Optimization of modified M iddlebrook 7 H 11 agar for isolation of M ycobacterium bovis from raw milk cheese

Reports have highlighted the absence of contemporary peer reviewed publications pertaining to M ycobacterium bovis culture from raw milk and cheese. By replicating traditional methods, cheese‐making methodology and equipment were devised to produce C heddar ( n  = 6) and C aerphilly ( n  = 3) artificially contaminated with M . bovis (three genotypes) under stringent laboratory‐containment guidelines for handling hazardous microbiological material. Middlebrook 7 H 11, modified for M . bovis isolation, was assessed for capacity to enumerate M . bovis despite changing cheese microflora and prolonged M . bovis exposure to the cheese matrix using maturing cheese test portions ( n  = 63; up to 16 weeks). Malachite green ( MG ) containing media isolated M . bovis at significantly ( P  < 0·05) lower levels than unmodified M iddlebrook 7 H 11 agar despite MG being a common adjunct of M iddlebrook 7 H 11 agar modified for M . bovis growth. Subsequently, a selective MG ‐free M iddlebrook 7 H 11 agar modified using haemolysed red cells and calf serum was demonstrated as the best performing ( P  < 0·05) medium for recovery of M . bovis from typical UK cheese types, C heddar and C aerphilly. Significance and Impact of the Study Following increased M . bovis infection of UK cattle, the risk posed to consumers from consumption of unpasteurized milk and dairy products has changed. Furthermore, published methods for the culture and molecular detection of M . bovis in raw milk products are limited. Cheese‐making protocols and M . bovis culture media reported here provide tools for further investigation of M . bovis survival during all stages of cheese manufacture and could inform future assessment of the risk to consumers from M . bovis contamination of unpasteurized dairy products.