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Objective:To identify factors that increase the microbial load in the operating room (OR) and recommend solutions to minimize the effect of these factors.Design:Observation and sampling study.Setting:Academic health center, public hospitals.Methods:We analyzed 4 videotaped orthopedic surgeries (15 hours in total) for door openings and staff movement. The data were translated into a script denoting a representative frequency and location of movements for each OR team member. These activities were then simulated for 30 minutes per trial in a functional operating room by the researchers re-enacting OR staff-member roles, while collecting bacteria and fungi using settle plates. To test the hypotheses on the influence of activity on microbial load, an experimental design was created in which each factor was tested at higher (and lower) than normal activity settings for a 30-minute period. These trials were conducted in 2 phases.Results:The frequency of door opening did not independently affect the microbial load in the OR. However, a longer duration and greater width of door opening led to increased microbial load in the OR. Increased staff movement also increased the microbial load. There was a significantly higher microbial load on the floor than at waist level.Conclusions:Movement of staff and the duration and width of door opening definitely affects the OR microbial load. However, further investigation is needed to determine how the number of staff affects the microbial load and how to reduce the microbial load at the surgical table.