Measuring Protozoan Biodiversity

Soil protozoa comprise four groups: the "naked" rapidly growing flagellates, amoebae, and ciliates, and the more slowly growing shelled amoebae, or testacea. The small size and flexibility of the first two groups allows them to exploit small pore spaces, and they furnish most of the protozoan numbers.

The more diverse and larger ciliates and testacea inhabit the larger pore spaces which are subject to desiccation and other stresses; consequently, both groups show a wide spectrum of species of r/K selection and degree of autochthonism (Wodarz et al., 1992). Ciliates are divided into pioneer r-selected Colpodida, competitive K-selected Polyhymenophora, and intermediate remaining taxa. Dividing the number of species of the first group by the second produces a C/P ratio, where C/P > 1.00 indicates a stressed soil of low productivity, and C/P < 1.00 a more productive soil with microarthropods and macrofauna (Foissner, 1987; Yeates et al., 1991). Among the testacea, certain species indicate soil acidity or alkalinity, and the shell conveys information about moisture fluctuations (Bonnet, 1964). Consequently, these two groups can serve as bioindicators of soil conditions.

Ideally, biodiversity studies measure both species and numbers per species. However, the small size and transparency of naked protozoa make them too difficult to find among soil particles; consequently, counting has been traditionally performed by the most probable number (MPN) technique of Singh (1946) or its modification by Darbyshire et al. (1974). There are criticisms of the method (Foissner, 1987), in response to which a second direct count method was developed by Griffiths and Ritz (1988) that separates the protozoa from soil particles by percoll phosphate gradient centrifugation and staining for fluorescent microscopy. This method is employed routinely to measure the protozoan component of the soil fauna in experimental field crop studies by the Technical University of Munich. The larger and more motile ciliates can be counted by examining a watered soil suspension drop-by-drop until at least 0.4 g of fresh soil has been examined (Foissner, 1987). The possession of a shell enables direct counting of testacea by this method, or by staining a soil suspension and mounting small samples on slides, providing a permanent record (Couteaux, 1967; Korgonova and Geltser, 1977). Combining the temporary and permanent methods provides a more complete census than either method alone.

Estimating species richness is best done by placing 10 to 50 g of sample in a petri dish and adding water until 5 to 20 ml will drain off when gently pressed with a finger. By placing several coverslips, each underlaid with a piece of lens paper on top of the sample, and examining after 1 day, a variety of flagellate species will be revealed. The culture is examined at 3 to 4 day intervals for a month to determine the succession of species of mainly ciliates and testacea (Foissner, 1987). Most amoebae will be found by streaking soil samples on bacterized water (non-nutrient) agar plates or by placing soil samples in wells cut in such plates. The amoebae migrate out from the soil particles (Bamforth, 1995a).

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