Simulated Use Test for High-Level Disinfectants is outstanding way to get a real world understanding of the efficacy of a disinfectant in an exposure condition that will test the more difficult aspect of the disinfect process. In order for a disinfectant to be effective it must come in contact with the microbe in order to kill it. This test will assess the efficacy while attempting to disinfect a hard to reach and more resistant vegetative microbial type, mycobacteria. This test is part of an FDA Premarket Notification 510(k) Submission.

Prior to initiation of the experiment, the active ingredient in the test substance is measured, the pH is taken and the concentration of the test substance is validated using titration analysis. Then, the test substance is diluted to the minimum recommended concentration (MRC) and confirmed again by titration analysis and a final pH is taken. Prior to initiation of the study, the test culture (generally Mycobacterium terrae) is grown on appropriate agar slants 21-25 days, resuspended in broth for 28-35 days, the culture is homogenized, supplemented with calf serum and serially diluted to the appropriate inoculum concentration. Generally, TEE probes are used in the assessment of this study. The probes are sterilized and prepared within 20 minutes of the testing.

In this study a negative control is ran using a sterile swab that has been dipped in neutralizer recovery media, then is used to swab each of the surface sites. The swab will then be cut and left in 10ml of recovery media, vortexed, contents filtered, then plating the filter on M7H9 agar supplemented with OADC additive for the incubation period. The test probes when inoculated with an aliquot of the test microbe are allowed to dry for more than 30 minutes. The recovery stage of the experiment is very similar to the negative control, where a swab dipped in neutralizer recovery medium is used to scrub the inoculated and dried surface site. The swabs are then cut into 10ml of recovery media. This is repeated with each site of the probe being tested. Along side the study, a low numbers control is also ran where three surface sites of a probe are inoculated with about 100 CFU/ 10ml. After the surface has dried, the three sites are swab recovered as before, vortexed, ran through a filter, plated and then incubated for 12-14 days. This low numbers control helps to show that even at a low inoculum concentration that the microbe is recoverable from the probe. In this study a positive control is also ran to ensure the appropriate inoculum concentration is reached in the study, this done by inoculating a 5-fold dilution of the microbe at three surface sites of the probe. The sites are then swab recovered, vortexed, serially diluted, ran through membrane filter, filter plated, incubated for 12-14 days and recorded to ensure the inoculum concentration is at least 106 CFU.

After the inoculum concentration has been confirmed, the test can begin by inoculating the probe in the three test sites as stated before. The probe can be tested either manually or using a disinfector. If manually, the probe is submerged in an appropriate sized container of test strength test substance for the sponsor's specified exposure time. If using a disinfector, the probe is placed into the apparatus with the test strength test substance in the appropriate reservoir (or container) and ran for full cycle exposing the probe to the test substance for the appropriate exposure time at the label temperature. However, the probe is then removed before the rinse cycle. Then in the case of manual and disinfector, the probe is dipped in bacteria-free water to stop action of the test substance and placed on sterile surface. The surface sites will then undergo swab recovery, filtration and plating as described before and then, final calculations will be made to establish the log10 reduction observed. The FDA deems a passing test will effectively eliminate at least 6 log10 (99.9999%) of the original test microbe in the claim contact time at the label claim temperature. Other controls that are typically ran in the test are also the neutralization validation, soil sterility and media sterility to help validate readings. All results will be calculated, graphed and presented in a single report and submitted to the study sponsor. This study is an outstanding way to get a full understanding of the capability of a test substance in a realistic exposure scenario with quantitative results.

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