Prepared by Miss Norzawani Jaffar Bsc Hons Biomedical Sciences Learning Outcomes Able to understand the principle and function of biochemical test Beta glucuronidase Bile solubility ID: 316359
Download Presentation The PPT/PDF document "BIOCHEMICAL TEST TO IDENTIFY BACTERIA" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
BIOCHEMICAL TEST TO IDENTIFY BACTERIA
Prepared by:
Miss
Norzawani
Jaffar
Bsc
(
Hons
) Biomedical SciencesSlide2
Learning Outcomes
Able to understand the principle and function of biochemical test;
Beta-
glucuronidase
Bile solubility
Catalase
Citrate utilization
Coagulase
DNA-
ase
Indole
Litmus milk
decolorization
Lysine
decarboxylase
Oxidase
UreaseSlide3
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.Slide4
TEST
PURPOSE
Beta-
glucuronidase
To identify
E.coli
Bile
Solubility
To differentiate S.
pneumoniae
from other alpha-
haemoytic
streptococci
Catalase
To differentiate staphylococci from streptococci
Citrate
utilization
To differentiate
enterobacteria
Coagulase
To identify S.
aureus
DNA-
ase
To help
identify S.
aureus
Indole
To differentiate
Gram negative rods, particularly
E.coliSlide5
TEST
PURPOSE
Litmus
milk
decolorization
To help identify
Enterococcus
and some
clostrodia
Oxidase
To help identify
Neisseria
,
Pasteurella
,
Vibrio
, Pseudomonas
Urease
To help identify Proteus,
Morganella
, Y.
enterocolitica
, H. pylori
Lysine
decarboxylase
To assist in the identification
os
Salmonella and
shigellaSlide6
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.Slide7Slide8
Catalase
test
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.Slide9
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.Slide10
Citrate Utilization Test
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
.Slide11
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.Slide12Slide13Slide14
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
.Slide15
DNA-
ase
test
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.Slide16
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. Slide17
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)-
dimethylamino-benzaldehyde
. 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
.Slide18
Result: Red
surfeca
layer------------ positive
indole
test
No red layer ------------------- negative
indole
test.Slide19
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.Slide20
Result: White or pale yellow-pink
colour
---------------suggestive of
Enterococcus
No change or a pink
colour
-------------- probably not
EnterococcusSlide21
Oxidase
Test
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
.Slide22
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.Slide23
Motility agar
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). Slide24Slide25
Urease
test
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
.Slide26Slide27
END
Q&ASlide28
Take note from
http://norazlicucst.weebly.com
MICROBIOLOGY II: TOPIC 2