Five replicates of each selleck products material were used in each test. Organisms from stock cultures were transferred to Tryptic Soy Broth and incubated for 24 hours at 35-37°C (25-30°C for ATCC 13048). Two loopfuls of culture were transferred consecutively daily for three days for the inoculation stocks and the pellicle of bacteria were aspirated. Daily transfers were done for at least 3 consecutive days but for no more than 10 days. To this culture 0.25 ml of heat-inactivated fetal bovine serum (FBS) and 0.05 ml of Triton X-100 were added to 4.7 ml bacteria suspension
to yield 5% FBS and 0.01% Triton X-100 organic soil load. The challenge microorganism titer was determined by serially diluting a final 48 hour culture using phosphate buffered solution (PBS) and selected dilutions were plated in duplicate 3-Methyladenine using Tryptic Soy Agar (TSA) pour plates. Carriers were inoculated with 0.02 ml of the 48 hour culture. The bacterial inoculum per experiment is detailed in Table 1. All control plates were incubated in parallel
to the test plates. The inoculum was spread to within ~1/8 inch of the control or test carrier before air drying for 20–40 minutes at 35-37°C and 38-42% relative humidity. After 120 minutes exposure at 21°C, the carriers were transferred to 20 ml neutralizer solution (2x Letheen broth ) and sonicated for 5 minutes and rotated to mix. Within one hour serial dilutions (10−1 to 10−4) were made Cell press in PBS and plated using TSA and incubated for 48 hours at 35-37°C for colony observation and enumeration, taking into account also the 20 fold dilution used to retrieve the bacteria from the carriers. The following controls were performed: culture purity control – each prepared culture was streaked using TSA for purity control; organic soil purity control – duplicate 1 ml aliquots of organic soil were plated in TSA pour plates for sterility control; neutralizer sterility control – a jar containing the neutralizer was incubated with the test plates and selleck chemical observed for growth or no growth; carrier sterility control – an
uninoculated test (per lot) and control carrier was put in independent jars containing the neutralizer, incubated and observed for growth or no growth; carrier viability control – for each challenge microorganism, a single inoculated control carrier was subcultured in a jar containing the neutralizer, incubated and the neutralizer observed for growth or no growth; and neutralization confirmation control – for each challenge microorganisms, per lot of the test article, a single sterile test carrier was put in individuals jars containing 20 ml of the neutralizer. To each jar a 1 ml aliquot of the diluted inoculum was added to reach ~100 colony forming units (CFU)/ml in the neutralizer. The jar was mixed and the 1 ml inoculum was removed and plated in duplicate.