This test is among a suite of IMViC Tests (Indole, Methyl-Red, Vogues-Proskauer, and Citrate) that are used to differentiate among the Gram-Negative bacilli in the family Enterobacteriaceae.
Principle of Citrate Utilization Test
Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy. The medium contains citrate as the sole carbon source and inorganic ammonium salts (NH4H2PO4) as the sole source of nitrogen.
Bacteria that can grow on this medium produce an enzyme, citrate-permease, capable of converting citrate to pyruvate. Pyruvate can then enter the organism’s metabolic cycle for the production of energy. Growth is indicative of utilization of citrate, an intermediate metabolite in the Krebs cycle.
When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from green to blue above pH 7.6.
Christensen developed an alternative citrate test medium that does not require the organism to use citrate as a sole carbon source. Christensen’s medium contains both peptone and cysteine. Thus citrate-negative bacteria can also grow on this medium. A positive reaction shows that the organism can use citrate but not necessarily as the sole carbon source.
Media used in Citrate Utilization Test
Simmon’s Citrate Agar
|Sodium Chloride||5.0 gm|
|Sodium Citrate (dehydrate)||2.0 gm|
|Ammonium Dihydrogen Phosphate||1.0 gm|
|Dipotassium Phosphate||1.0 gm|
|Magnesium Sulfate (heptahydrate)||0.2 gm|
|Bromothymol Blue||0.08 gm|
Deionized water = 1,000 ml
Final pH 6.9 +/- 0.2 at 25 degrees C.
- Dissolve above salts in deionized water.
- Adjust pH to 6.9.
- Add agar and Bromothymol blue.
- Gently heat, with mixing, to boiling until agar is dissolved.
- Dispense 4.0 to 5.0 ml into 16-mm tubes.
- Autoclave at 121 degree C under 15 psi pressure for 15 minutes.
- Cool in slanted position (long slant, shallow butt).
- Tubes should be stored in a refrigerator to ensure a shelf life of 6 to 8 weeks.
- The uninoculated medium will be a deep forest green due to the pH of the sample and the bromothymol blue.
Procedure of Citrate Utilization Test
- Streak the slant back and forth with a light inoculum picked from the center of a well-isolated colony.
- Incubate aerobically at 35 to 37C for up to 4-7 days.
- Observe a color change from green to blue along the slant.
Result Interpretation of Citrate Utilization Test
Positive Reaction: Growth with color change from green to intense blue along the slant.
Examples: Salmonella, Edwardsiella, Citrobacter, Klebsiella, Enterobacter, Serratia, Providencia, etc.
Negative Reaction: No growth and No color change; Slant remains green.
Examples: Escherichia, Shigella, Morganella, Yersinia etc.
Quality Control of Citrate Utilization Test
Citrate Positive: Klebsiella pneumoniae ATCC 13883 (growth; blue color)
Citrate Negative: Escherichia coli ATCC 25922 (no growth or trace of growth)
Limitations of Citrate Utilization Test
- Some organisms are capable of growth on citrate and do not produce a color change. Growth is considered a positive citrate utilization test, even in the absence of a color change.
- Tests with equivocal results should be repeated.
- The reactions of this medium alone are not sufﬁcient for identiﬁcation to the species level.
- MacWilliams, M. P. (2009). Citrate Test Protocol. Accessed 10th Sept, 2015. http://www.microbelibrary.org/component/resource/laboratory-test/3203-citrate-test-protocol
- Bailey & Scott’s Diagnostic Microbiology 13th Edition: Citrate Utilization
- Clinical Microbiology Procedures Handbook 2nd Edition: Citrate Utilization Test (Simmons)
- Microbe Online
- Hardy Diagnostics
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- University of Windsor