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SOIL FLORA STUDIES*

IV. NON-SPORE-FORMING BACTERIA IN SOIL.

H. JOEL CONN.

SUMMARY

I. The majority of colonies on culture plates made from soil are those of non-spore-forming bacteria.

2. The only non-spore-former that has been successfully identified with previously described species is Ps. Auorescens (Flügge) Migula. This organism is most abundant in freshly aerated soil or in soil to which organic matter has been recently added. It is known to cause ammonification in pure culture, and is therefore thought to take part in the decomposition of organic matter in soil. There are other liquefying non-spore-formers in soil that seem to be closely related to Ps. fluorescens. They increase in numbers under much the same conditions and are thought to have similar functions.

3. The great majority of the asporogenous bacteria of soil grow so poorly in ordinary laboratory media and produce such small noncharacteristic colonies on culture plates that little is known about them. One of the greatest present needs for a soil flora study is the development of methods by which the different species in this group may be identified.

4. The numbers of the non-spore-forming bacteria in soil show greater fluctuations than do those of spore-forming bacteria or of Actinomycetes (the two other large groups of soil bacteria). High numbers of non-spore-formers have been especially noticed in freshly aerated and in freshly manured soil. This indicates that they are among the most active soil micro-organisms.

INTRODUCTION. . In the first paper of this series ? (p. 99), the bacteria of soil were classified as follows:

Spore-producers;
Non-spore-producers:

Liquefaction rapid;
Liquefaction slow or none:
Rods:

Yellow chromogenic;

Non-chromogenic;

Cocci;
Actinomycetes.

1 Conn, H. J. General characteristics of the soil fora. N. Y. Agr. Exp. Sta., Tech. Bul. 57:7-17. 1917. Reprint of Technical Bulletin No. 59, March, 1917.

The second paper 2 dealt only with the methods by which these organisms had been studied. The third paper 3 took up the best known altho the least numerous of the three large groups of soil micro-organisms: the spore-forming bacteria. The two which remain to be considered, the non-spore-forming bacteria and the Actinomycetes, are much more numerous in soil than the sporeforming varieties; but much less is known about them.

It has so far proved impossible, indeed, to distinguish the different kinds of organisms in these two groups from each other, or to obtain definite knowledge as to their function in soil. For this reason the present paper on non-spore-forming bacteria and the following one on Actinomycetes are merely preliminary. Even less is known about the non-spore-forming bacteria than of the Actinomycetes; so the present paper can do no more than point out the lines along which their study must develop in the future. Its object is primarily to call attention to the probable importance of non-spore-forming bacteria in soil. It is hoped that this preliminary work may be followed by a more complete study, not only at this Station, but by investigators working elsewhere.

HISTORICAL.

Spore-forming bacteria and Actinomycetes have been studied considerably in the past; but except for the nitrifiers and some of the other organisms concerned in the transformations of nitrogen, scant attention has been given to any non-spore-forming bacteria found in soil. As an illustration of this fact, mention may be made of Löhnis' review of the subject in his “ Handbuch der landwirtschaftlichen Bakteriologie.” In his section (p. 514) entitled:

Allgemeines über die im Boden vorkommenden Arten von Mikroorganismen " he refers to non-spore-forming bacteria in just two sentences. In these two sentences he says that except for the fluorescent varieties, attention has been given only to certain yellow and blue chromogenic forms.

A further illustration of the scant attention given to them can be obtained from the work of Houston 4 and that of Chester 5 both referred to in the preceding bulletins of this series. Houston describes only one non-spore-forming organism that is not fluorescent; while

2 Conn, H. J. Methods best adapted to the study of the soil flora. Id.:18–42. 1917.

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

4 Houston, A. C. Chemical and bacteriological examination of soils. Local Gov't Board, An. Rept. 27 of Medical Off.:251–296. 1898.

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

Chester, among the three predominating micro-organisms in a certain soil sample, found only the least abundant one of the three to be without spores. In Chester's case, as already mentioned (Tech. Bul. 57, p. 33), this might have been due to a short incubation period. Houston's results might have been due in part to a short incubation; but as he mixed his soil with sterile water and allowed the mixture to stand an hour before plating, he furnished a good opportunity for those types to predominate (Ps. fluorescens, for example) that grow well in water.

In other words, the large group of non-chromogenic, asporogenous bacteria that produce punctiform colonies on agar or gelatin has been given very little attention, and the non-motile members of this group have been practically overlooked. Considering what a large proportion of the colonies on the plates are of this type, it seems strange at first thought that they have been overlooked; and yet the reason is not far to seek. These bacteria grow poorly in ordinary culture media. In laboratory culture, their activities are so insignificant that they can scarcely be measured by chemical tests. It has been assumed that organisms so inactive in the laboratory must also be inactive in soil.

CLASSIFICATION.

THE TWO PRINCIPAL GROUPS.

Two main groups of non-spore-formers have been recognized in this work. The first is the group that liquefies gelatin rapidly, the second the group that produces mere punctiform colonies in gelatin. The latter group contains some slow liquefiers and some non-liquefiers. At first thought it would seem as tho a much more natural division to make would be to distinguish liquefiers from non-liquefiers, but that has not proved to be the case. The bacteria found in the soil which liquefy gelatin slowly seem to be more nearly related to those which do not liquefy it at all than they do to those which liquefy rapidly.

This division has also proved easier to recognize in practice than it would have been to draw the line between liquefiers and nonliquefiers. Many slow-liquefiers do not begin to liquefy at the end of seven days' incubation, and cannot be distinguished from non-liquefiers by means of their colonies. The rapid-liquefying group, however, can always be distinguished by the fact that its colonies are over 1 cm. in diameter, and if given time often liquefy the entire plate; while the colonies of the other group never become as large as 1 cm. in diameter, however long the incubation.

The most important member of the rapidly liquefying group is Ps. fluorescens (Flügge) Migula. For convenience sake, therefore,

the group may be spoken of as the Ps. fluorescens group; but this expression must not be taken to mean that all the organisms are fluorescent. Many of them, indeed, never produce fluorescence, and non-fluorescent strains of Ps. fluorescens itself are often encountered. The rapid liquefaction of gelatin is the most striking common characteristic of this group. No non-liquefying fluorescent organisms have been encountered.

The other group of asporogenous bacteria here discussed probably does not contain any well-known organism. They grow so poorly in liquid media that all efforts to characterize species within the group have so far proved fruitless. They have been spoken of in previous publications 6 as the group of "slow-growers” because of their tiny colonies and their poor growth in laboratory media. This term, however, gives a false conception of their importance in soil; for, in soil, conditions probably favor their growth and there is reason to believe that they multiply rapidly there.

In Cornell Bulletin No. 338 the non-spore-formers were classified according to the tests called for by the classification card of the Society of American Bacteriologists.? Later work has indicated that except in the case of Ps. fluorescens, these tests are of little value. In the present bulletin, therefore, the slowly liquefying asporogenous organisms are merely classified into a few general groups.

RAPID-LIQUEFIERS. Ps. fluorescens (Flügge) Migula. The most striking characteristic of this type is its fluorescence, which is observed in broth, beefextract-peptone agar, and sometimes in gelatin. Ability to produce fluorescence is often lost, however, and then the type must be recognized by its other characteristics, such as: rapid liquefaction of gelatin, uniform turbidity in broth, cloudy, structureless colony in gelatin, and acid production from dextrose.

Morphology: Rods 0.4 to 0.8 by 0.8 to 1.5 microns, in old cultures nearly the same shape and size as in young cultures. Flagella 3 to 6, arranged in a clump at one pole. Motility great. Rods do not form chains.

Cultural characteristics: Good growth in broth; no surface growth, uniform turbidity, causing distinct cloudiness of medium; sediment scant or none. Gelatin colonies liquefying with great rapidity; round to irregular in shape, cloudy, structureless, occasionally fluorescent. Growth on agar streak cultures, smooth, soft, glistening, generally causing the medium to show a green fluorescence.

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

? For a discussion of this card see Technical Bulletin No. 57, pp. 33 to 40.

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Physiology: The typical group number is: Ps. 211.2332133. Fairly consistent results can be obtained in determining its group number, altho certain variations occur. Occasionally there is no acid from dextrose. Some of the cultures reduce nitrates. The power of producing fluorescence is often lost. These variations may indicate the existence of separate species that are now grouped under this one head. In Cornell Bulletin No. 338, several types (Nos. 10,

8 The group number, as used on the classification card of the Society of American Bacteriologists, is as follows: 100.

Endospores produced 200.

Endospores not produced 10.

Aerobic (strict) 20.

Facultative anaerobic 30.

Anaerobic (strict) 1.

Gelatin liquefied 2.

Gelatin not liquefied 0.1

Acid and gas from dextrose 0.2

Acid without gas from dextrose 0.3

No acid from dextrose 0.4

No growth with dextrose .01

Acid and gas from lactose .02

Acid without gas from lactose .03

No acid from lactose .04

No growth with lactose .001

Acid and gas from saccharose .002

Acid without gas from saccharose .003

No acid from saccharose .004

No growth with saccharose .0001

Nitrates reduced with evolution of gas .0002

Nitrates not reduced .0003

Nitrates reduced without gas formation
.00001

Fluorescent
.00002 Violet chromogens
.00003

Blue
100004

Green .00005

Yellow .00006 Orange .00007

Red .00008

Brown
.00009

Pink
.00000 Non-chromogenic
.000001 Diastasic action on potato starch, strong
.000002 Diastasic action on potato starch, feeble
.000003 Diastasic action on potato starch, absent
.0000001 Acid and gas from glycerin
0000002 Acid without gas from glycerin
.0000003 No acid from glycerin

.0000004 No growth with glycerin The genus according to the system of Migula is given its proper symbol which precedes the number thus: BACILLUS COLI (Esch.) Vig.

becomes B. 222.111102 For a more complete discussion of the group number see Technical Bulletin No. 57, pp. 33-40. (This report, pages 117-126.)

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