Clinical Preoperative Skin Preparation (PSPs) kits are the standard of care for skin disinfection prior to surgery and the most important method of infection prevention. However, we have discovered that the antiseptics contained in these kits do not eradicate the natural flora that live in the deep regions of the skin. FDA approved PSPs fail the 2-3 log10 reduction required by the FDA when full-thickness skin samples are used on Yorkshire pigs [1]. We hypothesized that the method with which the PSP are tested is directly correlated with the log reduction result. To test this, we compared two skin sampling methods: the FDA required process, the cup scrub method, and a new procedure called the tissue blend method. The cup scrub method consists of placing a sterile cylinder on the skin that has been cleaned with antiseptic. Broth is added to the cylinder and the skin is agitated within the cup. The bacteria in the broth is then quantified. In contrast, the tissue blend method is performed by cutting out full-thickness skin samples from the backs of pigs. These tissue samples are blended, and then the homogenized tissue is quantified. We performed the cup scrub and tissue blend methods on the backs of 7 Yorkshire pigs following alternating scrubs of 4% chlorhexidine gluconate (CHG) and alcohol (n=5/pig). The cup scrub and tissue blend methods were also performed on control skin as a relative baseline (n=5/pig). With 4 treatment groups, 20 samples were taken from each animal for a total of 140 samples. The average log reduction for the cup scrub and tissue blend methods were 1.57 +/- 0.45 CFU/cm2and 0.23 +/- 0.48 CFU/cm2, respectively. When analyzing the data, CHG appears to decrease the bioburden following PSP application when using the cup scrub method, however the tissue blend method shows that it is not killing bacteria dwelling deep in the skin, ultimately resulting in infection risk. This research is fundamental for the development of new PSP technology that eradicates deep dwelling flora.