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Short answer
The citrate utilization test shows whether a bacterial isolate can use citrate as its only carbon source. On Simmons citrate agar, growth with a bromothymol blue colour change from green to blue (pH above 7.6) is a positive. Little or no growth with no colour change is a negative. The result is one input among several identification tests, never a verdict on its own. This is laboratory identification background, not medical advice.
Definition and scope
The American Society for Microbiology (ASM) protocol describes the test as screening an isolate for the ability to use citrate as its carbon and energy source. It belongs to the IMViC group of tests that help distinguish members of the Enterobacteriaceae. ASM notes that commercial identification kits such as API-20E and Enterotube II include a citrate utilization medium. For the neighbouring tests in a typical workflow, see methyl red test, urease test results and triple sugar iron agar results.
How Simmons citrate agar works
Simmons medium offers citrate as the sole carbon source and ammonium dihydrogen phosphate as the sole fixed nitrogen source. An organism that can take up citrate (ASM names citrate permease) and cleave it (citrate lyase, giving oxaloacetate and acetate) can grow. Its metabolism produces alkaline by-products: carbon dioxide and sodium combine to sodium carbonate, and ammonium salts yield ammonium hydroxide. The pH rises.
The indicator is bromothymol blue, deep forest green at neutral pH and blue above pH 7.6. ASM’s figure shows green across pH 6.9 to 7.6. The FDA Bacteriological Analytical Manual (BAM) formulation, medium M138, per litre: sodium citrate 2 g, sodium chloride 5 g, dipotassium phosphate 1 g, ammonium dihydrogen phosphate 1 g, magnesium sulfate 0.2 g, bromothymol blue 0.08 g and agar 15 g, final pH 6.8 ± 0.2.
Reading a positive and a negative slant
- Positive: visible growth on the slant and the medium turning an intense Prussian blue (ASM).
- Negative: trace or no growth and no colour change. ASM says such a tube is virtually indistinguishable from an uninoculated slant.
FDA BAM Chapter 5 (Salmonella) words the positive slightly differently: growth, “usually accompanied by” a colour change from green to blue. A negative is no or very little growth and no colour change.
ASM and FDA BAM protocols compared
The two named protocols are written for different purposes: ASM for teaching and general identification, BAM for Salmonella confirmation in food testing. They differ in ways that change how a tube is set up and read.
| Point | ASM Citrate Test Protocol | FDA BAM Chapter 5 (Salmonella) |
|---|---|---|
| Inoculation | Lightly streak the surface of the slant from a single isolated colony, ideally with a needle | Streak the slant and stab the butt |
| Incubation | 35 °C (± 2) for 18 to 48 hours; some organisms may need up to 7 days | 96 ± 2 hours at 35 °C |
| Positive | Growth and an intense blue colour | Growth, usually accompanied by green-to-blue colour change |
| Negative | Trace or no growth, no colour change | No growth or very little growth, no colour change |
The practical point: a result read at 24 hours under the ASM approach and one read at 96 hours under the BAM approach are not interchangeable records, so note which protocol and which reading time a result came from. ASM also says to use a fresh 16 to 18 hour pure culture and to avoid liquid cultures as the inoculum, and to keep screw caps loose because citrate utilization needs oxygen.
Simmons vs Koser vs Christensen
ASM describes three media that all test citrate use, but a positive does not mean the same thing on each.
| Medium | What ASM states |
|---|---|
| Koser’s citrate broth | Citrate is the only carbon source, but there is no indicator and no agar; a heavy inoculum can be misconstrued as a positive, so it is more prone to misinterpretation |
| Simmons citrate agar | Adds agar and bromothymol blue to the Koser concept (Simmons, J Infect Dis 1926;39:209-214); the most commonly used in teaching labs as part of IMViC |
| Christensen’s citrate | Contains extras such as yeast extract, glucose and cysteine, with phenol red (yellow at pH 6.7, red when alkaline); negatives still grow, and a positive means citrate can be used but not necessarily as the sole carbon source |
Where it fits in an identification workflow
BAM places Simmons citrate in the Salmonella confirmation panel alongside other biochemical tests. In its Table 1, the Salmonella reaction for Simmons citrate is listed as variable (“v”), although the chapter text says most Salmonella cultures are citrate-positive. That is why ASM advises using multiple identification tests: some natural isolates give weak or nonstandard results, and rare citrate-positive E. coli variants and citrate-negative E. aerogenes exist. Read citrate together with lysine iron agar, SIM medium and the tests above rather than in isolation.
For labs that stock media, ASM notes Simmons medium is available both as premixed powder and as premade slants from a variety of suppliers. Record the lot and storage conditions of each batch with the result.
Common misunderstandings
- Heavy inoculum. A thick streak can produce apparent growth that is not true citrate use, which is why ASM stresses a light inoculum.
- Growth without a colour change. Under the ASM reading, a positive needs the blue colour; under BAM the colour change usually accompanies growth. Check which criterion your protocol applies and keep reading times consistent.
- Streak versus stab. ASM calls for a surface streak because the reaction needs oxygen; BAM adds a butt stab. Follow the protocol you are working under.
- Treating it as stand-alone. Citrate is variable for Salmonella as a genus, so it cannot decide an identification alone.
FAQ
What does a citrate utilization test positive mean?
The organism grew with citrate as its sole carbon source and made the medium alkaline, turning bromothymol blue from green to blue.
What colour is a negative Simmons citrate tube?
It stays green, with trace or no growth, and looks like an uninoculated slant (ASM).
How long should the tube be incubated?
ASM gives 18 to 48 hours at 35 °C (± 2), with up to 7 days for some organisms. FDA BAM specifies 96 ± 2 hours at 35 °C for Salmonella confirmation.
Sources
- American Society for Microbiology, Citrate Test Protocol (M. MacWilliams, 2016). Checked 2026-11-01.
- U.S. FDA, BAM Media M138: Simmons Citrate Agar. Checked 2026-11-01.
- U.S. FDA, Bacteriological Analytical Manual, Chapter 5: Salmonella. Checked 2026-11-01.
