Epifluorescent filter

Viability-based technologies Direct epifluorescent filter microscopy Membrane laser scanning Fluorescence cytometry Fluorescence flow cytometry... 

The DNA in the sections is denatured by treatment with 70% formamide/2 x SCC for 5 min at 80°C. Ten microliters of the probe solution (hybridization buffer 7 pd, probe 1 pi, and distilled water 2 pi) is placed on the slide and coverslipped. The slide is placed in a microwave oven (2.45 GHz, 300 W) and heated for 3 sec at 2-sec intervals for a total of 15 min at 42°C. DAPI II (4,6-diamidine-2-phenylindol) (125 ng/ml) is used for nuclear staining. The sections are promptly observed under a fluorescent microscope equipped with epifluorescence filters and a photometric CCD camera. The captured images are digitized and stored in an image analysis program. 

Direct counting can be improved by the use of fluorescent dyes, such as acridine, especially if combined with the recovery of cells by membrane filtration. Direct epifluorescent filter techniques (DEFT) are used in the milk and dairy industries to estimate both bacteria and fungi. They can produce results in less than 25 minutes which correlate closely with traditional methods. Further developments have automated the counting procedure by the use of image analysers thus removing the problem of operator fatigue. 

Novato, CA 94949, USA PHONE (415) 883-0128 FAX (415) 883-0572 WEB SITE http //www.sutter.com [ Digitally-controlled epifluorescence filter wheels]... 

With the need to prevent contaminated milk from being consumed or processed, rapid tests have been developed. The direct epifluorescent filter technique is an accepted ISO test, used routinely by some laboratories. A fluorescent marker dye is attached chemically to the nucleic acid of living and dead cells. Marked microorganisms that are viable fluoresce orange under UV light and are counted automatically by an image analyzer through a microscope. 

Tortorello, M. L., Stewart, D. S. (1994). Antibody-direct epifluorescent filter technique for rapid, direct enumeration of Escherichia coli 0157 H7 in beef. Applied Environmental Microbiology, 60(10) 3553-3559. PMCID PMC201854. 

Splittstoesser (1992) described a method using fluorescent dye (acridine orange) known as direct epifluorescence filter technique (DEFT), a method also applied by Divol and Lonvaud-Funel (2005). Divol and Lonvaud-Funel (2005) used a different substrate, fluoresceine diacetate, which is hydrolyzed by viable cells to form a fluorescent product, fluoresceine. However, Atlas and Bartha (1981) observed that cell population values can differ substantially between (epifluorescence and direct plating) methods (10 vs. 10 CFU), possibly due to the presence of viable-but-non-culturable cells. In addition, Meidell (1987) reported interference... 

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