The aim of this investigation was to determine the bacterial load on used instruments and to evaluate the relationship between the bacterial load and holding time prior to cleaning. Forty 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 surface area of 10cm 2 . After incubation at room temperature for 2, 4,6,8, 12, 24, 36 and 48 h, colonies were counted as compared to time zero. Bacterial counts were between 20 and 320 CFU/cm2, depending on the operation site. Bacterial load was found to have increased after 6 hrs. An increase of 3log10 CFU/cm2 was measured after 12 h. It is imperative to clean surgical instruments in the first 6 h to ensure effective disinfection and sterility. KEYWORDSSurgical Instruments, Bacterial Load, Bioburden, Holding Time, Contamination. HOW TO CITE THIS ARTICLE:Mohite ST, Mahesh Reddy S, Kshirsagar AY, et al. Effect of holding time on the bacterial load of surgical instruments.