results.
e.g. Staphyloccocus aureus.
Some anaerobic organisms will display (-) results, indicating that they do not produce
catalase to prevent oxygen accumulation. Why?
Because since oxygen is totally not used for survival of these organisms, they do not have
the ability to produce catalase.
• Purpose: The citrate utilization
test is used to determine the
ability of an organism, using the
enzyme citrase, to use citrate as
its sole carbon source
• Used occasionally to assist in the
identification of enterobacteria.
Coagulase Test
• This test is used to identify S. aureus which
produces the enzyme coagulase.
• PRINCIPLE:
– Coagulase cause plasma to clot by converting fibrinogen
to fibrin. Two types of coagulase are produced by most
strains of S.aureus
• Free coagulase which converts fibronogen to fibrin by
activating a coagulase-reacting factor present in plasma. Free
coagulase is detected by clotting in the test tube.
• Bound coagulase (clumping factor) which converts fibrinogen
directly to fibrin without requiring a coagulase-reacting factor.
It can be detected by the clumping pf bacterial cells in the
rapid slide test.
• A tube test must be performed when the result
of a slide test is not clear, or when the slide test
is negative and Staphylococcus has been isolated
from a serious infections.
• Before performing a coagulase test, examine a
Gram stained smear to confirm that the
organism is a Gram positive coccus.
• Used in the identification of S.aureus which produces
deoxyribonuclease (DNA-ase) enzymes. The DNA-ase test is
particularly useful when plasma is not available to performed
a coagulase test or when the results of a coagulase test are
difficult to interpret.
• PRINCIPLE:
– Deoxyribonuclease hydrolyzes deoxyribonucleic acid(DNA). The test
organism is cultured on a medium which contains DNA. After
overnight incubation, the colonies are tested for DNA-ase-production
by flooding the plate with a weak hydrochloric acid solution. The acid
precipitates unhydrolyzed DNA. DNA-ase producing colonies are
therefore surround by clear areas due to DNA hydrolysis.
Note there is breakdown of the DNA in the agar. There is a clear zone (arrow) around the
bacterial growth where there is no longer any DNA left in the agar to precipitate out of
solution after the HCl was added.
Indole Test
• Testing for indole production is important in the identification
of enterobacteria. Most strains of E. coli, P. vulgaris, P.
rettgeri, M. morganii, and Providencia species break down the
amino acid tryptophan with the release of indole.
• PRINCIPLE:
– The test organism is cultured in a medium which contains
tryptophan. Indole production is detected by Kovac’s or
Ehrlich’s reagent which contains 4 (p)-dimethylaminobenzaldehyde. This reacts with the indole to produce a red
coloured compound. Kovac’s reagent is recommended in
preference to Ehrlich’s reagent for the detection of indole
from enterobacteria.
Result: Red surfeca layer------------ positive indole test
No red layer ------------------- negative indole test.
Litmus Milk Decolorization test
• A rapid and inexpensive technique to assist in the
identification of enterococci.
• It is based on ability of most strains of Enterococcus
sp to reduce litmus milk by enzyme action as shown
by decolorization of the litmus.
• PRINCIPLE
– A heavy inoculum of the test organism is incubated
for up to 4 hours in a tube containing litmus milk.
Reduction of litmus milk is indicated by a change in
colour of the medium from mauve to white or pale
yellow.
Result: White or pale yellow-pink colour---------------suggestive of Enterococcus
No change or a pink colour-------------- probably not Enterococcus
• The oxidase test is used to assist in the
identification of Pseudomonas, Neisseria,
Vibrio, Brucella, Pasteurealla species, all of
which produce the enzyme cytochrome
oxidase.
• PRINCIPLE
– A piece of filter paper is soaked with a few drops of
oxidase reagent. A colony of the test organism is
then smeared on the filter paper.
– When the organism is oxidase-producing, the
phenylenediamine in the reagent will be oxidized to
a deep purple colour.
Result:
Blue-purple colour------------------positve oxidase test(within 10 seconds)
No blue-purple colour--------------Negative oxidase test (within 10 seconds)
Note!: Ignore any blue-purple colour that develops after 10 seconds.
• is a differential medium used to determine whether an
organism is equipped with flagella and thus capable of
swimming away from a stab mark.
• The results of motility agar are often difficult to
interpret.
• Generally, if the entire tube is turbid, this indicates that
the bacteria have moved away from the stab mark (are
motile).
• The organisms in the two tubes pictured on the right
are motile. If, however, the stab mark is clearly visible
and the rest of the tube is not turbid, the organism is
likely nonmotile (tube pictured on the left).
• This test is used to identify bacteria capable of
hydrolyzing urea using the enzyme urease.
• It is commonly used to distinguish the genus Proteus
from other enteric bacteria.
• The hydrolysis of urea forms the weak base, ammonia,
as one of its products.
• This weak base raises the pH of the media above 8.4
and the pH indicator, phenol red, turns from yellow to
pink.
• Proteus mirabilis is a rapid hydrolyzer of urea (center
tube pictured here). The tube on the far right was
inoculated with a urease negative organism and the
tube on the far left was uninoculated.
END
Q&A
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MICROBIOLOGY II: TOPIC 2