There is an extensive literature on approaches to extracting and counting protozoa. For much of the 20th century, researchers have been heavily influenced by the papers of Cutler (1920, 1923), Cutler et al. (1923), and Singh (1946). These authors favored the culture technique, in which small quantities of soil or soil suspensions from dilution series are incubated in small wells that are inoculated with a single species of bacteria as a food source. Based on presence or absence in each well, one can calculate the overall population density ("most probable number"). Other scientists, notably Couteaux (1972) and Foissner (1987a), espouse the direct count approach, in which one examines soil samples, in water, to see what organisms are present in the subsample. The advantages of this approach are that it is possible to observe the organisms, which are immediately present, and not have to rely on the palata-bility of the bacterium used to inoculate the series of wells in the culture technique. The disadvantage of the direct count method, as noted by Foissner (1987b), is that one usually employs only 5-30 milligrams (mg) of soil, so as not to be overwhelmed with total numbers. Unfortunately, this discriminates against some of the more rare forms of testaceans or ciliates, which occur only infrequently, but may have a significant impact, if they happen to be very large. Given rather limited research budgets, it is seldom possible to employ a small army of staff to scan literally hundreds of slides of soil from a single sample site.
An additional complication is the fact that the culture technique attempts to differentiate between active (trophozoite forms) and inactive (cystic) forms by the treatment of replicate samples with 2% hydrochloric acid overnight. The acid kills off the trophic forms, and then, after washing in dilute NaCl, the counting continues. This assumes that all of the cysts will excyst after this drastic process; sometimes the assumption is met, but not always.
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