Meat extract casein peptone agar - A novel culture medium for the enumeration of Bacillus endospores in commercial products

Abstract

Here we propose a novel culture medium, Meat Extract Casein Peptone (MECP) agar, to support the enumeration of Bacillus endospores in commercial products. The formulation is the result of screening eight different veterinary, pharmaceutical, and industrial grade peptones for the ability to support the formation of small, well-defined Bacillus colonies on solid culture medium. The impact of agar purity, agar formulation rate, and metal cation additives were examined in prototype medium batches prepared from preferred peptone inputs. A customized plate counting assay based on the resultant MECP agar formulation was compared with standardized pour-plate and spread-plate assays (ISO 4833) and flow cytometry for the ability to accurately enumerate five Bacillus-based biostimulants and biofertilizers. Estimations of Bacillus endospore concentration generated by the customized spread-plate assay were significantly higher than those produced by ISO 4833 pour-plate and spread-plate assays for four out of the five tested products and were in better agreement with flow cytometry values; however, flow cytometry values were numerically higher than values returned by both plating methods. Both flow cytometry and plating assays based on MECP or similar culture media represent potential candidates for standardization and validation through organizations such as ISO and AOAC International for the enumeration of Bacillus-based products.

Keywords: Bacillus; Enumeration; Flow cytometry; Peptone; Plate counting.