Citrate utilization test

Principle

• Tests whether an organism can use citrate as its only carbon source and ammonium salts as its only nitrogen source.
• Utilization of citrate and ammonium produces alkaline products, raising the pH.
• Bromothymol blue in Simmons citrate agar is green at neutral pH and turns blue above about pH 7.6.

Specimen

• Pure, well-isolated colony of a Gram-negative rod (18–24 h).

Reagents and equipment

• Simmons citrate agar slant (sodium citrate, ammonium dihydrogen phosphate, bromothymol blue).
• Straight needle or light loop.

Procedure

• Pick a light inoculum from the colony center, avoiding carryover of nutrients from the original medium.
• Streak the slant surface (a butt stab is optional per local procedure).
• Loosen cap for aeration and incubate at 35–37 °C for 18–24 h; hold up to 4 days before calling negative if required by procedure.

Results

• Positive: growth with a blue color change on the slant; growth on the slant without color change is also read as positive by many procedures.
• Negative: no growth and green medium unchanged.
• Positive examples: Klebsiella pneumoniae, Enterobacter, Citrobacter, Serratia, most Salmonella.
• Negative examples: E. coli, Shigella, Salmonella Typhi and Paratyphi A (usually).

Quality control and pitfalls

• Controls: K. pneumoniae or Enterobacter aerogenes (positive), E. coli (negative).
• Heavy inoculum carries over organic matter, causing false positives.
• Inoculate citrate before other media if using the same needle, to avoid carrying glucose or protein.

Clinical use

• Part of the IMViC series: E. coli is typically ++−− (indole, MR, VP, citrate) and Klebsiella/Enterobacter −−++.