Abstract Background Automated plating instruments have been shown to improve quality in Microbiology laboratories in a number of ways including consistent streaking patterns that provide better isolation of bacterial colonies compared to manual methods. The time per culture set up and automated camera imaging is dependent upon the amount of media set, such that more individual plates slow down turn around time. Conversely, smaller media surface area and the use of non-selective media may result in mixed colonies that require sub-culture for isolation before identification and susceptibilities can be performed. Based upon these findings, we attempted to reduce our media usage per culture with the implementation of automation, with the goal of using as few plates as possible while at the same time not negatively impacting the turn around time of the culture via the need to subculture poorly isolated colonies. We conducted a pre-and post-implementation study evaluating the costs per culture and turn around time delays related to subculturing in a subset. Methods In the pre-implementation period, from April-January 2023, three individual media plates (sheep blood agar, chocolate agar and CNA/MacConkey bi-plate) were routinely set for general aerobic bacterial culture sources including swabs and body fluids. In the post implementation period, from April-January 2024, a single sheep blood/chocolate agar bi-plate was set unless the gram stain showed evidence of mixed organisms (defined as more than one morphology) at which time a secondary selective media (CNA/MacConkey) was manually set. For urine cultures, in the pre-implementation period 2 plates were set (sheep blood and CNA/MacConkey bi-plate) and in the post-implementation period a single chromogenic agar/sheep blood bi-plate was set. Total numbers of cultures in both periods were collected as well as the number of cultures for which additional media were set based on gram stain results. Orders of subculture media in the laboratory information system were compared per positive cultures. Statistical analysis was performed in GraphPad Prism. Results In the pre- versus post-implementation periods, a total of 16,454 vs 17,782 general aerobic cultures and 50,680 vs 53,916 urine cultures were set, respectively. Only 3.6% of general cultures required additional media set based on gram stain. The difference in cost between pre- and post-implementation was approximately a two-thirds reduction in cost for general cultures and a 25% reduction for urine cultures, with a total savings of approximately $34,238 in media avoidance in the post-implementation period. The frequency of positive cultures requiring subculture was not significantly different between the pre- (average 36% of positive growth) and post-implementation (average 22% of positive growth) periods for both culture types and may actually have been reduced due to automation. Conclusions The implementation of reduced media concomitant with streaking via automation resulted in significant cost savings while not negatively impacting isolation, which in turn impacts culture turn around times. The automation may have reduced need for subculture, perhaps due to better isolation, thus subsequent analyses will explore this additional finding.