Citrate utilization test: Principle, procedure, result and interpretation, citrate

Citrate Utilization Test: Principle, Procedure, Result and Interpretation, Citrate Utilization Test Positive Bacteria and keynotes

Principle of Citrate Utilization Test

A citrate utilization test is used to determine the ability of an organism to utilize sodium citrate as its only carbon source and inorganic ammonium salts as its only nitrogen source. When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity turning the bromthymol blue indicator from green to blue.

Requirements for test

Simmons citrate agar slant
Cotton plug
Sterile Inoculating wire/ sticks
Test organism
Bunsen burner
Incubator
Test tube rack

Procedure of Citrate Utilization Test

Streak the slant back and forth with a light inoculum picked from the Center of a well-isolated colony.
Place cap loosely on the tube.
Incubate aerobically at 35°C to 37°C for 18 to 24 hours.
Observe a color change from green to blue along the slant.

Quality control

Quality Control strains used in citrate utilization test

Positive Control (PC)-Klebsiella pneumoniae ATCC 13883

Negative Control (NC)-Escherichia coli ATCC 25922

Observation

Positive: Growth on the medium with color change from green to intense blue.
Negative: Absence of growth and color change

Result and interpretation in citrate utilization test

Citrate utilization test positive: The growth will be visible on the slant surface and the medium will be an intense blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue.
Citrate utilization test negative: trace or no growth will be visible. No color change will occur; the medium will remain the green color of the uninoculated agar. Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium, thus a citrate utilization negative test culture will be virtually indistinguishable from an uninoculated slant.
Uninoculated (UN): No growth and no color change; slant remains green
Negative control: No growth and no color change; slant remains green
Positive control: Growth with color change from green to intense blue along the slant
Test organism/bacteria: Positive i.e. growth with color change from green to intense blue along the slant as shown above figure.

List of Bacteria which gives positive citrate utilization test

Klebsiella pneumoniae
Citrobacter freundii
Enterobacter cloacae (minority of strains gives negative result)
Salmonella other than Typhi and Paratyphi A
Serratia marcescens
Proteus mirabilis(a minority of strains gives negative result)
Providencia alcalifaeciens
Vibrio vulnificus
Euringella Americana
Achromobacter oxylosoxidans

Citrate Test: variable (different strains give different results)

Proteus vulgaris
Vibrio cholerae
Vibrio parahaemolyticus

Citrate test: negative

Escherichia coli
Shigella species
SalmonellaTyphi
SalmonellaParatyphi A
Morganella morganii
Yersinia enterocolitica
Edwardsiella tarda
Vibrio holisae

Keynotes on Citrate utilization test

Generally, incubate aerobically at 35°C to 37°C for 18 to 24 hours but if negative Some organisms may require up to 4 days of incubation due to their limited rate of growth on citrate medium.
Other methods of citrate utilization test available are-IMViC the test kit method and API test kit method
As you know, Escherichia coli is citrate utilization test negative; although uncommon, natural E. coli variants that are citrate positive have been isolated. Citrate-negative strains of E. aerogenes have also been found.
Other media used for citrate utilization test are-

Koser’s citrate medium

It is a liquid medium lacking agar.
It does not contain any indicator.
A positive test is shown by the presence of turbidity in the medium.

Christen’s citrate medium

Same as Simmons citrate medium in which bromothymol blue indicator is replaced by phenol red.

Limitations of Citrate utilization test

The limitations of this test are as follow:

Luxuriant growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue on further incubation, the test should be repeated with less inoculum.
Do not stab the slant, since the test requires an aerobic environment.
Do not inoculate from the broth culture, due to carryover of media.
Tests with equivocal results should be repeated.
To avoid false-positive reactions, use a light inoculum to prevent the carryover of t substances from the previous medium.
The reaction of this medium alone is not sufficient for identification at the species level.

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