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

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

Introduction of Citrate Utilization Test

Sodium citrate is the only carbon source in citrate agar composition. On the basis of citrate utilization, some bacteria can be differentiated. Klebsiella pneumoniae and Citrobacter freundii are citrate positive while Escherichia coli and Shigella species are negative.  Simmons Citrate Agar is recommended for differentiation of the members of Enterobacteriaceae as well as other Gram-Negative rods ( Pseudomonas, Acinetobacter) on the basis of citrate utilization from clinical and non-clinical samples.

Principle of Citrate Test

A citrate test or 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.

Composition of Simmons Citrate Agar

Ingredients Gms / Litre

  • Magnesium sulfate 0.2
  • Ammonium dihydrogen phosphate 1.0
  • Dipotassium phosphate 1.0
  • Sodium citrate 2.0
  • Sodium chloride 5.0
  • Bromothymol blue 0.08
  • Agar 15.0
  • Final pH (at 25°C) 6.8±0.2

Requirements for Test

  1. Simmons citrate agar slant
  2. Cotton plug
  3. Sterile Inoculating wire/ sticks
  4. Test organism
  5. Bunsen burner
  6. Incubator
  7. Test tube rack

Procedure of Citrate Test

  1. Streak the slant back and forth with a light inoculum picked from the Center of a well-isolated colony.
  2. Place cap loosely on the tube.
  3. Incubate aerobically at 35°C to 37°C for 18 to 24 hours.
  4. 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 of Citrate Test

  • Citrate 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 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 or negative

 List of Bacteria which gives positive citrate utilization test

  1. Klebsiella pneumoniae
  2. Citrobacter freundii
  3. Enterobacter cloacae (minority of strains gives negative result)
  4. Salmonella other than Typhi and Paratyphi A
  5. Serratia marcescens
  6. Proteus mirabilis(a minority of strains gives negative result)
  7. Providencia alcalifaeciens
  8. Vibrio vulnificus
  9. Euringella Americana
  10.  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  Test

  1. 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.
  2. Other methods of citrate utilization test available are-IMViC the test kit method and API test kit method
  3.  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.
  4. 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 Test 

The limitations of this test are as follow:

  1. 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.
  2. Do not stab the slant, since the test requires an aerobic environment.
  3. Do not inoculate from the broth culture, due to carryover of media.
  4. Tests with equivocal results should be repeated.
  5. To avoid false-positive reactions, use a light inoculum to prevent the carryover of t substances from the previous medium.
  6. The reaction of this medium alone is not sufficient for identification at the species level.

Further Readings

  1. Jean F. Mac Faddin Biochemical tests for Identification of Medical bacteria
  2. Bailey’s and Scott’s Diagnostic Microbiology-13th Edition
  3. Mackie and McCartney Practical Medical Microbiology-14th Edition
  4. ASM Clinical Microbiology Procedures Handbook-2nd Edition
  5. Monica Cheesbrough Distinct Laboratory Practice in Tropical Countries…2nd Edition
  6.  Textbook of practical Microbiology – Subhash Chandra Parija
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