Spectrophotometric determination of the hydrolysis of fluorescein diacetate (FDA) was shown to be a simple, sensitive, and rapid method for determining microbial activity in soil and litter. FDA hydrolysis was studied in soil and straw incubated for up to 3 h. Hydrolysis was found to increase linearly with soil addition. FDA hydrolysis by pure cultures of Fusarium culmorum increased linearly with mycelium addition both in shake cultures and after inoculation into sterile soil. FDA hydrolysis by Pseudomonas denitrificans increased linearly with biomass addition. The FDA hydrolytic activities in soil samples from different layers of an agricultural soil were correlated with respiration. Acetone was found to be suitable for terminating the reaction.

Fluorescein diacetate hydrolysis as a measure of total microbial activity in soil and litter.

With the increasing pressure to produce more food, fiber, and fuel to meet world demands on a limited land area, there is an unprecedented need to address global concerns about soil degradation. Understanding the underlying biological processes in tandem with identification of early warning indicators of ecosystem stress is needed to provide strategies and approaches for land resource managers and policymakers to promote long-term ecosystem sustainability. Biological and biochemically mediated processes in soils are fundamental to terrestrial ecosystem function. Ultimately, all members of the food web are dependent on the soil as a source of nutrients, and for degradation and cycling of complex organic compounds. Primary decomposers of organic matter provide energy that supports the activities of organisms from a number of trophic levels in soils. Historically, chemical and physical properties have been used as crude measures of soil productivity. Most notably, determination of soil organic matter has been related to general soil tilth. Soil organic matter changes very slowly, and therefore, many years may be required to measure changes resulting from perturbations. However, there is growing evidence that soil biological parameters may hold potential as early and sensitive indicators of soil ecological stress or restoration (Dick, 1992; Dick & Tabatabai, 1992). In Chapter 5 (this book) information is provided on soil quality in relation to soil microorganisms. In this chapter, soil enzyme activities will be discussed as a potential biochemical/biological indicator of soil quality.

Soil Enzyme Activities as Indicators of Soil Quality

With the increased interest in integrated soil bioecosystem studies, there is a need to have a method of measuring overall microbial activity potential. Hydrolysis of fluorescein diacetate [3',6'-diacetylfluorescein (FDA)] has been suggested as a possible method because the ubiquitous lipase, protease, and esterase enzymes are involved in the hydrolysis of FDA. Following hydrolysis of FDA, fluorescein is released and can be measured spectrophotometrically. Our objective was to optimize the assay for FDA hydrolytic activity in soil samples and determine the kinetic parameters involved in this reaction. The optimized method involves extraction and quantitative measurement of the fluorescein released when 1.0 g of soil is incubated with 50 ml of 60 mM Na-phosphate solution (buffered at pH 7.6) at 37 °C for 3 h. Results showed that FDA hydrolysis was optimum at buffer pH 7.6 and the soil enzymes were denatured at temperatures above 60 °C. Three soils were used to optimize this method: Heiden clay, Raub silt loam, and Cecil sandy loam. This procedure is simple, precise, and can be used in commercial soil testing laboratories to determine general microbial activity and as a soil quality indicator.

Assay for fluorescein diacetate hydrolytic activity: Optimization for soil samples

A new microdilution method has been developed for determining the minimum inhibitory concentration (MIC) of oil-based compounds. The redox dye resazurin was used to determine the MIC of a sample of the essential oil of Melaleuca alternifolia (tea tree) for a range of Gram-positive and -negative bacteria. Use of 0.15% (w/v) agar as a stabilizer overcame the problem of adequate contact between the oil and the test bacteria and obviated the need to employ a chemical emulsifier. A rapid version of the assay was also developed for use as a screening method. A comparison of visual and photometric reading of the microtitre plates showed that results could be assessed without instrumentation; moreover, if the rapid assay format was used, rigorous asepsis was not necessary. Accuracy of the resazurin method was confirmed by plate counting from microwells and MIC values were compared with results obtained using an agar dilution assay. The MIC results obtained by the resazurin method were slightly lower than those obtained by agar dilution.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2672.1998.00379.x

A new method for determining the minimum inhibitory concentration of essential oils

Organic matter in aquatic environments consists mostly of large compounds that cannot be taken up and utilized directly by microbial cells Prior to incorporation, polymeric materials undergo degradation by cell-bound and extracellular enzymes produced by these microbes; in fact, such enzymatic mobilization and transformation is the key process which regulates the turnover of organic as well as inorganic compounds in aquatic environments This volume brings together studies on enzymatic degradation processes from disciplines as diverse as water and sediment research, bacterial and algal aquatic ecophysiology, eutrophication, and nutrient cycling and biogeochemistry, in both freshwater and marine ecosystems Its scope extends from fundamental research exploring the contribution of microbial enzymatic processes to whole ecosystem functioning to practical applications in water biotechnology The first comprehensive publication providing an overview of this emerging field of enzymology, "Microbial Enzymes in Aquatic Environments" will be of interest both to ecologists and microbiologists This monograph on industrial application, biotechnology, environmental science, microbial ecology and enzymes is intended for researchers and students of microbial ecology, aquatic ecology, environmental chemistry and biogeochemistry

Microbial enzymes in aquatic environments

Fluorescein diacetate (FDA) was applied as a vital stain to soil bacteria in pure cultures, sterilized soil, and natural soils. The staining ability of FDA was tested on 111 isolates of soil bacteria in pure cultures in a diluted laboratory medium and in sterilized soil. Nearly 80% of the isolates were stained. In a growth experiment in sterilized soil, FDA was compared to acridine orange (AO). At a given time, when most bacteria were considered metabolically active, the number of FDA-stained bacteria exceeded that stained by AO. The staining efficiency and the simple procedure indicate that the FDA-method is useful for estimating the number of metabolically active bacteria in soil. This was confirmed by examination of ten different soils, where the FDA-method was compared to AO-staining, fluorescein isothiocyanate (FITC)-staining, and plate counts.

Fluorescein diacetate as a stain of metabolically active bacteria in soil [garden soil, sandy soil, sand dune soil, mull, soil from Pinus, Picea and Fagus forest, fluorescence technique, acridine orange staining, fluorescein isothiocyanate staining, plate counts]

B. Lundgren

Papers

Fluorescein diacetate as a stain of metabolically active bacteria in soil [garden soil, sandy soil, sand dune soil, mull, soil from Pinus, Picea and Fagus forest, fluorescence technique, acridine orange staining, fluorescein isothiocyanate staining, plate counts]