Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37 degrees C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H2SO4 both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5.5 resulted in lower mycobacterial counts than egg media at pH 6.5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green-cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6.5.