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

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.

Incubation temperature and culture medium formulation impact the accuracy of pour-plate techniques for the enumeration of industrial Bacillus assemblages.

Aerobic plate counting assays based on the pour-plate technique are frequently used to enumerate microbial products; however, colony swarming and merging at the agar surface can reduce the accuracy of these assays. Some plating methods mitigate this risk through the inclusion of strategies including agar overlays; however, these interventions may be inadequate to mitigate swarming and merging of certain Bacillus colonies. In the present study, we assessed the accuracy of several pour-plate techniques for the enumeration of a mixed-species Bacillus assemblage.

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.

Customized antimicrobial efficacy tests offer superior evaluation of growth inhibitor efficacy for liquid microbial products.

Antimicrobial effectiveness tests are common methods used to assess the risk of microbial contamination in pharmaceuticals and cosmetics. These assays may be inappropriate for endospore-based microbial products which often carry a similar - if not greater - risk of microbial contamination. In the present study, we compared the antimicrobial efficacy assessment provided by United States Pharmacopeia Chapter <51> Antimicrobial Effectiveness Testing with a modified test which utilized a customized bacterial challenge.

Nutrient germination improves DNA recovery from industrial endospores during qPCR enumeration assays.

Growth-independent microbial enumeration methods such as quantitative PCR require the efficient extraction of genomic DNA from targeted cells. endospores are popular inclusions in commercial products due to their hardiness and metabolic dormancy; however, this hardiness is known to render endospores resistant to traditional DNA isolation techniques. Metagenomic studies have sought to address this resistance through nutrient-based germination of bacterial endospores in environmental samples.

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.

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.