Fluorescein diacetate assay, determination

EPTC and Butvlate Fluorescein Diacetate Assay. Spectrophotometric determinations of the hydrolysis of fluorescein diacetate have been shown to be simple, rapid, and sensitive methods for determining microbial activity in soil (18). Essentially, the hydrolytic cleavage of diacetate from fluorescein is responsible for the reaction products including fluorescein, which may be detected spectrophotometrically at 490 nm. This method is somewhat nonspecific in that it is indicative of overall activity of several enzymes (protease, lipase, esterase) rather than of a specific class of enzymes. Enzyme activity may be influenced by subtle pH changes in the sample since abiotic hydrolysis of fluorescein diacetate may occur. Also, an associated lag phase in soil hydrolytic activity must be accounted for in each assay. 

Determined on the basis of loss of cell membrane permeability in response to a deliberate modification in culture conditions. Cell viability can be determined by either neutral red or fluorescein diacetate retention assays. Volume 1(14,15). 

A wide range of bacteria and fungi, and some protozoan, algal and mammalian cells have the ability to hydrolyse fluorescein diacetate. Recently Schnurer and Rosswall have used this substrate to develop an assay for esterase activity in soil and straw based on the spectrophotometric determination of extracted fluorescein. The amounts formed increased linearly with time (up to 3h). The rates of hydrolysis were directly proportional to the amounts of soil used, to the amounts of fungal mycelium (Fusarium culmorum) or bacterial biomass (Pseudomonas denitrifleans) in buffered (pH 7.6) pure culture suspensions, and, in the case of fungi, to the amounts added to autoclaved soil, which alone was inactive. Esterase activities correlated with respiratory (O2 uptake) activities for samples from different depths in the soil profile. 

Chrost, R. J. Gajewski, A. Siuda, W. Fluorescein-diacetate (FDA) assay for determining microbial esterase activity in lake water. Adv. Limnol. 1999, 54, 167-183. 

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