Mycobacteriology: Acid-Fast Staining, Culture, and Identification

Mycobacteria have a mycolic-acid-rich wall that resists acid-alcohol decolorization once dye has entered the cell. That property is acid-fastness, and it is why a dedicated acid-fast stain, not a Gram stain, is the smear method when mycobacterial infection is suspected. The same wall makes cells stain poorly with crystal violet or safranin, so they appear as faint, beaded, gram-neutral or ghost rods. Wright- and Papanicolaou-stained smears show similar ghost images.¹

Acid-fast staining

Spread two to three drops of concentrated sediment thinly, air-dry, and heat-fix (15 minutes at 80 °C, or 1-2 hours at 65-70 °C on a hot plate) before staining.

Fluorochrome screening covers more of the smear per unit time. Some rapidly growing mycobacteria stain poorly with fluorochrome dyes. When a rapid grower is likely, as in a postsurgical wound, restain a negative fluorochrome smear with carbol fuchsin before reporting it negative.

Acid-fast bacilli are purple-to-red (carbol fuchsin) slightly curved rods, about 1-10 μm by 0.2-0.6 μm, often beaded or banded. Morphology does not identify species, but a few patterns are suggestive: serpentine cording in broth smears for M. tuberculosis complex (MTBC); large, cross-barred “shepherd’s crook” bacilli for M. kansasii; and pleomorphic, sometimes coccobacillary, periodic acid-Schiff-positive cells for M. avium complex (MAC).

Smear specificity is at least 99%. Sensitivity is about 25% to 75%, and roughly 5,000-10,000 organisms/mL are needed for a positive smear, compared with about 10-100 organisms for culture. False-positive smears (positive smear, negative culture) follow nonviable organisms after therapy, over-decontamination, or staining cross-contamination. Sensitivity is higher with cavitary pulmonary disease, respiratory specimens, more specimens, higher burden, and species such as M. tuberculosis or M. kansasii that shed more organisms than some nontuberculous mycobacteria (NTM).¹

Smear results are reported semiquantitatively. CDC still uses the Kent and Kubica 1+-4+ scale. Fluorochrome counts are adjusted for the larger field:

A negative smear does not exclude tuberculosis. Acid-fast organisms other than MTBC also stain.¹

Mycobacterium tuberculosis complex

MTBC includes M. tuberculosis (the dominant human pathogen), M. bovis (cattle and other warm-blooded animals, including humans; BCG is derived from it and can cause disseminated disease), M. africanum (tropical Africa), M. caprae (mainly goats and cattle), M. microti (rodents), M. canettii, and M. pinnipedii (pinnipeds). CDC also lists M. mungi. Transmission is mainly inhalation of 1-5 μm droplet nuclei generated by cough, shout, or song; surface contact does not transmit MTBC. Direct inoculation of abraded skin is an occupational risk when infected tissue is handled. M. bovis also spreads through unpasteurized milk and from infected humans back to cattle via urine when genitourinary infection is present.¹

Close contact with known or suspected active tuberculosis, birth in or travel to a high-prevalence country, congregate settings, health-care work with at-risk patients, and drug or alcohol use disorder increase the chance of acquiring infection. HIV, age under 5 years, immunosuppressive therapy, infection acquired in the past 2 years, prior inadequate treatment, silicosis, diabetes, chronic renal failure, certain cancers, gastrectomy or jejunoileal bypass, weight under 90% of ideal, and cigarette smoking increase the chance of progression to disease.

Inhaled bacilli reach alveoli and are phagocytosed by macrophages that cannot kill them. Infected macrophages migrate to tracheobronchial nodes, present antigen, and seed the lung apices and distant sites (kidney, long-bone epiphyses, vertebral bodies, meninges). Cytokine-activated macrophages then drive caseation. A residual apical nidus can reactivate years later. The primary Ghon lesion is a 1-2 cm subpleural focus in the lower part of an upper lobe or the upper part of a lower lobe; together with the draining-node lesion it is the Ghon complex.

Pulmonary disease can be insidious, lingering, flu-like, hemoptysis, or pleuritic. Extrapulmonary disease is more often multiorgan and concentrates in young children, older adults, and immunocompromised persons, especially with HIV coinfection.

Nontuberculous mycobacteria

Runyon grouped NTM by pigment and growth rate on solid media: photochromogens pigment only after light exposure; scotochromogens pigment in dark or light; nonchromogens remain colorless; rapid growers form visible colonies in 7 days or less. The scheme has limits: M. kansasii is usually photochromogenic but not always; MAC is usually nonchromogenic; M. szulgai is scotochromogenic at 37 °C and photochromogenic at 25 °C; and liquid culture, which excludes light, cannot support the classification. Nearly 200 NTM species are now recognized.

M. leprae has never been cultured in vitro; it can be propagated only in the mouse footpad over about 6 months. Transmission is principally person-to-person via nasal aerosols from lepromatous disease. Diagnosis uses nasal secretion or skin-slit smear and lesion biopsy. M. lepromatosis is identified genomically.

Specimen collection and decontamination

CDC currently asks for at least three consecutive sputum specimens, each collected 8-24 hours apart, with at least one early-morning specimen. Collect them in an airborne-infection isolation room or other well-ventilated space. Sterile-site specimens may be inoculated directly. Specimens from sites with normal flora must be liquefied, decontaminated, and concentrated, or faster flora will mask mycobacteria. Process in a biological safety cabinet. Refrigerate specimens that cannot be processed immediately.²

The standard NALC-NaOH method uses an equal volume of freshly prepared N-acetyl-L-cysteine-2% sodium hydroxide (working digestant made from equal parts 4% NaOH and sodium citrate plus NALC). Vortex 15-30 seconds, then stand 15 minutes at room temperature (do not exceed about 20 minutes). Add phosphate buffer pH 6.8 to about 30-50 mL to stop decontamination, centrifuge, decant, and resuspend the sediment in 1.5-2 mL of sterile water or buffer for smear and culture. Over-exposure kills mycobacteria as well as contaminants. Gastric lavage should be processed promptly; if delayed, traditional practice neutralized it with 10% sodium hydroxide or 1% sodium bicarbonate, though later data question whether that step is required. Stool: dilute 1-2 g formed (or 5 mL liquid) to 10 mL, vortex, and filter through gauze before decontamination. Urine: split across 2-4 tubes, centrifuge 3,000-3,600 × g for 30 minutes, combine sediments, and bring to 10 mL before decontamination.

Culture methods

Inoculate both broth and solid media. Broth is more sensitive and faster; solid media show colony morphology and pigmentation. A broth-only approach is acceptable where local data show no extra yield from solid media. Culture remains the gold standard for laboratory confirmation of tuberculosis and is required for full susceptibility testing and genotyping even when NAAT is positive.²

Solid media are egg-based (Löwenstein-Jensen) and agar-based (Middlebrook 7H10, 7H11, selective 7H11). Cutaneous specimens need hemin for M. haemophilum and mycobactin J for M. genavense. Incubate at 37 °C in 5-10% CO₂; keep tubed media slanted with loose caps for at least the first week. Colonies often take 3-4 weeks.

Liquid systems use modified 7H9 broth with an oxygen-sensitive fluorescent indicator, incubated at 37 °C for at least 6 weeks, or fully automated instruments that track oxygen consumption or CO₂ production.

Identification methods

Biochemical panels take weeks and often fail to resolve species. Current methods are faster and more specific.

Direct-specimen NAAT is the fastest route to a pulmonary TB diagnosis. CDC recommends NAAT on at least one respiratory specimen from each patient with symptoms of pulmonary TB when the diagnosis is being considered and the result would change case management or control activities. Transcription-mediated amplification of rRNA reports positive, equivocal, or negative. Real-time PCR assays such as Xpert MTB/RIF detect MTBC and, in the same cartridge, rpoB mutations associated with rifampin resistance, usually in under 2 hours. Sensitivity is higher on smear-positive than smear-negative sputum. Neither format differentiates individual MTBC species. NAAT does not replace smear, culture, growth-based susceptibility testing, or genotyping. A result of MTBC detected / rifampin resistance detected has a high probability of rifampin resistance and needs additional rapid confirmation; MTBC detected / resistance not detected still needs growth-based first-line testing; an indeterminate rifampin result also needs growth-based testing.^2,3

Chemiluminescent DNA probes identify a species or complex within 1-2 hours of adequate growth; commercial probes exist for MTBC, MAC, M. avium, M. intracellulare, M. gordonae, and M. kansasii. Limitations include occasional failure to identify MAC grown in liquid medium, false-positive MAC results from broth aliquots, and rare false-positive MTBC results.

MALDI-TOF requires pre-extraction to disrupt the mycolic-acid-rich wall. Pre-extraction also inactivates MTBC, permitting analysis outside a BSL-3 environment. Most protocols use solid-medium growth. The method cannot resolve individual MTBC species or M. abscessus subspecies.

Line-probe reverse-hybridization assays hybridize biotinylated PCR product to immobilized probes. They work best from colonies or positive broth and can also be applied to AFB smear-positive specimens.

Sequencing has the highest identification accuracy among these methods. 16S rRNA is the usual first target; many species need additional loci (rpoB, secA, hsp65, gyrB, or the internal transcribed spacer). Whole-genome sequencing can add resistance markers but remains largely a reference-laboratory method; phenotypic susceptibility testing should still accompany it because uncatalogued mutations are missed.