ABSTRACTThe ASTM E1174-21 Health Care Personnel Handwash method is prescribed by the U.S. Food and Drug Administration (FDA) to demonstrate the efficacy of antiseptic handwashing products. The standardized method allows for marker bacteria to be collected from the hands by using either a bag or a glove. Two recent studies utilizing the different collection methods testing the same product showed substantial differences in results. We sponsored two independent studies to compare the bag and glove collection methods following contamination withSerratia marcescens. Overall, there was no difference between collection methods for bacteria recovered (P =0.603). The distribution of recovery for the bag method was slightly less variable than for the glove method. Statistical differences were observed within each lab based on the collection day. The day-to-day variability is critical to consider for future multiple-day studies. Additionally, hand size appears to impact recovery, especially for the glove method, with both small and medium hand sizes resulting in higher recovery than large and extralarge hand sizes (P =0.015), whereas hand size did not impact recovery with the bag method (P =0.315). While it appears that both the bag and glove methods can be used, our findings suggest that gloves may not be the best option for subjects with large to extra-large hands. Additional work looking at bacterial recovery following product treatment is warranted to understand the impact of large hands in the bag versus glove recovery method.IMPORTANCEAntiseptic hand wash products are evaluated using the standard ASTM E1174-21 to demonstrate their antibacterial efficacy. Often products are tested at multiple labs, and the need to understand variables that may play a role in the outcome of the study is important. This work allows us to evaluate the impact that the two collection methods, bags and gloves, have on bacteria recovery. If differences are observed, standardization to one method may be critical to ensure similar test results when planning studies at multiple labs.