14.2 Acid-Fast Reaction, Colony Morphology, and Growth Characteristics

14.2 Acid-Fast Reaction, Colony Morphology, and Growth Characteristics

Quick Answer: Mycobacteria are fully acid-fast; Nocardia is partially acid-fast on a modified stain. Rapid growers such as M. abscessus are visible in fewer than 7 days; M. tuberculosis, MAC, M. kansasii, and M. gordonae are slow. Photochromogen = M. kansasii (and M. marinum); scotochromogen = M. gordonae; nonphotochromogen = MAC and TB. Serpentine cords and rough buff colonies suggest M. tuberculosis. Incubate most AFB at 37 °C and add 30 °C for M. marinum. Use LJ or 7H11 plus liquid MGIT.

Outline III.A expects you to read an acid-fast reaction, a colony, and a clock. Identification algorithms start here: a 4-day cream colony from CF sputum is not worked like a 21-day rough buff slant, and a branching filament that destains with mycobacterial acid-alcohol is not reported as M. tuberculosis. Stain chemistry was taught under preanalytic I.C; this section is how those reactions, plus growth rate and pigment, sort the organisms named in 14.1.

Fully acid-fast versus modified acid-fast

Mycobacterial cell walls are packed with long-chain mycolic acids. Carbolfuchsin binds those lipids and resists acid-alcohol (classically about 3% HCl in ethanol). After destaining and a methylene-blue or malachite-green counterstain, mycobacteria remain red and often beaded. That is full acid-fastness. Rapid growers may stain more weakly or more irregularly than M. tuberculosis, but they are still mycobacteria, not Nocardia.

Nocardia has shorter mycolic acids. It destains with standard mycobacterial acid-alcohol (negative or only rare residual color) but retains carbolfuchsin when the decolorizer is milder (about 0.5–1% sulfuric acid). That is partial / modified acid-fastness. Pair it with the Gram stain: Nocardia is a branching Gram-positive filament, often beaded, that may show aerial hyphae on solid media. Mycobacteria are rods, not true branched filaments, and they Gram-stain poorly (ghosts or beaded Gram-positive rods).

Do not call a organism “acid-fast Nocardia” on a Ziehl–Neelsen or Kinyoun smear that used mycobacterial acid-alcohol. Do not call beaded fully acid-fast rods “Nocardia” because they look filamentous at low power. The decolorizer strength is the discriminator the exam uses.

Slow growers versus rapid growers

Rapid growers produce visible colonies in fewer than 7 days on solid media (often in 3–5 days, and sometimes on blood, chocolate, or MacConkey agar). The M-level rapid grower to own is the M. abscessus group (CF lung, chronic lung, post-procedure skin). M. fortuitum and M. chelonae are related rapid-grower names you may see on reports; the growth-rate rule is the same.

Slow growers take more than 7 days, commonly 10–21 days or longer. M. tuberculosis complex, MAC, M. kansasii, M. gordonae, and M. marinum sit in this bin. Liquid MGIT shortens time-to-detection compared with solid media but does not convert a slow grower into a rapid grower. Mycobacterial cultures are typically held 6–8 weeks before a final negative; a 48-hour “no growth” reading is a bacterial culture, not an AFB culture.

Runyon grouped nontuberculous mycobacteria by pigment and rate. The scheme is still a useful exam language even though modern labs identify species by MALDI or sequencing:

M. tuberculosis was not originally a Runyon NTM, but on the bench it behaves as a slow, nonphotochromogenic, often rough organism. Do not assign it to Group IV because a MGIT flags at day 8.

Photochromogen testing is practical: grow the isolate in the dark until colonies are visible, then expose one tube to light and incubate again. M. kansasii turns yellow. M. gordonae is already pigmented in the dark. MAC and TB stay buff. M. marinum is a photochromogen that you will miss if you never incubated the skin culture at 30 °C.

Cords, rough versus smooth, and temperature

Cord factor (trehalose 6,6'-dimycolate) makes M. tuberculosis align in serpentine cords—ropes of parallel AFB on smears from sputum or liquid culture. Cording is a strong clue to M. tuberculosis complex; it is not a species diagnosis. Some NTM can cord weakly. Report cords as a morphology comment and identify the isolate.

Colony texture tracks with that chemistry. M. tuberculosis on Löwenstein–Jensen is classically rough, dry, buff, and cauliflower-like. MAC is more often smooth, creamy, and smaller, though rough MAC exists. Rapid growers are typically smooth or waxy and appear on routine media. Nocardia is chalky, dry, and may pile into aerial hyphae—more like a moldlet than a mycobacterial buff dome.

Incubation temperature is a trap for skin specimens:

If the requisition says “hand nodule, aquarium,” set both 30 °C and 37 °C. M. haemophilum also prefers 30 °C and extra hemin, but that organism is SM-only as tested content—know it exists so you do not invent it as an M answer, then move on.

Solid LJ/7H11 versus liquid MGIT

Recovery is better when solid and liquid media are both inoculated after digestion-decontamination of sputum.

Malachite green on LJ suppresses some bacteria but does not replace NALC–NaOH. 7H11 is preferred when mixed NTM and TB might coexist because colonies are easier to pick. MGIT (Mycobacteria Growth Indicator Tube) detects oxygen consumption as a fluorescent signal; a positive tube is acid-fast stained immediately, because contaminant bacteria also consume oxygen. Never assume a MGIT flag is TB without a smear and an identification method.

Nocardia is recovered on sheep blood, chocolate, BCYE, and fungal media in a few days to two weeks; it may be inhibited by harsh sputum decontamination. If the Gram stain already showed branching filaments, do not wait for mycobacterial media to tell you what the bacterial plates already grew.

Put the pieces together: fully AFB + cords + rough buff at 37 °C in 2–3 weeks → work as TB at BSL-3. Fully AFB + smooth + no pigment + slow → MAC until identified. Fully AFB + yellow only after light → M. kansasii. Orange in the dark from a single sputum → think M. gordonae contaminant. Colonies in 4 days from CF sputum → M. abscessus group. 30 °C photochromogen from a fish-tank hand → M. marinum. Branching partial AF → Nocardia.