[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$fvNRxDMNkj0bjYWqPjywTvKfiI0V_WTZqxqx8jk1Oi4E":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":126},[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":47},"key-biochemical-methods-used-to-distinguish-mycobacterial-group","Biochemical Tests to identify Mycobacteria, NTM",null,"Acharya Tankeshwar","2013-07-22","2026-07-04",false,"biochemical-tests","Various biochemical tests can be performed for the identification of Mycobacteria and Non-tuberculous Mycobacteria. An overview of some of these biochemical tests is mentioned in this blog post.\n\n### Niacin accumulation test\n\n All species of *Mycobacterium* produce niacin (nicotinic acid) and *Mycobacterium tuberculosis* accumulates the most. A positive niacin test provides preliminary evidence that an organism that exhibits a buff-colored, slow-growing rough colony may be *M. tuberculosis*.\n\n![Biochemical test for Mycobacteria and NTM](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FBiochemical-tests-to-identify-NTM.png)\n\u003Cfigcaption>Figure: Biochemical test for Mycobacteria and NTM\u003C\u002Ffigcaption>\n\n### Nitrate Reduction test\n\n*Mycobacterium tuberculosis is a strongly nitrate-positive* organism. This test is valuable for the identification of *M. tuberculosis, M. kansasii, M. szulgai* and *M. fortuitum*.\n\nRapid growers such as *M. fortuitum* can be tested within 2 weeks, but slow growers should be tested after 3-4 weeks of luxuriant growth.  Both chemical procedures and commercially available nitrate strips are available. Control strains must be used while performing and interpreting the result.\n\n### Catalase test\n\n A semiquantitative [catalase test](\u002Fcatalase-test-principle-uses-procedure-results\u002F) is used for the identification of Mycobacteria. Catalase is an enzyme that splits hydrogen peroxide into water and oxygen and a positive catalase test is indicated by the formation of gas bubbles.  Most species of Mycobacteria, except for certain strains of *M. tuberculosis* complex (some isoniazid-resistant strains) and *M. gastri*, produce catalase enzyme.\n\n### Tween 80 hydrolysis test\n\n[Tween 80 hydrolysis test](\u002Ftween-80-hydrolysis-test-principle-procedure-results\u002F) is used to separate the species of Photochromogens, scotochromogens, and nonchromogens. Nonpathogenic slow-growing scotochromogens and nonchromogens produce a lipase that is able to hydrolyze Tween 80 (the detergent polyoxyethylene sorbitan monooleate) into oleic acid and polyoxyethylated sorbitol, whereas pathogenic species do not.\n\n### Tellurite reduction test\n\nThe ability of mycobacterial species to reduce tellurite in 3 to 4 days is used to distinguish members of *M. avium* complex from most other non-chromogenic species. All rapid growers reduce tellurite in 3 days.\n\n### Arylsulfatase test\n\nArylsulfatase enzyme is present in most mycobacteria. The rate by which arylsulfatase enzyme breaks down phenolphthalein disulfate into phenolphthalein (which forms a red color in the presence of sodium bicarbonate) and other salts is used to differentiate certain strains of Mycobacteria. Three-day arylsulfatase test is used to identify potentially pathogenic rapid growers such as *M. fortuitum*and *M. chelonae.* Slow-growing *M. marinum*and *M. szulgai*are positive in the 14-day arylsulfatase test.\n\n### Urea Hydrolysis test\n\nMany *Mycobacterium* species possess a urease enzyme that hydrolyzes urea to form carbon dioxide and ammonia. The ammonia released increases**the**pH of the medium turning the indicator pink.\n\n**References and further readings**\n\n- Gurpreet S. Bhalla, Manbeer S. Sarao, Dinesh Kalra, Kuntal Bandyopadhyay, Arun Ravi John, Methods of phenotypic identification of non-tuberculous mycobacteria, Practical Laboratory Medicine, Volume 12,2018, e00107, ISSN 2352-5517, \u003Chttps:\u002F\u002Fdoi.org\u002F10.1016\u002Fj.plabm.2018.e00107>. (\u003Chttps:\u002F\u002Fwww.sciencedirect.com\u002Fscience\u002Farticle\u002Fpii\u002FS2352551717300823>)",[],[46],"mycobacteria",[48,57,66,72,89,95,112,118],{"slug":49,"title":50,"description":50,"seoTitle":37,"seoDescription":37,"author":51,"createdDate":52,"lastUpdatedDate":53,"draft":41,"category":54,"image":37,"faq":55,"tags":56},"mycobacterium-tuberculosis-lab-diagnosis","Laboratory Diagnosis of Mycobacterium tuberculosis Infection","Nisha Rijal","2020-05-26","2026-07-19","bacteriology",[],[46],{"slug":58,"title":59,"description":60,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":61,"lastUpdatedDate":62,"draft":41,"category":63,"image":37,"faq":64,"tags":65},"preparation-uses-lowenstein-jensen-lj-medium","Löwenstein-Jensen (LJ) Medium: Principle, Preparation, Uses, and Colony Characteristics","Löwenstein-Jensen (LJ) medium is the standard solid culture medium for Mycobacterium tuberculosis. Learn its principle, preparation by inspissation, colony characteristics of M. tuberculosis and NTM, and how LJ compares to MGIT liquid culture.","2016-04-29","2026-07-05","culture-media",[],[46],{"slug":67,"title":68,"description":68,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":69,"lastUpdatedDate":62,"draft":41,"category":54,"image":37,"faq":70,"tags":71},"genexpert-mtbrif-assay-principle-procedure-results-interpretations","GeneXpert MTB\u002FRIF Assay","2016-01-04",[],[46],{"slug":73,"title":74,"description":75,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":76,"lastUpdatedDate":53,"draft":41,"category":77,"image":37,"faq":78,"tags":88},"auramine-rhodamine-fluorochrome-staining-principle-procedure-results-limitations","Auramine-Rhodamine Fluorochrome Staining: Principle, Procedure, and Results","Auramine-rhodamine is a WHO-recommended fluorochrome stain for detecting acid-fast bacilli — more sensitive than Ziehl-Neelsen and faster to screen. Learn the Truant method procedure, results grading, and when to confirm with ZN staining.","2015-04-03","staining-techniques",[79,82,85],{"question":80,"answer":81},"Why is auramine-rhodamine staining more sensitive than Ziehl-Neelsen for detecting acid-fast bacilli?","Auramine-rhodamine allows smear screening at 250x or 400x magnification — compared to 1,000x oil immersion required for ZN staining. At lower magnification, a much larger area of the slide can be examined per unit time (3-5 minutes vs 15-20 minutes per slide). This means more of the smear is examined, increasing the chance of detecting paucibacillary specimens. Studies consistently show auramine-rhodamine detects approximately 10% more positive cases than ZN in direct smear microscopy, which is why WHO recommends it as the preferred method where fluorescence microscopy is available.",{"question":83,"answer":84},"What is the two-step workflow for auramine-rhodamine results?","Positive auramine-rhodamine results should be confirmed by ZN staining of the same slide, as fluorescence artefacts (dust, fibres, non-AFB structures) can occasionally give false-positive fluorescence. Negative auramine-rhodamine results require examination of the minimum required number of fields before reporting — at 200-250x this is typically 30-100 fields. In high-suspicion patients, a negative fluorochrome result should prompt ZN confirmation and repeat specimen collection, as the minimum detection threshold for smear microscopy (approximately 5,000-10,000 AFB\u002FmL) means culture is more sensitive than any smear method.",{"question":86,"answer":87},"Can auramine-rhodamine staining detect organisms other than mycobacteria?","Yes. A modified fluorochrome method using a weaker decolouriser (0.5% sulphuric acid instead of 3% acid-alcohol) detects partially acid-fast organisms including Cryptosporidium parvum, Cyclospora cayetanensis, and Isospora belli oocysts in stool specimens, and Nocardia species in respiratory or wound specimens. These organisms share a partial acid-fast property with mycobacteria. The oocysts appear as bright yellow-orange fluorescent structures against a dark background. This application requires the modified decolouriser — the standard 3% acid-alcohol used for TB smears will over-decolourise these weakly acid-fast organisms.",[46],{"slug":90,"title":91,"description":91,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":92,"lastUpdatedDate":40,"draft":41,"category":54,"image":37,"faq":93,"tags":94},"short-notes-atypical-mycobacterial-infections","Atypical Mycobacterial Infections","2014-11-07",[],[46],{"slug":96,"title":97,"description":98,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":99,"lastUpdatedDate":100,"draft":41,"category":77,"image":37,"faq":101,"tags":111},"ziehl-neelsen-technique-principle-procedure-reporting","Ziehl-Neelsen Staining: Principle, Procedure, Grading, and Interpretation","Ziehl-Neelsen (ZN) staining detects acid-fast bacilli in sputum and other specimens. Learn the hot and cold methods, AFB grading scale, smear reporting, troubleshooting, and when to use fluorochrome staining instead.","2013-12-06","2026-07-07",[102,105,108],{"question":103,"answer":104},"Why does Ziehl-Neelsen staining require heat while other staining techniques do not?","Mycobacteria have a cell wall rich in mycolic acids — long-chain fatty acids that make the wall waxy, hydrophobic, and impermeable to most dyes at room temperature. Heat acts as a mordant by disrupting this waxy barrier and allowing carbol fuchsin to penetrate the cell wall. Once inside, the stain is held so tightly by the mycolic acids that even acid-alcohol — one of the strongest decolorisers used in microbiology — cannot remove it. This is why the stain is called 'acid-fast' — the organisms hold fast to the dye even after acid treatment.",{"question":106,"answer":107},"How is an AFB smear graded and what does the grade mean clinically?","AFB smears are graded using the WHO\u002FIUATLD scale: No AFB seen (after examining 300 fields); Scanty — 1-9 AFB per 100 fields (report exact count and request repeat); 1+ — 10-99 AFB per 100 fields; 2+ — 1-10 AFB per field in at least 50 fields; 3+ — more than 10 AFB per field in at least 20 fields. Higher grades indicate greater organism burden and greater infectiousness. Grade is recorded at treatment initiation and at months 2, 5, and 6 to monitor bacteriological response. Conversion from positive to negative smear during treatment indicates therapeutic response.",{"question":109,"answer":110},"What is the difference between Ziehl-Neelsen and Kinyoun (cold) acid-fast staining?","Both methods use carbolfuchsin as the primary stain and acid-alcohol for decolourisation, but they differ in how the dye penetrates the mycobacterial cell wall. Ziehl-Neelsen uses heat (the hot technique) — the slide is steamed to drive the dye through the waxy cell wall. Kinyoun's cold technique achieves penetration without heat by increasing the concentration of both carbolfuchsin and phenol and incorporating a wetting agent (Triton X-100 or similar). The results are equivalent. Kinyoun is preferred where open flames are unsafe or inconvenient, and for partial acid-fast organisms (Nocardia, Cryptosporidium) where lower decoloriser concentrations are needed.",[46],{"slug":113,"title":114,"description":114,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":115,"lastUpdatedDate":40,"draft":41,"category":54,"image":37,"faq":116,"tags":117},"introduction-transmission-pathogenesis-and-lab-diagnosis-of-leprosy-hansens-disease","Leprosy: Etiology, Pathogenesis, Lab Diagnosis","2012-05-15",[],[46],{"slug":119,"title":120,"description":121,"seoTitle":37,"seoDescription":37,"author":51,"createdDate":122,"lastUpdatedDate":53,"draft":41,"category":54,"image":123,"faq":124,"tags":125},"tuberculin-skin-test-mantoux-test-principle-procedure-results","Tuberculin Skin Test (Mantoux test): Principle, Procedure, Results","Details about Tuberculin Skin Test.","2023-02-02","https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FMantoux_tuberculin_skin_test-1.jpg",[],[46],[127,133,140,145,149,153,158,163,167,171],{"slug":128,"name":38,"description":129,"image":130,"body":131,"postCount":132},"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":134,"name":135,"description":136,"image":137,"body":138,"postCount":139},"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":141,"name":142,"description":143,"image":37,"body":37,"postCount":144},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":146,"name":147,"description":143,"image":37,"body":37,"postCount":148},"samikshya-acharya","Samikshya Acharya",20,{"slug":150,"name":151,"description":143,"image":37,"body":37,"postCount":152},"alisha-tripathi","Alisha Tripathi",6,{"slug":154,"name":155,"description":156,"image":37,"body":37,"postCount":157},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",10,{"slug":159,"name":160,"description":161,"image":37,"body":37,"postCount":162},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":164,"name":165,"description":143,"image":37,"body":37,"postCount":166},"srijana-khanal","Srijana Khanal",18,{"slug":168,"name":169,"description":161,"image":37,"body":37,"postCount":170},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":172,"name":51,"description":143,"image":37,"body":173,"postCount":174},"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]