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°C for 5 h to allow germination of microbial spores. T h e recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with N a O H or H2S04 both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with N a O H followed by oxalic acid. Egg media at p H 5.5 resulted in lower mycobacterial counts than egg media at p H 6.5 or Mycobacteria 7H11 agar. T h e numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. T h e 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 p H 6.5.

The effect of prolonged storage on mycobacteria and other heterotrophic bacteria in brook water samples was examined by determination of viable counts from fresh samples and again after water concentrates had been stored in nutrient broth at -75 degrees C for 15 months. The counts of mycobacteria were on average three times higher after storage (range of ratio 0.9-10.4). In contrast, the viable counts of other heterotrophic bacteria were reduced by 69%. The increase in mycobacterial counts was probably due to break-up of microcolonies or release of attached bacteria from particles. The possibility of cultivating mycobacteria from frozen samples is of practical help in large-scale field surveys.

Two decontamination methods and five media were compared for the isolation of mycobacteria from brook waters of different physical, chemical and bacteriological characteristics. The decontaminants used were: 0.7 mol 1‐1 NaOH followed by 50 g 1‐1 oxalic acid and 0.9 mol 1‐1 H2SO4 combined with 0.5 g 1‐1 cycloheximide. The media compared were: Mycobacteria 7H11 agar with OADC enrichment (pH 6.6), glycerol egg (pH 6.5 and 5.5), and pyruvate egg (pH 6.5 and 5.5). All media contained cycloheximide, 0.5 g 1‐1. The NaOH—oxalic acid method generally resulted in lower contamination and higher isolation of mycobacteria than the H2SO4‐cycloheximide method. With the NaOH—oxalic acid method, all five media were equal in positivity rates but contamination was a problem on Mycobacteria 7H11 agar. Of the four egg media tested, the highest positivity rate (92% of the samples) was obtained on the pyruvate modification (pH 6.5), and the highest mean colony count of mycobacteria (900 cfu 1‐1) on the glycerol modification (pH 6.5). Characteristics of water and sampling site had similar effects on the isolation frequencies of mycobacteria obtained by different combinations.