an organism in pure culture may lead to a very false conception of its function in soil; as seems to have been the case when it was assumed that the members of the B. subtilis group were the important soil ammonifiers. As the asporogenous bacteria have been shown to be active in soil, no misconception of this sort seems possible in regard to them; altho, while their activities still remain unknown, the question is naturally raised as to what may be their significance. This question must be the subject of future investigation.

SOIL FLORA STUDIES.*

V. ACTINOMYCETES IN SOIL.

H. JOEL CONN.

SUMMARY.

1. Ordinarily from twelve to fifty per ct. of the colonies on plate cultures made from soil are those of Actinomycetes.

2. Description of species of Actinomycetes is at present very difficult. The literature abounds with descriptions that have become invalidated as better methods of study have been developed.

3. About seventy different types have been found in the soils studied. Three of them are of fairly common occurrence. One of these three, A. pheochromogenus, n. sp., is considered distinct enough to be given a specific name. One of the other two types agrees in cultural characteristics with the potato-scab organism, but its pathogenicity has not yet been tested. The other common type probably is not a distinct species. Classification of the less common types is left for a more complete publication on these organisms which, it is hoped, will appear later.

4. The significance of these organisms in soil needs further investigation. There are good indications that they are active as well as numerically important.

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INTRODUCTION.

Of the previous papers of the present series of Soil Flora Studies, the first two 1 were introductory, and the next two 2 each considered one of the large groups of soil micro-organisms. Both of these two groups were groups of true bacteria. The present paper, however, takes up the third large group of soil micro-organisms, the Actinomycetes, which are generally spoken of as higher bacteria, but are sometimes considered to belong with the true fungi rather than with the bacteria.

1 Conn, H. J. The general characteristics of the microscopic flora of soil. N. Y. Agr. Exp. Sta., Tech. Bul. 57:1-17. 1917. Methods best adapted to the study of the soil flora. Id.:18-42. 1917.

2 Conn, H. J. Spore-forming bacteria in soil. N. Y. Agr. Exp. Sta., Tech. Bul. 58. 1917. Non-spore-forming bacteria in soil. N. Y. Agr. Exp. Sta., Tech. Bul. 59. 1917.

* Reprint of Technical Bulletin No. 60, March, 1917.

The present paper, like the preceding one (on non-spore-forming bacteria), is merely preliminary. It has not yet proved possible to distinguish species among the Actinomycetes or among the nonspore-forming bacteria with as much success as among the sporeforming bacteria. While three or four common species of sporeforming bacteria have been recognized with some degree of certainty, it has proved impossible to establish beyond question the identity of more than one species of Actinomycetes or of non-spore-forming bacteria. In regard to their activities, moreover, only enough has been learned to indicate that they are important in soil, but not enough to show what their importance may be.

There is great need, therefore, of more work on the Actinomycetes and non-spore-forming bacteria of soil. At the present time especial attention is being given at this Station to the Actinomycetes. A study is being made of their morphology, of their physiological activities in soil and in culture media, and of the constancy of the various characteristics that may be used for the separation of species. The present paper is merely a report of progress on this study. A more complete publication on the subject may be expected later.

GENERAL DESCRIPTION OF ACTINOMYCETES.

The genus Actinomyces Harz, em. Gasperini, is characterized by the possession of a mycelium composed of hyphae which show true branching, like that of the higher fungi. These hyphae are very delicate, however, in comparison with the hyphæ of higher fungi, (seldom measuring over 2 microns in diameter) and to judge by their staining reactions, resemble true bacteria in their protoplasmic properties. Their growth is not wholly within the agar or gelatin medium upon which they have been inoculated; for when conditions favor, an aerial mycelium is produced. In the aerial mycelium (( conidia are formed. These conidia are sometimes round, sometimes oval, and sometimes rod-shaped. They resemble bacteria closely in size, shape, and staining properties. They are generally between 0.6 and 1.5 microns in diameter, and if oval or rod-shaped, between 1 and 2 microns long. They stain readily with ordinary bacterial stains, and in a microscopic preparation which does not contain any hyphæ, often cannot be distinguished from true bacteria. In many cases deep-stained granules show at the poles, strongly suggestive of the metachromatic granules of the diphtheria organism. According to Sanfelice, some of the Actinomycetes are acid-fast like the tubercle organism. The diphtheria and tubercle organisms, moreover, sometimes produce branching forms, and some writers place these two organisms in the same group with Actinomyces.

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3 Sanfelice, F. Ueber die pathogene Wirkung einige Streptothrix- (Actinomyces-) Arten. Centbl. Bakt., I Abt., Orig., 36:355–367. 1904.

The growth of Actinomycetes on solid media is very characteristic. The mass of growth is generally of a tough, leathery consistency, sometimes smooth, sometimes wrinkled, and often piled high above the surface of the medium. Often the mass is brilliantly colored, and the color produced varies greatly with differences in the composition of the medium, but with constant composition of the medium, the color of the growth may be characteristic of the species. The aerial mycelium, often produced above this growth, may also be brilliantly colored and of an entirely different color from the mass of growth beneath it. Sometimes the aerial hyphæ are short and give the growth a chalky or mildewy appearance; but often they are long enough to cover the growth with a light, delicate nap, one or two millimeters thick. Some species produce pigments that diffuse thru the medium, coloring it gray, yellow, brown, red, blue or green. The color varies with the species and with the composition of the medium. It is not so definitely characteristic of the species as is the color of the growth itself or of the aerial mycelium; but with a medium of constant composition, the color produced is of considerable value in the recognition of species. On gelatin there is less diversity of growth than on agar. The growth is generally gray, brown or colorless; the aerial mycelium is often lacking, and if present is white, gray or colorless; and if the medium itself is colored, it generally becomes a reddish brown.

The growth in liquid media is also characteristic. The medium remains clear except for small colonies that may sink to the bottom, remain in suspension, float on the surface, or adhere to the walls of the tube. The surface colonies often grow together and become covered with a mass of aerial mycelium, sometimes forming a firm, wrinkled membrane that strongly suggests the surface membrane of the tubercle organism growing on broth. Pigments are often produced in liquid culture, the pigment varying with the composition of the medium and with the species growing in it.

The physiology of these organisms has never been thoroly studied. There is no agreement yet as to the media best suited for physiological studies of them, and each investigator has used media of his own. This fact, together with the confusion in nomenclature, has prevented concordant results. Nearly all liquefy gelatin and ammonify proteid, Münter 4 claiming that ammonification is their chief function. Nitrate-reduction has often been observed, as has the decomposition of cellulose. Some are animal pathogens and at least one a plant pathogen. Other important physiological activities will undoubtedly be worked out when the technic for studying them is further developed. It is not impossible that they are as diverse in physiology as are the true bacteria.

4 Münter, F. Ueber Stickstoffumsetzungen einiger Aktinomyceten. Centbl. Bakt., II Abt. 39:561-583. 1914.

One of the most characteristic features of many members of this group is their peculiar odor. It is a pungent, musty odor, difficult to describe, but impossible to mistake after once having it brought to the attention. It is sometimes spoken of as an earthy odor; but it would be more correct to say that soil often has an Actinomycesodor, as the odor of the cultures is much stronger than that of soil, and the soil odor is undoubtedly due to the Actinomycetes it contains. The odor seems to be associated with the aerial conidia, and does not seem to be produced by cultures that do not possess aerial mycelium. Not all species of Actinomyces have this odor, however, even when an abundant aerial mycelium is produced.

HISTORICAL.

THE QUESTION OF NOMENCLATURE.

A full account of previous work will have to be omitted in this preliminary paper. In Krainsky's recent paper 5 there is a very complete discussion of literature, which is easily available to anyone wishing a more complete review of the subject. For the present a brief resumé of the work bearing on nomenclature and classification must suffice, leaving a full review for the later paper.

The first member of this group to which a specific name was given was Streptothrix Foesteri Cohn 1875. This species was not described definitely enough for identification, a fact which undoubtedly explains why some have claimed it to be pathogenic, others non-pathogenic. The species Actinomyces bovis Harz 1877, on the other hand, has been definitely associated with bovine actinomycosis (" lumpy jaw ") and is generally regarded as a distinct species. Even in regard to A. bovis, however, there is some dispute, some having claimed it to be aerobic and others to be anaerobic.

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8

Great confusion has arisen as to the phylogenetic relationships of these organisms, Migula having placed them as bacteria in the genus Cladothrix, while Savageau and Radais placed them with Hyphomycetes in the genus Oospora. The opinion of Lehmann and Neumann 9 in this matter stands midway between these two views. They consider the group to stand between the Schizomycetes and Hyphomycetes, resembling the former in the size of its cells and staining properties of its protoplasm, but resembling the latter in forming

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

1914.

6 Cohn, F. Untersuchungen über Bacterien, II. Beitr. z. Biol. d. Pflanzen., 1:(Hft. 3):141-207. 1875.

7 Migula, W. System der Bakterien. 1897.

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

9 Lehmann, K. B., and Neumann, R. O. Atlas und Grundriss der Bakteriologie, Teil II. 1912. See especially p. 158.