We optimized the microbiocidal assay using both E. coli and S. aureus for four different species: coyote, house finch, garter snake, and newt. This range of species should provide an approximate starting point for new researchers utilizing this technique; however, any researcher replicating this protocol should perform a species validation. To optimize for different species we plated pooled plasma samples (3 pooled samples of 2 individuals each) for house finches, garter snakes, side-blotched lizards, and newts and individual samples (i.e., not pooled) for coyotes in the top row of 96 well microplates. We serially diluted each sample down the plate (from 1:1–1:128). Specifically, we added 18 µl of pooled plasma sample in triplicate and 18 µl PBS to the first row of the plate and then added 18 µl of PBS to all other wells on the plate (except for positive and negative controls). We mixed the plasma and PBS in row 1 using a multichannel pipette. We then removed 18 µl from row 1 and transfer to row 2 re-mixed the solution and repeated to each subsequent row to serially dilute down the plate (after row 8 the remaining 18 µl can be disposed) for least 8 dilutions. We then followed the same assay procedure as above. All plasma samples were incubated with bacteria (105 CFU/ml) for 30 min at 37°C and then for 12 hours at 37°C following the addition of tryptic soy broth. Assay results depict average response across replicate samples for each species.