Presentation on theme: "FDA hydrolysis activity test Made by: Zách Attila."— Presentation transcript:
FDA hydrolysis activity test Made by: Zách Attila
Introduction The FDA is the short form of fluorescein-diacetate (3’-6’ diacetyl-fluorescein). Many enzymes (proteases, lipases, esterases) are able to hydrolyze FDA. The product is fluorescein-diacetate. FDA is used for determine total microbial activity. This is a good general measure of organic matter turnover in natural habitats (reason: more than 90% of the energy flow passes through microbial decomposers).
A brief description of the method Determination of total microbial activity. Active fungi, bacteria quantitative determination, locate acetyl-esterase in living protist cells and examine the enzymatic activity of animal cells. Previous studies revealed that all fungi, most bacteria, protozoa and algae show some FDA hydrolytic activity. Rapid, sensitive and simple method. The result of fluorescein produced by microbial activity, and this is measured by fluorescence microscopy, fluorimetric, or by spectrophotometry.
Some examples for test materials FDA-hydrolysis activity of various samples could be examined. Soil: –the sample is taken from agricultural land, different various layers: topsoil sandy loam clay layer –Soil samples are sterilized by autoclaving at 120°C for 20 min on 2 consecutive days.
Straw: –barley straw –cut into 2 cm pieces, store at room temperature –Autoclave at 110 °C for 30 minutes Pure microorganism cultures, examples: –Pseudomonas denitrificans – Fusarium culmorum
Determination of FDA hydrolysis Dissolve FDA in acetone and store as a stock solution (2 mg/ml) at -20°C. Acetone is used for : –ensure the end of the enzymatic reaction –produce a clear solution with a low background absorbance –It helps to dissolve the cell-membranes –It is difficult to remove colloids from the supernatants of clay samples without acetone The hydrolyzed FDA is measured at absorbance of 490 nm, by a Beckman spectrophotometer If the samples’ absorbance is more than 0.8, these samples must be attenuated before the measurement Add 10 µg/ml FDA and 60mM sodium phosphate buffer to the samples, so the pH is set to 7.6, and the resulting mixture is incubated at 24 °C on a rotary shaker
In the experiments with straw, A490 values can be determined directly in buffer samples taken from the incubation flasks In the experiments of pure cultures: –P. denitrificans: cells are removed from the incubation solution by centrifugation for 5 min at 6,000 rpm, and the supernatant is filtered through Munktell no. 3 filter paper, after this procedure we can measure the absorbance –F. culmorum: the mycelium is removed by centrifugation at 6,000 rpm for 5 min followed by filtration of the supernatant through a 3 µm membrane filter, after this we can measure the absorbance Soil is removed from the incubation solution by centrifugation for 5 min at 6,000 rpm followed by filtration through Munktell no. 3 filter paper.
Evaluation Soil: –FDA hydrolysis activity increases linearly with time and the amount of soil –Autoclaved soils does not show any FDA activity –The sand and the clay samples show no FDA activity Straw: –Autoclaved sample shows less activity Pure cultures: –P. denitrificans: the concentration-absorbance relationship is linear –F. culmurum: the concentration-absorbance relationship is linear
Hydrolysis of FDA by different amounts of soil Source: Johan Schnürer and Thomas Rosswall, (1982 June), Fluorescein Diacetate Hydrolysis as a Measure of Total Microbial Activity in Soil and Litter, Applied and Environmental Microbiology, Vol 43(6), 1258
Hydrolysis of FDA by soil samples over time Source: Johan Schnürer and Thomas Rosswall, (1982 June), Fluorescein Diacetate Hydrolysis as a Measure of Total Microbial Activity in Soil and Litter, Applied and Environmental Microbiology, Vol 43(6), 1258
Conditions of this method Proper pH pH=7,6 by sodium-phosphate buffer the reason of the pH-sensitivity is that on low and high pH there are no FDA hydrolytic activity Proper temperature samples are incubated at 24 °C on a rotary shaker Respiration (O 2 -sensitivity) There is a relationship between O 2 and the FDA hydrolytic activity, the best activity of soil samples is in the topsoil, the activity decreases as the depth increases. respiratory rate, as the consumption of O 2 was defined at 15 °C, by Gilson respirometer
Disadvantages More suitable for determining the heterotrophic activity, as determine biomass quantification. –reason: the active biomass is not directly proportional to the measured FDA hydrolysis activity using the method described above –FDA microscope can just make difference between active and inactive biomass Proper and accurate preparation is required: –to avoid ATP hydrolysis –appropriate incubation, pH However, this is the only method for determining the total microbial activity, and it has the greatest promises.
Sources Schnürer, J. and Rosswall, T. (1982), Fluorescein Diacetate Hydrolysis as a Measure of Total Microbial Activity in Soil and Litter, Applied and Environmental Microbiology, Vol 43(6), p.1256-1261