Enumeration of industrial Bacillus assemblages in commercial products with customized plate-counting assays

The aerobic plate count assay remains among the most widespread methods for enumerating industrial Bacillus assemblages, as growth-independent methods are either cost prohibitive or unavailable in some areas. However, the standard plating assays used to verify the CFU count of Bacillus-based products are not tailored to Bacillus species and thus may not produce the most accurate possible estimations. Standard plating assays assume that established limits of quantification are applicable to Bacillus species whose colonies swarm on solid media, and that colonies of each species in a mixed-species assemblage form independently of one another on agar plates. In the present study, we examined the upper limit of quantification for an assemblage of swarming industrial Bacillus isolates by comparing plate count on medium with and without a swarming inhibitor with direct counts for spore suspensions of increasing endospore concentration. Additionally, we examined the impact of assemblage species composition on the evenness of colony distribution across replicate plates for four industrial Bacillus isolates. We compared the observed distribution of colonies across replicate plates to the expected Poisson distribution for axenic endospore suspensions, for a 3-species assemblage and a 4-species assemblage, respectively. Results suggest that customized plating assays may be more appropriate than standard protocols for the enumeration of Bacillus-based products, and that interactions between colonies on solid media should be considered when interpreting plating data for mixed-species Bacillus assemblages.