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.