The aim of this investigation was to determine the bacterial load on used instruments and to evaluate the relationship between the bacterial load and the holding time prior to cleaning. Thirty six sets were evaluated to establish the average number of bacteria per square centimeter. For the experimental study, three different bacteria were prepared in sheep blood and used to contaminate sterile stainless steel pieces with the surface of 10 cm(2). After incubation at room temperature for 2, 4, 6, 8, 12, 24, 36, and 48 h, colonies were counted and compared to time zero. Bacterial counts were between 10 and 250 CFU/cm(2), depending on the operation site. Bacterial load was found to have increased after 6 h. An increase of 3log(10) CFU/cm(2) was measured after 12 h. It is imperative to clean surgical instruments in the first 6 h to ensure effective disinfection and sterility.