Sterility was assessed by inoculating 5 g humidified soil onto 7H10 agar incubated at 37 uC under a 5% CO2 atmosphere for 4 weeks. Sterilized soil was used as a negative control in all experiments. Sterilized soil was then thoroughly mixed with each of the MTC organisms studied and suspended in sterile PBS to achieve a homogeneous inoculum of 108 c.f.u. (g soil)21, and 50 g of this preparation was placed in an opaque sterile plastic container that was incubated at room temperature for 12 months. Room temperature was monitored weekly and sterile distilled water (1 ml) was gently added twice per month to the container. Before each sampling, soil was mixed with a sterilized fork to ensure the homogeneity of the sample.