Chapter 4: Culture Media for Bacillus spp. and Related Genera Relevant to Foods

Bacillus and related genera are characterised by the ability to form vegetative cells and spores. Endospore-forming catalase-positive bacteria have been traditionally allocated to the genus Bacillus. These organisms are widespread within environmental niches and habitats. Their spores are resistant to heat and other means of sterilization, so spore-forming bacteria are a major concern to food microbiologists. Virtually any food can be colonized by these organisms due to their ubiquitous distribution, their physiological diversity and thus in their growth requirements. With respect to diagnostics, it is therefore not possible to design a single medium which allows growth of all or most species within this group. Numerous media have been described for the cultivation of individual species; however, most of these are not selective and only some of them are elective. Bacillus strains do not form endospores under all cultural conditions. Sporulation is inducible; e.g. by limitation of nutrient factors, including carbon or nitrogen. Further, most Bacillus strains form endospores in media supplemented with manganese salts. The only selective media for aerobic spore-forming bacteria in the mesophilic/neutrophilic range have been those developed for the so-called Bacillus cereus group. This group consists of genetically closely related species, B. anthracis, B. cereus, B. thuringiensis, B. weihenstephanensis, B. mycoides and B. pseudomycoides. Despite the variations in potential virulence, the differentiation of the B. cereus group members remains a difficult important task. The most widely used plating media for detection of B. cereus are mannitol egg-yolk polymyxin agar (MEYP or MYP) and polymyxin egg-yolk mannitol bromothymol blue agar (PEMBA). Procedures for reliable enumeration and identification of Bacillus cereus are included in international standards, e.g. EN ISO 7932:2004 and EN ISO 21871:2006. Recently, two new chromogenic media have been developed for the Bacillus cereus group, supplemented with 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate. Other simple methods of selection are to adjust media to certain pH values or to incubate at certain temperatures. This physiological approach has been successful e.g. with Alicyclobacillus spp., emerging food spoilage organisms in the fruit juice and fruit juice products industry.

In general it should be noted that aerobic spore-forming organisms are no longer just the traditional genus Bacillus, but fall into an increasing number of additional genera. Recent reports have described toxin-producing B. subtilis group members and the analysis of plasmids, which are very common in the B. cereus group, became a focal point of interest. As it cannot be assumed that novel species are of no relevance to food, it is sometimes advisable to perform a more thorough taxonomic identification to complement the diagnostic approach. Genomic studies and the development of more and better diagnostic media would probably aid the detection of aerobic spore forming bacteria in foods.