REFERENCES TO CHAPTERS II. AND III.

[1] Ashby, S. F., Journ. Agric. Sci., 1907, vol. ii., p. 35.

[2] Beijerinck, M. W., Centr. f. Bakt., 1900, Abt. II., Bd. 6, p. 1.

[3] Beijerinck, M. W., and Van Delden, A., Centr. f. Bakt., 1902, Abt. II., Bd. 9, p. 3.

[4] Bewley, W. F., and Hutchinson, H. B., Journ. Agric. Sci., 1920, vol. x., p. 144.

[5] Bonazzi, E., Journ. Bact., 1921, vol. vi., p. 331.

[6] Burrill, T. J., and Hansen, R., Illin. Exp. Sta., 1917, Bulletin 202.

[7] Christensen, H. R., Centralblatt. f. Bakt., 1915, Abt. II., Bd. 43, p. 1.

[8] Christensen, H. R., Centralblatt. f. Bakt., 1907, Abt. II., Bd. 17, pp. 109, 161.

[9] Christensen, H. R., and Larsen, O. H., Centralblatt. f. Bakt., 1911, Abt. II., Bd. 29, p. 347.

[10] Conn, H. J., Centralblatt. f. Bakt., 1910, Abt. II., Bd. 28, p. 422.

[11] Conn, H. J., Centralblatt. f. Bakt., 1914, Abt. II., Bd. 42, p. 510.

[12] Conn, H. J., Journ. Bact., 1916, vol. i., p. 187.

[13] Conn, H. J., Journ. Bact., 1917, vol. ii., p. 137.

[14] Conn, H. J., Journ. Bact., 1917, vol. ii., p. 35.

[15] Cramer, E., Arch. f. Hyg., 1893, Bd. 16, p. 151.

[16] Cutler, W., Crump, L. M., and Sandon, H., Phil. Trans. Roy. Soc., 1923, Series B, vol. ccxi., p. 317.

[17] Doryland, C. J. T., N. Dakota Agr. Exp. Sta., 1916, Bulletin 116.

[18] Fabricius, O., and Feilitzen, H., Centr. f. Bakt., 1905, Abt. II., Bd. 14, p. 161.

[19] Fisher, R. A., Thornton, H. G., and Mackenzie, W. A., Ann. Appl. Biol., 1922, vol. ix., p. 325.

[20] Fred, E. B., and Hart, E. B., Wisconsin Agr. Exp. Sta. Research, 1915, Bulletin 35.

[21] Gainey, P. L., Journ. Agric. Research, 1918, vol. xiv., p. 265.

[22] Golding, J., Journ. Agric. Sci., 1905, vol. i., p. 59.

[23] Greaves, J. E., Soil Sci., 1916, vol. ii., p. 443.

[24] Greaves, J. E., Journ. Agric. Res., 1916, vol. vi, p. 389.

[25] Greaves, J. E., Soil Sci., 1920, vol. x., p. 77.

[26] Greaves, J. E., and Lund, Y., Soil Sci., 1921, vol. xii., p. 163.

[27] Greaves, J. E., and Carter, E. G., Journ. Agric. Research, 1916, vol. vi., p. 889.

[28] Groenewege, J., Arch. Suikerindust., 1913, Bd. 21, p. 790.

[28b] Green, H. H., Union of S. Africa Dept. Agr., Rept. of Director Vet. Res., 1918, p. 592.

[29] Hutchinson, C. M., Rept. Agr. Res. Inst. and Col. of Pusa, 1912, p. 85.

[30] Hutchinson, H. B., and Clayton, J., Journ. Agric. Sci., 1919, vol. ix., p. 143.

[30b] Hutchinson, H. B., and Richards, H. H., Journ. Min. Agric., 1921, vol. xxviii., p. 398.

[31] Hanzawa, J., Centr. f. Bakt., 1914, Abt. II., Bd. 41, p. 573.

[32] Hopkins, C. G., and Whiting, A. L., Ill. Agr. Exp. Sta., 1916, Bulletin 190, p. 395.

[33] Hoppe-Seyler, G., Ztschr. Phys. Chem., 1886, vol. x, pp. 201, 401; 1887, vol. xi., p. 561.

[34] Hesselmann, H., Skogsvårdsför. Tidskr., 1917, No. 4, p. 321.

[35] Joshi, N. V., Mem. Dept. Agr. in India, Bact. Ser., 1920, vol. i., No. 9.

[36] Koch, R., Mitt. Kais. Gesundh., 1881, vol. i., p. 1.

[37] Kaserer, H., Centr. f. Bakt., 1906, Abt. II., Bd. 16, p. 681.

[38] Kaserer, H., Centr. f. Bakt., 1905, Abt. II., Bd. 15, p. 573.

[39] Krainskii, A. V., Centr. f. Bakt., 1910, Abt. II., Bd. 26, p. 231.

[40] Klimmer, M., and Kruger, R., Centr. f. Bakt., 1914, Abt. II., Bd. 40, p. 257.

[41] Krzeminiewski, S., Centr. f. Bakt., 1909, Abt. II., Bd. 23, p. 161.

[42] Koch, A., and Seydel, S., Centr. f. Bakt., 1912, Abt. II., Bd. 31, P. 570.

[43] Lipman, C. B., Bot. Gaz., 1909, vol. xlviii., p. 106.

[44] Lipman, C. B., and Burgess, P. S., Centr. f. Bakt., 1914, Abt. II., Bd. 41, p. 430.

[45] Lipman, C. B., and Burgess, P. S., Centr. f. Bakt., 1915, Abt. II., Bd. 44, p. 481.

[46] Lipman, C. B., and Waynick, D. O., Soil Sci., 1916, vol. i., p. 5.

[47] Löhnis, F., and Pillai, N. K., Centr. f. Bakt., 1908, Abt. II., Bd. 20, p. 781.

[47b] Löhnis, F., and Smith, T., Journ. Agric. Res., 1914, vol. vi., p. 675.

[48] Mackenna, J., Rept. Prog. Agric., India, 1917, p. 101.

[49] Marchal, E., Bull. Acad. Roy. Belgique, 1893, vol. xxv., p. 727.

[50] McBeth, I. G., and Scales, F. M., U.S. Dept. Ag., Bureau Plant Indus., 1913, Bulletin 266.

[51] Mockeridge, J., Biochem. Journ., 1915, vol. ix., p. 272.

[52] Nabokich, A. J., and Lebedeff, A. F., Centr. f. Bakt., 1906, Abt. II., Bd. 17, p. 350.

[53] Nagaoka, M., Bull. Coll. Agr., Tokyo, 1900, vol. vi., No. 3.

[54] Omelianski, W. L., Comptes Rendus Acad. Sci., 1895, vol. cxxi., p. 653; 1897, vol. cxxv., pp. 907, 1131; Arch. Sci. Bio., (St. Petersburg), 1899, vol. vii., p. 411.

[55] Omelianski, W. L., and Sohmskov, M., Arch. Sci. Biol., Publ. Inst. Imp. Med. Exp. (Petrograd), 1916, vol. xviii., pp. 327, 338, 459; vol. xix., p. 162.

[56] Prescott, J. A., Journ. Agr. Sci., 1920, vol. x., p. 177.

[57] Söhngen, N. L., Centr. f. Bakt., 1905, Abt. II., Bd. 15, p. 513.

[58] Sen Gupta, N., Journ. Agr. Sci., 1921, vol. xi., p. 136.

[59] Shearer, C., Journ. Hyg., 1919, vol. xviii., p. 337.

[60] Tappeiner, Ber. Deut. Chem. Gesell., 1883, vol. xvi., p. 1734; Zeitsch. Biol., 1884, vol. xx., p. 52.

[61] Thornton, H. G., Ann. Appl. Biol., 1922, vol. ix., p. 241.

[62] Wallin, I. E., Journ. Bact., 1922, vol. vii., p. 471.

[63] Waksman, S. A., and Joffe, J. S., Journ. Bact., 1922, vol. vii., p. 239.

[64] Wilson, J. K., Cornell Agric. Exp. Sta., 1917, Bulletin 386.

CHAPTER IV.
PROTOZOA OF THE SOIL, I.

That protozoa could be isolated from the soil was a matter of common knowledge to the biologists of the nineteenth century, but not until the early part of the present century was it suggested that these organisms might be playing some part in the general economy of the soil micro-population. Of recent years a great deal of our knowledge of the cytology of the different groups of protozoa, especially the Amœbæ, has been obtained from the study of representatives normally living in the soil; but unfortunately little or no knowledge has been gained of the biology of these animals in their natural habitat.

The view that the presence of these organisms in excessive numbers may lead to “soil sickness” was first put forward by Russell and Hutchinson in 1909, and elaborated in their further papers dealing with “Partial Sterilisation of the Soil.”

It is unnecessary to discuss in detail this important branch of agriculture, but to obtain a clear idea of the development of the study of soil protozoa it is necessary to give as briefly as possible the conclusions deduced by Russell and Hutchinson from their extensive experiments on soils treated with steam and various volatile antiseptics[21], [22]:—

“(1) Partial sterilisation of the soil causes first a fall, then a rise, in bacterial numbers, which goes on till the numbers considerably exceed those present in the original soil.

“(2) Simultaneously there is a marked increase in the rate of accumulation of ammonia which is formed from organic nitrogen compounds.

“(3) The increase in bacterial numbers is the result of improvement in the soil as a medium for bacterial growth, and not an improvement in the bacterial flora.

“(4) The improvement in the soil brought about by partial sterilisation is permanent, the high bacterial numbers being kept up even for 200 days or more. It is evident from (3) and (4) that the factor limiting bacterial numbers in ordinary soil is not bacterial, nor is it any product of bacterial activity, nor does it arise spontaneously in soils.

“(5) But if some of the untreated soil is introduced into partially sterilised soil, the bacterial numbers, after the initial rise, begin to fall. Thus the limiting factor can be reintroduced from untreated soils.

“(6) Evidence of the limiting factor in untreated soils is obtained by studying the effect of temperature on bacterial numbers. Untreated soils were maintained at 10°, 20°, 30° C. in a well-moistened aerated condition, and periodical counts were made of the numbers of bacteria per gram. Rise in temperature rarely caused any increase in bacterial numbers. But after the soil was partially sterilised the bacterial numbers showed the normal increase with increasing temperatures.

TABLE VI.

Temperature
of Storage.
°C.		Untreated Soil.Soil Treated with Toluene.
At
Start.		After
13
Days.		After
25
Days.		After
70
Days.		At
Start.		After
13
Days.		After
25
Days.		After
70
Days.
5°-12°656341328·5 73101137
20°654122238·5187128182
30°652750168·5197145 51
40°6514 9338·5148 52100

“(7) It is evident, therefore, that the limiting factor in the untreated soils is not the lack of anything, but the presence of something active. The properties of the limiting factor are:—

“(a) It is active and not a lack of something.

“(b) It is not bacterial.

“(c) It is extinguished by heat or poisons.

“(d) It can be re-introduced into soils from which it has been extinguished by the addition of a little untreated soil.

“(e) It develops more slowly than bacteria.

“(f) It is favoured by conditions favourable to trophic life in the soil, and finally becomes so active that the bacteria become unduly depressed.

“It is difficult to see what agent other than a living organism can fulfil these conditions. Search was therefore made for a larger organism capable of destroying bacteria, and considerable numbers of protozoa were found. The ciliates and amœbæ are killed by partial sterilisation. Whenever they are killed the detrimental factor is found to be put out of action; the bacterial numbers rise and maintain a high level. Whenever the detrimental factor is not put out of action, the protozoa are not killed. To these rules we have found no exception.”

From such premises as the above Russell and Hutchinson founded the “protozoa theory of partial sterilisation,” and at Rothamsted there was commenced the serious study of these soil organisms.

Goodey was one of the early workers on this new subject, and added considerably to our knowledge of the species living in normal soils, and of the chemical constitution of the cyst wall of ciliates. He also made investigations on the effects of various chemicals on the micro-population of soils, but was unable to draw very definite conclusions.[11]

One of the first criticisms raised against the protozoa theory of partial sterilisation was that the protozoa were not normal inhabitants of the soil, and were present only in small numbers, all of them in the cystic, quiescent condition. It was further held that these cysts were carried by the wind from dried-up ponds and streams. It is difficult to trace the origin of this view, since early observers, viz., Ehrenberg and Dujardin, in 1841, were of the opinion that the protozoa were living in the trophic active condition in the soil, and it was not until 1878 that Stein showed that free living protozoa can encyst. To Martin and Lewin, however, must be ascribed the distinction of first proving that the soil possesses an active protozoan population, for by a series of ingenious experiments these observers isolated several flagellates and amœbæ in a trophic condition from certain of the Rothamsted soils.[18] The more recent work in this country has been in the direction of devising new quantitative methods of research, since by this means alone is it possible to elucidate many fundamental questions.

In America and elsewhere experiments have been devised for testing the conclusions of Russell and Hutchinson. Cunningham and Löhnis,[2] in America, Truffaut and Bezssonoff,[24] in France, supply evidence in favour of the theory, but most of the American work is in opposition to it.

Sherman[23] is perhaps the most prominent in opposing the phagocytic action of protozoa on soil bacteria in spite of the fact that certain of his experimental results apparently show enormous decreases in bacterial numbers in the presence of protozoa. In many of his soil inoculation experiments, however, it was not demonstrated that his active cultures remained alive after entering the soil.

The experimental difficulties of dealing with soil protozoa are considerable, and without a thoroughly sound technique investigators may easily go astray.