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General Microbiology Laboratory
Biochemical tests
Citrate Utilization test
 Determine ability of an organism to use citrate as sole carbon
source.
 Simmon's citrate agar: sodium citrate as sole carbon source
and ammonium ion as the sole nitrogen source.
 Bromthymol blue (pH indicator), turns from green at neutral
pH (6.9) to blue at pH higher than 7.6 (basic or alkaline).
 Organisms that metabolize citrate utilize the ammonium salts
releasing ammonia and increasing the pH of the medium .
Bromthymol blue is present in the medium as the indicator
dye.
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 How to Perform Test: Inoculate slant with inoculating loop.
 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.
 Media and Reagents Used: Simmon’s Citrate Agar contains
sodium citrate (carbon source), ammonium ion (nitrogen
source), & pH indicator—bromthymol blue.
 Reading Results:
• A + result is blue (meaning the bacteria metabolised citrate
and produced an alkaline end product) and a – result
remains green
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Result
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Urease Test
 Some bacteria are able to produce an enzyme called urease that
attacks the nitrogen and carbon bond in amide compounds such as
urea, forming the end products ammonia, CO2, and water.
 Urease activity (the urease test) is detected by growing bacteria in
medium containing urea and using a pH indicator such as phenol red.
When urea is hydrolyzed, ammonia accumulates in the medium and
makes it alkaline. This increase in pH causes the indicator to change
from orange-red to deep pink or purplish red and is a positive test for
urea hydrolysis.
 This test is particularly useful in distinguishing the genus Proteus from
other enteric bacteria
 How to Perform Test: Inoculate Urea broth or urea slant agar with
inoculating loop.
 Property it tests for: This test is done to determine a bacteria’s ability to
hydrolyze urea to make ammonia using the enzyme urease.
 Media and Reagents Used:

Urea Disks or Tablets
Urea broth or Urea slants contains a yeast extract, monopotassium
phosphate, disodium phosphate, urea, and phenol red indicator.
 Reading Results: Urea slant is a yellow color. The enzyme urease will
be used to hydrolyze urea to make ammonia. If ammonia is made, the
broth turns a bright pink color, and is positive. If test is negative, broth has
no color change and no ammonia is made.
Limitations of the procedure
Some bacteria have a delayed urease reaction
that may require an incubation period longer
than 48 hours.
Urease-Producing Enterobacteriaceae
•
•
•
•
Proteus
Klebsiella pneumoniae
Enterobacter cloacae
Yersinia enterocolitica
End of lecture
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