Direct epifluorescence filter technique

The total viable count is an important parameter in industrial fermentations. Traditional methods for total viable count started with the cultivation method of counting the colonies. Later methylene blue and Ponceau S stain came into use for microscopic examinations, which give a direct count (Kunkee Neradt, 1974). Today, systems are available with smart filtering and concentration steps and sensitive fluorescence stains, and a result can be obtained in less than lOmin. This method cannot be applied to highly viscous or particulate materials. 

It is important not to mix up vitality and viability of yeast cells. The vitality is the condition of the physiological capabilities of the cell, while viability describes if a cell is alive or dead. The viability is reported as a percentage of live cells (so live and dead cells are counted), whereas the vitality gives the status of the metabolic function (Report of Subcommittee, 2003 Van Zandycke, Simal, Gualdoni, Smart, 2003). 

1 Principle of the method (fluorescence stains, differentiation of live and dead cells) 

Damaged cells Primuline 340 nm X 425 nm Primuline is used for visualization of permeabiUzed or damaged cells. It binds noncovalently to lipid structures. 

Viable damaged cells Acridine orange solution A9231 Sigma Ag SOOnm ABm526nrn (bound to DNA)Uex 460nm Aem 650 nm (bound to RNA) 

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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. 

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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