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Pure culture studies of this sort have been made in the course of the present work of about a thousand cultures isolated from soil. Various methods have been used in these studies. The classification card of the Society of American Bacteriologists has proved quite well adapted to the study of the spore-formers; and has proved of value in making a preliminary study of other organisms before learning what special tests were best adapted to them. In studying the Actinomycetes, however, none of the tests included in the card study have proved of value, the most important information having been obtained from a study of their growth on various special agar media containing little or no protein material. No methods have yet been found which prove satisfactory for the qualitative study of the non-spore-formers.

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SOIL FLORA STUDIES.

III. SPORE-FORMING BACTERIA IN SOIL.

H. JOEL CONN.

SUMMARY 1. Certain spore-forming bacteria are always found in soil, but in comparatively small numbers.

2. The three most abundant spore-formers found in the soils investigated are: B. megatherium DeBary, B.mycoides Flügge, and B. cereus Frankland. B. simplex Gottheil occurs in somewhat smaller numbers. Occasionally other spore-forming bacteria have been encountered, but not in large enough numbers to be considered important.

3. In the past, the spore-forming bacteria have been considered important soil bacteria. The technic used in this work, however, shows that they comprise but a small part of the flora and apparently occur in normal soil only in the form of spores. This indicates that they are ordinarily inactive in soil.

INTRODUCTION. The present bulletin is the third of a series of five papers grouped together under the heading of Soil Flora Studies. The first two papers (I. The General Characteristics of the Miscroscopic Flora of Soil; II. Methods Best Adapted to the Study of the Soil Flora) were published in Technical Bulletin No. 57. They constitute an introduction to the series of papers. The remaining two papers of this series are to be entitled: IV. Non-spore-forming Bacteria in Soil. (Technical Bulletin No.

59.) V. Actinomycetes in Soil. (Technical Bulletin No. 60.)

HISTORICAL. No soil micro-organisms have been studied more frequently or more completely than the group of spore-formers. They are constantly present in soil, and are always found on gelatin or agar plates made from soil, producing the largest and most striking of the colonies that develop on such plates. They grow rapidly on ordinary media and if a short period of incubation is used they are among the most numerous of all the kinds of bacteria developing on the plates.

In Houston's' paper mentioned in the preceding bulletin the numbers of the different organisms of this group developing on gelatin plates were determined in so nearly the same way as has been done in the present work that the paper is of a good deal of interest, altho now twenty years old. Houston found four common sporeforming bacteria in his soil, which he called B. mycoides, B. subtilis, B. mesentericus, and a "Granular Bacillus." His descriptions of these organisms are careful enough so that it is possible to conclude that his identification of B. mycoides was correct, that his B. subtilis was actually B. cereus, and his "granular bacillus" B. megatherium, while his B. mesentericus probably contained a number of different, ill-defined, small-spored organisms. As will be shown later, these findings agree very well with those of the present work.

1 Houston, A. C. Chemical and bacteriological examination of soils. Local Gov't Board, An. Rept. of Medical Off., 27:251-296. 1898. * Reprint of Technical Bulletin No. 58, March, 1917.

Houston, however, states that B. mycoides is present in soil both in the vegetative form and as spores, whereas the writer has found them present only in the form of spores. Houston's method of determining whether these organisms were present as spores or as vegetative forms was essentially the same as that used by the writer, with the one exception that after mixing the soil with sterile water Houston allowed the infusion to stand one hour before plating in order to secure a thoro mixture. This one difference in technic makes it impossible to accept Houston's conclusions in this particular, as he gives no proof that none of the spores of B. mycoides germinated during the hour in which they were suspended in water.

Except for Houston's paper, there is very little reference in the literature to the relative numbers of the different kinds of sporeformers in soil. In general, literature relating to this group of organisms has been concerned either with their activities or with the taxonomy of these organisms. In view of the present doubt as to whether they occur in soil in vegetative form or as spores, their activity in soil is questionable. For this reason the literature concerning them is of interest from the taxonomic rather than from the practical standpoint, and there seems to be no need of reviewing that which relates to their activities. The taxonomic studies of this group, however, are well worth reviewing; for there is no group of bacteria, except that which includes the colon organism, which has been given such a thoro study from the taxonomic point of view.

The importance of studying the botanical relationships of these bacteria was first emphasized by Arthur Meyer and his students, who laid great stress on the need of accurate measurements and careful descriptions. The first important contribution to the taxonomy of the group was by Gottheil, one of Meyer's students.

2 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.

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

The value of Gottheil's work arises from his thoro study of the bacteria and his painstaking descriptions of their morphology. A few years later Neide published a continuation of Gottheil's work, which he took up where it had been dropped by Gottheil. Neide described about as many species as had been described by Gottheil. Both of these pieces of work were done in the same laboratory by the same methods, and with the cultures of one investigator available to the other. For this reason Gottheil's and Neide's papers may be regarded as two installments of a single investigation. Later a further contribution from Meyer's laboratory was published by Holzmüller; 5 but as his paper was concerned only with B. mycoides and certain closely related forms, it is not such an important contribution to the general taxonomy of the spore-forming bacteria.

Almost simultaneously with Neide's publication, an independent piece of work was published by Chester 6 Chester had obtained Gottheil's cultures as well as others from other sources, and published the results of his study of them. He greatly simplified Gottheil's descriptions, making it much easier for anyone else to follow them, and helped remove some of the previous confusion. Unfortunately, however, Neide's paper appeared at so nearly the same time as Chester's that neither author knew of the other's work, and as a result the two pieces of work do not harmonize. No attempt has been made, until within the last year, to harmonize Chester's work with that of Meyer's students.

The work which has finally brought order into the previous chaos is that of Ford and his collaborators. They obtained a larger number of cultures than had been studied by any previous investigators. Most of these cultures were of their own isolation; but they also had for comparison many cultures obtained from other investigators, from Kral's collection, and from the collection of the American Museum of Natural History. With such a large number of cultures it was possible to study correlations of characteristics and to observe differences to better advantage than had been done previously, and their findings have a correspondingly greater authority Their descriptions of species, moreover, are so carefully and concisely drawn up that they can be quite readily followed others. Thanks to this work, future investigators need not be as puzzled over the relationships of these bacteria as past workers have been.

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

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

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

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

CLASSIFICATION.

THE THREE MOST COMMON SPECIES.

Occurrence in soil.Three types of spore-forming bacteria have been found in all the soils studied and have always been observed on the plates, unless too great dilutions were used. They have been satisfactorily identified with B. megatherium DeBary, B. mycoides Flügge and B. cereus Frankland. In each case the identification has been regarded as correct, because the cultures isolated from these soils agree well with the published descriptions given by Chester and by Ford, and also because when representative cultures were sent to Ford for identification, he assigned to them the same names as those already decided upon by the writer. The constant occurrence of these three species in soil and their greater abundance per gram than the other spore-formers make it well to take them up in greater detail than the others.

Of these three species, B. megatherium has proved to be slightly more abundant than the other two, plate counts averaging about 375,000 per gram. Plate counts of B. mycoides have averaged about 225,000 per gram, and of B. cereus about 180,000. It must be recognized that these are not counts of active individuals, but probably they are fairly accurate counts of the spores. Microscopic examinations 8 of various soils have shown that spores of these organisms seldom occur in clumps or chains, so that probably the individual spores are ordinarily separate from each other when they are poured into the plates. Also these three organisms all grow well in gelatin, so that probably every living spore produces a colony. For these reasons it seems safe to conclude that these three organisms do not occur in normal soil to the extent of more than 1,000,000 per gram.

It is interesting to notice that Houston, who made a similar enumeration of the bacteria developing on gelatin plates, did not find as many colonies of the spore-formers; for of B. mycoides, which occurred in greatest numbers, he did not find in any case over 100,000 per gram. Nevertheless the numbers of B. mycoides which he reports are more nearly equal to the numbers found in the present work than his total counts are to the total counts in the present work. Considering that B. mycoides grows more vigorously on gelatin than most soil bacteria, this suggests that Houston's low counts were due to short periods of incubation. In regard to incubation, he merely states that he counted his plates “at as late a date as the liquefaction of the gelatin or the crowding of the micro-organisms allowed. This probably was not a very long period, as he apparently employed room temperature for incubation,

8 For the technic used, see: Conn, H. J. The direct microscopic examination of bacteria in soil. Abstr of Bact. 1: No. 1. 1917.

9 See footnote 1.

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