Measurement of bacterial production by 3H-thymidine incorporation
During the fast ten years, numerous methods have been proposed in the literature to mesure the activity of heterotrophic bacteria in aquatic ecosystems. Among these methods, the measurement of tritiated thymidine incorporation proposed by FUHRMAN and AZAM (1980) seems to be, at the present time, the most useful. It offers in fact the advantage of its specificity for bacteria and its simple experimental procedure. This method is based on the fact that, in bacteria, DNA synthesis is directly proportional to the division rate. The close relation between growth and DNA synthesis means that measurement of the rate of DNA synthesis is a good way to measure the bacterial growth rate. The DNA synthesis rate is estimated from the incorporation rate of methyl-3H thymidine. Thymidine is one of the four nucleoside precursors of DNA, but it is not a precursor of RNA. At the nanomolar concentrations of (methyl-3H) thymidine used in this experiment only heterotrophic bacteria utilize exogenous thymidine and all active heterotrophic bacteria utilize thymidine. The usual experimental procedure used by the various authors working with this method is that of the FUHRMAN and AZAM (1982). A 5 nM thymidine concentration has been recommended by these authors for the marine environment, but it has been shown that in more eutrophic ecosystems higher concentrations are needed to saturate the incorporation process. In fact, the conversion of experimental data into bacterial production raises some problems. Among these, two questions are important : - Which is the relative part of exogenous and endogenous thymidine used for DNA synthesis? In other words, which is the isotopic dilution factor? - The cold trichloroacetic acid (TCA) fraction collected in this experiment includes, besides DNA, proteins and RNA. As some catabolic products of thymidine could be incorporated into RNA or proteins, how mach radioactivity is really incorporated into DNA? Some theoretical and experimental answers can be given to these questions. - Comparing their results of thymidine and H32PO4= incorporation in DNA of marine bacteria, FUHRMAN and AZAM (1982) found an isotopic dilution factor in the 3-7 range. MORIARTY and POLLARD (1981) have proposed a kinetic approach for estimating the internal pool of thymidine but the accuracy of this method was critized by RIEMANN et al., (1982) and FUHRMAN and AZAM (1982). - Usually, in order to determine the part of radioactivity incorporated into DNA, RNA and proteins, the biochemical procedure of LURIA (1960) is used. SERVAIS et al., (1987), after testing this procedure with labelled macromolecules, have concluded that this method is not accurate for such determinations. These authors have proposed an enzymatic procedure based on the use of DNase tell breakage. With this method, they have showed important fluctuations in the percentage of thymidine incorporated into DNA from one ecosystem to another. The first authors using thymidine incorporation calculated conversion factors based on theoretical data and various assumptions to convert thymidine incorporation data into bacterial cell production. These factors were in the range of 0.2 to 4 1018 bacteria produced per mole of thymidine incorporated in the cold TCA insoluble material. More recently, because of the uncertainty or the difficulty in estimating some of the parameters (isotopic dilution, part of thymidine incorporated into DNA, part of thymidine residues in DNA, quantity of DNA per bacterial cells) required for the calculation of the conversion factor, most of the authors have used an experimental conversion factor. It was estimated from the comparison of cell number increase and thymidine incorporation in sterilized, and reinoculated samples. These experimental conversion factors were usually in the range of 0.5 to 10 1018 bacteria produced per mole of thymidine incorporated. The conversion of cells production into bacterial biomass production expressed in carbon - which is finally the important flux to know for the study of the first trophic levels dynamic - requires a knowledge of the average bacterial biovolume and the carbon content par unit of cell volume. The first parameters can be estimated from the observation of bacteria by epifluorescence microscopy. Most authors use 1.2 10-13 gC.μm-3 to calculate carbon content of bacteria proposed by WATSON et al., (1977). The use of the tritiated thymidine incorporation method is not limited to measure bacterial production in the water column; it is also used to measure the activity of fixed bacteria, to study the grazing of bacteria by microzooplankton and in ecotoxicological studies.