Spore-Forming Bacteria Associated with Dairy Powders Can Be Found in Bacteriological Grade Agar–Agar Supply

Thermophilic spore-forming bacteria are found ubiquitously in natural environments and, therefore, are present in a number of agricultural food products. Spores produced by these bacteria can survive harsh environmental conditions encountered during food processing and have been implicated in food spoilage. During research efforts to develop a standardized method for enumerating spores in dairy powders, the dairy powder-associated thermophilic sporeformer Anoxybacillus flavithermus was discovered growing in uninoculated control plates of tryptic soy agar (TSA) supplemented with 1% (w/v) starch, after incubation at thermophilic (55°C) growth temperatures. This article reports the investigation into the source of this thermophilic sporeformer in TSA medium components and characterization of the bacterial isolates collected. Aqueous solutions of tryptic soy broth powder from four suppliers and four agar-agar powders (two manufacturing lots from one supplier [agar A_1 and agar A_2] and two from separate suppliers [agar B and agar C]) were subjected to two different autoclave cycle times (121°C for 15 min or 121°C for 30 min) and then prepared as TSA. After incubation at 55°C for 48 h, bacterial growth was observed only in media prepared from both lots of agar A agar-agar powder, and only when they were subjected to a 15-min autoclave cycle, implicating these powders as a source of the sporeformer contamination. Genetic characterization of 49 isolates obtained indicated the presence of five unique rpoB allelic types of the thermophilic sporeformer Geobacillus spp. in agar-agar powder from agar A. These results not only highlight the importance of microbiological controls but also alert researchers to the potential for survival of thermophilic sporeformers such as Anoxybacillus and Geobacillus in microbiological media used for detection and enumeration of these same thermophilic sporeformers in products such as dairy powders. HIGHLIGHTS