Culturebased Studies

Culturing a soil organism involves transferring its propagules to a nutrient medium conducive to its growth. Culture-based techniques allow specific soil microorganisms to be isolated from a wide range of soils. Culturing techniques are selective and designed to detect microorganisms with particular growth forms or biochemical capabilities. Bacterial cells from surfaces of soil particles and aggregates are liberated in sterile water, Windogradsky's solution, or physiological saline (0.85% NaCl). After extraction soil suspensions are diluted to an appropriate concentration. The degree of dilution required is related to the initial number of organisms (or propagules) in the soil. Serial dilutions, usually 1:10, stepwise, are made, beginning with a known weight of wet soil. Aggregates are broken by brief mixing in a Waring blender, often in the presence of a dispersing agent such as Na4P2O7, prior to performing serial dilutions. Replicate 0.1-ml portions of appropriate suspensions are transferred to solid or liquid medium in which individual propagules develop into visible growth. After appropriate incubation, single colonies on solid media are counted, and each colony is equated with a single propagule in the soil suspension (colony-forming unit).

The plate count of bacteria in soil, water, and sediment usually represents 1 to 5% of the number determined by direct microscopy, leading to many discussions concerning viable but nonculturable bacteria in nature (Kjelleberg, 1993) and the usefulness of this method for quantitative soil microbiology. Plate count methods are not suitable for enumerating fungal populations or densities because both individual spores and fragments of hyphae develop into colonies that are counted. Due to these strong limitations, many researchers doubt whether plate counts can be used for quantification of soil microbiota.

Population densities of various groups of bacteria can be estimated by the most probable number (MPN) technique. This method uses a liquid medium to support growth of soil microorganisms. The resulting dilution count is based on determining the highest soil dilution that will still provide visible growth in a suitable medium. An actual count of single cells or colonies is not necessary. Inoculating replicate tubes (10 usually, 5 minimum) from each of three successive serial dilutions at the estimated extinction boundary for growth enables resultant visual growth to be converted to numbers within statistical limits of reliability (Gerhardt et al., 1994). Computer software has been developed to determine MPN and related confidence limits as well as to correct for biases (Klee, 1993). MPN methodology is useful because it allows a bacterial population to be estimated based on process-related attributes such as nitrification by Nitrosomonas and Nitrobacter spp., denitrification by deni-trifiers, or nitrogen fixation by free-living aerobic and microaerophilic N2-fixing bacteria. The MPN technique requires an appropriate choice of growth medium and accurate serial dilutions to obtain quantitative data. In general, the results are usually less precise than those obtained with direct plating methods and suffer from similar biases due to the ability of cells to grow in artificial media.

Worm Farming

Worm Farming

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