Isolation and enumeration of aerobic mesophilic Bacillus spp. in milk by heating in the presence of ethanol

Colonies obtained from the current standard test for the enumeration of aerobic spore-forming bacteria (Bacillus) in milk (including a heat treatment of 80 degrees C for 10 min) were examined microscopically and identified as spore-forming or non-spore-forming, It was found that only 0-4% of the colonies obtained by this method were produced by spore-forming bacteria. The remaining colonies were produced by heat-resistant coryneform organisms. Therefore, the use of this test to enumerate spore-forming organisms in milk overestimates the actual numbers of spores present. The optimal growth temperatures of these two types of organisms were investigated. It was found that simply incubating spore-selective plates at 37 degrees C rather than 30 degrees C after a heat treatment of 80 degrees C for 10 min resulted in the growth of a much lower number of non-spore-forming organisms and a slightly higher number of spores. An alternative method for the accurate enumeration of aerobic spore-forming bacteria was developed, in which ethanol was used to kill the vegetative coryneform bacteria. Heating milk which has been diluted in a pre-heated ethanol solution to give a final ethanol concentration of 20%, followed by a heat treatment of 80 degrees C for 10 min, completely eliminates non-spore-forming organisms from the spore-selective test.