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Contaminated operating room surfaces can increase the risk of orthopaedic infections, particularly after procedures in which hardware implantation and instrumentation are used. The question arises as to how surgeons can measure surface cleanliness to detect increased levels of bioburden. This study aims to highlight the utility of adenosine triphosphate (ATP) bioluminescence technology as a novel technique in detecting the degree of contamination within the sterile operating room environment.What orthopaedic operating room surfaces are contaminated with bioburden?When energy is required for cellular work, ATP breaks down into adenosine biphosphate (ADP) and phosphate (P) and in that process releases energy. This process is inherent to all living things and can be detected as light emission with the use of bioluminescence assays. On a given day, six different orthopaedic surgery operating rooms (two adult reconstruction, two trauma, two spine) were tested before surgery with an ATP bioluminescence assay kit. All of the cases were considered clean surgery without infection, and this included the previously performed cases in each sampled room. These rooms had been cleaned and prepped for surgery but the patients had not been physically brought into the room. A total of 13 different surfaces were sampled once in each room: the operating room (OR) preparation table (both pre- and postdraping), OR light handles, Bovie machine buttons, supply closet countertops, the inside of the Bair Hugger™ hose, Bair Hugger™ buttons, right side of the OR table headboard, tourniquet machine buttons, the Clark-socket attachment, and patient positioners used for total hip and spine positioning. The relative light units (RLUs) obtained from each sample were recorded and data were compiled and averaged for analysis. These values were compared with previously published ATP benchmark values of 250 to 500 RLUs to define cleanliness in both the hospital and restaurant industries.All surfaces had bioburden. The ATP RLUs (mean ± SD) are reported for each surface in ascending order: the OR preparation table (postdraping; 8.3 ± 3.4), inside the sterilized pan (9.2 ± 5.5), the inside of the Bair Hugger™ hose (212.5 ± 155.7), supply closet countertops (281.7 ± 236.7), OR light handles (647.8 ± 903.7), the OR preparation table (predraping; 1054 ± 387.5), the Clark-socket attachment (1135.7 ± 705.3), patient positioners used for total hip and spine positioning (1201.7 ± 1144.9), Bovie machine buttons (1264.5 ± 638.8), Bair Hugger™ buttons (1340.8 ± 1064.1), tourniquet machine buttons (1666.5 ± 2144.9), computer keyboard (1810.8 ± 929.6), and the right side of the OR table headboard (2539 ± 5635.8).ATP bioluminescence is a novel method to measure cleanliness within the orthopaedic OR and can help identify environmental trouble spots that can potentially lead to increased infection rates. Future studies correlating ATP bioluminescence findings with microbiology cultures could add to the clinical utility of this technology.Surfaces such as the undersurface of the OR table headboard, Bair Hugger™ buttons, and tourniquet machine buttons should be routinely cleansed as part of an institutional protocol. Although correlation between ATP bioluminescence and clinical infection was not evaluated in this study, it is the subject of future research. Specifically, evaluating microbiology samples taken from these environmental surfaces and correlating them with increased bioburden found with ATP bioluminescence technology can help promote improved surgical cleaning practices.