![]() coli C and 2.5 mL molten top agar was added to the assay fluid and poured it onto bottom agar plates for each experimental setup. Then, 200 μL of a log-phase culture of E. 3 Some test were run with the bacteriophage in vomit, which survived a 1-minute duration, but this would not be comparable with previous work or represent a worst-case clinically relevant exposure.Ī total of 500 μL collected assay fluid was used for the plaque assay. The vomit test soil rendered the φχ174 nonviable for the 60 minutes required by the standard test cycle. The penetration cell was swirled for approximately 1 minute, after which the assay fluid was removed and assayed for the presence of φχ174. At the conclusion of the test, the observed side of the test material was rinsed with assay fluid (5 mL sterile NB) to ensure proper contact of the fluid with the entire viewing surface of the test specimen. If liquid penetration was not visible at this point, the specimen was observed again after an additional 54 minutes. Next, the system was pressurized to 2 psi for 1 minute, then the pressure was turned off and the apparatus was monitored for visual leaks. The penetration cell was filled with 60 mL of either blood or vomit artificial test soil with the above generated φχ174 bacteriophage challenge suspension (2.5 × 10 10 pfu/mL), and the apparatus was observed for any leaks for 5 minutes. The protein concentration was assessed by using Lowry reagent (Bio-Rad, Richmond, CA) on a 96-well plate format (Molecular Devices, San Jose, CA). The pellet was resuspended in 200 μL cold PBS. The φχ174 was spun at 118,000 × g in an MLS 50 swinging bucket rotor for 2.5 hours at 4☌ in Beckman Optima MAX-XP Benchtop Ultracentrifuge. A total of 4.5 mL bacteriophage was overlaid on 500 μL sucrose cushion. φχ74 was purified by centrifugation on a 20% sucrose cushion prepared in PBS and filter sterilized with a 0.22-μm filter. The supernatant was retrieved, and the filter sterilized using a 0.22-μm pore size membrane filter (Millipore). The culture was clarified by spinning at 10,000 × g in a Sorval ST 16 centrifuge. coli C bacteria were inoculated with φχ174 and incubated for 4 hours. coli C was added to 125 mL NB and grown to an optical density of 0.26 at 600 nm. To produce φχ174 for antibody production, E. coli C on plates with NA for the bottom layer and agar with NB on top. Φχ174 was propagated on a lawn of permissive E. Nonetheless, the method presented in this investigation is a substantial improvement of a standard method for viral challenge testing of PPE materials. However, using the dot-blot system, which uses antibodies to detect the bacteriophage and signal amplification, does not indicate if virus viability or infectivity is retained, whereas the ASTM F1671 method indicates both. ![]() The dot-blot method described here is less labor intensive and faster than the ASTM F1671 method. The results indicated that ASTM F1671 and the dot-blot apparatus methods were equivalent. This study then compared the standard ASTM F1671 (using bacteriophage φχ174) with a modified dot-blot method to assess viral penetration of PPE materials. In this study, modification of the nutrient broth provided consistently higher titers of virus and the use of the top agar in smaller increments prevented premature solidification. However, an alteration of the bacteriophage propagation method detailed in the standard was necessary to obtain consistent titers of virus. ASTM test method F903, which specifies the test method setup also used in ASTM F1670 and F1671, has been used for decades to test liquid (ASTM F1670) or viral (ASTM F1671) penetration resistance of PPE fabrics. Appropriate test systems and test soils are needed to adequately evaluate PPE. Effective personal protective equipment (PPE) is critical in preventing the spread of infectious diseases.
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