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. Tested modifications included a customized culture medium formulation, agar overlays, decreased incubation times and increased incubation temperature. Methods which produced countable plates were assessed for agreement with a Bacillus-specific plate counting assay and with total cell counts rendered by flow cytometry. While all tested pour-plate methods underestimated Bacillus endospore concentrations relative to flow cytometry and customized spread-plating, our results suggest that increasing incubation temperature and the inclusion of bile salts into culture medium formulations can improve the accuracy of pour-plate techniques when used to enumerate Bacillus assemblages by decreasing the incidence of spreading colonies. As Bacillus endospore preparations become more ubiquitous in the market, familiar enumeration methods such as the pour-plate technique may require methodological modifications to ensure that the cGMP compliance of Bacillus-based microbial products is assessed accurately.

基于倾板技术的需氧平板计数测定常用于计数微生物产品；然而，琼脂表面的菌落聚集和合并会降低这些测定的准确性。一些电镀方法通过包含包括琼脂覆盖在内的策略来降低这种风险；然而，这些干预措施可能不足以减轻某些芽孢杆菌菌落的聚集和合并。在本研究中，我们评估了几种浇注板技术对混合种芽孢杆菌组合计数的准确性。测试的修改包括定制的培养基配方、琼脂覆盖、减少的孵育时间并提高孵化温度。评估产生可计数平板的方法与芽孢杆菌特异性平板计数测定和流式细胞术提供的总细胞计数的一致性。虽然所有测试的倾板方法都低估了相对于流式细胞术和定制铺板的芽孢杆菌内生孢子浓度，但我们的结果表明，提高孵育温度和将胆汁盐加入培养基配方中可以提高倾板技术的准确性。通过降低散布菌落的发生率来列举芽孢杆菌组合。作为芽孢杆菌随着内生孢子制剂在市场上变得越来越普遍，熟悉的计数方法（如浇注板技术）可能需要对方法进行修改，以确保准确评估基于芽孢杆菌的微生物产品的 cGMP 合规性。