[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$f-Td0o7vFFxRLYn8o2Be0QutCpeknemrkzuYUc3kECQ4":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":63},[4,8,12,16,20,24,28],{"title":5,"slug":6,"path":7},"About Microbeonline.com","about-microbeonline-com","\u002Fabout-microbeonline-com\u002F",{"title":9,"slug":10,"path":11},"About Me","about-me","\u002Fabout-microbeonline-com\u002Fabout-me\u002F",{"title":13,"slug":14,"path":15},"Advertise with Us","advertise-us","\u002Fadvertise-us\u002F",{"title":17,"slug":18,"path":19},"Privacy Policy","privacy-policy","\u002Fprivacy-policy\u002F",{"title":21,"slug":22,"path":23},"Abbreviations","abbreviations","\u002Fabbreviations\u002F",{"title":25,"slug":26,"path":27},"Microbes","microbes","\u002Fmicrobes\u002F",{"title":29,"slug":30,"path":31},"Books","recommended-books","\u002Frecommended-books\u002F",{"type":33,"data":34},"blog",{"slug":35,"title":36,"description":37,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":40,"lastUpdatedDate":41,"draft":42,"category":43,"image":38,"body":44,"faq":45,"tags":61,"related":62},"citrate-utilization-test","Citrate Utilization Test: Principle, Procedure, Results & Citrate-Positive Organisms","Citrate test principle and procedure — including the one rule most students miss: growth alone counts as positive, even without the blue color change.",null,"Acharya Tankeshwar","2013-05-17","2026-07-11",false,"biochemical-tests","The citrate utilization test determines whether an organism can use sodium citrate as its sole carbon source and ammonium salts as its sole nitrogen source. It's the fourth and final test in the [IMViC panel](https:\u002F\u002Fmicrobeonline.com\u002Fimvic-tests-principle-procedure-and-results\u002F).\n\n![](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FCitrate-utilization-test-204x300.jpg)Figure: Citrate Test Right: Negative Left: Positive\n\n## Why It Matters\n\nThis test has a direct public-health pedigree: it's one of the classic ways to distinguish *Klebsiella aerogenes* (formerly *Enterobacter aerogenes*) — citrate-positive, naturally found in soil and water — from *E. coli* — citrate-negative, whose presence in a water sample specifically signals fecal contamination. Where indole and MR\u002FVP separate organisms by *what* they ferment, citrate separates them by a different question entirely: can this organism survive on citrate as its only carbon source at all?\n\nThat's a transport-protein question first: without citrate permease to carry citrate across the membrane, the intracellular machinery never gets a substrate to work on. Many Enterobacteriaceae possess the downstream enzymes but lack the permease, so they test negative despite being biochemically capable on paper. This is why citrate doesn't always track the same way MR\u002FVP does, and why it's run as a fourth, independent check rather than a confirmation of the first three. The Principle section below follows the citrate from the permease that admits it to the lyase that cleaves it.\n\nClinically, the same logic applies at the bench: *Klebsiella pneumoniae* (citrate-positive) and *E. coli* (citrate-negative) can look identical on a lactose-fermenting MacConkey colony; citrate is one of the fastest ways to split them apart before final identification comes back, which matters because *Klebsiella* carries a meaningfully different resistance profile than *E. coli* in most local antibiograms.\n\n## Principle\n\nCitrate is an intermediate in the Krebs cycle. When an organism uses it as its carbon and energy source, the breakdown produces alkaline carbonates and bicarbonates, raising the medium's pH. This pH shift is what the test actually detects  not citrate utilization directly, but its downstream alkalinizing effect.\n\n![](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FPrinciple-of-Citrate-Utilization.jpg)Figure: Citrate Utilization Test Chemistry\n\n### **The chemistry:**\n\nThe chemistry runs on two tracks, and the second one matters more than students are usually told.\n\n**Carbon track.** Citrate permease first transports citrate into the cell. Inside, citrate lyase cleaves it to oxaloacetate and acetate, oxaloacetate is decarboxylated to pyruvate, and CO₂ is released. Some of that CO₂ combines with sodium in the medium to form sodium carbonate, which is mildly alkaline.\n\n**Nitrogen track, the dominant one.** The medium's only nitrogen source is an ammonium salt (ammonium dihydrogen phosphate). To grow, the organism must strip nitrogen from it, releasing ammonia. Ammonia dissolves to ammonium hydroxide, and this is what drives most of the pH rise past 7.6.\n\nSo the blue color is really a report on **two** simultaneous demands being met: the organism is using citrate as its sole carbon source *and* the ammonium salt as its sole nitrogen source. The nitrogen half is why an organism that merely tolerates citrate will not turn the slant blue, while one that genuinely lives on this medium will.\n\nThe medium's bromothymol blue indicator is **forest green at neutral pH** and shifts to **Prussian blue above pH 7.6**.\n\n## Procedure\n\nSimmons citrate agar is poured as a slant; this is an **aerobic surface test**, not a stab test.\n\n1. Touch the tip of a needle to a young colony (18–24 hours old).\n2. **Streak the slant surface lightly. Do not stab.** This test requires an aerobic environment; stabbing creates an anaerobic pocket that won't read correctly.\n3. Cap loosely and incubate aerobically at 35–37°C for 18–24 hours.\n4. Some slow-growing organisms may need up to **7 days** before a positive shows.\n\n![](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002Fcitrate-utilization-test-225x300.jpg)Figure: Citrate utilization test A: Negative B: Positive\\\n(Image source: Microbe library)\n\n### Where students actually get confused\n\n- **Don't stab the slant.** If you've just run a TSI or SIM tube, the instinct to stab is automatic — citrate is the opposite: surface streak only, aerobic incubation, no stab.\n- **Growth alone is a positive result, even without color change.** This is the single most missed rule in this test. Some citrate-utilizing organisms grow visibly on the slant without ever turning it blue. If you see growth, it's positive — don't wait for blue that may never come.\n- **No growth at all = negative**, and it will look almost identical to an uninoculated slant — deep forest green, no visible colonies. That's the actual negative result, not \"green but with some growth.\"\n- **Don't call it negative too early.** Some organisms are slow citrate utilizers and need up to 7 days of incubation. A 24-hour negative isn't final for every organism.\n- **Use a light inoculum from a young colony, never from a broth culture.** Carryover nutrients from broth or an overly heavy inoculum can produce a false-positive by feeding growth that has nothing to do with citrate utilization.\n\n## Quality Control\n\nInspect agar for freezing damage, contamination, cracks, dehydration, or bubbles before use. Discard any tube that's already blue before inoculation.\n\n| Organism | Expected result |\n| --- | --- |\n| *Klebsiella pneumoniae* ATCC 13883 | Citrate positive (growth, blue color) |\n| *Escherichia coli* ATCC 25922 | Citrate negative (no growth, no color change) |\n\n## Result Interpretation\n\n| Result | Appearance |\n| --- | --- |\n| Positive | Growth on the slant — color may or may not turn Prussian blue |\n| Negative | No or trace growth, remains deep forest green, indistinguishable from an uninoculated slant |\n| Equivocal | Repeat the test |\n\n### Citrate-positive organisms\n\n*Klebsiella pneumoniae*, *Enterobacter* species (minority negative), *Citrobacter freundii*, *Salmonella* (other than Typhi\u002FParatyphi A), *Serratia marcescens*, *Proteus mirabilis* (minority negative), *Providencia*\n\n### Citrate-variable\n\n*Proteus vulgaris*, *Vibrio cholerae*, *Vibrio parahaemolyticus*\n\n### Citrate-negative organisms\n\n*Escherichia coli*, *Shigella* species, *Salmonella* Typhi, *Salmonella* Paratyphi A, *Morganella morganii*, *Yersinia enterocolitica*\n\n> Exceptions exist on both sides: rare citrate-positive *E. coli* variants and citrate-negative *E. aerogenes* strains have been isolated — a single citrate result should never be the sole basis for species-level identification.\n\n## Uses\n\nCitrate is part of standard multitest identification kits, including [API-20E](https:\u002F\u002Fmicrobeonline.com\u002Fapi-20e-test-system-introduction-procedure-results-interpretations\u002F) and Enterotube II, alongside the rest of the IMViC battery.\n\n## Limitations\n\n1. Luxuriant growth without color change can still indicate a positive — but if the slant never turns blue even after extended incubation, repeat with a smaller inoculum.\n2. Never inoculate from a broth culture — carryover media can produce a false positive.\n3. Use a light inoculum to avoid carryover-driven false positives.\n4. Citrate results alone are not sufficient for species-level identification.\n\n### How to remember\n\n**\"Citrate turns the sky blue over KECSS.\"** The reliable citrate-positive Enterobacteriaceae:\n\n- **K** — *Klebsiella*\n- **E** — *Enterobacter*\n- **C** — *Citrobacter*\n- **S** — *Salmonella* (except Typhi and Paratyphi A)\n- **S** — *Serratia*\n\nAnd the anchor that matters clinically: **E. coli lives in the gut, not on citrate.** A citrate-negative result is part of what flags *E. coli* as a fecal indicator in water testing, while citrate-positive *Klebsiella aerogenes* is at home in soil and water. If citrate comes back positive on what you assumed was a fecal coliform, question the assumption.\n\n**References and further reading**\n\n1. Leber AL, editor. *Clinical Microbiology Procedures Handbook.* 4th ed. Washington, DC: ASM Press; 2016. doi:10.1128\u002F9781555818814\n2. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. *Koneman's Color Atlas and Textbook of Diagnostic Microbiology.* 7th ed. Philadelphia: Wolters Kluwer; 2017.\n3. MacFaddin JF. *Biochemical Tests for Identification of Medical Bacteria.* 3rd ed. Philadelphia: Lippincott Williams & Wilkins; 2000.",[46,49,52,55,58],{"question":47,"answer":48},"I see growth on the slant but it's still green — is that positive or negative?","Positive. Growth itself is the result that matters; the blue color change is common but not universal. If you see visible growth on the slant, report it as citrate-positive even without a color shift.",{"question":50,"answer":51},"Why did I get a false positive on my citrate test?","The most common cause is too heavy an inoculum, or inoculating directly from a broth culture instead of a young colony — both carry over nutrients that can support growth unrelated to citrate utilization.",{"question":53,"answer":54},"Can I stab the citrate slant like I do for TSI?","No. Citrate utilization requires an aerobic environment, so the slant is streaked on the surface only — stabbing creates an anaerobic pocket that interferes with the reaction.",{"question":56,"answer":57},"Does the blue color come from the citrate breaking down?","Not mainly. The test detects a pH rise, and the largest part of that rise comes from ammonia, not from citrate itself. The medium's only nitrogen source is an ammonium salt, and to grow the organism must strip ammonia from it, forming ammonium hydroxide and driving the pH past 7.6. The carbon dioxide released from citrate breakdown contributes some alkalinity as sodium carbonate, but the ammonia from the nitrogen source is the dominant driver. This is why growth on the slant, which signals the organism is using both the citrate and the ammonium salt, counts as positive even before any blue appears.",{"question":59,"answer":60},"Does the citrate test detect citrate permease or citrate lyase?","Both are involved, in sequence. Citrate permease is the gatekeeper: it transports citrate across the cell membrane. Citrate lyase then cleaves the citrate inside the cell. The discriminating step among Enterobacteriaceae is usually the permease, because many organisms possess the downstream enzymes but lack the transporter, so they test negative despite being biochemically capable on paper. A positive test means the organism has both the permease to admit citrate and the machinery to live on it.",[],[],[64,70,77,82,86,90,95,100,104,108],{"slug":65,"name":39,"description":66,"image":67,"body":68,"postCount":69},"acharya-tankeshwar","Editor-in-chief","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Ftankeshwar-acharya-author-microbeonline.jpg","***Tankeshwar Acharya, MSc (Medical Microbiology)***\n\n*Tankeshwar Acharya is an Assistant Professor in the Department of Microbiology at Patan Academy of Health Sciences (PAHS), Nepal, where he has been teaching and practicing clinical microbiology for over 14 years. He is the founder of Microbe Online, one of the leading free microbiology education resources on the web, covering bacteriology, mycology, parasitology, immunology, and clinical laboratory diagnostics written from direct experience in both the classroom and the diagnostic laboratory.*",433,{"slug":71,"name":72,"description":73,"image":74,"body":75,"postCount":76},"ashma-shrestha","Ashma Shrestha","SEO Copywriter and Science Communicator\nKathmandu, Nepal","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Fashma-shrestha.png","Ashma Shrestha holds a Master of Science in Medical Microbiology from the Institute of Science and Technology (IOST), Tribhuvan University, Nepal, where she developed a strong foundation in virology, molecular biology, and diagnostic microbiology.\n\nShe now works as an SEO Copywriter at Resolution Digital, where she combines her scientific training with research-driven content strategy. She is certified in Google Analytics and Google Business Profile (GBP), and brings a data-informed approach to science communication writing content that is not only accurate but structured to reach and serve the students who need it most.\n\nAt microbeonline, Ashma contributes articles primarily in virology and molecular biology, areas she finds most compelling for their mechanistic depth and their growing clinical relevance. Her writing reflects the same standard the site is built on: factual rigor, clear explanation of the *why* behind microbiology concepts, and content that helps students move from memorization to genuine understanding.\n\nShe is passionate about making complex microbiological concepts accessible without sacrificing accuracy; a skill that sits at the intersection of her scientific training and her professional work in content and SEO.",81,{"slug":78,"name":79,"description":80,"image":38,"body":38,"postCount":81},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":83,"name":84,"description":80,"image":38,"body":38,"postCount":85},"samikshya-acharya","Samikshya Acharya",20,{"slug":87,"name":88,"description":80,"image":38,"body":38,"postCount":89},"alisha-tripathi","Alisha Tripathi",6,{"slug":91,"name":92,"description":93,"image":38,"body":38,"postCount":94},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",10,{"slug":96,"name":97,"description":98,"image":38,"body":38,"postCount":99},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":101,"name":102,"description":80,"image":38,"body":38,"postCount":103},"srijana-khanal","Srijana Khanal",18,{"slug":105,"name":106,"description":98,"image":38,"body":38,"postCount":107},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":109,"name":110,"description":80,"image":38,"body":111,"postCount":112},"nisha-rijal","Nisha Rijal","**Nisha Rijal** is a microbiologist and quality assurance specialist. She served for nearly 12 years as a microbiologist at the National Public Health Laboratory (NPHL), Nepal's national reference laboratory, and continues to work as a consultant microbiologist in international public health organization. ",51]