[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$f8T8JAIIf6hCkUSvifozLC5llZAF57UO0xiq5ZgZyVFk":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":57},[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":55,"related":56},"hippurate-hydrolysis-test-principle-procedure-uses-results","Hippurate Hydrolysis Test: Principle, Procedure, Results","Hippurate hydrolysis test principle, classical and rapid ninhydrin procedures, for identifying Streptococcus agalactiae and other organisms.",null,"Acharya Tankeshwar","2015-03-14","2026-07-11",false,"biochemical-tests","## Why It Matters\n\nLate in pregnancy, usually around 36 to 37 weeks, women are screened for one organism carried harmlessly in the vagina and rectum: **Group B Streptococcus**, *Streptococcus agalactiae*. The mother is unaffected. The concern is the newborn. If GBS is present during delivery it can pass to the baby and cause early-onset neonatal sepsis, pneumonia, and meningitis, and it remains a leading cause of life-threatening infection in the first week of life. A positive screen means intrapartum antibiotics, which sharply reduce that risk.\n\nSo a lab that grows a beta-hemolytic *Streptococcus* from a vaginal-rectal swab needs to answer one question fast: **is this Group B Strep?** Hippurate hydrolysis is one of the classic ways to answer it. *S. agalactiae* hydrolyzes hippurate; the other beta-hemolytic streptococci (Groups A, C, F, G) do not.\n\nThe test does a second job among the curved Gram-negative rods that cause gastroenteritis: *Campylobacter jejuni* hydrolyzes hippurate and *C. coli* does not, making hippurate the standard way to separate the two commonest *Campylobacter* species. Both calls are presumptive, confirmed by further testing, but both are the kind of fast answer that changes what happens next.\n\n![Hippurate hydrolysis test-Left (positive tube), Right (Negative tube) - Hippurate hydrolysis test-Left (positive tube), Right (Negative tube)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FHippuratee-hydrolysis-test.jpg)Figure: Hippurate hydrolysis test-Left (positive tube), Right (Negative tube)\n\nHippurate hydrolysis occurs in an organism that can form glycine and benzoic acid.\n\nIn the classical test (48 hours), the production of benzoic acid was detected using ferric chloride as an indicator. The [gas-liquid chromatography (GLC)](\u002Fgas-chromatography-gc-principle-parts-and-uses\u002F) method is also available to detect benzoic acid.\n\nIn contrast, the rapid test (2 hours) detects glycine, the second byproduct of Hippurate hydrolysis. It uses ninhydrin as the indicator. Rapid Hippurate hydrolysis is more specific and as sensitive as the classical method.\n\n## Principle of Hippurate Hydrolysis\n\nHippurate is a single molecule made of two parts joined by an amide bond: **benzoic acid** and the amino acid **glycine**. Think of it as benzoic acid with a glycine attached. Hippuricase cuts that bond and releases both halves. This is why there are two detection methods that look completely different but test the same thing: the classical ferric chloride method watches for the **benzoic acid** half, and the rapid ninhydrin method watches for the **glycine** half. Two windows onto one cut.\n\nHippuric acid is hydrolyzed to benzoic acid and glycine by the enzymatic action of hippuricase. The addition of a ninhydrin reagent detects glycine. Ninhydrin reacts with glycine to form a deep blue or purple color (Ruhemann’s purple). Hwang and Ederer described this [rapid test for detecting glycine.](http:\u002F\u002Fwww.ncbi.nlm.nih.gov\u002Fpmc\u002Farticles\u002FPMC274963\u002Fpdf\u002Fjcm00233-0126.pdf)\n\nSimilarly, the traditional method detects benzoic acid using ferric chloride. The ferric chloride dissolves the benzoic acid present as residue after centrifugation of hippurate solution with the test organism.\n\n## Uses of Hippurate Hydrolysis\n\nThe hippurate test identifies *Campylobacter jejuni, Listeria monocytogenes, Gardnerella vaginalis*, and *Streptococcus agalactiae.*\n\n1. The hippurate hydrolysis test is critical for separating *Campylobacter jejuni* (hydrolyzes hippurate) and *Campylobacter coli* (negative) strains.\n2. It also aids in differentiating β-hemolytic *Streptococcus agalactiae* from other β-hemolytic streptococci.\n\n## Test Procedure\n\n### Classical Method\n\n- Firstly, take or prepare sterile sodium hippurate broth and inoculate it with the test organism.\n- After that incubate it overnight at 35°C.\n- Then, [centrifuge](\u002Fcentrifuge-parts-types-handling\u002F) the broth and remove the sediment.\n- Finally, add ferric chloride reagent to the supernatant.\n- If the residue remains after 10 minutes, benzoic acid is present. The test is positive for hippurate hydrolysis.\n\n**Rapid Method (Ninhydrin)**\n\n- Add 0.2-0.4 mL of sodium hippurate reagent to a sterile tube.\n- Make a heavy suspension of the test organism from an 18-24 hour culture, taking care not to pick up agar, which contains protein that can interfere with the reaction.\n- Cap and incubate at 35-37°C for 2 hours, a water bath is preferred for even heating.\n- Add 2-4 drops (about 0.2 mL) of ninhydrin reagent.\n- Re-incubate at 35-37°C for an additional 10-30 minutes.\n- Observe at 10-minute intervals for a deep blue or purple color (Ruhemann's purple).\n\n**Positive:** deep blue\u002Fpurple color within 30 minutes.\n\n**Negative:** no color change, or a faint blue-gray tint.\n\n## Quality Control used in Hippurate Hydrolysis Test\n\nTest each new lot or shipment of the reagent with known positive and negative controls, and retest at least monthly. Discard all reagents and prepare new ones if the reagents do not pass QC. Grow the [ATCC strains](\u002Fquality-control-strains-uses\u002F) in sodium hippurate broth for quality control and look for the results.\n\n1. *Streptococcus agalactiae* (ATCC 4768): Moderate to heavy growth and hydrolysis of hippurate.\n2. *Streptococcus pyogenes* (ATCC 19615): Moderate to heavy growth; hippurate not hydrolyzed.\n\n**Hippurate Hydrolysis Test Positive Organisms :**\n\nThe hippurate hydrolysis test is used in the presumptive identification of various bacteria. Organisms giving positive (+ve) tests are:\n\n- *Gardnerella vaginalis,*\n- *Campylobacter jejuni,*\n- *Listeria monocytogenes* and\n- Group B streptococci (*Streptococcus agalactiae*)\n\n## Limitations\n\n- False-positive results can occur if incubation with ninhydrin exceeds 30 min.\n- A negative test does not rule out the identification of *G. vaginalis* since the biotypes that cause bacterial vaginosis can be hippurate negative.\n- Viridans group streptococci can be hippurate positive; performing other tests to confirm the identification of non-hemolytic colonies is essential.\n- A small number of enterococci are beta-hemolytic and may hydrolyze hippurate, but they are [pyrrolidinyl-β-naphthylamide (PYR)](\u002Fpyrrolidonyl-arylamidase-pyr-test-principle-procedure-results\u002F) positive (*S. agalactiae* is PYR negative)\n- Furthermore a small percentage of *C. jejuni* organisms are hippurate negative and use of other methods for complete identification is necessary.\n\n**Where students actually get confused**\n\n- **The rapid method is more sensitive with enterococci than the classical method, not less.** A published evaluation found 95.4% of enterococcal Group D strains positive by the rapid method versus only 9.3% by the classical method. If you're using the rapid method, expect more Group D positives, that's the method being accurate, not a contamination problem.\n- **A weak hippurate-positive Group D streptococcus isn't automatically GBS.** Bile esculin is the test that separates them, Group D hydrolyzes esculin, GBS doesn't.\n- **Viridans streptococci and PYR are the other tiebreaker.** A small number of enterococci hydrolyze hippurate too, but they're PYR-positive while S. agalactiae is PYR-negative, exactly the same logic already covered on the PYR test page.\n- **Ninhydrin timing is not flexible.** Reading past 30 minutes risks a false positive. Set a timer rather than checking back \"whenever convenient.\"\n\n### Key exam facts in one table\n\n| Feature | Detail | Memory hook |\n| --- | --- | --- |\n| Detects | Hippuricase activity | Splits hippurate into glycine + benzoic acid |\n| Classical method | Ferric chloride detects benzoic acid | 48 hours |\n| Rapid method | Ninhydrin detects glycine | 2 hours, deep blue\u002Fpurple |\n| Identifies | S. agalactiae, C. jejuni, L. monocytogenes, G. vaginalis |  |\n| GAS\u002FGBS panel role | The one result that doesn't flip | GBS positive, almost everything else negative |\n| Group D false positive | Resolved by bile esculin | Group D hydrolyzes esculin, GBS doesn't |\n| Enterococcus false positive | Resolved by PYR | Enterococcus PYR-positive, GBS PYR-negative |\n| Ninhydrin read window | Within 30 minutes | Longer risks false positive |\n\n**References and further readings**\n\n1. Hwang MN, Ederer GM. Rapid hippurate hydrolysis method for presumptive identification of group B streptococci. *J Clin Microbiol.* 1975;1(1):114-115. doi:10.1128\u002Fjcm.1.1.114-115.1975\n2. Wilkinson HW, Thacker LG, Facklam RR. Evaluation of the rapid hippurate hydrolysis test with enterococcal group D streptococci. *J Clin Microbiol.* 1977;5(3):290-292. doi:10.1128\u002Fjcm.5.3.290-292.1977\n3. Leber AL, editor. *Clinical Microbiology Procedures Handbook.* 4th ed. Washington, DC: ASM Press; 2016. doi:10.1128\u002F9781555818814\n4. 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.\n5. Tille PM. *Bailey and Scott's Diagnostic Microbiology.* 15th ed. St. Louis: Elsevier; 2022.",[46,49,52],{"question":47,"answer":48},"Why do the classical and rapid hippurate methods detect two different products from the same reaction?","Because hippurate is one molecule made of two parts joined by an amide bond: benzoic acid and the amino acid glycine. Hippuricase cuts that bond and releases both. The classical ferric chloride method detects the benzoic acid half over 48 hours, while the rapid ninhydrin method detects the glycine half in 2 hours, forming a blue-purple color. They look completely different but are two windows onto the same cleavage.",{"question":50,"answer":51},"A Group D streptococcus tests weakly hippurate positive by the rapid method. How do I tell whether it is actually Group B Strep?","Two follow-up tests resolve it. Bile esculin separates them: Group D streptococci hydrolyze esculin, while Group B Strep does not. PYR is the other tiebreaker: enterococci are PYR positive, while Streptococcus agalactiae is PYR negative. A hippurate-positive, bile-esculin-negative, PYR-negative beta-hemolytic coccus fits Group B Strep.",{"question":53,"answer":54},"Why does switching from the classical to the rapid hippurate method change how often enterococci appear positive?","Because the rapid ninhydrin method is far more sensitive with enterococcal Group D strains than the classical method. A published evaluation found about 95 percent of enterococcal Group D strains positive by the rapid method versus about 9 percent by the classical method. So seeing more Group D positives on the rapid method is the method being accurate, not a contamination problem.",[],[],[58,64,71,76,80,84,89,94,98,102],{"slug":59,"name":39,"description":60,"image":61,"body":62,"postCount":63},"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":65,"name":66,"description":67,"image":68,"body":69,"postCount":70},"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":72,"name":73,"description":74,"image":38,"body":38,"postCount":75},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":77,"name":78,"description":74,"image":38,"body":38,"postCount":79},"samikshya-acharya","Samikshya Acharya",20,{"slug":81,"name":82,"description":74,"image":38,"body":38,"postCount":83},"alisha-tripathi","Alisha Tripathi",6,{"slug":85,"name":86,"description":87,"image":38,"body":38,"postCount":88},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",10,{"slug":90,"name":91,"description":92,"image":38,"body":38,"postCount":93},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":95,"name":96,"description":74,"image":38,"body":38,"postCount":97},"srijana-khanal","Srijana Khanal",18,{"slug":99,"name":100,"description":92,"image":38,"body":38,"postCount":101},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":103,"name":104,"description":74,"image":38,"body":105,"postCount":106},"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]