SOIL FLORA STUDIES.*

I. THE GENERAL CHARACTERISTICS OF THE MICROSCOPIC FLORA OF SOIL.

II. METHODS BEST ADAPTED TO THE STUDY OF THE SOIL FLORA.

1. A study has been New York State soils. eight years.

H. JOEL CONN.

SUMMARY.

made of the microscopic flora of various The work has been in progress for about

2. Determinations of the total number of bacteria have been made by the plate method. Little stress has been laid on the quantitative results, however, as the method is considered to be inaccurate.

3. The chief use made of the plate method has been to learn the relative numbers of different kinds of organisms in the soil, so far as it is possible to distinguish them from each other by the appearance of their colonies on the plates.

4. About one thousand pure cultures of these different organisms have been isolated and studied by various methods to learn their characteristics.

5. Non-spore-forming bacteria (mostly immotile rods) have been found to be the most abundant of all soil micro-organisms. Next to them in abundance are the various types of Actinomyces. 6. Certain spore-forming bacteria have also been found, but not in great numbers; and apparently they are not of great importance in normal soil.

7. Soil bacteria are quite different from those found in milk, cheese, or water. Evidently soil has its own characteristic bacterial flora. I. GENERAL CHARACTERISTICS OF THE SOIL FLORA.

PREVIOUS INVESTIGATIONS.

Soil flora studies in the past and the same fact is true concerning flora studies of milk or water- have developed in two different directions, the first extensive, the second intensive. Extensive studies have resulted in learning a little about many kinds

of soil organisms; intensive studies in learning much about a few, small, selected groups of organisms. Both lines of work must be harmonized in order to obtain a comprehensive knowledge of the soil flora.

An early contribution to the extensive flora studies of soil was made by Houston. This line of work was further developed by Chester in 1903, when he outlined the principles involved in such a study and the methods necessary. In an earlier publication 2 he had given rather full descriptions of miscellaneous bacteria isolated from soil; but it was in 1903 that he first emphasized the importance of determining the predominating soil organisms and selecting them for special study. In 1904,3 he carried this line of investigation to an extreme in which he has not been followed by other investigators. At this time he proposed a formula (coefficient of "zymotic efficiency") for expressing the results of qualitative bacterial analysis of soil. No subsequent worker has expressed his results in the terms proposed by Chester; but this does not alter the fact that important principles underlie Chester's work. He was handicapped by the lack of intensive work on the various groups of soil organisms (a handicap which he realized and did his best to overcome by intensive studies of his own), and also by the crudity of bacteriological methods at the time when his work was done. In spite of these handicaps, Chester's work still stands as the most complete extensive study yet made of the soil flora; and it is unfortunate that he was forced to drop this line of work before it had progressed far enough to have practical results.

4

At about the same time that Chester was making these studies, Hiltner and Störmer published a piece of work which did not go as far as Chester's work in classifying and describing the different kinds of bacteria, yet which has proved as valuable to later investigators as Chester's contributions. Instead of using the meager data at their command as a basis for establishing species, they merely pointed out three large groups of soil micro-organisms that can be readily distinguished on gelatin plates and showed that the relative

1 Houston, A. C. Chemical and bacteriological examination of soils. Local Gov't Board, Rept. 27:251–296. 1898. The results of this investigation are considered primarily in their relation to pathogenic organisms and the general conclusions are of less interest than the details of the work. The paper will therefore be more fully discussed in Tech. Buls. 58-60.

14 Chester, F. D. Study of the predominating bacteria in a soil sample. Del. Agr. Exp. Sta., Rept. 14:52-66. 1903.

2 Chester, F. D. Description of certain species of bacteria isolated from cultivated soil. Del. Agr. Exp. Sta., Rept. 11:52-75. 1900.

3 Chester, F. D. Bacteriological analysis of soils. Del. Agr. Exp. Sta., Bul. 65. 1904.

4 Hiltner, L., and Störmer, K. Studien über die Bakterienflora des Ackerbodens, mit besonderer Berücksichtigung ihres Verhaltens nach einer Behandlung mit Schwefelkohlenstoff und nach Brache. Kaiserl. Gesundheitsamte, Biol. Abt. Land- u. Forstw., 3:445-545.

1903.

abundance of these three groups is nearly constant in normal soil. From their data it appeared that any external influence which disturbed the equilibrium of the soil flora would be indicated by a change in the relative abundance of these three groups. This conception of the soil micro-organisms as being normally in a state of equilibrium has proved of considerable value in interpreting soil phenomena.

Meanwhile intensive work on special groups of soil bacteria has been accumulating more rapidly than extensive work on the general flora. Part of this work has been done on the group of sporeforming bacteria, and part of it on Actinomyces. Chester, himself, contributed to the study of the spore-formers.5 Also a series of articles on this group has been appearing from time to time representing the work of Meyer and his students at Marburg (Gottheil, 7 Neide and Holzmüller 9). 8 Certain disagreements between Chester's work and the Marburg work have caused some confusion for others who have wished to study the group; so the recent work of Ford and his associates,10 which harmonizes previous investigations, is an especially welcome contribution.

6

The Actinomycetes have been less carefully studied. Nearly all the work done on this group has been on the pathogenic types, and the early systematic work (e. g. that of Sauvageau and Radais,11 Gasperini,12 Lachner-Sandoval 13 and Sanfelice 14) left much to be desired. Recently, however, an important article by Krainsky 15 has opened the field for further investigations of this group. His methods are already beginning to be used by other investigators. A more complete discussion of the literature on spore-formers and Actinomycetes will follow in the two later papers of the present series which are to take up these two groups of organisms.

5 Chester, F. D. Observations on an important group of soil bacteria: Organisms related to Bacillus subtilis. Del. Exp. Sta. Rept. 15:42-96. 1904. A review of the Bacillus subtilis group of bacteria. Centbl. Bakt., II Abt., 13:737–752. 1904.

6 Meyer, A. Practicum der botanischen Bakterienkunde. Jena. 1903.

7 Gottheil, O. Botanische Beschreibung einiger Bodenbakterien. Centbl. Bakt., II Abt., 7:430, 449, 481, 529, 582, 627, 680, 717. 1901.

8 Neide, E. Botanische Beschreibung einige sporenbildenden Bakterien. Centbl. Bakt., II Abt., 12:1, 161, 337, 539. 1904.

9 Holzmüller, K. Die Gruppe des Bacillus mycoides Flügge. Centbl. Bakt., II Abt., 23:304–354. 1909.

10 Ford, W. W., et al. Studies on aerobic spore-bearing non-pathogenic bacteria. Jour. Bact., 1: 273-320, 493-534. 1916.

11 Sauvageau and Radais. Sur les genres Cladothrix, Streptothrix, Actinomyces. Ann. Inst. Past., 6:242-273. 1892.

12 Gasperini, G. Versuche über das Genus Actinomyces. Abstract in Centbl. Bakt., I Abt., 15:684-686. 1894.

13 Lachner-Sandoval. Ueber Strahlenpilze. Inaug. Diss. Strassburg. 1898. 14 Sanfelice, F. Ueber die pathogene Wirkung einige Streptothrix- (Actinomyces-) Arten. Centbl. Bakt., I Abt., Orig., 36:355–367. 1904.

15 Krainsky, A. Die Aktinomyceten und ihre Bedeutung in der Natur. Centbl. Bakt., II Abt., 41:649-688. 1914.

Thanks to the intensive work which has been done on these two groups, extensive work on the general soil flora can be undertaken today with a much greater chance of success than in earlier years. Flora studies have already proved of great value in dairy work; as shown by the investigations of H. W. Conn, Esten and Stocking, 16 Harding and Prucha,17 and Hastings, Evans and Hart, 18 which have resulted in showing the significance of the lactic acid types in milk and cheese (in milk the common lactic acid type, and in cheese the common type and also the Bulgarian type). Knowledge of equal importance in regard to soil may easily result from a better acquaintance with the soil flora.

Realizing this, several years ago the writer criticised the prevailing methods of soil bacteriological investigations, pointing out the importance of making a flora study, and concluded with the words: "In the future, if we wish to make any great advance in our knowledge, we must learn first to distinguish as rapidly as possible the different bacteria present in any given soil and then must study the significance of the forms we have learned to recognize." 19 Following up the line of work indicated in this statement, a preliminary grouping of the bacteria in a certain soil was published a few years ago. 20 This study included all the forms obtained upon gelatin plates incubated in the presence of air. About thirty-five types of bacteria were mentioned that could be distinguished by means of certain cultural and physiological characteristics. It was expected that some of these types could later be broken up into two or more species, while others would prove to be separated by characteristics not sufficiently constant to furnish actual distinctions. This study has been continued for five years since the work already published was brought to an end; and now considerable information is at hand to show which of these types are distinct, which must be grouped together, and which require further subdivision.

During the course of this study, several minor points have been investigated and discussions of them have been published from time to time. The surprisingly high plate counts of frozen soil, for example, were noticed early in the work, and as data accumulated on this subject,

16 Conn, H. W., Esten, W. M., and Stocking, W. A. A classification of dairy bacteria. Storrs (Conn.) Agr. Exp. Sta., Rept. 1906:91–203.

17 Harding, H. A., and Prucha, M. J. The bacterial flora of cheddar cheese. N. Y. Agr. Exp. Sta., Tech. Bul. 8. 1908.

18 Hastings, E. G., Evans, Alice C., and Hart, E. B. Studies on the factors concerned in the ripening of cheddar cheese. Wis. Agr. Exp. Sta., Res. Bul. 25. 1912.

Bacteria concerned in the production of the characteristic flavor in cheese of the cheddar type. J. Agr. Res., 2:167-192. 1914.

19 Conn, H. J. Future methods of soil bacteriological investigations. Centbl. Bakt., II Abt., 35:454-457. 1909.

20 Conn, H. J. A classification of the bacteria in two soil plats of unequal productivity. Cornell Agr. Exp. Sta., Bul. 338:65–113. 1913.

they were published.21 Gradually, as the work progressed, evidence was obtained of the comparative value of different culture media for soil bacteriological work.22 These minor publications all had to do with quantitative work alone (altho the main line of investigation was primarily qualitative), because results could be obtained with greater ease in quantitative work. It is only recently that the qualitative side of the work has been advanced sufficiently for publication. Two short articles have been published within the last year, one dealing with the spore-formers of soil 23 and the other with Actinomycetes.24 The work published in all these papers formed an integral part of the general soil flora study; so it is all summarized in the present series of publications.

SOILS INVESTIGATED.

In the course of this investigation, about five hundred samples have been taken from as great a variety of soils as could be obtained in the neighborhood of the Experiment Station. This included muck, clay loams, loams, sandy loams and fine sand. With the exception of the muck, these soils were all of glacial origin or of post-glacial alluvial derivation. They are, for the large part, delta deposits in one of the glacial lakes of central New York. Described according to the system of the Bureau of Soils of the U. S. Department of Agriculture,25 these soils are as follows:

21 Conn, H. J. Bacteria of frozen soil. Centbl. Bakt., II Abt., 28:422-434, 1910; 32:70-97, 1912; 42:310-519, 1914. N. Y. Agr. Exp. Sta., Tech. Bul. 33, 1914. 22 Conn, H. J. Culture media for use in the plate method of counting soil bacteria. Sta., Tech: Bul. 38. 1914.

N. Y. Agr. Exp. 23 Conn, H. J. Are spore-forming bacteria of any significance in soil under normal conditions? N. Y. Agr. Exp. Sta., Tech. Bul. 51. 1916. Jour. Bact., 1:187-195. 1916.

24 Conn, H. J. A possible function of Actinomycetes in soil. N. Y. Agr. Exp. Sta., Tech. Bul. 52. 1916. Jour. Bact., 1: 197-207. 1916.

25 U. S. Dept. Agr. Bureau of Soils Bul. 96, pp. 1-791. 1913. See also Survey of Ontario County, New York, published by this Bureau, 1912; pp. 1–55.