Mycobacteriology and Nocardia
This domain tests decontamination chemistry, growth timelines, acid-fast staining behavior, and the distinction between Mycobacterium and Nocardia.
Specimens and processing
- Collect three early-morning sputa on separate days; use induced sputum or BAL when smears are negative; gastric aspirate in children (neutralize promptly); first-morning urine; CSF; fresh tissue in saline, never formalin.
- Decontaminate with NALC–2% NaOH for about 15 minutes at room temperature, neutralize with phosphate buffer to pH 6.8, and centrifuge at 3,000 × g for 15 minutes.
- Inoculate egg-based Lowenstein-Jensen and a liquid system such as MGIT; hold solid media 6–8 weeks.
Stains and identification
- Ziehl-Neelsen uses carbolfuchsin with acid-alcohol decolorizer; auramine O fluorescence is the more sensitive screening stain.
- Confirm M. tuberculosis complex with a nucleic acid amplification test; identify nontuberculous mycobacteria by HPLC or sequencing.
Nocardia
- Aerobic, gram-positive, branching, beaded filaments that are weakly acid-fast with modified Kinyoun using 1% sulfuric acid - not the 3% acid-alcohol of Ziehl-Neelsen.
- Grows in 3–7 days on blood agar as chalky, dry, umbilicate colonies; catalase positive. Identification by MALDI-TOF or 16S sequencing; susceptibility by broth microdilution (TMP-SMX is often active, but N. farcinica is resistant).
Worked example
A day-5 sputum culture grows dry, chalky white colonies. Gram stain shows branching gram-positive rods; modified acid-fast is positive with 1% sulfuric acid but negative by Ziehl-Neelsen. Reasoning: weak acid-fastness plus branching plus aerobic growth points to Nocardia, not Mycobacterium (strongly acid-fast) and not Actinomyces (anaerobic, not acid-fast). Identify and send for susceptibility.
Traps
Submitting tissue in formalin; discarding mycobacterial plates at two weeks; equating a positive AFB smear with tuberculosis; dismissing Nocardia as a diphtheroid contaminant.