The bacterial strains used in the study were stored at -20°C prior to use and were then used to aseptically inoculate 100 ml of tryptone soya broth (TSB). The TSB was incubated in a shaking incubator for 24 hours at 37°C (48 hours in the case of M. parafortuitum). The concentration of each culture was determined by serial dilution followed by inoculation of 0.1 ml onto tryptone soya agar plates. Following incubation at 37°C for 24 or 48 hours the number of colonies was counted and the concentration of bacteria in the original culture was determined. Based on this information appropriate dilutions of the cultures were carried out to give a concentration of approximately 3000 cfu/ml. Sterile tryptone soya agar (TSA) plates were subsequently inoculated with 0.1 ml of this dilution, which was spread evenly over the surface of the media with a sterile spreader, to yield approximately 300 colonies per plate. The plates were then allowed to dry at room temperature before being used in the ion exposure experiments. For each exposure period five identical replicate plates were prepared.
Following ion exposure the agar plates were incubated at 37°C for a period of 24 or 48 hours after which time the number of colonies on each plate was counted. Control plates were treated in an identical manner with the exception of exposure to the ion source.