colony like each of the three more common species and does not occur in great enough abundance to be encountered regularly when chance isolations are made. In a series of chance isolations, B. simplex has been found about one-fifth as often as B. megatherium, B. mycoides or B. cereus a fact which gives some idea as to its frequency in soil. The cultures of B. simplex obtained from the soils studied agree fairly well with Chester's and with Ford's descriptions of this species; and the identification has been confirmed by Ford, in the case of a typical culture of this type sent to him.

B. simplex Gottheil, 1901.- This type is characterized by its rather long cylindrical spores, produced only in old cultures (or often not at all), by the occurrence of filaments and long chains of rods, often lightly stained, and by its rather slow growth in gelatin.

Morphology: Young rods are 0.8 to 1.2 by 2.5 to 5 microns (Fig. 4, c). Rods often occur in long chains, and filaments are frequently observed 15 microns in length. In cultures a few days old, these filaments lose their power of staining deeply and become

20000 000

the "shadow forms" characteristic of the species. (Fig. 4, b). Young rods are plainly motile; flagella peritrichic, but not very many have been observed on each rod (Fig. 4, a). Spores are seldom produced before the third day and sometimes not until the culture is a week or more old, and are always relatively scarce in comparison with the vegetative forms. Cultures are sometimes obtained that do not produce spores but agree with B. simplex in other respects; so that they are thought to be strains of this organism that have lost their power of producing spores, altho without the spores to study it is impossible to be sure of the identity of the organism. The spores are cylindrical, elongate, 0.6 to 1.0 by 1.5 to 3 microns (Fig. 4, d). (It is hard to understand the difference between these measurements and those published by Ford and Lawrence, unless the shape of the spores varies greatly in different strains. The culture sent to Ford and identified by him as B. simplex is one that often produces strikingly long spores about 3 microns in length.)

Cultural characteristics: In broth, slight turbidity and sediment with no surface growth. Gelatin colonies punctiform, slowly liquefying. Growth on agar streak culture, soft, glistening, translucent to almost transparent, with indistinct edges.

Physiology: The typical group number is B. 111.44430?4, the same as that of B. megatherium except for the figure referring to nitrate-reduction. As neither B. megatherium or B. simplex grow well in nitrate broth, it is possible that their lack of agreement in this one respect may have been brought about by irregularities due to their poor growth. Altho, as indicated by the group number,

this organism makes no growth in sugar broth, it does grow on litmus agar containing sugars, and it has been found that a vigorously growing culture produces a trace of acid under such conditions from dextrose and sucrose, but not from lactose.

(This type was not recognized at the time when Cornell Bul. 338 was written. The type denoted as "B. simplex (?)" in that bulletin was not correctly identified, its actual identity being unknown because the cultures were lost. It is known that some examples of true B. simplex were found in that work, as two cultures of it isolated then, are still alive; but at the time that bulletin was published they were not distinguished from the other spore-formers. They were probably included in types 6 and 7.)

Other types. Besides the four species just described, a number of cultures have been obtained that are plainly different organisms. One or two small-spored organisms have been found that closely resemble B. mesentericus (Flügge) Migula; two cultures have been obtained of a round-terminal-spored organism, probably B. fusiformis Gottheil; and also several others that have proved difficult to identify. Very little study has been given them, however, and no serious attempt has been made to establish their identity, because they have been found so seldom that they are probably of no importance in soil and, indeed, may have been mere contaminations on the plates, coming from the air or some other source.

Certain spore-formers have been found which are small short rods that grow poorly on ordinary media and may not be closely related to those described above. They have never been found on plates made in the ordinary way. They have occurred only when the soil infusion has been heated to 75° C. before plating. On such plates, presumably, the exclusion of the non-spore forms allows the growth of certain slow-growing spore-producers that are crowded out of the ordinary plates. No study of them has been made, as they are not believed to be very abundant in soil.

GELATIN COLONIES OF THE SPORE-FORMING BACTERIA.

In making a flora study of soil, it is quite important to know the relative abundance of the different types of organisms studied. About the only way to determine this is by means of the relative numbers of their colonies which appear on plate cultures made from soil; and in order to count the colonies of any type, it is necessary that they can be readily distinguished from the colonies of other bacteria. As stated in the preceding paper, 15 gelatin has proved the best medium upon which to recognize the spore-forming bacteria. This is because each of the three most common sporeformers produces a characteristic colony on gelatin (altho B. simplex and the other less common types do not).

15 N. Y. Agrl. Expt. Sta. Tech. Bul. 57, p. 30.

Unfortunately it is not possible to describe the colonies of these bacteria so that they can be recognized with certainty by other investigators. Differences in technic make so much difference in the appearance of the colonies that the results in different laboratories are likely to be quite divergent. It is possible, however, for one man so to standardize his own technic that he can recognize the colonies of these three spore-formers with some degree of accuracy, after he has once learned their characteristics upon his own plates. The descriptions of the colonies given here may not agree perfectly with those which other investigators may find; but they are given for whatever they may be worth.

It has been found in the present work that all of the colonies on 12 per ct. Gold Label gelatin that have liquefied appreciably (i. e. have become more than one or two millimeters in diameter after seven days at a temperature of 18° C.) are either Pseudomonas forms (principally Ps. fluorescens) or spore-formers. The Pseudomonas colonies are generally structureless, Ps. fluorescens producing a rapidly liquefying, almost clear colony, that will liquefy the entire plate if given time. Next to Ps. fluorescens, the most rapid liquefier has been found to be B. mycoides, which produces large, filamentous to rhizoid colonies. B. cereus is almost as rapid a liquefier as B. mycoides, and under certain conditions produces colonies that cannot be distinguished from it; but on plates from normal soil, the B. cereus colonies are round with entire edges, with a surface membrane containing granules that tend to be arranged concentrically. B. megatherium liquefies much more slowly. Its largest colonies are seldom over 1 cm. in diameter. They are characterized by a flocculent center composed of white opaque granules, surrounded by a zone of clear liquefied gelatin. The smaller B. megatherium colonies. are often under 2 mm. in diameter with no surrounding clear zone, and can be recognized only because of their granular nature, all other punctiform colonies appearing round to the naked eye.

Using these criteria, it has proved possible to obtain a fairly good estimate of the numbers of each of these three species in the different samples of soil investigated. In this way the figures given on p. 6 were obtained. The data recently given,16 showing that heating the soil infusion did not decrease the number of colonies of spore-formers, depended upon the fact that certain spore-formers produce colonies on gelatin plates that can be easily recognized. SIGNIFICANCE OF THE SPORE-FORMING BACTERIA

IN SOIL.

The great attention that has been given in the past to the sporeforming bacteria of soil is undoubtedly due to their constant presence in soil, their large, striking colonies on agar or gelatin plates, and 16 See footnote 2.

their rapid growth in laboratory media. Chester, 17 for example, used fairly short periods of incubation, and because of the rapid growth of these organisms, found them to comprise nearly half of the colonies on the plates. Hiltner and Störmer, 18 on the other hand, incubated their plates until no more new colonies appeared (about ten days), and found only 5 per ct. of the colonies to be liquefiers (which category includes nearly all, if not all, of the sporeforming bacteria).

Long incubations, however, have only recently come into general use. The use of short incubations has led to an exaggerated idea of the abundance and importance of spore-forming bacteria in soil. Because of their relative abundance on the plates, they were thoroly studied and it was found that they were vigorous ammonifiers in laboratory culture media. Naturally it was assumed that they were also vigorous ammonifiers in soil. This idea has persisted in spite of repeated observations as to their relatively small abundance. Lipman,19 for instance, states: "The more important ammonifiers, including members of the B. subtilis group and certain Streptothrices, are numerically important in all arable soils." Similarly Stevens and Withers20 used cultures of B. subtilis for inoculating different soils in order to determine their ammonifying capacity, thus very evidently making the assumption that B. subtilis is a typical and important soil ammonifier. These two cases are mentioned merely by way of illustration. Writers have frequently made similar statements; and still other writers, who with more caution have refrained from stating that the spore-forming bacteria actually cause ammonification in soil, have referred to their ammonifying powers and left the reader to assume that such must be their activity in soil.

At the present time there is even less justification for such statements than before. It was recently pointed out by the writer21 that in normal soil even tho a large quantity of fresh horse manure had been added - there was no evidence of the spore-forming bacteria being present in vegetative form. Since writing that paper, further information on the subject has been obtained by means of the microscopic examination of soil; and it has entirely corroborated this conclusion.

The recent work of Ford and associates, 22 indeed, makes it doubtful if these bacteria should be considered soil organisms in

17 Chester, F. D. Study of the predominating bacteria in a soil sample. Del. Agr. Exp. Sta., Rept. 14:52-63. 1903.

18 Hiltner, L., and Störmer, K. Studien über die Bakterienflora des Ackerbodens. Kaiserl. Gesundheitsamte, Biol. Abt. Land- u. Forstw., 3:445-545. 1903.

19 Lipman, J. G. In Marshall's Microbiology (2nd edition), p. 325.

20 Stevens, F. L., Withers, W. A., et al. Studies in Soil Bacteriology III. Centbl. Bakt. II Abt., 25:64-80. 1909.

21 See footnote 2.

22 See footnote 7.

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any true sense. These writers found practically the same kinds of spore-formers not only in milk, in water, and in soil, but also in dust, and the species found most frequently in soil were to a striking extent the most abundant forms in milk, dust, and water as well. No one would suggest for a moment that their results indicate that these bacteria are active in dust. The probable explanation is that their spores are omnipresent, and become active only when conditions are favorable.

Just what conditions do favor the growth of these bacteria, it still remains to be learned. B. mycoides and B. cereus seem to grow best in liquids of high organic content, but the other sporeformers have not been observed to grow rapidly under laboratory conditions except on the surface of agar. It is not impossible that the reason why these species have developed the power of producing spores is because the conditions necessary for their growth are so rarely met in nature that their continued existence would be impossible without some resting stage. In soil, conditions favorable to their growth probably occur just often enough so that the spores do not diminish in number; and when conditions are unfavorable to them, the organisms are inactive.