The citrate utilization test is used to differentiate between members of the Enterobacteriaceae family based on their ability to use citrate as a sole carbon source. Some bacteria like Enterobacter and Klebsiella are able to convert citrate to oxaloacetate and acetate through metabolic pathways that produce carbon dioxide. The test uses Simmon's citrate agar with citrate as the sole carbon source and a pH indicator. A color change in the medium from orange to blue indicates positive test results and the ability to use citrate. E. coli tested negative while Klebsiella pneumoniae tested positive on the citrate utilization test.

1. CITRATE UTILIZATION TEST
SNEKHA. K
B.PHARM 2nd YEAR

2. • The citrate test is performed along with the other IMViC tests
to differentiate Gram-negative bacilli of the
Enterobacteriaceae family.
• It is an important test that allows the species-level
identification of the members of the Enterobacteriaceae
family.
• The citrate utilization test is a part of the IMViC test (Indole,
Methyl Red, Vogues-Proskauer, and Citrate Test) that
differentiates organisms on the basis of their ability to use
citrate as a sole source of energy.

3. • Bacteria such as Enterobacter aerogens, Klebsiella
pneumoniae can convert citrate to oxaloacetate and
acetate. The oxaloacetate is then metabolized to pyruvate
and CO2.
• Utilization of exogenous citrate requires the presence of
citrate transport proteins (permeases).
• Citrate = acetate + oxaloacetate
• Oxaloacetate = pyruvate + CO2

5. Principle of Citrate Utilization Test
• Citrate agar is used to test the ability of an organism to
utilize citrate as a source of energy.
• The agar medium contains citrate as the sole carbon
source and inorganic ammonium salts as the sole source
of nitrogen.
• Bromothymol blue is considered as a pH indicator.
• For the citrate utilization test oxygen is required and the
process is done on the slants.

6. • The citrate is oxidized from bacteria and is extracted with
the release of CO2 from the medium.
• Sodium from sodium citrate combines with CO2 and water,
to form an alkaline product (sodium carbonate).
• The change in the pH of the solution gives details about the
presence and absence of the test.
• Colour change of the solution to blue indicates the positive
citrate test.
• If there is no colour shift, that means there is no growth in
the medium, which indicates the absence of the citrate test.

7. Simmon’s Citrate Agar Composition

8. Requirements:
• Simmon’s citrate agar slant
• Given bacterial samples ( E. coli and Klebseilla)
• Inoculating loop
Procedure:
• Inoculate the given sample of organism were on the slant
of the media using sterile wire and label the tubes
• Incubate the tubes at 37°C for 24 -48 hours.
• Observe the color change in the medium

9. Result:
• Negative: Escherichia coli
• Positive: Klebsiella
pneumoniae

10. Thank you