Direct Measures of Numbers and Biomass

Total counts of microbes are made by preparation of soil (about 10 milligrams [mg]) suspensions spread in thin agar films on microscope slides (Jones and Mollison, 1948). The films are then stained, often with fluorescent dyes, and scanned. More recently, there have been improvements to the direct count technique such as the membrane filtration technique, which enables one to quickly count fungal hyphae against the filter or a stained background. This approach is generally much faster and easier than the more laborious agar film technique. Other more classical techniques such as viable counts on nutrient-containing agar media are discussed by Parkinson et al. (1971) and Parkinson and Coleman (1991). The viable culture techniques usually recover only 1% or less of the total viable cells, so they are useful only for comparative purposes when one is focusing on a few readily culturable species ofbacteria.

Other direct measures include sampling for extractable DNA (Torsvik et al., 1990a, 1990b; Torsvik et al., 1994; Torsvik and 0vreas, 2002), and using the PCR to multiply specific genes (such as the 16S rRNA) to determine the identities of the organisms of interest. Another approach to microbial community analysis uses signature lipid bio-

markers (SLB). This technique, pioneered by Dr. David White and colleagues at the University of Tennessee (Tunlid and White, 1992), measures ester-linked polar lipid fatty acids and steroids to determine microbial biomass and community structure. Further comments on these techniques are given in Paul and Clark (1989, 1996). For microbial community characterizations, various biomarkers are used. Prominent among these are the PLFAs. Phospholipids are found in the membranes of all living cells but not in the storage products of microorganisms. They may be extracted and characterized using GC/MS (gas chromatogra-phy/mass spectrometry) (Zelles and Alef, 1995).

A variety of techniques has been developed for the isolation and identification of DNA from soil. Techniques include the cell extraction method and the direct lysis method (Saano and Lindstrom, 1995, Zhou et al., 1996). In recent studies of microbial community makeup in agroe-cosystem field soils, Furlong et al. (2002) compared the microbial community composition of earthworm- and non-earthworm-influenced soils at the Horseshoe Bend field site in Athens, GA. The objective was to compare microbial communities from worm casts and open soil; this was done by creating clone "libraries" of the 16S rRNA genes, which were prepared from DNA isolated directly from the soil and earthworm casts. In the cast soils, representatives of the genus Pseudomonas, as well as the Actinobacteria and Firmicutes, increased in number (Furlong et al., 2002). The results were consistent with a model where a large portion of the microbial population in soils passed through the gastrointestinal tract of the earthworm unchanged while representatives of some bacterial phyla increased in abundance. In Chapter 4, we will consider the various faunal groups in soil, and their life-history attributes that have impact on microbial community makeup and turnover.

As noted in the introduction to prokaryotes above, we are only now becoming aware of the phylogenetic richness of archaea in soil communities. PCR amplification using primers specific for archaeal 16S rRNA genes allows detection of archaea in diverse habitats (Bomberg et al., 2003). The abundance of crenarchaeal (one of the two kingdoms comprising the archaeal domain) 16S rRNA in both cultivated and native field soils has been estimated to be from 1 to 2% of the total 16S rRNAin these soils (Buckley et al., 1998).

The above procedures are primarily used in determining bacterial community structure. For fungi, several recent studies have made use of the fact that ergosterols are specific to fungi, and the amounts of ergos-terols can be quantified to determine the amount of fungal tissues (biomass) present in soils (Newell and Fallon, 1991; Eash et al., 1994, Zelles and Alef, 1995). Molecular phylogeny has been an equally powerful tool for describing fungal communities (see, for example, Husband et al., 2002).

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