Culture medium brand choice impacts colony swarming behavior among industrial Bacillus isolates and the accuracy of aerobic plate counts.

Aerobic plate count assays are an industry standard method for the enumeration of microbial products. Colony swarming among industrial Bacillus isolates on solid medium can impact a counting technician's interpretation of colony count, promote inter-technician variance and reduce the agreement of plate counts with growth-independent enumeration methods. In the present study, we examined swarming behavior among four industrial Bacillus species as a function of culture medium brand choice. Colony diameter for three Bacillus species was found to be influenced by culture medium brand, as was colony count interpretation among three out of four plate counting technicians. Estimations of Bacillus endospore concentration were likewise influenced by culture medium brand, leading to an increased incidence of QC failure for replicate samples of a Bacillus-based microbial product as a function of brand choice. Results suggest that culture medium brand choice may be an additional source of variance when plate counting is used for the enumeration of Bacillus-based microbial products. We recommend that plating medium brand availability in testing laboratories be considered by microbial product manufacturers when considering sources of variance in customer and regulatory laboratories.

A comparison of methods for enumerating bacteria in direct fed microbials for animal feed.

Aerobic plate counts are the standard enumeration method for probiotic-containing products. This counting method is limited by the ability of many cells to enter a viable but non-culturable (VBNC) state upon exposure to stressful conditions like dehydration and heating commonly used in probiotic product preparation. Alternative enumeration methods are available including flow cytometry (FC) which counts total live/dead cells by assessing cellular integrity and/or metabolic activity, and quantitative polymerase chain reaction (qPCR) in which enumeration is correlated with the quantity of a nucleic acid target. These three methods were compared for enumerating three lactic acid bacteria (LAB): Pediococcus acidilactici, Pediococcus pentosaceus, and Lactobacillus plantarum, and a Bacillus subtilis related strain in twenty samples of a mixed probiotic product ranging in age from one to 825 days post-production. Flow cytometry and qPCR enumerations were similar and much higher compared to plate counts at later storage times, suggesting that some strains in the population were entering the VBNC state and were only countable by FC and qPCR. We propose the use of FC and/or qPCR as an alternative to plate counts for more accurate enumeration of bacteria in probiotic products.