Agricultural Bacteriology

Front matter

AGRICULTURAL BACTERIOLOGY

CONN

BY THE SAME AUTHOR

Preface To Second Edition

Since the publication of the first edition of this work advance along all lines of bacteriology has been very rapid. Scarcely a phase of the relation of bacteria to agriculture has failed to receive substantial contributions. Much new information has been obtained, the relative importance of different subjects has been changed, and in a few points our previous conclusions have been corrected. These' changes have been so considerable that in preparing this second edition it has been found necessary to rewrite the whole book in order to bring it up to the times. The subject has grown so large that it is difficult to include within the limits of one volume even the fundamental facts of the rapidly growing science, and many subjects of importance have been treated very briefly.

The growing recognition of the importance of the subject to students of agriculture has caused agricultural schools and colleges to give to it an increasing amount of attention. For this reason this edition has been planned with special reference to its use by classes; and some changes in method of presentation have been adopted in order to make it more useful to students. For the same reason there has been added a somewhat extended set of experiments for elementary laboratory work. These laboratory directions are far from exhaustive and are designed simply to introduce the student to the methods of bacteriological work.

The close relation of the functions of the higher fungi to the functions of bacteria has come to be fully realized to-day and has made it necessary to include more extended references to the higher fungi in this review. The only other important addition in this

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PREFACE TO SECOND EDITION.

edition is a considerable extension of the treatment of bacterial diseases of plants that have received so much investigation in the last ten years. With these additions it is hoped that the present edition will be a fair summary of our present information upon the relation of germ life to agriculture.

Middletown, Conn.

June 15, 1909.

H. W. C.

Preface To First Edition

To set any exact limits to Agricultural Bacteriology is difficult. Primarily the subject includes only phenomena produced by bacteria, and phenomena that especially affect agriculture. But some agricultural processes are so closely bound with other industrial phenomena that they cannot be separated. Agriculture grades by imperceptible degrees into numerous secondary industries. Quite a number of the phenomena which will be considered in these pages have a closer relation to these secondary industries than they do to agriculture proper, but nevertheless they do have at least an incidental relation to the farm and must, therefore, be included in a discussion of Agricultural Bacteriology.

It has, moreover, in recent years, been a growing conviction that a considerable number of phenomena, hitherto attributed to bacteria, are directly due to a class of chemical ferments called enzymes. These enzymes are sometimes produced by bacteria, but in other cases by organisms totally unrelated to bacteria. When the latter is the case the fermentations produced by them have, of course, nothing to do with bacteriology proper. But we do not know as yet how commonly these enzymes, or chemical ferments, are concerned in agricultural processes, and even where they do occur it is found that, in some cases, they are intimately associated with true bacteriological action. It is impossible to separate chemical from biological fermentations by a hard and sharp line, nor can we tell to-day how far both of them may be concerned in any particular type of fermentations. In the following pages, therefore, it will be necessary to consider, to a certain extent, both types of fermenta-

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PREFACE TO FIRST EDITION.

tion. While both must be described and discussed, the bacteriological fermentations will demand most of our attention. For all of these reasons the limits which we shall draw to the subject of agricultural bacteriology are somewhat arbitrary, and some of the topics here considered may not be regarded as belonging strictly to the subject. All of them, however, have at least an incidental relation to the farmer and his industry.

Middlktown, Conn.

H. W. C.

PART 1. General Nature of Microorganisms and Their Activities

CHAPTER PAGE

I. — The General Characters of Microorganisms .... i

II. — ^Fermentation, Putrefaction, and Decay 22

PART II. Bacteria in Soil and Water

III. — Nature’s Food Supply. The Carbon Cycle .... 37

IV. — Decomposition of Nitrogenous Compounds .... 47

V. — Nitrification and Denitrification 57

VI. — The Manure Heap and Sewage 69

VII. — Reclaiming Lost Nitrogen 90

VIII.^ — ^Bacteria and Soil Minerals iii

IX. — Some Practical Lessons from Soil Bacteriology. . . 118

X. — Bacteria in Water 127

GENERAL NATURE OF MICROORGANISMS AND THEIR ACTIVITIES.

Chapter I. The General Characters Of Microorganisms

MICROORGANISMS AND FARM LIFE.

The successful farmer of to-morrow will be the one who most skillfully regulates the growth of microorganisms. Though he may not be conscious of it, much of the work that the farmer is carrying on even now is, as we shall see, really directed toward the control of germ life. In the consideration of the microorganisms related to farm life we are concerned with three different types of plants: Bacteria,Yeasts and Higher Fungi. Although the latter are plants of considerable size, and hence hardly microorganisms, in many respects they are related to the microscopic bacteria and yeasts, and the functions of all three in farm life are so similar that all must properly be considered together. The molds, being plants of considerable size, have been known a long time, although only recently has their relation to nature’s processes been understood. Yeasts were used, under the name of leaven, far back in history; but it was not till 1680 that the Dutch microscopist, Leeuwenhoek, showed with his microscope that yeast consists of minute globules; and it was 150 years later when Schwann and Caignard-Latour proved yeast to be a living plant. Leeuwenhoek was also first to see bacteria, and studied them as early as 1695. His descriptions,

I

THE GENERAL CHARACTERS OF MICROORGANISMS.

considering the fact that he had only simple lenses to work with, were remarkably correct. Even his suggestions concerning their nature sound quite modern and were certainly superior to much of the speculation that followed. He intimated that they might be the cause of disease. But for 150 years after Leeuwenhoek, although the microscope became a familiar plaything, it was hardly thought that these minute organisms offered a subject for serious study. For a century they were simply objects of speculation, and many were the exclamations which they excited as to the wonders of nature, with here and there a suggestion as to their possible importance in producing certain natural phenomena.

Relation to Disease. — Not until toward the middle of the nineteenth century was it conceived that the microscopic organisms, at first grouped together under the general head of animalcula, could have more than scientific import. At that time there began to appear suggestions as to their possible relations to certain diseases, and almost simultaneously they were thought of as causing /gfwmtations. Even before it was known what yeast was, it was recognized as in some way associated with alcoholic fermentation; but not till about 1838 was it clearly proved that yeast plants are the cause of the fermentation of sugar. The development of a knowledge of bacteria followed a little later. One of the first real contributions to a knowledge of their significance was the demonstration in 1840, of the fact that certain microscopic organisms cause blue milk. Thus, at the very beginning of the modern study of bacteria, they were associated with peculiar agricultural phenomena, an interesting fact when we notice that, in the next quarter of a century or more, the chief investigations, and all the interest in them, centered around the question of their agency in producing disease. Bacteria are still suffering in reputation from the fact that, for thirty years, they were studied by microscopists chiefly from the standpoint of their agency in the production of disease. It was quite early suggested, and soon demonstrated, that these little plants have the power of producing certain dreaded diseases, and the reputation which they thus obtained still clings to them. The very word bacteria, or germs, has become, in the minds of some, almost synonymous with

MICROORGANISMS AND FARM LIFE.

disease. Their relation to the medical profession was soon recognized, and more or less extended courses in bacteriology have rapidly made their appearance in medical schools. Health boards and sanitary officers have recognized that their primary duty is to deal with bacteria; and most of the regulations for the preservation of the public health have been directed toward the destruction or control of these organisms.

As more information has accumulated during the last twenty years or so, it has become evident that microorganisms, including bacteria, do not deserve all the ill repute that they have acquired. It has been learned that there are hundreds and even thousands of kinds of bacteria, and that, while certain species are the cause of disease, others are harmless, some are beneficial in the body, and many perform functions of the highest significance and value. Although the disease side of the bacteria story was the first to be studied, it is only a small part of the subject. Among the many hundred kinds of bacteria known, only a few, less than two score, are as yet definitely known to have any power of causing disease in man. As bacteriologists have widened their views and looked outside of the human body, they have found that these organisms are not only excessively abundant in nature, but have relations to the phenomena of living things which were wholly unsuspected. Within the last twenty years a larger and larger amount of attention has been directed to the part played in nature by microorganisms which are never parasitic and have no relations to human disease. As a result there has developed a new branch of bacteriology which deals with phenomena wholly separate from disease.

Relation to Agriculture. — In particular it has been shown that bacteria are related to agriculture. Not only is it true that they are the cause of certain animal and plant diseases with which the farmer has to contend, but it is becoming manifest that they are intimately associated with many normal processes which are going on in the soil, water, and elsewhere, and that they are fundamental to the processes of agriculture.

The agricultural side of bacteriology is, if possible, more impor-

THE GENERAL CHARACTERS OF MICROORGANISMS.

tant than the pathological side. If the medical student needs to know something of these organisms and their relations to disease, even more does the agriculturist need to understand their relations to his industry. These microorganisms play such a fundamental part in the processes of nature that the life phenomena of animals and plants are inextricably bound up in the functions of bacteria, and without them life processes must soon cease. The physician, in the curing of disease, gains a certain advantage from his knowledge of bacteria; but the farmer is obliged to make use of these agents in a large number of his farming processes; hence, it is a matter of necessity that the agriculturist of the future should have a practical knowledge of the general phases of bacteriology. The solution of the most vital agricultural problems, like that of continued soil fertility, involves bacteriology. From beginning to end the occupations of the farmer are concerned in the attempt to obtain the aid of these microorganisms where they may be of advantage, and to prevent their action in places where they would be a detriment. The farm cannot be properly tilled unless the farmer has, in addition to his seed crop and cattle, a stock of the proper kind of bacteria to aid him in preparing the soil and in curing the crops. Farming without the aid of bacteria would he an impossibility, for the soil would yield no crops.

The relation of microorganisms to farm life is one of the most recent branches of science. Scarce twenty years have elapsed since the first steps in this direction were taken, and some of our scientists, who are still young, have seen practically the whole development of the subject from its starting-point in the early eighties. With a science as young as this, it is inevitable that many questions remain unsolved. Scientific discovery usually precedes any practical application, and in these early years of the development of agricultural bacteriology we must expect to find the theoretical side of the subject proceeding rapidly, while the application of the facts to farm methods lags behind and is, in many respects, hesitating, tentative, or even unsatisfactory. Nevertheless, the discoveries made have already revolutionized agricultural processes. Changes in agricultural methods, due to bacteriology, have been

WHAT ARE MICROORGANISMS?

largely adopted all over the world; but they have generally been adopted by farmers in ignorance that they are benefiting from bacteriological research. That these practical applications of bacteriology to agricultural processes will increase with the next few years is certain. Successful agriculture of the future is indissolubly bound up in the problem of the proper handling of microorganisms. We have reached a point where every advanced farmer, who wishes to put himself into a proper condition to make the best use of the means at his disposal and to profit by discoveries as they are made, must at least have a general knowledge of the fundamental factors of bacteriology as they are related to agriculture.

WHAT ARE MICROORGANISMS?

In studying the relation of germ life to the farm we are concerned with a class of phenomena called fermentation, putrefaction, decay, decomposition, and the like (see Chapter II). These phenomena are all caused by living bodies that are frequently called microorganisms. This term strictly means animals or plants of microscopic size. But this conception of the term is at once too narrow and too broad to cover the organisms we are to study. Some microscopic organisms have no particular relation to the classes of phenomena which we are considering. A great host of microscopic, green water-plants and also many microscopic animals have nothing to do with our subject, though they might properly be called microorganisms. On the other hand, some plants of large size, like molds and toadstools, have a part to play in producing the decomposition of organic structures very similar to that played by bacteria. These cannot properly be called microorganisms, but nevertheless they must be included with the study of bacteria and yeasts, since they perform similar or closely allied functions in nature. A better term to cover the organisms which we must study might be the rather broad term of Fungi, for all of the organisms with which we are concerned belong to this general class of plants. But this term is also unsatisfactory, since it fails to convey the idea that the organisms are largely microscopic. To most people the term fungus gives at

THE GENERAL CHARACTERS OF MICROORGANISMS.

once the impression of a large plant; and we are chiefly concerned with microscopic forms. We shall, therefore, still use the term microorganism, although some of the plants that we shall refer to are not microscopic. • In this discussion we are concerned chiefly with Bacteria, secondarily with Yeasts, and to a less degree with the Higher Fungi.

The Fungi. — All of the plants with which we are here concerned belong to the class that botanists call Fungi. There is one characteristic common to all Fungi — they all lack green coloring matter. This green coloring material in ordinary plants makes it possible for them to live upon the mineral ingredients in the soil; and green plants only can be thus nourished. The colorless plants are unable to obtain nourishment from the mineral world. The Fungi, since they are all colorless, must live upon food furnished them by other plants or animals. It is this fact that gives them their significance in nature, and explains their important relations to farm life. Fungi are very abundant everywhere, and there are thousands of different kinds. For the purpose of our study, we may recognize three groups:

I. Higher Fungi. — Under this general name we will include a large number of colorless plants, mostly of large size. It includes such plants as molds, mushrooms, toadstools, tree fungi, and hosts of others less commonly known. Some of them are of great importance in farm life, especially as agents in bringing about the decomposition of vegetable matter, so that it may be incorporated into the soil to be used again; here they play a part secondary only to bacteria. They are of endless variety, and it would be manifestly impossible here to attempt any consideration of their classification. One point concerning them must be understood. In all the higher Fungi with which we are concerned the body of the plant consists of a mass of delicate threads which grow into a dense, usually white mass (Fig. i). Sometimes the threads are large enough to be seen easily and sometimes they are so delicate that a microscope is required to see the individual threads, though the mass of threads may be of considerable size. The mass of threads grows on the surface or in the substance upon which the plant is feeding. This thread is able

WHAT ARE MICROORGANISMS?

in many cases, by growing, to force its way into the solid mass of hard substances, like wood, and to push itself between the wood fibers. Thus it is a primary agent in effecting the destruction of wood. Such a mass of branching threads is called a mycelium, and b

Fig. I. — One of the higher fungi, the common bread mold, Penicillium glaucum. a, the whole plant; b, one of the spore-bearing branches more highly magnified.

is found in all the Fungi of this class. So far as the mycelium is concerned, most of these plants are much alike. But the different species have many different methods of reproduction, and it is chiefly upon their reproductive

Fig. 2. — Mucor, a common mold, showing mycelium and spore formation.

bodies that botanists rely to distin-

Fig. 3. — Aspergillus, a common mold, showing mycelium and spore formation.

guish the different species. After the mycelium has grown for a little time, it commonly sends up into the air small or large branches that produce spores, or reproductive bodies. The method of spore production differs sufficiently in the different fungi to make it

THE GENERAL CHARACTERS OF MICROORGANISMS.

possible to classify them. Frequently only the spore-producing part of the plant is seen, and it may be the only part known, except to botanists. For example, the toad stool is only the reproducing portion of a fungus; it has a mycelium wholly under ground or buried within the hard mass of the trunk of a tree. It is the myce lium, however, that does the work for which these fungi are re sponsible, and not the spore-producing part that we see. Figs, i to 3 show the general appearance of some of these fungi and their methods of forming spores. With these methods of reproduction and classification we are not concerned in this work, and only such types as are related to our subject will be mentioned later in their proper places.

2. Saccharomyces {Yeasts, Budding Fungi) . — These immensely important plants are all microscopic in size. While varying somewhat, an average size is about 1/4000 of an inch in diameter. They are usually spherical or oval in shape, though sometimes slightly elongated (Fig. 4, a). They form no mycelium and cannot force their way into hard substances. Their chief characteristic is their method of reproduction by a process called budding. There appears on the side of the yeast cell a minute bud, which continues to increase in size until it becomes as large as the cell from which it has grown. Then the two cells may break apart at once; or each may in turn produce buds before they separate. In either case, two or more cells are produced from the one, and although they may remain attached so as to form irregular masses of several cells, (Fig. 4, h), each cell is really complete in itself. Eventually they break apart. This budding takes place rapidly, though not so rapidly as the division of bacteria, which will be mentioned later.

A second important character of yeasts is the nature of the fermentation they produce. They have an action especially upon sugars, which they break up into carbonic acid and alcohol. This action makes them play a large part in nature’s processes, quite distinct from that of bacteria.

Fig. 4. — Yeast plants, showing method of growth by budding, a, single cells; ft, budding cells.

GENERAL CHARACTERS OF BACTERIA.

Any further classification of yeasts is quite unnecessary for our purposes.

3. Schizomycetes {Fission Fungi, or Bacteria). — This group comprises the bacteria proper; it is certainly the most abundant of the three, and in some respects it is the most important. It is with the bacteria that we are chiefly concerned in this work. Bacteria have sometimes much the same shape as yeasts. The chief distinction between them is their method of multiplication. Instead of budding they multiply by fission. The bacterium elongates a little, and then divides into two equal ^ ^ — n

halves at once (Fig. 5). Hence the name fission ^ ^

fungi. Bacteria are also, as a rule, smaller than yeasts, frequently not more than 1/25000 of an inch in diameter. The size would make it possible for 8,000,000,000 to be crowded into a mass no

CO larger than a pinhead; and we can, therefore, easily s£wing~mSTd understand that there may be 100,000,000 in a of division by drop of milk. Occasionally, however, there are larger bacteria and smaller yeast cells. While the size is no sure criterion between the two, when one finds, under the microscope, rather large round or oval plants, he is pretty safe in calling them yeasts, while the smaller ones he may call bacteria. But it is necessary, in some cases, to study the method of reproduction before one can with certainty distinguish yeasts from bacteria.

This group of bacteria is of such primary importance to our study that we must learn further facts concerning their classification and characters.

GENERAL CHARACTERS OF BACTERIA.

Colonies. — Bacteria are so minute that they cannot be handled as individuals, but must be treated in masses. One of the primary difficulties in the study of these organisms has been to get masses of bacteria that would be large enough to handle, and yet would contain only one kind of bacteria. Such masses are called pure cultures, and it was this difficulty in procuring pure cultures that lO

THE GENERAL CHARACTERS OF MICROORGANISMS.

for a long time prevented the development of the science. Bacteriological study to-day commonly begins with cultivating the bacteria, i.e., allowing them to grow in some medium adapted to them until they become abundant enough to be handled in bulk. To prevent the mixing of the different kinds that may be in the material we are studying, they are usually grown in a solid or jelly-like medium, which holds the individuals fast in one spot. As the individuals multiply in. this solid medium, they are unable to separate from each other; so they remain in little clusters which in time become large enough to be seen without a microscope.

Such clusters are called colonies, and figures of some of them are shown in Fig. 6. The shape and appearance of the colonies produced by different kinds of bacteria are often very different, showing, indeed, greater Fig. 6.— Colonies of bacteria, varieties than can be seen in the bacteria themselves with a microscope. As a result the shape and appearance of these colonies are often used to separate the numerous bacteria from each other and to classify them. A colony, when it comes from the multiplication of a single individual bacterium, is made of one kind of bacteria only. This colony may easily be picked out with a sterile needle, and when properly placed in another culture medium it becomes a pure culture. The starting-point in practical bacteriological study is thus the colony rather than the individual bacterium (see Laboratory Work).

Form of Bacteria. — Bacteria are of three quite different shapes, but are all very simple, i. Simple spheres (see Fig. 7, a). Such spherical forms are called Cocci. In common microscopical preparations no internal structure can be seen, the bacteria appearing as deeply stained balls. The Cocci, however, differ somewhat in their method of growth, thus enabling the microscopist to distinguish different kinds, as will be mentioned presently. 2. The rodformed bacteria (Fig. 7, b). These organisms are longer than they are broad, sometimes only slightly so, but at other times very much longer, forming, indeed, long, slender threads. 3. The spiralformed bacteria. These are either long, coiled spirals, or very

GENERAL CHARACTERS OF BACTERIA.

II short ones, with only a'single turn (Fig. 7, c). This type is of less importance than the others.

Motility of Bacteria. — The next point of distinction among bacteria is based upon their motility. Some bacteria are capable of an active swimming motion, others are stationary. The motion is produced by minute, extremely delicate, vibrating hairs, called flagella (Fig. 8). The flagella are so delicate that they cannot often be seen in the living bacteria, and they do not stain by the ordinary method of staining. Therefore, they are never seen in the usual microscopic preparations. They may be seen by special methods, but these are so difficult that the beginner cannot use them satisfactorily. The question of their motility is, however, usually determ-

Fig. 8. — Showing bacteria with flagella; a, peritrichic; &, lophotrichic; c, monotrichic.

ined without staining, by the study of the living bacteria (Experiment No. 8). These flagella are differently distributed upon different bacteria. Sometimes there is a single one on the end of a rod (Fig. 8, c) — monotrichic; sometimes a small tuft at one or both ends of a rod (Fig. 8, h) — lophotrichic; and sometimes there is a covering of flagella over the whole body of the bacterium (Fig. 4, a) — peritrichic.

a

Fig. 7. — General shape of bacteria, a, spheres; b, rods; c, spirals.

THE GENERAL CHARACTERS OF MICROORGANISMS.

Classification. — Based upon the distinctions thus mentioned, the bacteria are divided into groups:

Spherical Bacteria:

Dividing in one plain, so as to form chains (Fig. 9, a), Streptococcus.

Dividing in two plains, and not forming chains (Fig. 9, h).

Micrococcus.

Dividing in three plains, and forming cubical masses (Fig. 9, c), Sarcina.

Rod-shaped Bacteria:

With flagella and consequently

motile (Fig. 8)*, Bacillus.

Without flagella and consequently

Fig. 9.— Showing different non-motile (Fig. 7, h), Bactcriimi.

types of cocci, a, &, and c, Micro- -inn j

cocci; d, Streptococci; e, Sar- With a Single flagellum, PSCUGO-

monas.

Spiral Bacteria (Fig. 3, a). Spirillum.

The genus Bacillus is further divided as follows:

Bacilli with only one flagellum (Fig. 8, c) are named Monotrichic Bacilli, or Pseudomonas.

Bacilli with one flagellum at each end, Microsporon.

Bacilli with a tuft of flagella at one end (Fig. 8, h), are called Lophotrichic Bacilli.

Bacilli with flagella over the whole body (Fig. 8, a) are called

Peritrichic Bacilli.

Higher Bacteria {Cladothrix, Leptothrix, Streptothrix, Actinomyces) (Fig. 10).

Under this head are included a few forms of fungi which resemble other bacteria in some respects, but differ in others. They are composed of threads which are commonly larger than the

^Unfortunately bacteriologists are not agreed to-day in regard to the use of the terms above given. The names Bacillus and Bacterium are not always used as here stated, and recent classification of the spherical forms recognizes, in addition to the names given,three others, Diplococcus, Metacoccus, and Ascococcus. This variation in nomenclature results in great confusion. In the absence of any well-accepted classification to-day, we shall in this book use the names as above defined.

GENERAL CHARACTERS OF BACTERIA.

threads of bacteria, an'd which may show frequent branching, a characteristic not usual in bacteria. They also have a peculiar method of forming reproducing bodies. The group is not one of very great importance. One type of Streptothrix is extremely abundant in soil and appears as round, white opaque colonies with an extensive brown halo upon the plates described in Experiment No. 24.

Thus it will be seen that the term bacteria applies to the whole group of organisms that multiply by division, the study of which constitutes the study of bacteriology, while the term Bacterium refers to a single division of the group, viz.: the non-motile, rod forms.

The term Bacillus should apply to motile forms only. The names Bacillus and Bacterium are sometimes confused; for example, the tubercle bacillus, according to the above classifiication, is a Bacterium, since it is non-motile; and indeed recent study indicates that it belongs to the group of higher fungi; but the name bacillus was given it years before the above distinctions were recognized, and we will still use the common name. Some other bacteria, named twenty years ago, retain their earlier names in some books, but they are slowly having their names brought into ces. o, a small colony;

harmony with the above distinctions. &, single rods {Bostrom).

The term Coccus is applied to any spherical organism of the group bacteria.

This classification gives only what are recognized as the genera of bacteria. A further classification of the group into species is at the present time in a condition of the greatest confusion. Many hundred varieties have been described by different bacteriologists, but there is great difficulty in giving any distinctive description of such minute organisms, which have so few characters; and it is quite uncertain whether these many hundred described species represent distinct forms or whether they should be reduced to a