Status and Future of the Direct Epifluorescent Filter Technique

Abstract

The direct epifluorescent filter technique (DEFT) was originally developed for counting bacteria in raw milk at the National Institute for Research in Dairying, Reading (Pettipher et al. 1980; Pettipher 1983). The method, which uses membrane filtration and epifluorescence microscopy, takes less than 30 min to complete and does not suffer from many of the disadvantages of other microscopic methods. Pretreatment of the milk with a proteolytic enzyme and surfactant at 50°C lyses somatic cells and makes fat globules sufficiently fluid to enable 2 ml of milk to be filtered routinely through a 25-mm diameter membrane filter. Filtration concentrates and distributes the bacteria in a manner that makes counting easier and the techniques ca. 100 times more sensitive than the Breed smear. The use of a fluorescent stain and an epifluorescence microscope produces wellstained micro-organisms that are easily distinguishable from the small amounts of fluorescent debris. A polycarbonate membrane filter is used, the flat surface of which is better suited to microscopy than the uneven surface of cellulose acetate membranes. Micro-organisms on DEFT slides can be counted automatically, thereby considerably reducing operator fatigue. DEFT can be used rapidly to enumerate micro-organisms in urines, beverages and, with additional sample pretreatment, a variety of foods, in addition to milk.