Fluorescein+diacetate+(FDA)+hydrolysis

=Purpose and Description= ====Fluorescein diacetate (FDA) hydrolysis is a technique that is used to measure microbial enzyme activity in a sample, as well as to reveal how much bacteria is present in the overall sample. It is considered to be non-specific because it is sensitive to the activity of several enzyme classes including lipases, esterases, and proteases. This process is usually used in measuring soil microbial activity. The enzymes present in the sample catalyze the hydrolysis of the FDA. After the FDA has been hydrolyzed, fluorescein is released and its intensity can then be measured with a spectroflourometer (Green, 2005). This leads to the hydrolytic cleavage of colorless FDA to produce a bright yellow glow when enzyme activity is greatest. This process can also be used in conjunction with propidium iodine (PI) to ascertain the viability in eukaryotic cells through the process of fluorescent microscopy. Living cells convert the non-fluorescent FDA into fluorescein which is green in color, which confirms viability, while a red color signifies cell death of the nucleus membrane compromised cells. This method is used commonly in the assessment of human pancreatic islet viability to determine suitability for transplant. FDA solution may be prepared by dissolving 5mg of fluorescein diacetate in 1ml acetone and sucrose. This solution has to be kept in the dark to prevent it from losing its ability. This process is simple and cheap and is suggested as a method as opposed to more expensive alternatives.====

=History= ====Even with extensive research, the origin and history of this process could not be found. However, in my readings I found articles dating back to 1982 that utilized the process so this indicates that it has been a part of scientific procedure for quite some time.====

=Recent Research= ====Kumar et al in their paper used FDA hydrolysis to determine the microbial activity in a range of soil samples and types. They required the process to determine the hydrolase activity in natural soils as opposed to degraded soils. They found that the activities were more predominant in natural soils and these were dependent on soil pH, effect of incubation time, termination reaction, substrate concentration and temperature.==== ====In the second article (Sanchez-Mondero, 2008), use the process to determine microbial activity of soil that had been amended with organic components and measured and compared by biomass and CO2 evolution.==== ====Finally, in Li et al’s research on the “Applicability of the fluorescein diacetate assay for metabolic activity measurement of Microcystic aeruginosa (Chroococcales, Cyanobacteria)”, the FDA assay was used in the study of cyanobacterial Microcystic aeruginosa Kütz cells under stress conditions which included nitrogen or phosphorus deficiency, as well as darkness and low temperature. This was studied by monitoring the change in FDA fluorescence values. The results revealed that the esterase activity decreased in dark-stressed cells while the cells lost photosynthetic activity, but had the highest esterase activity in the other three conditions (especially at low temperature). However the findings contrasted with assay criteria which require that the expression of a stain should correlate with the change of photosynthetic activity, and also that stressed cells have lower staining intensity. Their results thus showed that the esterase activity response was dependent on environmental factors. And finally, that higher fluorescence intensity did not indicate higher metabolic activity, but a value which indicated severe stress. ====

=References= > ====3. Li, J., Ou, D., Zheng, L., Gan, N. and Song, L. (2011), Applicability of the fluorescein diacetate assay for metabolic activity measurement of Microcystis aeruginosa (Chroococcales, Cyanobacteria). Phycological Research, 59: 200–207. ====
 * 1) ====Green, V.S., Stott, D.E., Diack, M. 2006. Assay for fluorescein diacetate hydrolytic activity: optimization for soil samples. Journal of Soil Biology and Biochemistry. 38:693-701.====
 * 2) ====Sánchez-Monedero M.A., Mondini C., Cayuela M.L., Roig A., Contin M., De Nobili M. Fluorescein diacetate hydrolysis, respiration and microbial biomass in freshly amended soils. Biology and Fertility of Soils 04/2012; 44(6):885-890.====