This protocol tests the disinfection efficacy of hot water laundering. It can help assess if a washer and dryer are suitable for cleaning clothing contaminated by a virus. The main advantage of this laundering technique is that it can be replicated in other labs at a smaller scale using procedures that have been through the quality assurance process.
Demonstrating the procedure will be Ahmed Abdel-Hady, a microbiologist, Mariela Monge and Jonathan Sawyer, research scientists, and others from our laboratory. To begin, prepare the modified tryptic soy agar by weighing and mixing the ingredients in deionized water as described in the text. Then, add one milliliter of undiluted, concentrated Phi6 aliquot to soft agar and 100 microliters of a log phase pseudomonas syringae culture.
Next, pour soft agar onto the surface of a solidified modified tryptic soy agar plate having a diameter of 100 millimeters. Prevent bubbles or spillage by swirling the plates gently and distributing the soft agar evenly over the solid agar surface. After overnight incubation at room temperature, gently scrape the contents of the three plates with a sterile cell spreader into a sterile 50 milliliter chronicle tube containing 15 milliliters of SM buffer.
Vortex the tubes at maximum setting for one to two minutes to break the agar and then centrifuge them for 15 minutes at 7, 000 times G.Then collect the supernatant and filter it through a 0.2 micron syringe filter. Store the filtered supernatant as one milliliter aliquots in cryovials at 80 degrees Celsius until further use. Perform pre-test visual assessment by measuring and recording the length and width of the unwashed personal protective equipments or PPE items at various locations.
For inoculation, prepare a 10%beef extract solution by dissolving one gram of beef extract in a total volume of 10 milliliters of 1X PBS. Filter sterilize the entire volume of the solution using a 0.2 micron syringe filter. Next inside the bio safety cabinet, inoculate the test and positive control coupons with approximately 10 to the seventh plaque forming units per sample by pipetting a 10 microliter amount of the solution onto the PPE item as multiple droplets and spreading the droplet using the tip of the pipette.
Warm the laundering solution to 50 degrees Celsius using a hot plate and stir bar. Then measure and record the pH and temperature of the laundering solution and collect 10 milliliters of solution for sterility check. Then, pour 3.25 liters of laundering solution into a sterilized washer.
Place PPE items, one inoculated face covering, and four non-contaminated film masks without coupons in the washer. After washing PPE items for 18 minutes, drain the washer and triple rinse with five liters of room temperature tap water each time to remove the foam. Add 3.25 liters of room temperature sterilized tap water into the washer and perform a nine minute long rinse cycle.
Next, move the PPE items into the washer spin side to spin for five minutes. Dry the PPE items for 80 minutes on the high heat setting of 93 degrees Celsius in the dryer. Then move the PPE items from the dryer to the sterile workspace.
Aseptically remove the coupons from the inoculated items and place them in conical tubes. Next, extract the coupons in 10 milliliters of 10%Dey-Engley neutralizing broth by vortexing for two minutes at the maximum setting of the equipment. To plate the extracts using a conventional soft top agar overlay method, add test sample aliquots to the soft agar tube containing six milliliters of soft agar and 100 microliters of the log phase P syringae.
Next, pour the soft agar onto the surface of a solidified modified tryptic soy agar plate and evenly distribute the soft agar over the solid agar surface by swirling the plate. After overnight incubation at room temperature, manually enumerate the platforming units on each plate. The systematic laboratory testing to assess the laundering effectiveness of viral disinfection from full sized PPE or clothing, showed that the platforming unit or PFU plate count and the extracted sample volume enabled the calculation of the number of platforming units per test coupon.
The results of the representative viral recovery were obtained for a face covering test. The laundering tests provided information about the material properties like the stretching or shrinkage of the clothing items and quality controlled data of the protocol. Efficacy of hot water laundering at disinfecting face coverings, denim, and scrub PPE materials from Phi6 was demonstrated in this graph where the stars denote the full disinfection.
It is important to use aseptic techniques throughout this procedure to prevent cross-contamination. Also, inoculum drying times for coupons are unique for each material and are established before testing. Other complimentary methods include microscopy and liquid penetration tests to explore material degradation or further size assessments ensuring the barrier protection still fits the wearer.
This technique provided the basis to scale up laundering efficacy and material testing for full scale clothing and PPE items. It can also aid in establishing effective laundering procedures during another contagion event or pandemic.
