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Propionibacterium acnes, which normally resides in the skin, is known to play a role in surgical site infection in orthopaedic surgery. Studies have suggested a persistence of propionibacteria on the skin surface, with rates of positive cultures ranging from 7% to 29% after surgical preparation. However, as Propionibacterium organisms normally reside in the dermal layer, these studies may underestimate the true prevalence of propionibacteria after surgical skin preparation. We hypothesized that, after surgical skin preparation, viable Propionibacterium remains in the dermis at a much higher rate than previously reported.Ten healthy male volunteers underwent skin preparation of the upper back with ChloraPrep (2% chlorhexidine gluconate and 70% isopropyl alcohol). Two 3-mm dermal punch biopsy specimens were obtained through the prepared skin and specifically cultured for P. acnes.Seven volunteers had positive findings for Propionibacterium on dermal cultures after ChloraPrep skin preparation. The average time to positive cultures was 6.78 days.This study found that Propionibacterium persists in the dermal tissue even after surface skin preparation with ChloraPrep. The 70% rate of persistence of propionibacteria after skin preparation is substantially higher than previously reported.Propionibacteria are increasingly discussed as having an association with infection, implant failure, and degenerative disease. This study confirms the possibility that the dermal layer of skin may be the source of the bacteria.