From the beginning great difficulty has been encountered in finding means for counting protozoa; and most of the early results have been obtained by the use of one of the following methods: (1) direct counts in a known volume of soil suspension by means of a microscope; (2) dilution method as used for counting bacteria, and suggested by Rahn, who made dilutions of the soil and determined, by examination at periodic intervals, the one above which protozoa did not grow; (3) Agar plating as used by Killer; (4) counting per standard loop of suspension as devised by Müller. Of these the two last have been little used, and for various reasons are now discarded by most workers. Direct methods have been used extensively in the United States by Koch[13] and others,[16] who claim to have got satisfactory results; they are, however, highly inaccurate and should be discontinued. The present writer[3] has shown that there exists a surface energy relationship between the soil particles and the protozoa, so that the two are always in intimate contact; thus rendering it impossible to count under the microscope the number of organisms in a given weight of soil suspension ([Fig. 9]). Further, in a clay soil, such as is found at Rothamsted, the clay particles alone make it very difficult to use such methods.
The demonstration of this surface energy relationship affords an effective rejoinder to the criticism made against Russell and Hutchinson’s hypothesis, viz., that soil protozoa must be very few in numbers, since it was impossible to see them on examining soil under the microscope.
Fig. 9.—Showing the number of amœbæ and flagellates withdrawn from suspensions of varying strengths by different types of solid matter. A = clay: B = partially sterilized soil: C = ignited soil: D = fine sand: E = waste sand. Since complete withdrawal occurs when the numbers of organisms added are less than the capacity of the solid matter, the first part of each of the above curves is coincident with the ordinate. The numbers of organisms are given in thousands. (From Journ. Agric. Soc., vol. ix.)
X-axis: Number of Organisms per c.c. left in Solution.
Y-axis: Number of Organisms per c.c. taken up by Solid Matter.
The second or dilution method is the one, therefore, that has been most extensively developed.
Cunningham obtained concordant results in this way, and his method, modified by L. M. Crump, was as follows: 10 grams of soil were added to 125 c.c. of sterile tap-water and shaken for three minutes. This gives a 1 in 12·5 dilution. From it further dilutions were made until a sufficiently high one was obtained. Petri dishes, containing nutrient agar, were inoculated with 1 c.c. of each of the dilutions and incubated. At intervals covering 28 days the plates were examined and the presence or absence of protozoa on each recorded. In this way the approximate number of organisms per gram of soil could be found.
By methods essentially similar to this numerous counts have been made of the bacteria and protozoa in field soil and in partially sterilized soils. They were, however, inconclusive; thus, on the one hand, Goodey and several American observers, found no correlation between the numbers of protozoa and bacteria, while Miss Crump and Cunningham obtained evidence pointing to the reverse conclusion.
Such divergence of opinion was probably mainly due to two causes: firstly, that the time elapsing between the successive counts was too long, for it has been shown recently that the number of bacteria and protozoa fluctuate very rapidly; and secondly, the method was not completely satisfactory since only the total numbers of protozoa were considered, no means having been found of differentiating between the cystic and active forms. This was a particularly serious source of error for it is possible for soil to contain large numbers of bacteria and protozoa, of which a high percentage of the latter are in the form of cysts. A count made on such a soil would give results apparently opposed to the theory that protozoa act as depressors of bacteria.