Download citrate & oxidase test

Islamic University_Gaza
Medical Technology Department
Introduction
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Tests for the ability of bacteria to convert
citrate (an intermediate of the Krebs
cycle) into oxaloacetate (another
intermediate of the Krebs cycle).
In this media, citrate is the only carbon
source available to the bacteria.
Principle Of Citrate Test
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To determine if an organism is capable of
utilizing citrate as sole carbon source for
metabolism with resulting alkalinity.
If organisms utilize citrate and ammonium
salts, the medium becomes more basic and the
bromthymol blue changes from green to blue
as a result of an increase in the pH of the
media.
If it can not use citrate then it will not grow.
Significance Of Citrate Test
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A differential cultural test to identify
genera within the bacterial family
Enterobacteriaceae that are able to utilize
sodium citrate as a sole source of carbon.
Materials
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1.
2.
3.
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Simmons Citrate agar
which contains :
sodium citrate (carbon
source) .
ammonium salts (nitrogen
source).
Bromthymol blue indicator.
Inoculating needle .
Method
1.
2.
Streak one
organism over the
surface of the agar
slant, then stab the
butt.
Incubate the tube
at37°C for 48 hours
.
Result
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Examine for growth
(+) .
Growth on the
medium is
accompanied by a rise
in pH to change the
medium from its
initial green color to
deep blue.
Result
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Some intestinal bacteria are (+)citrate as:
Klebsiella .
Citrobacter.
Enterbacter aerogenes .
while others is(-) citrate as: E. coli
Summary
Klebsiella
Enterbacter
aerogenes
Citrobacter
E. coli
(+)
(+)
(+)
(-)
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This laboratory test is based on detecting the
production of the enzyme cytochrome oxidase by
Gram-negative bacteria.
It is a hallmark test for the Neiserria
.It is also used to discriminate between aerobic
Gram-negative organisms like Pseudomonas
aeruginosa and other Enterobacteriaciae
Cytochrome oxidase, part of an intracellular system
that perform the final step in electron transport,
reducing oxygen to water.
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Pick a colony for testing using a sterile wooden toothpick.
Transfer the colony to the surface of one of the four grid
areas on the Oxidase Test slide .
The test slide surface is impregnated with the reagent
tetramethyl-p-phenylenediamine dihydrochloride. This
reagent causes a dark purple color to appear in the presence
of cytochrome oxidase .
Observe the color change. The reaction color will change
from pink to maroon to dark purple .
Read the test results within 20 seconds. Some organisms
may show slight positive reactions after this period and such
results are NOT considered definitive.
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Don't use an Iron inoculating loop to transfer
cells; this will cause false positives.
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The oxidase test is negative for all the
Enterobacteriaceae. It is positive for the oxidative
non-fermenters ,Pseudomonas and Burkholderia ,
Neisseria, Hemophilus and Moraxella,Vibrio,
Campylobacter, and Helicobacter are other oxidasepositive bacteria .