Evaluation of a direct epifluorescent filter technique (DEFT) for the enumeration of bacterial spores in raw milk

Abstract

Abstract A modified direct epifluorescent filter technique (DEFT), incorporating heat activation followed by a germination stage with l -alanine, was evaluated for the enumeration of Bacillus spores with differing germination rates. A Good correlation ( r = 0·98) was found between the pour plate spore count and the combined number of orange and green fluorescing cells determined using the DEFT for all three spore types. A good correlation ( r = 0·98) was also found between the number of green fluorescing cells, in the DEFT, and the number of viable, but ungerminated spores (all three spore types), determined by a plate count method. Although there is no direct experimental evidence, this implies that the number of orange fluorescing cells, in the DEFT, could be used as an estimate of the number of germinated spores. The work reported here indicates that DEFT can be used to determine rapidly the number of germinated, ungerminated and total number of Bacillus spores in raw milk. However, further research is required to improve its sensitivity (approximately 10 4 spores/ml) before it can have widespread application.