Fluorescein diacetate (FDA) hydrolysis assays can be used to measure the
enzyme activity of microbes in a sample. A bright yellow-green glow is produced and is strongest when enzymatic activity is greatest. This can be quantified using a
spectrofluorometer A spectrofluorometer is an instrument which takes advantage of fluorescent properties of some compounds in order to provide information regarding their concentration and chemical environment in a sample. A certain excitation wavelength is selected, ...
or a
spectrophotometer.
Applications
FDA hydrolysis is often used to measure activity in soil and compost samples; however, it may not give an accurate reading if microbes with lower activity phases, such as esterases, cleave the
fluorescein
Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used ...
first.
It is also used in combination with
propidium iodide
Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. When in an aqueous solution, PI has a fluo ...
(PI) to
determine viability in eukaryotic cells. Living cells will actively convert the non-fluorescent FDA into the green fluorescent compound fluorescein, a sign of viability; while nucleus of membrane-compromised cells will fluoresce red, a sign of cell death. Currently FDA/PI staining is the standard assessment of human pancreatic islet viability with suitability for transplantation when viability score is above 70%.
Preparation
FDA stock solution is prepared by dissolving 5 mg of fluorescein diacetate in 1 ml
acetone
Acetone (2-propanone or dimethyl ketone), is an organic compound with the formula . It is the simplest and smallest ketone (). It is a colorless, highly volatile and flammable liquid with a characteristic pungent odour.
Acetone is miscib ...
, and
sucrose
Sucrose, a disaccharide, is a sugar composed of glucose and fructose subunits. It is produced naturally in plants and is the main constituent of white sugar. It has the molecular formula .
For human consumption, sucrose is extracted and refined ...
may be added for live cell viability testing. FDA stain must be kept in the dark at 4°C or it will spoil.
References
3. "Fluorescein diacetate hydrolysis assay" http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/FDA%20assay.pdf
United States Department of Agriculture (USDA): Assay for Fluorescein Diacetate Hydrolytic Activity for soil samplesFluorescein Diacetate: A Potential Biological Indicator for Arid Soils
Chemical tests
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