Comparison of methods for isolation of mycobacteria from water

Abstract

Twelve methods for the isolation of mycobacteria were compared by applying them in parallel to 26 samples of surface water and 109 samples of treated water. Each method was defined by a particular combination of decontamination method, growth medium, and incubation temperature. For the decontamination of surface water, we used cetylpyridinium chloride (CPC) (30 min, 0.05%), as well as sample preincubation in tryptic soy broth (TSB) followed by decontamination with a cocktail of NaOH, cycloheximide, and malachite green. Treated water was decontaminated with 0.005 and 0.05% CPC (30 min). After enrichment by filtration, all samples were incubated on Lowenstein-Jensen medium (LJ), Ogawa egg yolk medium (OEY), and Ogawa whole-egg medium containing ofloxacin and ethambutol (OEOE) at temperatures of 30 and 37(deg)C. The efficacy of each method was determined by calculating the positivity rate, negativity rate, contamination rate, mean number of mycobacterial colonies grown, and mean number of different mycobacterial strains isolated. The last value was determined by subjecting the isolates to PCR restriction analysis and mycolic acid thin-layer chromatography. Statistical analysis demonstrated that both the TSB method and 0.05% CPC were appropriate for the decontamination of surface water. Decontamination with 0.005% CPC was best for treated water. The results for incubation on LJ were at least equal to those for incubation on OEY and always superior to the results with OEOE. At an incubation temperature of 30(deg)C, all methods achieved higher yields than at 37(deg)C.