[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$fLtggIcj3XXD5Z7eV2cv6ZyMMCRanz3pOeYcdYYCrlM0":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":125},[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":46,"related":48},"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.",null,"Acharya Tankeshwar","2016-04-29","2026-07-05",false,"culture-media","A 34-year-old man presents with three weeks of productive cough, night sweats, and weight loss. His sputum smear is negative for acid-fast bacilli on Ziehl-Neelsen staining — but his clinician's index of suspicion remains high. In a laboratory with access to [GeneXpert](https:\u002F\u002Fmicrobeonline.com\u002Fgenexpert-mtbrif-assay-principle-procedure-results-interpretations\u002F), the molecular result comes back within two hours: *MTB detected, rifampicin resistance not detected.* But the treating physician also wants to know which other drugs the organism is susceptible to. That requires growing the organism in culture, testing it against a panel of anti-TB drugs, and waiting.\n\nThe culture medium that will grow *M. tuberculosis* from that smear-negative sputum, and sustain it long enough for drug susceptibility testing, is Löwenstein-Jensen medium. It may take three to six weeks to produce a visible result — but in the absence of liquid culture systems, it is irreplaceable.\n\n## Principle\n\n*Mycobacterium tuberculosis* is an obligate aerobe with a generation time of 16–24 hours — approximately 10 to 20 times slower than common bacteria like *E. coli*. It has a lipid-rich cell wall that makes it hydrophobic, resistant to many conventional media components, and unusually dependent on specific nutrients for primary isolation. Standard bacteriological media (nutrient agar, blood agar, TSA) will not support its growth.\n\nLöwenstein-Jensen medium was developed to meet these specific requirements. Each component serves a defined purpose:\n\n| Component | Role |\n| --- | --- |\n| **Whole eggs (inspissated)** | Primary nutrient source — provide albumin, lipids, carbohydrates, vitamins, and minerals. Coagulation by inspissation (heating at 85°C) solidifies the egg protein into a firm slope without agar |\n| **Asparagine** | Preferred nitrogen source for *M. tuberculosis*; stimulates growth and enhances colony formation |\n| **Glycerol** | Carbon and energy source; specifically stimulates growth of *M. tuberculosis*; *M. bovis* does NOT utilise glycerol and grows poorly on glycerol-containing LJ |\n| **Pyruvate (in M. bovis variant)** | Replaces glycerol; required for *M. bovis* growth; LJ with pyruvate should be used in regions where bovine TB or *M. bovis* infection is endemic |\n| **Malachite green** | **Selective agent** — bacteriostatic dye that inhibits the growth of most contaminating bacteria from sputum and other specimens, while *Mycobacterium* species are resistant to its inhibitory effect due to their lipid-rich cell wall |\n| **Mineral salts (KH₂PO₄, MgSO₄, magnesium citrate)** | Provide essential ions for bacterial metabolism |\n\n**Key physical property:** LJ medium contains no agar. It solidifies entirely through the thermal coagulation of egg albumin during inspissation at 85°C. This is why the temperature and duration of inspissation are critical — undercoagulation produces a liquid medium, overcoagulation degrades nutrients and produces a brittle, cracked surface.\n\n**Selective but not highly selective:** Malachite green suppresses most contaminating bacteria but does not eliminate all of them. Respiratory specimens (sputum, BAL) require **decontamination** before culture — typically using N-acetyl-L-cysteine and sodium hydroxide (NALC-NaOH method) — to reduce contaminating bacterial load before inoculation onto LJ slopes.\n\n## Uses of Löwenstein-Jensen Medium\n\n**1. Primary isolation of *Mycobacterium tuberculosis* from clinical specimens** LJ medium is inoculated with processed (decontaminated and concentrated) specimens from suspected TB cases:\n\n- Sputum (the most common specimen; three morning samples recommended)\n- Bronchoalveolar lavage (BAL) for smear-negative cases or suspected pulmonary TB\n- Gastric lavage or induced sputum in children (who cannot produce sputum)\n- CSF, urine, tissue biopsies, and pleural fluid in extrapulmonary TB\n\n**2. Drug susceptibility testing (DST) — indirect method** After primary isolation, the *M. tuberculosis* culture is used to perform drug susceptibility testing. The proportion method on LJ medium remains the reference standard in many resource-limited settings: the organism is inoculated onto LJ slopes containing individual anti-TB drugs at critical concentrations, and the proportion of colonies growing in the presence of drug versus drug-free control determines susceptibility or resistance.\n\n**3. Species identification within the *Mycobacterium* genus** Colony characteristics on LJ medium, combined with growth rate, pigmentation patterns (Runyon classification), and biochemical tests (niacin, nitrate reduction, catalase), allow presumptive identification of *M. tuberculosis* complex and differentiation from non-tuberculous mycobacteria (NTM). See the colony characteristics table below.\n\n**4. *M. bovis* differentiation** By inoculating the same specimen onto both glycerol-LJ and pyruvate-LJ slopes, growth on pyruvate-LJ only (or markedly better growth on pyruvate-LJ) suggests *M. bovis* — relevant in regions where bovine TB is endemic and zoonotic transmission occurs.\n\n### LJ Medium vs. MGIT Liquid Culture\n\nModern TB diagnostic guidelines increasingly recommend **liquid culture systems** — particularly the Mycobacterium Growth Indicator Tube (MGIT, Becton Dickinson) — as the primary culture method for *M. tuberculosis* because:\n\n- Time to detection is 9–14 days on average (vs. 3–6 weeks on LJ)\n- Sensitivity is higher (detects smaller inocula)\n- Automated systems (BACTEC MGIT 960) can process large numbers of specimens\n\nHowever, LJ medium retains a critical role in many settings:\n\n| Feature | LJ Solid Medium | MGIT Liquid Culture |\n| --- | --- | --- |\n| Time to detection | 3–6 weeks | 9–14 days (average) |\n| Sensitivity | Moderate | Higher |\n| Equipment required | Inspissator, incubator | Automated BACTEC system |\n| Cost | Low | Higher |\n| Colony morphology visible | Yes — important for identification | No |\n| Drug susceptibility testing | Proportion method (reference standard) | MGIT DST (WHO-endorsed) |\n| *M. bovis* differentiation | Yes (glycerol vs. pyruvate slopes) | Requires additional testing |\n| Resource-limited settings | **Suitable** | Often not feasible |\n\n**In Nepal and similar settings:** LJ medium remains the primary culture method in most district and provincial laboratories. GeneXpert\u002FXpert MTB-RIF has reduced dependence on culture for initial *M. tuberculosis* detection, but culture on LJ remains essential for drug susceptibility testing beyond rifampicin and for extrapulmonary specimens where GeneXpert sensitivity is lower.\n\n## Preparation of Löwenstein–Jensen Media\n\n**Ingredients**\n\n**A.  Mineral salt solution**\n\n- Potassium dihydrogen phosphate anhydrous (KH2PO4): 2.4 g\n- Magnesium sulphate anhydrous: (MgSo4.7H20): 0.24 g\n- Magnesium citrate: 0.6 g\n- Asparagine: 3.6 g\n- Glycerol (reagent grade): 12 ml\n- Distilled water: 600 ml\n\nDissolve the ingredients**in order**in the distilled water by heating. [Autoclave at 121°C for 30 minutes to sterilize](\u002Fautoclave-principle-procedure-types-and-uses\u002F). Cool to room temperature. This solution keeps indefinitely and may be stored in suitable amounts in the refrigerator.\n\n**B. Malachite green solution 2%**\n\n- Malachite green dye: 2.0 g\n- Distilled water: 100 mlDissolve the dye in the distilled water completely. Filter and store in the refrigerator.\n\n**C. Homogenized whole eggs**\n\n- Get a fresh (those are not more than seven days old), hen’s eggs\n- Clean the eggs by scrubbing thoroughly with a hand brush in water and soap.\n- Let the eggs soak for 30 minutes in a soap solution.\n- Rinse eggs thoroughly in running water and soak them in 70% ethanol for 15 minutes.\n- Before handling the clean dry eggs scrub and wash the hands with a disinfectant.\n- Crack the eggs with the edge of the beaker into a sterile flask and beat them in a sterile blender for 30 seconds to one minute.\n\n**Preparation of complete medium**\n\nAseptically pool the following reagents in a large, sterile flask and mixed well:\n\n- Mineral salt solution: 600ml\n- Malachite green: 20 ml\n- Homogenised eggs (25-30 eggs, depending on size): 1000ml\n\nThe complete egg medium is distributed in 6-8ml volumes in sterile universal containers or culture bottles  (14 ml or 28 ml) and the caps tightly closed and inspissated without delay to prevent sedimentation of heavier ingredients.\n\n> Cultures are usually made in bottles rather than in Petri dishes because of the long incubation time required. Use of bottle limits both chances of contamination and drying of the culture media (if the caps are tightly closed).\n\n**Coagulation of medium**\n\n- Heat the inspissator to 85°C to quicken the build-up of the temperature before loading.\n- Place the bottles in a slanted position in the inspissator and coagulate the medium for 50 minutes at 85°C (since the medium has been prepared with sterile precautions this heating is to solidify the medium, not to sterilize it).\n\n> The quality of egg media deteriorates when coagulation is done at too high a temperature or for too long. Discoloration of the coagulated medium may be due to excessive temperature. The appearance of little holes or bubbles on the surface of the medium also indicates faulty coagulation procedures.\n\n#### Sterility check\n\n![Löwenstein–Jensen Medium - Lowenstein Jensen Medium](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FLowenstein-Jensen-Medium.png)Figure: Lowenstein Jensen Medium\n\n- After inspissation, the whole media batch of the media bottles should be incubated at 35°C-37°C for 24 hours as a check for bacterial sterility.\n- After 24 hours 5% of the slopes should be picked up randomly and continued for incubation for 14 days to check for fungal sterility.\n- In both cases the contamination rate should not be > 10 %.\n\n**Storage**\n\nThe Löwenstein–Jensen medium should be dated and stored with the batch number in the refrigerator and can keep for up to 4 weeks if the **caps are tightly closed to prevent drying of the medium**.\n\n## Inoculation and Incubation\n\nTwo slopes of LJ medium should be inoculated per specimen (an additional one slope with pyruvate in *M. bovis* endemic areas).\n\n- Remove the condensed moisture before inoculation.\n- Inoculate each slope with 0.2-0.4 ml (2-4 drops or 2-4 loopful) of the centrifuged sediment, distributed over the surface.\n- Incubate the cultures at 35-37°C  until growth is observed or discarded as negative after eight weeks.\n\n## Examination Schedule\n\nAll cultures should be examined 72 hours after inoculation to check that liquid has completely evaporated, to tighten the caps in order to prevent drying out of media and to detect contaminants. Thereafter, cultures are examined weekly, or if this is not operationally feasible, on at least three occasions, viz\n\n- **after one week** to detect rapidly growing mycobacteria which may be mistaken for *M. tuberculosis*\n- **after 3-4 weeks** to detect positive cultures of *M. tuberculosis* as well as other slow-growing mycobacteria which may be either harmless saprophytes or potential pathogens\n- **after 8 weeks** to detect very slow-growing mycobacteria, including *M. tuberculosis*, before judging the culture to be negative\n\n## Results\n\nVisible colonies are usually produced 2-3 weeks after incubation (*M. tuberculosis* is a SLOW GROWERS,  do not grow in primary culture in less than one week and may take 3-4 weeks to give visible growth), but cultures should be incubated for up to 8 weeks before being discarded.\n\nWhen cultured on Lowenstein Jensen (LJ) medium at 35-37°C, *M.tuberculosis* produces rough (having the appearance of bread crumbs or cauliflower), raised, dry, non-pigmented (cream\u002Fbuff colored) colonies.\n\n![Close view of Mycobacterium tuberculosis colonies  - Close view ofMycobacterium tuberculosiscolonies(Image source: CDC\u002F Dr. George Kubica)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FClose-view-of-colony-of-Mycobacterium-tuberculosisPHIL_4428_lores.jpg)Figure: Close view of *Mycobacterium tuberculosis* colonies(Image source: CDC\u002F Dr. George Kubica)\n\n> With doubtful cultures or when less experienced staff read cultures, the acid fastness of the isolate should be confirmed by [Ziehl-Neelsen (ZN) staining](https:\u002F\u002Fmicrobeonline.com\u002Fziehl-neelsen-technique-principle-procedure-reporting\u002F).\n\n## Colony Characteristics of Mycobacteria on LJ Medium\n\nThe **Runyon classification** groups non-tuberculous mycobacteria by growth rate and pigmentation — both directly observable on LJ medium.\n\n| Organism | Growth rate | Pigmentation | Colony morphology | Clinical significance |\n| --- | --- | --- | --- | --- |\n| *M. tuberculosis* | Slow (3–6 weeks) | Non-pigmented (cream\u002Fbuff) | Rough, dry, raised, \"breadcrumb\" or \"cauliflower\" surface; not easily emulsified | Pulmonary TB; extrapulmonary TB |\n| *M. bovis* | Slow (5–8 weeks) | Non-pigmented | Smooth, flat, dysgonic; grows better on pyruvate-LJ | Zoonotic TB; intestinal TB via unpasteurised milk |\n| *M. kansasii* (Runyon I — photochromogen) | Slow | Non-pigmented in dark; **yellow-orange in light** | Smooth | Pulmonary disease in COPD patients |\n| *M. scrofulaceum* (Runyon II — scotochromogen) | Slow | **Yellow-orange in dark AND light** | Smooth | Cervical lymphadenitis in children |\n| *M. gordonae* (Runyon II) | Slow | Yellow-orange in dark | Smooth; very common tap water contaminant | Rarely pathogenic; common culture contaminant |\n| *M. avium* complex (Runyon III — non-chromogen) | Slow | Non-pigmented | Smooth, flat, translucent | Disseminated disease in [HIV](https:\u002F\u002Fmicrobeonline.com\u002Fhiv-structure-laboratory-diagnosis-and-natural-resistance); pulmonary disease |\n| *M. fortuitum* (Runyon IV — rapid grower) | **Rapid (3–7 days)** | Variable | Rough or smooth; grows within 1 week | Wound infections, catheter infections, post-surgical |\n| *M. chelonae* (Runyon IV) | **Rapid (3–7 days)** | Non-pigmented | Smooth, small | Skin and soft tissue infections; keratitis |\n\n> **Key exam point — Runyon classification:**\n>\n> - Group I (Photochromogens): Pigment only in light — \"photo\" = light\n> - Group II (Scotochromogens): Pigment in dark and light — \"scoto\" = dark\n> - Group III (Non-chromogens): No pigment in either condition\n> - Group IV (Rapid growers): Colonies visible within 7 days\n\n> **Rapid growers are the key differentiator.** If colonies appear on LJ medium within the first week of incubation, the organism is a rapid grower and cannot be *M. tuberculosis*. Record the day of first visible growth on every LJ slope — this single observation rules in or rules out *M. tuberculosis* complex.\n\n## How to Remember\n\n**LJ medium has three defining features — each one critical:**\n\n1. **Eggs, not agar** — LJ solidifies by inspissation of egg albumin at 85°C, not by agar. This is why temperature control during preparation is so important: too hot and the egg protein degrades; too cool and the slopes stay liquid.\n2. **Malachite green selects for mycobacteria** — the lipid-rich mycobacterial cell wall resists the bacteriostatic effect of malachite green that kills most contaminating bacteria. Think of it as the mycobacterium's armour.\n3. **Glycerol for MTB, pyruvate for M. bovis** — always inoculate both slope types when *M. bovis* is a clinical possibility (cattle farmers, veterinarians, patients with unexplained intestinal TB).\n\n**Growth timeline — the three checkpoints:**\n\n- **72 hours:** Check liquid has evaporated; tighten caps; look for contaminants\n- **1 week:** Rule out rapid growers (Runyon IV); if growth already visible, not *M. tuberculosis*\n- **3–4 weeks:** First positive *M. tuberculosis* cultures typically appear here\n- **8 weeks:** Final read; discard as negative only after 8 full weeks\n\n**Inspissation problems and what they mean:**\n\n- Discoloured medium (brown\u002Fdark) = temperature too high during inspissation\n- Holes or bubbles on slope surface = faulty coagulation technique\n- Liquid medium that did not solidify = temperature too low or duration too short\n\n**Where LJ fits in the diagnostic pyramid:**\n\n> Smear microscopy (minutes) → GeneXpert (2 hours) → LJ culture (weeks) → DST on LJ (weeks more) Each step answers a different question. LJ answers: \"Is the organism alive and culturable? What drugs will kill it?\"\n\n**Identification**\n\n*Mycobacterium tuberculosis* can be identified presumptively on the basis of its colony characteristics. Though there is not a completely reliable single test that will differentiate *M. tuberculosis* from other mycobacteria.\n\nThe following tests when used in combination help to identify the *M.tuberculosis* strains.\n\n- Niacin Test\n- **Nitrate Reduction Test**\n- **Catalase Test**\n\n**References and Further Reading**\n\n- World Health Organization. (1998). *Laboratory Services in Tuberculosis Control, Part III: Culture.* WHO\u002FTB\u002F98.258. Geneva: WHO. Available at: [https:\u002F\u002Fapps.who.int\u002Firis\u002Fbitstream\u002F10665\u002F65942\u002F3\u002FWHO_TB_98.258\\\\\\_(part3).pdf](https:\u002F\u002Fapps.who.int\u002Firis\u002Fbitstream\u002F10665\u002F65942\u002F3\u002FWHO_TB_98.258%5C_\\(part3\\).pdf)\n- Cheesbrough, M. (2006). *District Laboratory Practice in Tropical Countries, Part 2* (2nd ed.). Cambridge University Press.\n- World Health Organization. (2014). *Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis.* Geneva: WHO.\n- Tille, P. M. (2017). *Bailey and Scott's Diagnostic Microbiology* (14th ed.). Elsevier.\n- Camus, J. C., Pryor, M. J., Médigue, C., & Cole, S. T. (2002). Re-annotation of the genome sequence of *Mycobacterium tuberculosis* H37Rv. *Microbiology*, 148(10), 2967–2973.",[],[47],"mycobacteria",[49,58,64,82,88,104,111,117],{"slug":50,"title":51,"description":51,"seoTitle":38,"seoDescription":38,"author":52,"createdDate":53,"lastUpdatedDate":54,"draft":42,"category":55,"image":38,"faq":56,"tags":57},"mycobacterium-tuberculosis-lab-diagnosis","Laboratory Diagnosis of Mycobacterium tuberculosis Infection","Nisha Rijal","2020-05-26","2026-07-04","bacteriology",[],[47],{"slug":59,"title":60,"description":60,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":61,"lastUpdatedDate":41,"draft":42,"category":55,"image":38,"faq":62,"tags":63},"genexpert-mtbrif-assay-principle-procedure-results-interpretations","GeneXpert MTB\u002FRIF Assay","2016-01-04",[],[47],{"slug":65,"title":66,"description":67,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":68,"lastUpdatedDate":69,"draft":42,"category":70,"image":38,"faq":71,"tags":81},"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","2026-07-07","staining-techniques",[72,75,78],{"question":73,"answer":74},"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":76,"answer":77},"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":79,"answer":80},"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.",[47],{"slug":83,"title":84,"description":84,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":85,"lastUpdatedDate":54,"draft":42,"category":55,"image":38,"faq":86,"tags":87},"short-notes-atypical-mycobacterial-infections","Atypical Mycobacterial Infections","2014-11-07",[],[47],{"slug":89,"title":90,"description":91,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":92,"lastUpdatedDate":69,"draft":42,"category":70,"image":38,"faq":93,"tags":103},"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",[94,97,100],{"question":95,"answer":96},"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":98,"answer":99},"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":101,"answer":102},"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.",[47],{"slug":105,"title":106,"description":106,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":107,"lastUpdatedDate":54,"draft":42,"category":108,"image":38,"faq":109,"tags":110},"key-biochemical-methods-used-to-distinguish-mycobacterial-group","Biochemical Tests to identify Mycobacteria, NTM","2013-07-22","biochemical-tests",[],[47],{"slug":112,"title":113,"description":113,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":114,"lastUpdatedDate":54,"draft":42,"category":55,"image":38,"faq":115,"tags":116},"introduction-transmission-pathogenesis-and-lab-diagnosis-of-leprosy-hansens-disease","Leprosy: Etiology, Pathogenesis, Lab Diagnosis","2012-05-15",[],[47],{"slug":118,"title":119,"description":120,"seoTitle":38,"seoDescription":38,"author":52,"createdDate":121,"lastUpdatedDate":69,"draft":42,"category":55,"image":122,"faq":123,"tags":124},"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",[],[47],[126,132,139,144,148,152,157,162,166,170],{"slug":127,"name":39,"description":128,"image":129,"body":130,"postCount":131},"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":133,"name":134,"description":135,"image":136,"body":137,"postCount":138},"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":140,"name":141,"description":142,"image":38,"body":38,"postCount":143},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":145,"name":146,"description":142,"image":38,"body":38,"postCount":147},"samikshya-acharya","Samikshya Acharya",20,{"slug":149,"name":150,"description":142,"image":38,"body":38,"postCount":151},"alisha-tripathi","Alisha Tripathi",6,{"slug":153,"name":154,"description":155,"image":38,"body":38,"postCount":156},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",10,{"slug":158,"name":159,"description":160,"image":38,"body":38,"postCount":161},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":163,"name":164,"description":142,"image":38,"body":38,"postCount":165},"srijana-khanal","Srijana Khanal",18,{"slug":167,"name":168,"description":160,"image":38,"body":38,"postCount":169},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":171,"name":52,"description":142,"image":38,"body":172,"postCount":173},"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]