It is evident, therefore, that the protozoa must be regarded as constituting part of the normal micro-organic population of soils, and as such are probably playing an important rôle. Unfortunately our knowledge of the physiology of these organisms is extremely scant, and much of future research must be directed towards elucidating their functions and their responses to varying environmental conditions. CHAPTER V. PROTOZOA OF THE SOIL, II. In the preceding chapter an outline has been given of the development of the study of soil protozoa, with especial reference to its qualitative aspects. Here it is proposed to deal with the quantitative methods which have been devised for studying these organisms and the results obtained. 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. [Illustration: FIG. 9.--Showing the number of amœbæ and flagellates withdrawn from suspensions of varying strengths by different types of solid matter.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.]