[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$fzT8oHq-_mxVQlD0Pe1apiQx78LRxQNL3Oi3rpuyht1M":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":47},[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":36,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":39,"lastUpdatedDate":40,"draft":41,"category":42,"image":37,"body":43,"faq":44,"tags":45,"related":46},"nitrocefin-test-principle-procedure-uses-limitations","Nitrocefin test: Principle, Procedure, Uses",null,"Acharya Tankeshwar","2015-04-23","2025-12-29",false,"biochemical-tests","The nitrocefin biochemical test is a sensitive technique for detecting beta-lactamase-producing strains of *N. gonorrhoeae*, *H. influenzae, Staphylococcus spp, Enterococcus spp,* and [Moraxella (Branhamella) catarrhalis](\u002Fmoraxella-catarrhalis\u002F). Nitrocefin is the only reliable test for detecting beta-lactamase-producing *Enterococcus* spp.\n\nIt provides the most sensitive test for most β-lactamases, exceptions being staphylococcal penicillinase and ROB-1, an uncommon plasmid-mediated enzyme of haemophili. Another chromogenic cephalosporin\\*, PADAC, exists but is less sensitive and is not readily available.\\* Rapid beta-lactamase tests can yield clinically relevant information earlier than a [MIC](\u002Fminimum-inhibitory-concentration-and-minimum-bactericidal-concentration-mbc\u002F) or [disk diffusion test](\u002Fantimicrobial-susceptibility-testing-procedure-modified-kirby-bauer-method\u002F).\n\nNitrocefin is a **chromogenic cephalosporin** that changes from **yellow to red** when the amide bond in the beta-lactam ring is hydrolyzed **by beta-lactamase**.   It is sensitive to hydrolysis by all known lactamases produced by Gram-positive and Gram-negative bacteria.\n\nAlthough the test can be performed by using a nitrocefin solution, nitrocefin is very expensive, light sensitive, and not easily obtained. Most laboratories find it more convenient to use a commercially available nitrocefin test kit.\n\n#### Beta-lactase detection using Nitrocefin solution (Powder)\n\n1. A 0.5 mM nitrocefin solution is prepared by dissolving 2.58 mg of pure powder in 0.5 mL of dimethylsulphoxide (DMSO) then diluting with 9.5 mL of 0.1 M phosphate buffer, pH 7.0. **Note:**\n\n- This solution is stable for 10 days at 4°C in a foil-wrapped bottle.\n- Glass containers should be used, since DMSO degrades plastics.\n\n1. Colonies of the test isolates are scraped from [nutrient agar plates](\u002Fnutrient-agar-composition-preparation-uses\u002F) and are suspended in 20 µL volumes of 0.1 M phosphate buffer pH 7.0, to produce a dense suspension on a glass slide, and 20 µL amounts of the nitrocefin solution are added.\n2. β-Lactamase activity is indicated by **a red colour within 1-2 min.**\n3. Weak activities may take longer to appear, but reactions taking >10 min should be treated with skepticism, as they may reflect the secondary ß-lactamase activity of those penicillin-binding proteins that form unstable acyl complexes.\n\n## Beta-lactase detection using Nitrocefin disk\n\n1. Using sterile forceps, remove a Nitrocefin disk from the vial and place it on an empty petri dish or microscopic slide. Immediately place the remaining unused disks into the [freezer](\u002Flaboratory-freezers-temperature-range-and-inventory-management\u002F).\n2. Prior to inoculation, allow the Nitrocefin disk to equilibrate to room temperature.\n3. Moisten each disk with one drop of sterile deionized water. Do not over-saturate the disk, which could dilute the reagent.\n\nNote: Water is critical to the development of the color reaction, if the disk begins to dry out it may be necessary to rehydrate the reaction area of the Nitrocefin disk with a small amount of water.\n\n1. With a sterilized loop or applicator stick remove a well-isolated colony and spread it on the disk surface.\n2. Observe the inoculated disk for the development of an orange\u002Fred color\n\n![Fig: Nitrocefin test result (Left Positive), Right (Negative). - Fig: Nitrocefin test result (Left Positive), Right (Negative).](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FNitrocefin-test.jpg)Figure: Fig: Nitrocefin test result (Left Positive), Right (Negative).\n\n## Test Results\n\n1. Left *(H. influenzae):* The orange\u002Fred color development was indicative as beta-lactamase positive.\n2. Right (*M. catarrhalis*): The yellow color development was indicative of beta-lactamase negative.\n\n## Other techniques\n\nRehydrate the nitrocefin as directed, and use this solution in the following ways:\n\n1. **Direct Plate Method** \\\n   Add one drop of the nitrocefin solution on to the surface of the colony. If the isolate is a high beta-lactamase producer then the colony and the surrounding **area will quickly turn red**. To detect a weak beta-lactamase producer the plate should then be **incubated for 30 minutes before being reported as negative.**\n2. **Slide Method** \\\n   Add one drop of the nitrocefin solution on to a clean glass slide. Using a sterile loop, pick one colony from the plate and emulsify it into the nitrocefin drop. Report as positive if the colour changes **from yellow to red within 30 minutes** (protect the slide from desiccation during the waiting period).\n3. **Broth Method** \\\n   Add four drops of nitrocefin solution to 1ml of the grown culture. Report as positive if the colour **changes to red within 30 minutes.**\n4. **Broken Cell Method** \\\n   Sonicate 1ml of the culture in order to break open the cells. Add 4 drops of nitrocefin solution. Report as positive if the **colour changes to red within 30 minutes**.\n5. **Paper Disc Spot Test** \\\n   A Whatman No.1 filter paper disc (diameter 7cm) is placed in a petri dish and impregnated with nitrocefin solution (0-5ml). This impregnated paper is generally usable for one day, but should be kept away from light to avoid spontaneous degradation.  An isolated colony is applied to the impregnated paper with a loop; a **pink to red reaction developing within 15 minutes indicates beta-lactamase presence.**\n\n**References**\n\n1. Ghavami, A., Labbé, G., Brem, J., Goodfellow, V. J., Marrone, L., Tanner, C. A., King, D. T., Lam, M., Strynadka, N. C., Pillai, D. R., Siemann, S., Spencer, J., Schofield, C. J., & Dmitrienko, G. I. (2015). Assay for drug discovery: Synthesis and testing of nitrocefin analogues for use as β-lactamase substrates. *Analytical biochemistry*, *486*, 75–77. \u003Chttps:\u002F\u002Fdoi.org\u002F10.1016\u002Fj.ab.2015.06.032>\n2. Uri J. V. (1985). Detection of beta-lactamase activity with nitrocefin of multiple strains of various microbial genera. *Acta microbiologica Hungarica*, *32*(2), 133–145.\n3. Skov, R., Lonsway, D. R., Larsen, J., Larsen, A. R., Samulioniené, J., & Limbago, B. M. (2021). Evaluation of methods for detection of β-lactamase production in MSSA. *The Journal of antimicrobial chemotherapy*, *76*(6), 1487–1494. \u003Chttps:\u002F\u002Fdoi.org\u002F10.1093\u002Fjac\u002Fdkab032>",[],[],[],[48,54,61,66,70,74,79,84,88,92],{"slug":49,"name":38,"description":50,"image":51,"body":52,"postCount":53},"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":55,"name":56,"description":57,"image":58,"body":59,"postCount":60},"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":62,"name":63,"description":64,"image":37,"body":37,"postCount":65},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":67,"name":68,"description":64,"image":37,"body":37,"postCount":69},"samikshya-acharya","Samikshya Acharya",20,{"slug":71,"name":72,"description":64,"image":37,"body":37,"postCount":73},"alisha-tripathi","Alisha Tripathi",6,{"slug":75,"name":76,"description":77,"image":37,"body":37,"postCount":78},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",10,{"slug":80,"name":81,"description":82,"image":37,"body":37,"postCount":83},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":85,"name":86,"description":64,"image":37,"body":37,"postCount":87},"srijana-khanal","Srijana Khanal",18,{"slug":89,"name":90,"description":82,"image":37,"body":37,"postCount":91},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":93,"name":94,"description":64,"image":37,"body":95,"postCount":96},"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]