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These soils were mostly agricultural soils, but some of the muck samples were taken from woodland. The samples were taken from soils in various states of cultivation, including sod of various ages and cultivated soil under various crops. Many of the samples were so taken as to furnish a comparison between sod and cultivated portions of the same soil.

KINDS OF MICRO-ORGANISMS IN SOIL.

Limitations of the study.— At present it would be a hopeless task to make a complete study, or even a complete classification, of the various types of micro-organisms occurring in soil. Because of the necessity of setting some limit to the work, only those forms have been studied that grow in the presence of air and occur in appreciable numbers on plates inoculated with soil infusion diluted 100,000 times. This excludes a number of the soil bacteria that have been described by others.

Foremost among the soil bacteria that are considered important, yet which are not mentioned in the present work, are those concerned with the transformations of nitrogen. The nitrifiers, for instance (which convert ammonium salts into nitrites and nitrates), do not grow on the ordinary organic laboratory media, and for that matter have never been found in soil in sufficient numbers to occur on plates dilute enough to show the more abundant organisms. The denitrifiers (that reduce nitrate to elementary nitrogen) and the nitrogen-fixing bacteria (that are able to assimilate free atmospheric nitrogen) likewise occur in too small numbers to be found by the methods employed in this work.

The bacteria that produce nodules upon the roots of legumes and enable these plants to utilize atmospheric nitrogen are excluded from this study, altho they can be obtained upon plates made directly from soil in the dilutions employed in the present work (as shown by Lipman and Fowler 26). They have not been encountered in the course of this work, however, probably because no special search has been made for them and because the media used have not been particularly adapted to them.

As all of the plates were incubated in the presence of air, strict anaerobes, if such occur in the soil, have been overlooked. The importance of strict anaerobes in soil, however, is somewhat questionable. The peculiar structure of soil seems to make it pre-eminently a medium for the growth of aerobic bacteria. It has often been claimed in the past that water-logged soil furnishes favorable conditions for anaerobes, particularly in the deeper layers. This is not at all improbable; but normal soil seems to furnish more favorable conditions for aerobic growth than any other natural medium inhabited by bacteria.

26 Lipman, C. B., and Fowler, L. W. Isolation of Bacillus radicicola from soil. Sci., N. S., 41:256-259. 1915.

Evidence has been obtained which supports this conclusion. Plates grown in the absence of oxygen always show a very scanty flora, a few thousand per gram; and none of the cultures isolated from the colonies on these anaerobic plates have proved to be strict anaerobes. Not enough evidence of this nature has been obtained to justify a positive statement; but the few results at hand are at least suggestive. They are borne out by the fact that the flora on ordinary aerobic plates consists almost wholly of strict aerobes, although the growth of facultative anaerobes should not be prevented by the presence of oxygen. The only facultative anaerobes encountered on aerobic plates are the spore-forming bacteria, and apparently they are not active in normal soil. If anaerobic growth were common in soil, it hardly seems as if there would be such a preponderance of strict aerobes on the plates. There is no reason, in view of the data obtained, for believing strict anaerobes to be of any real significance in normal soil.

Two other classes of organisms that may be important in soil, which have been excluded from the present work, are higher fungi and protozoa. The importance of protozoa in soil is not yet established, but a study of them would have been made except for the necessity of drawing some limits to the field investigated. Higher fungi have always been known to live in the soil, and their spores are numerous there; but it has never been shown whether mold hyphae are abundant enough in soil for their activities to compare with those of bacteria. The writer 27 has already called attention to this fact, and has stated that the microscope does not show the presence of mold hyphae in ordinary soil, altho it reveals the presence of bacteria in large numbers. Higher fungi certainly live in soil; but there is reason to doubt whether they are abundant enough to be of significance in comparison with bacteria.

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A further limitation of the work has resulted from the fact that a complete classification of a flora as complex as that of soil even that part of it which develops on aerobic gelatin plates offers difficulties that are almost insurmountable at the present day. Bacteriological methods are too crude as yet to allow the establishment of species, with any degree of certainty, even among pathogenic organisms and the bacteria found in milk and water. Among the bacteria of soil, which have been much less thoroly studied, there is still less certainty in regard to species. It is, therefore, much too soon to expect an exhaustive classification of the soil flora.

The most that can be looked for at present is a preliminary classification. This preliminary classification should include descriptions of known species that can be recognized without question;

Conn, H. J. Relative importance of fungi and bacteria in soil. Sci., N. S., 44:857-858. 1916.

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but to describe new species seems unwise. The literature relating to soil bacteriology is full of names of species in regard to which too little information is given to make recognition possible. It seems unwise to increase this confusion by adding more new names. The best plan seems to be to classify the soil micro-organisms into groups or types as small as present methods allow, each of which may be the subject of future investigation. Some of these types are undoubtedly species; others are larger groups, and must be further subdivided in the future.

Peculiarities of the soil flora.- One of the most striking characteristics of the soil flora that has come to light in the course of this work is its uniformity. Quantitatively this uniformity is particularly striking, as the number of colonies developing on gelatin or agar plates incubated at 18° C. for seven days is ordinarily between five and fifty million per gram of soil. Qualitatively there is also considerable uniformity, as pointed out some time ago.28 It was stated then that the predominating types of bacteria in every kind of soil studied had proved to be the same, so far as they could be determined by the methods used. There is no reason yet for modifying that statement, altho since that paper was written new methods of study have allowed further classification of the Actinomycetes, and some of the types picked up in some particular soils have not yet been encountered elsewhere. It is perfectly possible that the same fact may prove to be true of other groups of soil organisms as better methods are adopted for their classification; but the statement can safely be made that the same general types of bacteria occur in all of the soils studied. The greatest difference yet observed has been in the relative abundance of the different groups present. This fact gives still more weight to Hiltner and Störmer's conception of an equilibrium among soil micro-organisms.

Another fact which has been noticed in making a study of the soil flora is that the organisms encountered are strikingly different from those found in other natural media inhabited by bacteria. The short rods and cocci that produce acid from sugars, which are so abundant in milk, are entirely lacking in soil. The large sporeforming bacteria with peritrichic flagella, which seem to be characteristic of water, occur in soil, it is true, but comprise only a small and apparently unimportant part of the total flora. The bacteria found in soil in the course of this investigation have proved to be mainly of two classes: (1) Actinomycetes, a group occurring in milk and water so rarely that its occasional presence always seems accidental; (2) small immotile rods without spores, that do not

28 Conn, H. J. The distribution of bacteria in various soil types. Jr. Amer. Soc. Agron., 5:218-221. 1913.

acidify sugars and grow slowly in all of the ordinary bacteriological media (liquefying gelatin slowly if at all), a. group that does not occur in milk and has never been mentioned as occurring in water. The former group generally comprises 12-50 per ct. of the soil flora, the latter 40-75 per ct.

Groups of micro-organisms found.- Besides these two predominating groups, certain other bacteria have been found quite regularly but in smaller numbers. They are: Spores of the peritrichic sporeformers (often called the B. subtilis group); rapidly liquefying rods with polar flagella (principally Ps. fluorescens); and slow-growing cocci that liquefy gelatin slowly or not at all and do not ordinarily acidify sugars. A grouping of the soil bacteria which includes all these forms may be drawn up as follows:

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The three large groups, "rapid liquefiers," "slow growers" and Actinomycetes, given in the right-hand column of the above table, have been mentioned in several previous publications.29 Except for the Actinomycetes, they are purely artificial groups, used because of the convenience with which they can be recognized from gelatin plates. Hiltner and Störmer 30 used a similar classification of their gelatin colonies. They state that gelatin plates from soil normally show 5 per ct. liquefiers, 70 per ct. non-liquefiers, and 20 per ct. Streptothrix. Their 5 per ct. liquefiers include the B. subtilis and Ps. fluorescens groups—the "rapid liquefiers." It is true that usually more than 5 per ct. of the colonies have been found to be rapid liquefiers in the present work; but Hiltner and Störmer do not state the percentage of gelatin used in their work, and a sufficient concentration of gelatin might easily have lowered the numbers to 5 per ct. in the present work. Hiltner and Störmer's "non-liquefiers" are undoubtedly the same as the writer's "slow growers," in other words the organisms that produce punctiform colonies on gelatin. The term "non-liquefiers,' however, is misleading because many of them do liquefy gelatin in stab culture if given time enough and even produce liquefied colonies on the plates if the concentration of the gelatin is not too great. The average proportion of the total flora comprised by the organ

29 See footnotes 20 and 21.

30 Loc. cit., footnote 4.

isms that produce punctiform colonies is not far from the 70 per ct. which Hiltner and Störmer find to be non-liquefiers. Their third group, "Streptothrix," is without question the same as the Actinomycetes. Streptothrix is, however, a poor term to use, as it was applied to another genus before it was used for this group of organisms. In cultivated soils, such as Hiltner and Störmer were studying, the Actinomycetes have been found to average almost exactly 20 per ct. as shown in a recent publication.31

The soil flora may also be characterized by the kinds of organisms that are lacking. No non-liquefiers producing acid from sugar have been observed. Organisms producing gas from sugar may possibly be present in small numbers, 32 but are found only rarely by the methods used in this work. The only organisms observed that are not strictly aerobic are some of the spore-formers, and they do not seem to be active in normal soil.

Importance of these different groups. Unfortunately, little is known of the importance of these groups of soil organisms. Conclusions drawn from the relative abundance of the colonies on the plates are likely to be misleading. In the past, for example, when methods of plating were such as to prevent large numbers of the non-spore-formers and Actinomycetes from developing on the plates, the relative abundance of the spore-formers seemed to be quite high, and it was concluded that they must be important in the soil. The present work, however, as pointed out in a previous publication,33 shows that they are actually present in comparatively small numbers and, even at that, occur ordinarily only in the form of spores. In just the same way future improvements in methods may show that the organisms apparently most abundant are not actually the most important micro-organisms in soil.

Nevertheless, there is good reason to believe that non-motile non-spore-forming bacteria, which show up in such abundance on gelatin plates, are actually, as well as apparently, the most numerous soil micro-organisms. It is also safe to conclude that the Actinomycetes are one of the more abundant groups of organisms in soil. If abundance bears any relation to importance, these are probably the two most important groups of soil micro-organisms; and indeed (as mentioned in a previous publication 34 and as will be further discussed in a later paper of this series), the relative numbers of these two groups of organisms show such a close relation to certain soil conditions that there can be little question as to their activity and importance in soil.

31 Loc. cit., footnote 24.

32 They have been isolated from soil by others, as for example Chester's Bact. fermentationis (Del. Agr. Exp. Sta., Rept. 11, p. 53); but neither Chester nor anyone else has stated them to be among the predominating types.

33 Loc. cit., footnote 23.

34 Loc. cit., footnote 24.

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