Development of a sensitive and rapid method for the measurement of total microbial activity using fluorescein diacetate (FDA) in a range of soils

Fluorescein diacetate (FDA) hydrolysis is widely accepted as an accurate an d simple method for measuring total microbial activity in a range of enviro nmental samples, including soils. Colourless fluorescein diacetate is hydro lysed by both free and membrane bound enzymes, releasing a coloured end pro duct fluorescein which can be measured by spectrophotometry. The current me thod for measuring FDA hydrolysis in soils is limited in its application. F DA activity was very low in sandy and clayey soils. The low activity observ ed for these soil types was made difficult to measure by the original autho rs choice of solvent for terminating the hydrolysis reaction. Acetone (50% v/v) was found to be most efficient at stopping the hydrolysis reaction. Du ring this study acetone (50% v/v) was found to cause a decrease of approxim ately 37% in the absorbance of fluorescein produced by the soil samples mea sured. Although this colour loss is independent of initial fluorescein conc entration, it makes the measurement of FDA hydrolytic activity extremely di fficult in soils with low microbial activity i.e. sandy and/or clayey soils . Chloroform/methanol (2:1 v/v) was found to successfully stop the hydrolys is reaction for up to 50 min in a range of soil samples without causing the loss of colour observed with acetone. By changing the solvent used for ter minating the hydrolysis reaction, low activity soils could be measured succ essfully. Other parameters of the hydrolysis reaction were optimised for th e measurement of soil samples including effect of pH. optimum temperature o f incubation, amount of soil, time of incubation, amount of substrate and p reparation of suitable standards. A new, more sensitive method is proposed adapted from the original method, which provides a more accurate determinat ion of FDA hydrolysis in a wide range of soils. (C) 2001 Elsevier Science L td. All rights reserved.