Place the labeled TSA and SAB settling plates near the work area but in an area that will not interfere with testing. Document the sampling start time. Aseptically, open both plates, placing the lids on a clean surface in the biological safety cabinet away from the work area for up to four hours to prevent the agar from drying out.
At the end of the processing close the lids of the plates and document the sampling stop time. Loosely, bag the plates and incubate the cultures. For gloved fingertip sampling, obtain two labeled TSALT plates.
Aseptically remove the lid of the plates and place it on a clean work surface away from the sampling area. Gently roll the pad of each finger and thumb on the surface of the plate. Use sufficient force to make slight depressions on the agar without cracking the agar surface.
Aseptically place the lid back onto the plate. After sanitizing the hands at the completion of sampling, loosely bag the plates and incubate the cultures. For direct inoculation, following the United States Pharmacopeia method, place the TSA and SAB settling plates in the biosafety cabinet.
After preparing the cabinet and staging the materials, document the sampling start time. Obtain the test article and the associated tryptic soy broth and fluid thioglycollate medium bottles. If the bottles have a septum, remove the protective caps from each bottle and wipe the septum with a sterile alcohol wipe.
Vortex the test article to ensure a homogenous suspension and an inoculate the bottles with a validated volume of the test article using a sterile syringe and a hypodermic needle. Wipe the septum with a sterile alcohol wipe and insert a sterile venting unit to allow for air exchange during incubation. Repeat the steps for each test article of that session.
Aseptically close the TSA and SAB settling plates and document the sampling end time before performing the gloved fingertip sampling. Wipe the gloves with 70%sterile isopropyl alcohol, loosely bag the plates and incubate the cultures. Transfer all the test materials out of the biological safety cabinet.
Similarly, for direct inoculation, following the NIH alternative sterility testing method, place the TSA and SAB settling plates in the biosafety cabinet and document the sampling start time. Obtain the test article and the associated IFA Plus labeled, IFN Plus labeled and SAB plate. Remove the protective caps from the IFA Plus and IFN Plus bottles and wipe the septum with a sterile alcohol wipe.
After vortexing the test article, inoculate the bottles with validated volume of the test article using a sterile syringe and a hypodermic needle and wipe the septum again. Incubate the bottles on the BACT/ALERT Dual T instrument. Using a pipetter, inoculate the SAB plate with a validated amount of the test article and streak for isolation using a sterile loop.
Let stand for 10 minutes to ensure the sample does not run onto the plate lid when inverted for incubation Place each SAB plate inoculated with a test article into a plastic bag. Tie them loosely. Aseptically close the TSA and SAB settling plates.
Document the sampling end time on the plates, and perform the gloved fingertip sampling. After sampling, wipe the gloves with 70%alcohol. Loosely bag the plates after wiping the gloves with alcohol and incubate the cultures.
A TSA air settling plate illustrating a single colony of a contaminant cultured during passive air process monitoring in the biological safety cabinet is shown here. The TSALT surface sampling plate with three colony forming units of two distinct colony morphologies is shown in this figure. The TSALT surface culture with a single colony on the plate's edge indicates poor aseptic handling during the sampling process.
The correct method for obtaining gloved fingertip samples using the largest surface area of each finger or thumb is shown in this image. The incorrect process where only the fingertip is sampled is shown here. An example of mold balls visible to the naked eye that failed to be automatically detected by the BACT/ALERT system is shown here.
Based on these findings, terminal visual inspection of all BACT/ALERT bottles and the addition of the SAB plate for fungal culturing using the NIH alternative sterility testing method is recommended.
