Culture, Microscopic, and Sampling Methods 221 appropriately Following growth, the colonies are enumerated Alternatively, a DMC can be made In this case, the organisms collected on the membrane are viewed and counted microscopically following appropriate staining, washing, and treatment of the membrane to render it transparent These methods are especially suited for samples that contain low numbers of bacteria Although relatively large volumes of water can be passed through a membrane without clogging it, only small samples of dilute homogenates from certain foods can be used for a single membrane The overall efficiency of membrane filter methods for determining microbial numbers by the DMC has been improved by the introduction of fluorescent dyes The use of fluorescent dyes and epifluorescent microscopes to enumerate bacteria in waters has been employed rather widely since the early 1970s Cellulose filters were among the earliest used; however, polycarbonate Nucleopore filters offer the advantage of retaining all bacteria on top of the filter When lake and ocean waters were examined using the two kinds of membranes, counts were twice as high with Nucleopore membranes as with cellulose membranes.52 Direct Epifluorescent Filter Technique This membrane filter technique may be viewed as an improved modification of the basic method The direct epifluorescent filter technique (DEFT) employs fluorescent dyes and fluorescent microscopy,52 and it has been evaluated by a number of investigators as a rapid method for microorganisms in foods Typically, a diluted food homogenate is filtered through a 5-µm nylon filter, and the filtrate is collected and treated with ml of Triton X-100 and 0.5 ml of trypsin The latter reagents are used to lyse somatic cells and to prevent clogging of filters After incubation, the treated filtrate is passed through a 0.6-µm Nucleopore polycarbonate membrane, and the filter is stained with acridine orange After drying, the stained cells are enumerated by epifluorescence microscopy, and the number of cells per gram is calculated by multiplying the average number per field by the microscope factor Results can be obtained in 25–30 minutes, and numbers as low as around 6,000 cfu/g can be obtained from meats and milk products DEFT has been employed on milk97 and found to compare favorably with results obtained by aerobic plate count (APC), and standard Breed DMC on raw milk that contained between × 103 and × 108 bacteria per milliliter It has been adapted to the enumeration of viable Gram-negative and all Gram-positive bacteria in milk in about 10 minutes.108 As few as 5,700 bacteria per milliliter could be detected in heat-treated milk and milk products in about 20 minutes.98 In a collaborative study by six laboratories that compared DEFT and APC, the correlation coefficient was generally above 0.9, but the repeatability of DEFT was 1.5 times worse than APC, and reproducibility was only three times that for APC.96 Solid foods can be examined by DEFT after proper filtrations, and