Direct Epifluorescent Technique

A second membrane filter method, known as direct epiflu-orescent microscopy technique (DEFT), made use of fluorescent staining of cells trapped on the surface of a membrane filter followed by examination of the filter using a fluorescent microscope (9). This procedure overcame the limited counting range of standard membrane filtration by eliminating colony development. However, differentiation of viable and dead cells was not fully reliable, and reading the filters was tedious. Introduction of automated DEFT instruments addressed the latter problem (10,11). However, according to the developer of the technique, DEFT could not be applied to highly particulate food suspensions, nor could it be adapted to differential or selective enumerations (12).

Several researchers have improved on the original DEFT procedures by using prefiltration and enzyme digestion steps to reduce particle interference (13,14); adding short enrichment steps to enhance sensitivity (15); incubating the filters for a few hours after filtration to allow development of microcolonies, thus ensuring a true viable cell count and allowing for differential enumeration based on the culture media used (16,17); and using fluorescent antibody labeling in conjunction with DEFT to detect or enumerate specific target populations (18,19).

DEFT methods that do not incorporate enrichment or microcolony development steps offer the advantage of very fast results, with as little as 20 min required between analysis initiation and completion (14). Reliability of DEFT procedures has been evaluated for a variety of foods including raw minced meat (20), raw ham, ground beef and raw fish (21), raw lamb (22), and raw milk (14).

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