A method is described for the rapid and objective estimation of the amount of carbon in the living, non-resting microbial biomass of soils. The method, which is based on the initial respiratory response of microbial populations to amendment with an excess of a carbon and energy source, was quantified using an expanded version of Jenkinson's technique.

The simultaneous application of the two methods to 50 soil samples showed a highly significant correlation (r = 0.96) between both. From this correlation it could be deduced that at 22°C, a substrate-induced maximal respiratory rate of 1 ml CO2· h−1 corresponds to c. 40 mg microbial biomass C. Evidence supporting these results was obtained from pure culture studies. The various soil types investigated were collected from agricultural as well as forest sites and they contained between 15 and 240 mg microbial C·100g dry soil−1. The respiratory method provides reproducible estimates of biomass size within 1–3 h after soil amendment. It can be combined without difficulty with a selective inhibition method for determination of bacterial and fungal contributions to soil metabolism.

A physiological method for the quantitative measurement of microbial biomass in soils

http://www.fsl.orst.edu/ltep/Biscuit/Biscuit_files/Refs/Baldock%20OG2002%20fire.pdf

Chemical composition and bioavailability of thermally altered Pinus resinosa (Red pine) wood

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 x 10 cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1-mug DNA sample.

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DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community

Experiments were performed to evaluate the effectiveness of two different methodological approaches for recovering DNA from soil and sediment bacterial communities: cell extraction followed by lysis and DNA recovery (cell extraction method) versus direct cell lysis and alkaline extraction to recover DNA (direct lysis method). Efficiency of DNA recovery by each method was determined by spectrophotometric absorbance and using a tritiated thymidine tracer. With both procedures, the use of polyvinylpolypyrrolidone was important for the removal of humic compounds to improve the purity of the recovered DNA; without extensive purification, various restriction enzymes failed to cut added target DNA. Milligram quantities of high-purity DNA were recovered from 100-g samples of both soils and sediments by the direct lysis method, which was a greater than 1-order-of-magnitude-higher yield than by the cell extraction method. The ratio of labeled thymidine to total DNA, however, was higher in the DNA recovered by the cell extraction method. than by the direct lysis method, suggesting that the DNA recovered by the cell extraction method came primarily from active bacterial cells, whereas that recovered by the direct lysis method may have contained DNA from other sources.

Recovery of DNA from soils and sediments.

Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA.

A simplified procedure is presented for releasing and concentrating indigenous microbial cells from soil for viewing by transmission electron microscopy as thin sections or replicas of frozen-etched preparations. This procedure is compared with two others reported earlier, and their relative merits are discussed as concerns the choice of procedure for the cellular information desired from the soil. Freeze-etching showed that the cell types and size distributions for cells which have been released and concentrated from soil are in general agreement with those for cells in a crude soil slurry in which no attempt to release and concentrate cells was made. Microcolonies were present both in the crude slurry and in the discard soil debris centrifugation pellets from the cell release and concentration procedures. In contrast to the historic assumptions, these microcolonies, as well as some individual cells embedded in soil debris could not be broken up and (or) dislodged so that they would be washed from the soil. The relative numbers of these cells remaining with the soil debris, however, could not be quantitated in the present study.

Simplified procedures for releasing and concentrating microorganisms from soil for transmission electron microscopy viewing as thin-sectioned and frozen-etched preparations.

Soil was sterilized by various procedures, and then the resident microorganisms were physically separated and concentrated from the soil for viewing by transmission electron microscopy as thin sections and frozen-etched preparations. Remaining cell viability in the soil was tested by conventional plating before and after enrichment culture. The soil proved to be sterile after treatment with 60Co radiation, prolonged autoclaving, prolonged dry heat application at 200C, or glutaraldehyde (if followed by subsequent mild heating), and could be considered sterile after OsO4 treatment. Treatment with glutaraldehyde alone, or 160C dry heat for 3 h, did not sterilize the soil. Cellular fine structure was altered or destroyed by the heat treatments, but was not affected to any extent by any of the other treatments including glutaraldehyde followed by mild heating. These findings are considered in relation to the residual biological information observable by electron microscopy in soil samples which have been steri...

D. P. Labeda

Papers

Simplified procedures for releasing and concentrating microorganisms from soil for transmission electron microscopy viewing as thin-sectioned and frozen-etched preparations.

Soil sterilization effects on in situ indigenous microbial cells in soil.