Membrane filtration method for enumeration and isolation of spores of Bacillus cereus from milk

A method was developed for detection of Bacillus cereus spores in milk. It includes heat treatment at 72 degrees C, followed by treatment with trypsin and Triton-X100 at 55 degrees C, and membrane filtration. Colonies of B. cereus could be recognised by their typical colony morphology and the zone of hemolysis surrounding the colonies following incubation on blood agar with 10 ppm polymyxin. Blood agar was superior to MYP and PEMBA for detection. Psychrotrophic as well as mesophilic strains of B. cereus gave clearly visible micro-colonies after 48 h of incubation at 20 degrees C. At 20 degrees C interference by growth of other hemolytic Bacillus sp, was strongly reduced compared to incubation at 30 degrees C. Heat treatment of the milk at 72 degrees C for 5 min as compared to 75 degrees C for 15 min improved the filterability of the milk and resulted in significantly higher recovery of psychrotrophic spores. Al least 100 mt and up to 250 mt of milk could be filtered. Recovery of spores was typically 85 +/- 13% (SEM) with a mean coefficient of variation for duplicate analyses of 18%. (C) 1998 Elsevier Science Ltd. All rights reserved.