m^Xd^jii aad HASflhv^i.,; (TorneU TUntversit^ OF THE IRewlPorF? State College of Hariculture ft^M)^ 3i3;Ufa Cornell University Library QR 51.R8 Agricultural bacteriology for students i 3 1924 003 198 961 Cornell University Library The original of tliis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003198961 AGRICULTURAL BACTERIOLOGY FOR STUDENTS IN GENERAL AGRICULTURE H. L. RUSSELL Dean of The College of Aghiculture •University of Wisconsin E. G. HASTINGS Professor of Agricultural Bacteriology The College of Agriculture University of Wisconsin Madison, Wisconsin H. L. RUSSELL 1915 i L,, RS Copyright, 1915 BY H. L. RUSSELL and E. G. HASTINGS CANTWELL PRINTING COMPANY MADISON. WIS. FOREWORD The art of agriculture has long been practiced but the science of this subject is of comparatively recent Origin. To a scientific understanding of agriculture many other sciences, such as chemistry, physics, and biology have made most important contribution's. Among the biological sciences, the field of bacteriology has of late years assumed a most important relation- ship. The early researches of Pasteur, Koch, and their successors opened a field of inquiry as to the cause of animal disease, but since their day exact knowledge of the influence of microorganisms on soil processes, dairying, and foods in general has been greatly ex- tended. It is of the utmost importance for the farmer and the student of agriculture to have a proper con- ception of these relations and the text here presented is designed to give a summary but comprehensive treatment that will aid in a more rational under- standing of the problems of farm life. CONTENTS PART I Properties of Microorganisms Chapter Page I. The Relation of Living Things to Organic Matter.. 1 II. The Morphology and Physiology of Microorgan- isms 7 III. The Cultivation of Microorganisms 26 PART II Soil Bacteriology IV. TheRelalionof Microorganisms to Soil Fertility .... 33 V. The Decomposition of Organic Matter in the Soil.. 41 VI. The Action of Bacteria on the Minerals of the Soil 49 VII. The Cycle of Nitrogen 54 VIII. ■ Barnyard Manures and Sewage Disposal 68 IX. The Fixation of Nitrogen ' 77 PART III The Relation of Microorganisms to Foods X. The Contamination of Foods with Microorgan- isms 90 XI. The Contamination of Foods with Pathogenic Bac- teria , 109 XII. The Preservation of Foods 121 XIII. The Fermentations Occurring in Food Products 140 XIV. The Relation of Bacteria Lo Butler and Cheese 153 XV. The Control of Foods 166 VI Contents PART IV Transmissible Diseases Chapter Page XVI. The Relation of Microorganisms' to Diseases of Animals 179 XVII. Anthrax, Black Leg, Hemorrhagic Septicaemia, and Corn Stalk Disease 192 XVIII. Tuberculosis 204 XIX. Texas Fever, Contagious Abortion and Foot and Mouth Disease 232 XX. Rabies and Actinomycosis 247 XXI. Glanders and Tetanus 256 XXII. Hog Cholera 264 XXIII. Diseases of Fowls 277 XXIV. Bacterial Diseases of Plants 283 XXV. Disinfection 289 PART I PROPERTIES OF MICROORGANISMS CHAPTER I THE RELATION OF LIVING THINGS TO ORGANIC MATTER All living things may be divided into two groups, the basis of division being the nature of the sub- stances used as food. The first group is represented by the green plant, the food of which consists of simple, inorganic substances that are devoid of en- ergy and are fully oxidized. The green plant through the agency of the chlorophyll, the green coloring substance found mainly in the leaves, is able to make use of the radiant energy of the sun. It builds from the simple, inorganic substances a great va- riety of organic compounds in which is stored a sup- ply of energy. The second group may be represented by the higher animals that live on the organic compounds that have been formed by the green plant. The ani- mal may consume the vegetable matter directly or after it has been built up into the tissues of another animal. Through the processes of digestion and assimilation, the energy stored in the organic matter is set free and is used by the animal for all of its life processes.. The animal is thus to be looked upon as an agency concerned in the destruction of organic la 2 Agricultural Bacteriology matter, since the amount of energy contained in its tissues and in all the by-products formed during its life is less than the energy content of the food consumed in the same period. The work of the ani- mal is analytic in character; that of the green plant is synthetic. The green plant is the only form of hfe which is building complex compounds from the most simple, but the animal is not the only agency concerned in the destruction of these complex organic substances. Supplementing the work of animals is that of a gro'up of plants which are devoid of chlorophyll, and hence are unable to make use of the sun's energy. They must have organic matter for food, or at least substances that still contain a supply of energy which they can use in their life processes. This group of plants is most often called fungi or fungous plants and includes many familiar forms of life, such as the mushrooms, rusts, smuts, and others that are less well known on account of their microscopic size. If a handful of hay or other vegetable matter is placed in water, the liquid will soon become turbid, and a membrane may form on the surface. No living thing is visible to the naked eye but if a drop of the water is examined under a powerful microscope, it will be found to be teeming with a host of living organisms of varying size and form; the observer is thus introduced to the world of microorganisms. The organic matter in the water gradually becomes softer and slowly disappears. It has undergone decomposi- tion. In the process a great variety of compounds is formed, and the final result is that all the organic material is changed to inorganic compounds, or to minerals. The process is frequently termed the min- eralization of organic matter. Relation of Living Things 3 In the soil and air there is but a limited supply in an available form of many of the elements needed by the green plant, and it is a necessary condition for the continued growth of plant life on the earth that the elements contained in the tissues of the plant be returned to the soil so that they can again be used by succeeding crops of vegetable life. The plant derives all of its carbon from the carbon diox- ide of the air. This compound is thus constantly being removed from the air and a necessary condi- tion for the continued growth of plants is the return of this substance to the air. The complex series of chemical transformations by which the carbon of the carbon dioxide is built into organic compounds, and by which this carbon is again made available to the green plant, is called the carbon cycle. The other chemical elements contained in organic matter, as in vegetable matter undergoing decomposition in the water, are gradually being re- turned to a form in which they will again be avail- able to succeeding plant life. Matter is indestructi- ble and an atom of an element can thus pass through the tissues of plants or animals an unlimited num- ber of times. The organisms which have been seen in the drop of hay infusion examined under the microscope are ainong the principal agents concerned in the cycle of the elements, and, as they carry on their work, they influence the daily life of man in many ways. They decompose the organic matter added to the soil, and hence are important in determining its fer- tility. They hasten the decomposition of materials that man desires to store for food purposes. They form the basis of the great industrial fermentations, such as the alcoholic fermentation so important in 4 Agricultural Bacteriology the making of all alcoholic beverages and bread. They may also cause the decomposition of mate- rials within the body of the hving plant or animal and thus produce disease. They bear as intimate a relation to the life of man as do the green plants or the animals, and some knowledge concerning them is almost a necessity of modern life. If the material undergoing decomposition consists largely of carbohydrates such as sugars and starches, the word fermentation is often applied to the process. It is marked by the formation of acids, alcohols, and gases. The term is derived from the Latin word "fevere," to boil, the evolution of gas from the fer- menting material reminding one of a boiling liquid. The term putrefaction is usually applied to decom- posing proteins, such as eggs, and meat. It is marked by the production of offensive odors. Both of these decomposition processes can go on to some extent in the total absence of free oxygen. Many of the microorganisms are able to grow in materials that are totally devoid of free oxygen, which is essential for the life of the green plants and animals. It should be easy to show that the hay infusion is devoid of this gas. If the organic matter is to be completely decomposed, and all of the ele- ments returned to a form in which the green plant can use them, free oxygen must be available since the microorganisms that complete the process can grow only in the presence of free oxygen. When the decomposition goes on in the air so that the latter class of organisms can grow, the of- fensive smelling compounds are destroyed as fast as formed and no marked odor will be noted. The term decay is often applied to decomposition that takes place under such conditions. Relation of Living Things 5 Before detailed knowledge concerning microor- ganisms had been obtained, it was currently believed that the organisms found in infusions and in other decomposing material were developed from non- living matter; that in some mysterious way life had originated. This process was termed spontaneous generation. The French bacteriologist, Pasteur, com- pletely exploded this notion, and showed that spon- taneous generation does not occur but that the an- cestors of the microorganisms found in decomposing matter are the progeny of similar forms which in one way or other have been brought in contact with the organic matter. Occurrences which were most mysterious fifty years ago are now easily explained because of the knowledge that has been gained of the world of microorganisms. A great variety of microorganisms will be found in the hay infusion. Some will be classed as animals because they ingest solid matter as food; others are considered as plants, largely because they absorb their food in solution. The microorganisms are chiefly unicellular while the higher plants and ani- mals are multicellular. In the case of the multicellu- lar animals and plants there is a division of labor between the different groups of cells. The work of each group is essential to the life of the individual, and the sum of the work of all the groups is the life of the organism. In the case of the unicellular or- ganism all the complicated life processes are carried on within the limits of the single cell. The animal forms in the infusion are called pro- tozoa; the plant forms belong to three groups, the bacteria, the yeasts, and the molds. Since the bac- teria are of much greater practical importance to agriculture than the other groups of microorgan- 6 Agricultural Bacteriology isms, the consideration here given is devoted almost entirely to a discussion of their activities. The chemical transformations taking place in the hay infusion by which the elements contained in organic matter are prepared for reuse by the green plant are to be looked upon as an example of what is taking place in the soil, in water, and in many other places in nature under the action of microorganisms. CHAPTER II THE MORPHOLOGY AND PHYSIOLOGY OF MICROORGANISMS Forms of bacteria. — The plant cell is always surrounded by a firm wall which makes it impossible for the cell to change its shape as some of the lower animal forms can do. This, together with the sim- plicity of structure among the bacteria, makes the the form-types noted in this group of microorganisms comparatively few. They may be placed in three divisions as far as the shape of the cell is concerned, — the spherical bacteria, the rod-shaped, and the curved rod. The term coccus (plural cocci) is applied to c FIG. 1 — FORMS OF BACTERIA A spherical organism is termed a coccus; a rod-shaped one, a bacillus; a curved rod is called a spirillum. the spherical form, bacillus (plural bacilli) to the rods, and spirillum (plural spirilla) to the curved rods. The rod-like types may be long and slender or short and plump, the spirilla may have but the slightest curve or they may be true spirals with a number of turns of the spiral. The cocci can, of course, vary only in size. Each cell is a complete individual and need not depend on any other for aid in its hfe processes. The cells may, however, occur in more or less 8 Agricultural Bacteriology characteristic aggregations as, for example, the cocci may occur in chains; to this form of cell aggregation the term streptococcus (chain coccus) is applied. Other spherical forms may occur in masses that re- mind one of a packet or bale of twine. These are called sarcina. Deviations from these normal forms are often seen. They seem to arise when the conditions are not favorable for normal growth, as, for example, when the food supply is not well adapted to the FIG. 2 — ARRANGEMENT OF BACTERIA A spherical organism that occurs in chains is called a streptococcus; one that occurs in packets, a sarcina. When the cells are found in irregu- lar masses, the term staphylococcus is applied. needs of the organism. These are called involution forms. In the case of the bacilli, club, x, and y, shapes may be seen as well as other abnormal forms. These are generally regarded as degenerate cells and not capable of continued growth. Size of bacteria. — The unit of measurement in speaking of objects of microscopic size is the micron which is one one-thousandth of a millimeter or one twenty-five thousandth of an inch. The average size of the spherical bacteria is about one micron in diameter; the bacilh average about one micron in diameter and 3 to 6 microns long. There are, how- ever, great variations from these average sizes. Some of the rods are but 0.2 to 0.3 of a micron in di- Morphology and Physiology 9 ameter. An object less than 0.2 of a micron can not be seen even with a bacteriologically equipped microscope. Not a few microorganisms that are of importance in the production of diseases as foot and mouth disease and pleuro-pneumonia, are smaller than is possible to determine microscopically. Such organisms are known as ultramicroscopic. It is difTicult to comprehend the minuteness of bacteria since the ordinary standards of size that apply to daily life afford no parallel. The numbers found in some of our foods give a better idea of their minuteness. In a single teaspoonful of milk there may be ten to one hundred million organisms with- out their presence being evident by the change in odor or taste of the milk. A teaspoonful of bacteria of average size would contain three thousand billion individuals. Often it takes a hundred or more to equal in thickness an ordinary sheet of writing paper. It is one of the marvels of modern science that the bacteriologist can, by his laboratory methods, determine the number of such organisms in any substance with any degree of accuracy. Structure of bacteria. — All of the bacteria possess a definite cell wall which is firm and probably serves as a protecting membrane. Within the cell wall is found the protoplasm, a clear semi-liquid mass, the outer layer of which controls the kinds of materials that pass in and out of the living cell. The nucleus found in the cells of higher plants and animals 's not found in the bacterial cell. Granules of various kinds are often noted in the cells of the different kinds of bacteria. The outer layer of the cell wall may be sharply defined, or it may merge into a gelatinous layer of varying thickness, which, when sufficiently developed to be noticeable, is called 10 Agricultural Bacteriology a capsule. When the capsules are especially marked, the mass of bacterial growth may be jelly-like in consistency. Many of the bacilli and spirilla are capable of independent motion in liquids. The organs of loco- motion are delicate whip-like appendages which vary in number and in distribution on the surface of the cell in different kinds of bacteria. The flagella FIG. 3 — A PHOTOMICROGRAPH OF ANTHRAX BACILLI The chain-like arrangement is characleristie of this organism. Each member of the chain is an individual Ijacilhis. Magnified 1,000 diameters or cilia are not visible since they are beyond the hmits of microscopic vision, but by precipitating staining substances on these cilia, the diameter may be increased until Lhey become visible. The average rate of motion is about one-twentieth of an inch per minute. When one considers the size of the organism and compares this rate to that of known objects, it will be seen that the motion is very rapid. A man Morphology and Phsyiology 1 1 running at the rate of 80 miles per hour would be traveling at a comparable rate. Reproduction of bacteria. — The reproduction among the bacteria is by the division of the cell into two daughter cells, a process known as fission. The same type of cell increase takes place in the bodies of growing plants and animals. The division in the case of the bacilli and spirilla is at right angles to the long axis of the cell. In the cocci the plane of division may vary. In the streptococci the planes of division are parallel, in the sarcina cell division occurs in a regular sequence which is successively in the three planes at right angles to each other. ^^ GQ QO FIG. 4— REPRODUCTION OF BACTERIA The bacteria increase in numbers by the division of each cell into two cells. With some forms, under most favorable conditions, the process of division may take place in twenty minutes. Some of the bacilli form within the cells resistant bodies known as spores. Cell division or vegetative growth continues as long as nutritive conditions are favorable, but when the food becomes exhausted, or waste products accumulate, cell division among the spore-bearing bacteria stops, and spores are formed within the cells. On account of the position within the cell, they are often known as endospores. The most marked characteristic of the bacterial spore is its resistance to heat, desiccation and action of chemicals. Spores have been found alive after 12 Agricultural Bacteriology being dried for many years, and some can endure the temperature of boiling water for an hour or more. This resistance is of great practical importance in the preservation of foods and in the prevention of disease. A single spore is formed in the cell and since spore formation is always followed by the death of the cell, it can not be looked upon as a method of increase in numbers, but rather as an effort of the organism to live through unfavorable conditions, r^ ^m r=^ /<^ FIG. 5 — SPORE FORMATION AND GERMINATION A cell produces but one spore. Spore formation is followed by death and dissolution of the cell. Under favorable conditions the spore germ- inates and a cell similar to the one that produced the spore is developed. Vegetative reproduction then takes place. and in this respect is comparable to the seed pro- duction in the higher plants and to the encysted forms that occur in the lower animals. When the bacterial spores are placed in a nutrient solution under favorable temperature conditions, germination of the spore takes place and a cell identical to that which formed the spore is produced. Spore formation is thus a method of reproduction that enables the organism to be perpetuated from one growing, period to another, but does not result in actual numerical cell increase. Morphology and Physiology 13 Yeasts. — The yeasts are unicellular organisms like the bacteria, but the structure of the cell is more complex since a definite nucleus is present. Within the cell vacuoles, granules of reserve food and oil globules may often be seen. The yeast cells which are never motile are usually oval in shape and are FIG. 6 — REPRODUCTION OF YEASTS The yeast cell develops a bud that continues to grow until it reaches the size of the mother cell. It is separated from the mother cell by the formation of a wall. much larger than the bacterial cell, the diameter of the more common forms ranging from 2 to 12 microns. The vegetative reproduction is by the formation of buds which develop on one side of the mother cell. The bud enlarges and is separated from the mother cell by a constriction. The kinds known as the true yeasts also reproduce by the formation of spores within the cell, endospores, usually from 2 to 8 spores 14 Agricultural Bacteriology are formed by the cell. The resistance of the yeast spore to heat and chemicals is much less than that of the bacterial spores and not much different from that of the vegetative yeast cell. Molds. — The molds are multicellular and, hence, have a much more complex morphology than the FIG. 7 — REPRODUCTION OF MOLDS The reproduction is by the formation of spores that may be formed at the ends of special filaments, as in the case of the aspergillus and penicilLium groups, or within a spore case, as in the mucor group. The method of spore production on aerial filaments favors their distribution by air currents. bacteria or yeasts. The vegetative growth is in the form of branched threads which are within the food substance or on its surface. The threads or hyphae, as they are called, may be divided into cells each of which has a nucleus and a definite cell wall. Morphology and Physiology 15 -Reproduction is by means of spores which are usually produced on filaments that extend from the food medium into the air. The exact method by which the spores are produced varies with the differ- ent kinds of molds. The production of enormous numbers of spores on the aerial filaments renders their dispersal by air currents an easy matter. The spores or conidia are the cause of the color noted in many molds, as the green mold of bread and cheese. The mold spores, like those of the yeasts, are not as resistant to heat and chemicals as are the bacterial spores. Turgidity of microorganisms. — The cell main- tains its form because there is an internal pressure on the cell wall. The water in the cell contains more materials in solution than the water in which the cell is growing. The materials in solution within the cell differ also in kind from those in the liquid outside. The layer of protoplasm in contact with the wall of the living cell has the property of allowing certain substances to pass through it easily, but of not allowing the passage of certain other substances. It is a semi-permeable membrane. Water easily passes through it, while some of the soluble sub- stances in the cell sap can not. The density or the osmotic pressure of the liquid inside and outside the cell the same tends to be equalized. Since the material inside can not pass out, the only way of establishing such an equilibrium is for water to pass into the cell. This it does until the pressure on the cell wall is equal to the force drawing the water in. Growth of the cell can take place only when it has an internal pressure or is turgid. When the cell is placed in a solution that is more concentrated than the liquid inside the cell, the passage of water will 16 Agricultural Bacteriology be out. The cell will be wilted or flaccid, and in this condition can not grow. It is also said to be plas- molysed. This method of preventing growth is made use of in food preservation when the materials are placed in solutions of sugar or salt. Decomposition. — In the hay infusion referred to, as throughout nature, the bacteria are found in far greater numbers and are more widely distributed than yeasts and molds. The bacteria are capable of living and growing through a wider range of con- ditions than other microorganisms. The con- ditions for the growth of any one kind of bacteria may be relatively narrow, but the different species grow under divergent conditions, as will be seen later. The destruction or decomposition of organic matter is not caused by a single form of microbic life, but by a succession of different types, the by- products of one species forming the food supply of a succeeding group. The process of decomposition thus goes on step by step, each type concerned deriving not only the elements needed for its own cell sustenance and growth from the material, but a supply of energy to carry on its life processes. The resulting compounds thus produced become more simple, contain less and less available energy, until finally such stable end products are reached, as water, carbon dioxide, and mineral salts. These compounds are so stable chemically that they can not be utihzed by anything but the green plant which, by virtue of the energy absorbed from sun light, is able to decompose these stable compounds and build them up again into living tissues. This sequence of living forms concerned in the decompo- sition of organic matter is called metabiosis. The Morphology and Physiology 17 fermentations occurring in fruit juices exemplify the nature of the process. In extracting fruit juice, it inevitably becomes seeded with various forms of microorganisms. The conditions are especially favorable for the growth of yeasts which attack the sugars in the fruit juice, forming alcohol and carbon dioxide gas. The alcohol is an excellent food supply for certain classes of bacteria which oxidize it to acetic acid, and the cider turns to vinegar. Later the acetic acid of the vinegar may be changed to carbon dioxide and water by still other bacteria. In the decomposition of most organic compounds the sequence of life is more complicated and can not be duphcated in the laboratory. Conditions for growth. — Continued decompo- sition of organic matter can take place only when growth of microorganisms is going on. In daily life two problems are constantly presented; the first has to do with the prevention or the retardation of growth, the second with the stimulation of growth of microorganisms. Relation to moisture. — The bacteria and yeasts are to be looked upon as aquatic plants; the molds are air forms. It is evident from what has been said that none of these plants can grow in the absence of considerable amounts of water since the food must be in solution so that it can pass through the cell wall, and it must not be in too great con- centration else the water will be withdrawn from the cell and a flaccid condition produced. Again the water in the medium may be so held by the solids therein that it can not be used by the organisms. Thus in the soil when the water content reaches a certain minimum, depending on the size of the soil 2a 18 Agricultural Bacteriology grains or the amount of organic matter, it is no longer available for the growth of microorganisms or higher plants. This condition is known as a state of physiological dryness. The soil may actually contain several per cent of water, but it is held by adsorption or absorption and can not reach the cells. It is impossible to make any definite statements concerning the amount of water that will permit of growth, since it will vary widely with other con- ditions. It may be said that, as a rule, mold growth will take place in the presence of less n;ioisture than will yeast growth and that the yeasts will grow in far more concentrated solutions than will bacteria. Mold growth will occur in solutions containing 60-70 per cent of cane sugar while, as a rule, 15-30 per cent will restrain bacterial growth. The moisture conditions determine in many cases the group of microorganisms that will be concerned in the spoiling of different foods. Bread, cakes and jellies mold, but do not generally undergo bacterial decomposition. The spores of all the microorganisms are resistant to desiccation; the vegetative cells vary widely in this respect. Some of the bacteria are quickly destroyed by drying while others will resist for long periods of time. The rapidity with which cell death takes place is important in the case of the disease- producing bacteria. Desiccation of organic matter is the most important method of protecting it from the action of microorganisms. Relation to temperature. — The temperature- zone that is most favorable for bacterial growth varies with the different organisms. The pathogenic bacteria grow best at the temperature of the animal body; others find more favorable conditions at Morphology and Physiology 19 higher temperatures while still others grow best at low temperatures. The former are called the ihermophilic organisms, the latter the psychrophilic. Bacteria as a group can grow through a wider range of temperature than any other known group of organisms. Growth can occur from several degrees below freezing, if the liquid is uncongealed, to about 160° F., a temperature that is rapidly fatal to most forms of life. The temperature zone in which growth can take place may be very narrow as in the case of the pathogenic bacteria, or it may be wide as in the instance of most of the forms concerned in the decomposition processes occurring in nature. For any kind the temperature at which growth stops is only slightly above that at which growth is most rapid. The minimum growth temperature is usually far below the optimum. In any medium which is actually frozen it is impossible to conceive of growth taking place. If the freezing point has been lowered by the addition of soluble substances such as sugar and salt, growth can go on below the freezing point of pure water. In a frozen condition most microorganisms gradually die. The destruction is not rapid or complete enough so that it can be used in a practical way. Foods removed from cold storage spoil almost as quickly as if they had not been frozen. Freezing can not be relied upon to purify water, hence ice should not be brought in contact with foods unless it is obtained from sources from which water would be used. High temperatures destroy the vitality of all microorganisms by the coagulation of the cell-proto- plasm. No definite temperature can be given at which this chemical change will occur since it will 20 Agricultural Bacteriology vary widely with different organisms and under different environmental conditions. With decreas- ing water content of the medium in which the cells are found and hence of the cells themselves, the coagulation temperature of the protoplasm increases. Thus organisms in a dried or partially dried condi- tion are more difficult to destroy by heat than those with the normal content of water. It is beheved that the water content of the bacterial spore is low, and that this is the explanation of its remarkable resistance to heat. Some of 'the spores will resist the boiling temperature for hours. The vegetative cells of all the microorganisms are quickly destroyed at 212° F. In the preservation of foods, tempera- tures as low as 140° F are used since the majority of the vegetative cells of all microorganisms are destroyed at this temperature. Relation to oxygen. — Every form of life demands oxygen for the process of respiration. The individ- ual plant and animal uses the free oxygen of the air. In the case of the higher animals the oxygen taken into the lungs is combined with the haemoglobin of the blood and in this form is carried to every cell of the body. It is thus evident that the individ- ual cell of the animal body is not using free oxygen but combined oxygen. Many of the bacteria de- mand the free oxygen of the air while others may derive their supply from that contained in such compounds as sugars and other organic and inor- ganic substances. Those that can make use of combined oxygen are called anaerobes while those that are confined to the free oxygen are termed aerobes. A great host of bacteria can grow either in the absence or presence of free oxygen. These are termed facultative bacteria. Morphology and Physiology 21 As far as is known no kind of bacteria demand the total absence of free oxygen for their growth, but the strict anaerobes must have an environment that contains but minute quantities such as one- tenth of that found in the atmosphere. The range of oxygen that will permit of growth is wide in the case of most of the bacteria. Forms are known that will grow in the total absence of free oxygen and will also grow in an atmosphere that contains twenty times that found in normal air. None of the molds can grow in the absence of free oxygen although many can do so when a very small amount of oxygen is available. The molds are thus to be classed as aerobic. Their inability to grow in liquids is connected with the need of free oxygen. None of the yeasts can continue to grow for long periods in the absence of free oxygen as can the anaerobic bacteria. Growth may go on for a time but not indefinitely. Relation to acidity and alkalinity. — The bac- teria as a group grow best in nutrient solutions that have a neutral or a slightly alkahne reaction while the molds and yeasts prefer an acid medium. The reaction of any substance is an important factor in determining the groups of microorganisms that are to be concerned in its decomposition. Fruits and fruit juices are most likely to be attacked by yeasts and molds rather than by bacteria while the reverse is true in the case of meats and animal tissues in general. Relation to light. — The green plants that are to live on foods that contain no energy must of course obtain a supply from the sun and thus can grow only in the presence of an abundant supply of light while the fungus plants that live on materials 22 Agricultural Bacteriology that contain energy do not need the hght for their life processes and, indeed, are injured by it. The direct rays of the sun are especially harmful, de- stroying in a short time most of the bacteria. The injurious action is less marked in the case of the yeasts and the molds. Metabolism. — Every living thing must carry on two processes. The constituents of the cells must be built up from the food materials. This synthetic process is termed anabolism and is most marked in the green plant. The same process goes on in the animal body but here the reverse process or the breaking down of some of the con- stituents of the cells with the elimination of the waste products is most marked. This process is called katabolism while both processes are included in the term metabolism. The bacteria in growing in any food material form but little cell-substance, while their by-products are much greater in amount. In other words their work is primarily analytic in character and the process of katabolism is more marked than that of anabolism. Enzymes. — The solid food ingested by the animal must be brought into solution in the alimentary tract before it can pass into the tissues proper and be used by them. This solvent action is accom- plished by the aid of enzymes that are elaborated by different glands. For example in the saliva is found the enzyme ptyalin that has the property of changing starches to sugars; the enzyme trypsin, which changes proteins into soluble compounds, occurs in the intestinal juice. Enzymes also occur in the animal body that will decompose fat into fatty acids and glycerine. Morphology and Physiology 23 It is evident that, if the bacteria are to cause the decomposition of all kinds of organic matter, they must be able to use insoluble substances, and before doing so must in some way bring them into solution. This is accomplished by enzymes that are very similar to those elaborated by the animal body. Many of the bacteria form proteolytic enzymes, those capable of changing proteins into soluble substances; others form the lipases that act on fats, while others elaborate other kinds of enzymes, hence the work of decomposition carried on by one organism may be limited chiefly to insoluble proteins because it is equipped with the appropriate enzymes while another is fitted to act on celluloses. Food. — The bacteria differ widely in the demands as to the kind of food. Many are omniverous, grow- ing on all kinds of organic matter. The forms that are so actively engaged in the decomposition of organic matter are termed saprophytes. The parasitic bacteria are those that grow only in the body of the living plant or animal. They may or may not be harmful to the organism in which they are growing. If not harmful, they are called commensal bacteria; if they produce disease, they are called pathogenic bacteria. Some of the parasitic forms are very narrow in the food demands and find favorable conditions for growth only in the bodies of a certain kind of animal, for example the organism causing typhoid fever grows under natural conditions only in the human body. Occurrence in nature. — It is evident from what has been said that microorgamisms will be found in nature wherever organic matter is present and tem- perature and moisture conditions are such as to 24 Agricultural Bacteriology permit of growth. An immense amount of both plant and animal matter is added to the soil each year. This food supply under favorable environmental conditions, allows the rapid growth of microorgan- isms of the most varied kinds. The greater number occur in the upper layers of the soil, since here is found the major portion of the food supply, and temperature and moisture conditions are suitable for growth. The unfavorable environment limits the growth in the lower levels of the soil. It might be thought the percolating water would carry the microorganisms with it into the deeper layers, but such is not the case for the soil acts as an efTicient filter, and hence the water found in the depths of the earth is free from microorganisms. The water that enters a deep well probably contains no bacteria, but in removing it from the well it becomes con- taminated with them. Food supplies are also found in water in varying amounts and hence bacteria find here one of their homes in nature. The number of bacteria is largely determined by the food supply. In waters that receive the wash from cultivated fields and waste matters, such as sewage, large numbers of bacteria will be found, while in the nonpolluted waters the number will be less. Under natural conditions the number of bacteria found will vary inversely with the size of the body of water. It is of course evident that when the water is turbid because of the presence of soil, bacteria will abound. Microorganisms can not grow in the air but they occur in varying numbers depending on the amount of dust present. The quantity of dust is dependent on the nature of the soil, whether cultivated or not, the rapidity of the wind currents, and the frequency Morphology and Physiology 25 of rains. In the air of cities the greatest number of bacteria will be found, due to the presence of a large amount of street dust. The alimentary tract of the animal is free from microorganisms at time of birth but within a few hours they have invaded the whole length of the tract. They find here abundant food supplies and favorable temperature and moisture conditions. The products which they form are being rapidly removed. All these factors cause a most rapid growth. It has been found that from 25 to 50 per cent of the dry matter of human feces consists of bacterial cells. In the feces of animals consuming food which is more indigestible than that of man, the proportion of the feces consisting of bacteria is smaller. It is certain that no other natural substance contains such numbers of bacteria as do the animal feces. Microorganisms are to be found on the surfaces of all objects since they are constantly exposed to dust from one or the other of the natural homes of the bacteria. CHAPTER III THE CULTIVATION OF MICROORGAINSMS For the detailed study of any organism as to its form and its properties, it is necessary to separate it from all other types and to have available a con- siderable number of cells so as to determine its mass characters. Culture media. — Since such masses of micro- organisms do not occur in nature in a pure condition, it is necessary to cultivate them in the laboratory on appropriate foods. For such purposes materials of animal origin are usually employed such as milk, eggs, and blood serum. One of the standard culture media of the laboratory is made from commercial beef extract or from an infusion of fresh meat. In order to supply a larger amount of soluble nitrog- enous materials peptone is added. Vegetable in- fusions are also employed or the vegetable itself, as potato, carrots, and beets. The culture media prepared from animal materials may be so amended as to make them more suitable for the growth of many bacteria. As animal media are deficient in carbohydrates, dextrose is frequently added since this sugar is most easily utilized by most bacteria. Glycerine is often added to media of either animal or vegetable origin. With some of the strictly parasitic organisms, such animal fluids as blood serum are sometimes employed. The .chemical reaction of the culture media is of material importance, the bacteria growing better in Cultivation of Microorganisms 27 media made alkaline to litmus, while molds and yeasts thrive more luxuriantly when the reaction is acid. Liquefiable solid culture media. — Of much value in the isolation or separation of different types of bacteria are certain animal or vegetable substances which are added to the nutritive medium, and which possess the property of a liquid at certain tempera- tures, while assuming a solid condition at lower temperatures. The two substances most commonly employed-fof this purpose are gelatin, an animal prod- uct, and agar, a plant jelly derived from certain sea weeds. Gelatin dissolves in warm water and remains fluid at'temperatures above 80° F, but sohdifies as it cools^.-It, however, has the disadvantage that certaili of the digesting bacteria act upon it in such a way that decomposition changes set in that render it permanently liquid. Agar, on the other hand, does not melt until heated to 205° F, but when once melted, it does not solidify again until the tempera- ture falls to 100° F. It differs radically from gelatin in that bacteria, even of the digesting type, are unable to liquefy or digest it. Therefore it is of greatest service in the separation of different species. Colonies. — If material containing bacteria is mixed with one of these liquefiable solid media while it is in a fluid condition, and the same is then allowed to soUdify, the bacteria will be held in place. In a proper food medium and under favorable tempera- ture conditions, growth of all living cells takes place, each after its kind, and soon, within two or three days, the cell masses become visible to the naked eye in tiny spots known as colonies. The colony of each kind of bacteria has certain more or less marked characteristics which permit usually of at least 28 Agriculti-ral Bactkriolociv partial differentiation of the organisms into groups. In preparing the culture for the separation of one kind of organism from others in the mixture, it is usual to pour the melted medium into a shallow, flat, glass dish so that it forms a thin layer over the bottom, in which case it is spoken of as a plate FHl. S— A PI.A'rii CIU.TURI-: i'^ach (if the dots is a colony lliat has been lormed by the firowth of a bacterial ceil embeddecl in the solid nicdiuni. By couiiLinii the colonics the number of bacteria in the material with which the culture was seeded can be determined. culture. With Lhis arrangement the masses of growth, or colonies, can be easily examined. Deterininiiifj the number of hacleria. — The number of bacteria in any substance can be dcIiniLely determined by adding a known quantity to Ihe nulrienl medium, Ihe niiml)er of colonies ch^veloping Cultivation of Microorganisms 29 being assumed as an index of the number of bacteria per unit quantity of the material added. It is ordinarily assumed that each colony is the result of the growth of a single cell. This may or may not be true, but it is certain that there were as many bacteria in the substance as there are colonies on the plate culture, but there may have been more. Since it is impossible to establish conditions in any one medium that will permit of the growth of all the varied kinds of bacteria that may be found in any such natural material as the soil, it is impossible to determine in this way the actual number of bacteria in any substance that contains a mixture of kinds. In actual work the attempt is made to establish conditions which will permit of the growth of the kinds of bacteria that we wish to discover. For example, in the examination of milk the effort is made to have conditions that will favor the growth of those kinds that are concerned in the spoiling of milk. This limitation of the plate culture as a means of determining the total number of bacteria is not so important as it might at first seem. When the substance contains but a single form and the conditions that favor its growth are known, it is possible to determine the number of living cells with great accuracy. In fact the methods of the bac- teriologist rival the most delicate methods employed by the chemist. Pure cultures. — Since each colony has resulted from the growth of a single cell or from a number of cells of the same kind, it will contain only a single kind or type of organism, and is therefore known as a pure culture. A minute fragment of the colony may be transferred to other nutrient material in tubes, and by making repeated transfers from time 30 Agricultural Bacteriology to time so as to keep the organism supplied with fresh food and to remove it from its own waste products, a culture may be maintained in a pure condition for a long time. With this pure strain the bacteriologist can now determine all of the essen- tial characteristics and properties of the type in the different food media. Sterilization. — In the determination of the num- ber of bacteria in any substance or in the isolation and maintenance of pure cultures, it is essential that the food medium and every object which may come in contact with the culture shall be free from living organisms, i.e., it shall be sterile. This condition can be attained in the case of glassware and metal objects by heating them in an oven at a temperature of 300° F. for an hour or two, which treatment is sufficient to destroy the vitality not only of the growing cells but also the spores of all micro- organisms. For organic materials such as culture media, dry heat can not be used without injury. Such materials are rendered sterile by sterilizing in streaming steam or by boiling. By these methods it is impossible to obtain temperatures above 212° F. As noted before the spores of bacteria are not readily killed at this temperature. In order to insure the complete sterility of culture media by heating to 212° F., the following method which is known as intermittent sterilization is employed. The substance is first heated to 212° F. for a few moments in order to kill the vegetating cells; it is then incu- bated at a temperature that is favorable to the germination of the remaining spores. After a lapse of 24 hours when most of the spores have germinated it is heated again. This process depends on the germination of all the spores between the applica- Cultivation of Microorganisms 31 tions of heat. This method consumes much time and is not used so extensively in the laboratory or in practice as the method of sterilization under steam pressure. If water is boiled in an air-tight recep- tacle, the temperature is raised as the steam pressure is increased. A temperature of 248° F. exerts a much more deleterious effect on germ life than the ordinary boiUng point, 212° F. If such a tempera- ture is maintained for 15 or 20 minutes, not only the vegetating cells but also all bacterial spores will be destroyed. The heating is usually done by in- jecting steam from a boiler into a tightly closed vessel. The same method is used in the sterih- zation of dressings in hospitals, or the preparation of canned food supplies. Moist heat at a tempera- ture of 248° F. is as effective in destroying life as is a temperature of 350° to 390° F. when dry heat is employed. Metal objects such as the wires that are used in the laboratory for the transfer of pure cultures are sterilized by direct heating in a gas flame. The culture media is protected from air contami- nation by inserting a plug of cotton in the tube or flask before sterilization. This allows the air to pass freely in and out but effectually prevents the entrance of all dust and bacteria. It is possible to remove all microorganisms from such hquids as water by passing them through a filter of unglazed porcelain. This method may be used to sterilize such culture media as contains albumen and other compounds that would be in- jured by heating. A sterile or germ-free condition can also be attained by the use of chemical agents, but this method is not applicable to the preparation 32 Agricultural Bacteriology of culture media since the presence of the chemical agent destroys the food value of the medium. Identification of bacteria. — The biologist work- ing with the higher forms of plants and animals relies on the form and the structure of the organism when attempting to classify or identify different types. But the form or morphology of the bacteria is so very simple that it does not suffice for purposes of identification. The bacteriologist is therefore driven to adopt certain culture characteristics, such as are to be noted on different culture media, also the ability of the organism to cause chemical changes in the media in which it may be growing, or the biochemical properties of the organism for purposes of identification. To determine its pathogenicity or disease-producing qualities, it may be necessary to introduce the organism into the bodies of experi- mental animals. The bacteriologist is often driven to employ every means at his command to identify an organism that has been isolated from any partic- ular source. The technical descriptions of organ- isms, however, have no place in a manual of this character and concern only the professional bac- teriologist. PART II SOIL BACTERIOLOGY CHAPTER IV THE RELATION OF MICROORGANISMS TO SOIL FERTILITY Farming is primarily the production of plants of economic value. In many portions of the world animal industry is also an important phase of agriculture. Where civilization is the oldest and the population most dense, the production of animal food is relatively unimportant as it must always become when the maximum number of people is to be supported from the land. As before indicated, animal life is destructive in that it consumes large quantities of organic matter as food while furnishing small amounts in return. Much of the grain that is fed to domestic animals, under a different system could be used as a source of human food, but it is also true that animal life consumes much plant material which is not utilizable as human food, and thus does not diminish the supply available for man. The chief interest of the farmer is in the soil as the breeding ground of the plant he desires to culti- vate. The ability of the plant to grow is much influenced by the condition of the soil, as to its texture, moisture, and the amount and availability of food supply. On the texture depends whether 3a 34 Agricultural Bacteriology the plant roots can penetrate the soil readily for food and moisture. The water content, which is also of greatest importance in favoring or retarding plant growth, is also largely dependent upon the physical texture of the soil. The supply and availabihty of plant food are determining factors that often decide the yield and are therefore of greatest practical significance. Unavailable plant food. — The soil is commonly looked upon as a store house of plant food. It is usually assumed that there is sufTicient food in a fertile soil to produce a large number of crops, as experience well demonstrates in the older regions of the world where crops have been grown for thousands of years. The plant, to absorb its food through the roots, must be supplied with compounds that are soluble in the soil water, but if all plant food was stored in the soil in a soluble and available form, much of it would be removed in the drainage water. As a matter of fact, plant food is not stored in the soil in the form of soluble compounds, but mainly in insoluble combinations that are gradually made available to the plant through the agency of micro- organisms that are active in the soil. The amount of the necessary elements, such as nitrogen, that is found in the soil at the beginning of the growing season in an available form is frequently'insuITicient to meet the needs of a single crop. Available plant food. — The plant must have for its normal growth carbon, nitrogen, hydrogen, oxygen, phosphorus, sulphur, magnesium, calcium, iron, and potassium. The supply of carbon is found in the carbon dioxide of the air; the hydrogen and oxygen are obtained from water. Oxygen, needed by the plant for its respiratory processes, is also Relation to Soil Fertility 35 obtained from the air in the form of free oxygen. Iron is required in such small quantities that prac- tically all normal soils contain enough for the needs of plant life. Magnesium and calcium, the elements found in limestones, are usually present in soils in sufficient quantities to meet the food requirements of most crops. The remaining elements, nitrogen, phosphorus, potash, and sulphur, are less abundant, and the lack of any one of these in available form may be the limiting factor in the growth of the crop. Nitrogen is the element that is most likely to be low in amount. It is not sufficient that the various elements be in soluble form, but they must be present in the soil in certain definite chemical combinations before they are available to the plant. The fertility of any soil as far as the plant food is concerned depends on the amount and kind of raw food, and the number and efficiency of the microorganisms that are manu- facturing these raw materials into the finished food product. It is as essential to know the microorgan- isms concerned and the conditions that favor their development, as it is to know something of the nature and demands of the crop to be grown. The latter can never flourish until the microscopic crop has had opportunity to develop and to yield its harvest of suitable plant food. The visible crop is dependent on the invisible one. In the older portions of the world the inherent fertility of the soil, or rather the store of plant food in it, is not so important as the ability of the soil to decompose the organic matter added to it, and thus release the elements for the use of the plant. The work of the microorganisms of the soil in carrying on 36 Agricultural Bacteriology the cycles of carbon, nitrogen, sulphur, and phos- phorus is of special importance. Moisture and air. — The same factors influence the growth of microorganisms in the soil as influence the growth of the green plant. If the soil is filled with water, only the anaerobic bacteria will find favorable conditions for growth, and certain im- portant processes like nitrate building can not go on. As the water is reduced in volume, the pore spaces in the soil are emptied and the air enters, establishing a favorable environment for the arobic organisms. If the water content is reduced too much, the growth of organisms ceases. Some water always remains in the soil, but when present in very small quantities, it is so held by the soil particles that it can not be used by any living organism, and a state of physio- logical dryness results. The amount of water essential for growth is dependent on the size of the soil particles. In coarse-grained soils, as sand, much less moisture is necessary to form the water-film that surrounds each soil particle than in such fine grained soils, as clays; consequently a lower percentage of water is sufficient for development. It is probable that the soil in humid regions never becomes so dry as to destroy any considerable pro- portion of the non-spore-forming organisms by desiccation. The air supply of the soil is in an inverse relation to its water content. As the water is removed by drainage or evaporation, the pore spaces become filled with air. When organic matter decomposes, the oxygen supply is consumed and the carbon dioxide content may rise from a few hundredths of one per cent to as high as five or six per cent. The ventilation of close-textured soils, due to variation Relation to Soil Fertility 37 in atmospheric pressure and to wind, is not sufficient to cause any rapid replacement of the soil air. Under most favorable conditions aeration of such soils is insufficient to facilitate rapid growth of aerobic organisms, except at or near the surface; consequently at any considerable depth in such soils the decomposition of organic matter is slow and often incomplete, while in the open-textured, porous soils, decomposition may be too rapid for the most advantageous use of the plant food by the green plant. The ventilation of the more open soils may be limited by compacting the soil through packing or rolling, while that of dense soils is im- proved by all process of cultivation and by drainage. Temperature. — ^The temperature of the soil is also an important factor in the development of germ life. Most saprophytic organisms grow most rapidly at temperatures from 70° to 100° F. Some forms may grow at much lower temperatures, yet such growth is slow. There is a limited amount of available plant food in the soil at the beginning of the growing season, and before a plant can make any marked growth, the microorganisms must perform their work of changing the raw material into finished form for plant food. This can only occur where temperature conditions become favorable for the growth of microorganisms. The slowly responding soils, often known as cold soils, are those of a dense, close texture which retain much water, that, on account of its high specific heat, demands the absorption of a large amount of heat before the soil temperature can be raised to a point permitting rapid growth of microorganisms. By the removal of the water through drainage, less heat is required to warm the soil, and hence, decomposition processes 38 Agricultural Bacteriology start more quickly: Sandy soils warm up quickly, and are therefore better adapted for the growth of early crops, such as vegetables. Reaction. — The reaction of the soil also influences materially the rate and kind of decomposition processes that occur. A neutral or alkahne reaction favors the growth of bacteria while an acid sub- stratum is more fitting for molds and yeasts. As the more important part of decomposition processes are due to bacterial activity, it is therefore important to maintain a soil reaction that favors this type of life. The addition of lime to soils that have become acid because of the incomplete decomposition of the organic matter and the accumulation of organic acids, or to soils that have become acid on account of long cultivation will greatly increase the rate of decomposition processes. Number of organisms in soil. — But little can be said concerning the number of bacteria to be found in any soil since it will vary widely with changing environment. As a general rule the number varies directly as the fertility, since the latter is dependent on the former. The bacterial content of a fertile field soil is likely to be several millions of organisms per gram as measured by the ordinary plate culture method. It is to be remembered that by this means only those bacteria are determined that are concerned in the first stages in the decompo- sition of organic matter, while those that demand special conditions for growth escape attention. In all probability not more than a limited proportion of all living microorganisms in any soil are revealed by any single analytical process now in use. Little is known with reference to molds and other fungi in the soil or of the effect they produce. Relation to Soil Fertility 39 It is certain that an important part of the decompo- sition of organic matter is due to these forms. Another class of microscopic life, the green algae, are responsible for other physiological processes which are comparable to the activity of the higher, green plants. During the warm season when the surface of the soil is kept moist by abundant pre- cipitation, the growth may be so marked as to impart a distinct green color to the surface of the soil. The organic matter thus built up undergoes decomposition by the fungous plants. The action of microscopic animals is likewise a comparatively unknown field in its relation to soil fertility. These are present in the soil in numbers often amounting to several thousand per gram. When one compares their size with that of the bac- teria, it is evident that they may have much to do in the decomposition of the organic matter. Many of them live on bacteria. It has been claimed that one of the causes of reduced fertility of the soil is the destruction of certain classes of bacteria that are essential to the changes involved in the nitrogen cycle by the soil protozoa. It is certain that the protozoa living in water are of great importance in the sequence of life in the same. The macroscopic lower animal forms in and on the soil are likewise abundant and also function in the decomposition of organic matter. It has been shown that ten to fifteen million insects of macroscopic size may be found on a single acre of meadow land. Within the soil the common earth worm occurs in varying numbers. Determinations indicate that as high as 350 pounds per acre of these organisms may be found in the more fertile soils. All of these forms are living on complex, organic compounds, 40 Agricultural Bacteriology and are giving off simple, waste products. They also function in the pulverization of organic matter, and thus make it more easily attacked by micro- organisms. The earth worm is also of importance in the aeration of the soil through its burrows. The soil is passed through the alimentary tract of the earth worm and is brought by them to the surface in their "castings." This reworking of the soil particles materially improves the texture of the soil. When land is plowed that has been under grass for a number of years, it will be found to possess a granular structure, due to the action of earthworms and the roots of the higher plants. CHAPTER V THE DECOMPOSITION OF ORGANIC MATTER IN THE SOIL Almost every conceivable organic compound is added to the soil in the plant and animal matter that finds its way therein. All is grist for the mill of the microorganisms of the soil. So far as is known no substance is able to resist their action; even such resistant substances as chitin, horn, and gums ultimately disappear, .as do the bones of animals that are placed in the upper layers of the soil. If, in some way, they have been deposited in the lower layers of the earth or in a place where they are protected from the action of microorganisms, they will persist for ages. From the standpoint of. the action of the decomposing matter on the fertility of the soil and from the viewpoint of the farmer, all organic matter may be divided into two great groups : First, those compounds that contain only carbon, hydrogen, and oxygen of which the sugars and starches are examples : Second, the nitrogen-con- taining substances that are best illustrated by the proteins. These latter compounds also contain sul- phur and phosphorus, the cycles of which are of interest to the farmer. The decomposition of all these substances is an intricate process both from the standpoint of the chemical reactions concerned and the organisms in- volved. Though knowledge of this subject is in- complete, the main steps by which the elements in 42 Agricultural Bacteriology organic matter are once more rendered available for plant life are known, as are some of the organisms concerned. Humus. — The carbon of organic compounds ulti- mately appears as carbon dioxide, the hydrogen as water, the nitrogen as nitrate or free nitrogen, the sulphur as sulphate and the phosphorus as phos- phate. It must not be supposed that all of these changes reach their completion in a short period. A residue of organic matter or rather of partially decomposed organic matter tends to collect in the soil. To this is applied the term humus. It is a most important constituent of the soil, influencing its texture and having a marked effect on the water- holding capacity, as well as representing the main store of nitrogenous plant food. The different soils have been formed from the original rock and at first contained no organic mat- ter. Plants began to grow thereon and to undergo decomposition in the soil. This process has been going on for countless ages. The slowness with which humus accumulates is shown by the fact that the soil after this long period contains but a rela- tively small amount. A light, sandy soil may con- tain three to four per cent, the finer textured loam and clay soils from five to ten per cent. This greater accumulation in closer grained soils is due to the less perfect aeration of such soils, which condition is less favorable to the development of the aerobic organisms that are essential to the total decomposition of organic matter. Humus therefore accumulates most rapidly in soils containing a mini- mum amount of air, as in the water-logged soils of marshy land. The accumulation of peat is the re- Decomposition of Organic Matter 43 suit of the partial decomposition of the plants that have grown under these conditions. In cultivated lands there is little tendency for humus to accumulate, because the crop is generally removed, and l^e processes of cultivation, as ex- pressed in drainage and tillage, aerate the soil far better than would be the case under natural condi- tions. The growth of the aerobic organisms is therefore favored and the humus and the fresh, organic matter added to the soil are more rapidly attacked. The maintenance of the humus content of the soil is one of the important problems of the farmer in the older regions of the \yorld. The type of farming has much influence on this. If grass growing is the chief industry, no organic matter is returned to the soil and the humus content will be constantly depleted. The same is true of grain farming when no effort is made to return the straw to the soil, as is the case in the grain growing sections of this country where the straw is burned. If the farm crops are fed to animals, and what they produce sold in the form of dairy products, hve stock, wool and the like, while the manure is returned to the land, the tendency will be to maintain the humus content of the soil. The crop grown on most cultivated lands is larger than thie land would produce under natural condi- tions. In other words man by his tillage operations establishes more favorable conditions for the action of microorganisms in the soil than obtains in nature. This results in the more rapid change of the unavail- able to available plant food, a process that tends to reduce the humus content. The difference in the rate and completeness of the decomposition of organic matter in the various types of soils is shown when manure is added to them. 44 Agricultural Bacteriology An application of barnyard manure to a sandy field may show its effect in an increased crop only during the season in which it was appHed, while in the case of a clay soil its effect may be noted for two or three years. Again, when it is desired to use land for the disposal of organic matter, as in the case of sewage, the best conditions are found in an open, sandy soil in which the process will go on rapidly and completely with little or no accumulation of humus. In denser soils, a much larger area is required as the applica- tions can be made with less frequency, or the soil becomes clogged with the accumulation of organic matter which can not be decomposed by reason of the lack of growth of aerobic organisms. The dis- posal of sewage by conducting it onto the land as is done with the water in our irrigated districts is used only when areas of sandy land are available. Carbon. — The need of the elements being returned to the soil in a form in which they can be again used by the green plant is well illustrated in the case of carbon. Air contains 0.03 per cent of carbon dioxide. During the growing season an immense quantity of this gas is taken up by plant life and the carbon built up into various organic compounds. If no natural process existed by which the carbon was again returned to the air in the same form, the growth of green plants would ultimately cease, and all forms of life disappear since the green plant is the only form that can make use of the energy from the sun and is thus to be looked upon as the propelling force of the world. The respiratory processes of both plants and animals result in the formation of carbon dioxide. The artificial process of combustion carried on by man likewise results in the oxidation of carbon to Decomposition of Organic Matter 45 carbon dioxide. Under natural conditions the great mass of carbon locked up in organic matter is oxidized to carbon dioxide by the action of micro- organisms. The process can be followed in its completeness only in the case of simple compounds, as starch and sugars. The detailed chemical changes are illustrated in the following equations. 2 Ce Hio Os + H2 = C12 H22 On Starch + Water = Maltose C12 H22 0„ + H2 = C5 H12 Oe + Ce H12 Oe Maltose + Water =z Dextrose + Dextrose Ce Hx2 Oe = 2 C2 Hs H + 2CO2 Dextrose = Ethyl Alcohol + Carbon dioxide C2 He H + O2 = C2 H4 O2 + H2 Ethyl Alcohol + Oxygen = Acetic acid + Water C2 H4 O2 + 2O2 = 2CO2 + 2H2 Acetic Acid + Oxygen = Carbon dioxide + Water it IS to be noted that the first step is accomplished by the addition of a molecule of water to two of starch with the formation of two molecules of a sugar, maltose, which has the same chemical formula as cane sugar. This process is known as hydrolysis and is a very common one in the decomposition of organic matter. The complex sugar is next hydroly- zed to a simple sugar which is more available to organisms than are the more complex ones. The next step is a splitting process, alcohol and carbon dioxide being formed. All of the above steps may go on in the absence of oxygen, but the next is one of oxidation and can be carried on only by aerobic organisms. Alcohol is oxidized to acetic acid and water, the acid then being further oxidized to carbon dioxide and water. The store of energy contained in starch has been used by the different organisms 46 Agricultural Bacteriology concerned in the processes of decomposition for their life processes. The end products are completely oxidized and can be used only by the green plant. In the decomposition of carbohydrates sugars are first formed and from these organic acids. These can be used as food by some form of life. The reaction of the decomposing mass is likely to be acid due to the organic acids formed. The acids most frequently produced are lactic, acetic, formic, butyric and propionic acids. Cellulose decomposition. — The decomposition of certain carbohydrates is of special importance to the farmer. The great mass of plant tissue consists largely of cellulose, a compound that is insoluble in water and resistant to decomposition. The plant fibers such as cotton and hemp which are used for industrial purposes consist of cellulose. The larger portion of organic matter in barnyard manure also consists of cellulose. It is believed that cellulose is acted on by microorganisms with the formation of sugars, as in the case of starch. In the soil these are at once changed to still simpler compounds. Quantities of cellulose are contained in the rough feed, hay and grass, consumed by animals. It has been found that ruminants can digest about 75 per cent of the cellulose contained in the feed, horses about 50 per cent, man about 25 per cent of that contained in young, tender plants, while the dog can not digest cellulose at all. As has been previously stated all insoluble foods ingested by the animal are supposed to be changed to soluble compounds by the action of enzymes elaborated by the animal body. No enzyme capable of acting on cellulose has, as yet, been demonstrated in the animal body. The only explanation that can be offered is that bacteria, that Decomposition of Organic Matter 47 change the cellulose to sugars, are present in the alimentary tracts of animals and that these «ugars are utilized by the animal. The animals that can digest cellulose most completely are those in which the food is retained in the body for a long period of time. In the case of the cow and sheep this extends to six or seven days. In the laboratory the decom- position of cellulose is slow, but the conditions are not so favorable as in the animal where the tempera- ture and moisture conditions are at the optim], the constant removal of the by-products J^gaj the utmost importance in determining which any biological process will be main! Whether this is the only service to the animal whicK is rendered by the immense numbers of microorgan- isms growing in the intestinal tract of animals may well be doubted. Many experiments have been made with the hope of determining whether the life of the higher animals would be possible without the presence of bacteria in the alimentary tract. It is very difficult to maintain an animal in a perfectly sterile condition and still keep it otherwise normal. The general conclusion to be drawn from such experi- ments as have been most successful is that, while the bacteria may not be necessary for the life of the higher animals, they are of great importance in aiding the animal to utilize its food, and that probably such action is not confined to the celluloses. The relation existing between the animal and the bacteria of the intestinal tract is one in which the animal is deriving some benefit. There is a more or less characteristic flora for each kind of animal. If this flora is replaced by an abnormal one, the helpful relation may be changed to one in which the animal is injured. It is believed that the condition known as autointoxication 48 Agricultural Bacteriology in man is due to the replacement of the normal acid- producing flora by one that acts primarily on proteins with the production of poisonous substances which are absorbed from the alimentary tract and exert a cumulative effect on the animal. Metchnikoff, the Russian bacteriologist, also asserts that what are usually regarded as symptoms of old age are due to the gradual replacement of the normal flora by a harmful one. . ... , Pectin decomposition. — In the preparation of tlifi fibers from such plants as hemp and flax, it is necessary to decompose the binding substances that hold the fibers together. These bodies are carbohydrates and are known as pectins. In the rotting or "retting" process, it is essential that the cellulose fibers shall not be acted on so as to weaken their strength. In the case of flax, the straw may be immersed in water for a short time, or it may be allowed to lie on the surface of the ground. The bacteria and molds quickly decompose the pectins, and by breaking the straw into short pieces, the fiber may then be freed from the binding material. In some parts of the world certain streams have been found to be very favorable for the retting of flax. The river Lys in Belgium is a famous place for flax retting. The content of the water in pectin-decom- posing organisms is supposed to be the explanation of the favorable action of this river. Efforts have been made to isolate pure cultures of the pectin- fermenting organisms, and to use them in the retting of flax in tanks, instead of exposing it in natural waters. CHAPTER VI THE ACTION OF BACTERIA ON THE MINERALS OF THE SOIL The mineral portion of the soil is made up of a great number of compounds which are insoluble in water. All of the soluble compounds are of course quickly removed by the drainage water. The water that falls on the soil in the form of rain contains no mineral matter, or at most only traces, but if drain- age or well water is examined it will be found to contain considerable quantities of mineral matter in solution. It is thus evident that processes are at work in the soil by which some of the minerals of the soil are being converted into soluble compounds. In all ground waters will be found carbonates, sulphates, nitrates and chlorides of calcium, mag- nesium, potassium and sodium. Probably none of the minerals of the soil is abso- lutely insoluble in water, but most of them are so slightly affected as to be classed as insoluble. If water is not pure but contains gases or other com- pounds in solution, its action on minerals may be modified. For example water containing carbon dioxide exerts a much higher solvent property than pure water. As organic matter undergoes decomposition in the soil, soluble compounds, such as the organic acids, are formed which change the action of the water. Calcium. — The limestone that is found in the soil was removed from the water of the sea by the 4a 50 Agricultural Bacteriology action of the shell-forming animals. At the death of the animal the shell was deposited on the floor of the sea and in time limestone deposits were produced. By some movement of the earth's crust the floor of the sea was lifted above the water level and subjected to weathering. These deposits were also ground by the action of the ice in the great glaciers that swept over a large part of the country, or they were disintegrated by the weather, so that a great mass of limestone found its way into the soil. The carbon dioxide dissolved in the soil water increases the solvent action of the water on the calcium carbonate, bringing it into solution in the form of calcium bicarbonate, which is removed in the drainage water. Thus ultimately all of the limestone that was formed in the sea is returned to it, largely by virtue of the indirect action of soil organisms. It is estimated that lime is being removed from our soils at the rate of 400 to 1000 pounds per acre per year. The increased amount of decompo- sition of organic matter due to cultivation of the soil hastens the removal of the limestone therefrom. Ultimately the lime is removed to such an extent that the soils tend to become acid and limestone must be added. This again favors decomposition of the organic matter and the removal of the lime is hastened. The stronger organic acids formed in the decom- position of the organic matter also exert a solvent action on the lime carbonate as does the nitric acid formed in the process of nitrification. It is probably true that the calcium contained in organic matter appears as calcium carbonate at the end of the decomposition process. The Action of Bacteria on Minerals 51 amount thus formed is but a small fraction of that removed from the soil. Lime is added to the soil in the form of ground limestone (carbonate of calcium, marl, or burned lime, calcium oxide). It is essential that lime be in as fine a state of division as possible so that it can be mixed thoroughly with the soil and be brought in intimate contact with each soil grain. Phosphorus. — Phosphorus is another element that is needed by the growing plant, and is often found in such small amounts in an available form as to limit the yield of the crop. It occurs in the soil in the form of calcium phosphate, iron and aluminum phosphate, and in organic matter. All these forms are insoluble in water. In the southern states there are great deposits of calcium phosphate, or rock phosphate as it is often called. This rock is ground to a very fine powder which is sold as floats. This source of phosphatic fertilizer is the most important one. Superphos- phate which represents a more soluble form of phosphate is often used. It is produced by treating the ground rock phosphate with sulphuric acid, forming the acid phosphate. Phosphoric acid contains three atoms of hydrogen that can be replaced by such a base as calcium. The acid phosphates that are obtained when but one or two of the hydrogen atoms are replaced by calcium are quite soluble in water while the normal phosphate is not. Superphosphate is added when a quick acting phosphatic fertilizer is desired. If the insoluble phosphates in the soil are to be made available to the green plant, they must be rendered soluble by processes similar to those used by the manufacturer of superphosphate. The acids 52 Agricultural Bacteriology to accomplish the change must be those formed in the decomposition of the organic matter added to the soil. It is generally recoinmended that ground rock phosphate be mixed with barnyard manure or that it be applied directly to the land when the green manuring process is to be tried. It would be useless to add rock phosphate to a light sandy soil that is quite devoid of organic matter. Some of the phosphorus is also utilized by the bacteria for the formation of the cells, and on the death and decay of the organisms in some way becomes avail- able to the plant. Potassium. — Potassium is found in the soil largely in the form of an insoluble silicate, which also contains aluminum. A soil may contain thousands of pounds of this potassium compound per acre, and yet may respond to the application of such a fertilizer as potassium chloride or kainit. It is believed that the by-products of bacterial action have an effect upon the insoluble compounds, tending to bring some of the potassium into soluble form. For example the bicarbonate of lime is supposed to react with the aluminum-potassium salt forming potassium bicarbonate. It is probable that stronger organic acids formed in the decomposition of organic matter exert a solvent action on the potassium com- pounds of the soil. It is essential to have soluble potassium com- pounds not only for the green plants but for some of the important classes of bacteria in the soil. Sulphur. — Sulphur is an element found in the protoplasm of all cells. While comparatively small amounts of it are needed by the growing plant, it is certain that many soils respond to the application of a fertihzer containing sulphur. It is probable Action of Bacteria on Minerals 53 that the favorable action of superphosphate is some- times due to the sulphur it contains. The plant must have soluble compounds of sulphur for its needs, such as the sulphates of sodium, potassium, and calcium. In the decomposition of organic matter the sulphur is largely changed to sulphides, especially hydrogen sulphide. When the decomposing matter is high in sulphur, as in the case of the egg, the odor of this gas is very apparent. Hydrogen sulphide is in- jurious to plants. It is changed by what are called the sulphur bacteria first to free sulphur, and then to sulphuric acid which combines with bases to form sulphates in which compounds it is again available to the plant. The bacteria obtain energy for their life processes in this chemical change which is an oxidation process. They occur wherever hydrogen sulphide is found in nature as in sulphur springs. It is quite probable that the soil bacteria influence the tilth of the soil; that they influence the loss of moisture from it; and that they are the cause of the earthy odor that is so characteristic. This is due to volatile substances formed in the decomposi- tion of the organic matter in the soil. CHAPTER VII THE CYCLE OF NITROGEN While it is perhaps impossible to characterize any one process as more essential than some other, where all are necessary steps in a complex relation- ship, it is undoubtedly true that the cycle which nitrogen undergoes in nature is invested with the greatest interest of any of the elements, because of the intricacy of the changes involved, their de- pendence on each other, the completeness with which the various steps have been traced, and the possibility of controlling by scientific knowledge the progress of these changes. Nitrogen is constantly being removed from the soil by growing plants and it is essential that in some manner the nitrogen be returned to the soil and again made available to the plant. In the dis- cussion of the cycle of carbon it was shown that not only were microorganisms instrumental in the re- turn of the carbon of organic matter to a form in which the plant can again make use of it, but that both animals and plants are giving off carbon dioxide as a product of their respiration. In the case of nitrogen it will be seen that microorganisms are the only agents by which the nitrogen in plant and animal matter can be made available to the green plant. The amount of free nitrogen in the world is enor- mous. The air contains 80 per cent of this element. Over every acre there are 35,000 tons of this gas. The Cycle of Nitrogen 55 Nitrogen is an inert element and does not readily enter into combination with other elements, such as the oxygen of the air, but there are both chemical and biological methods of bringing nitrogen into combination. The difficulty of bringing about these reactions is indicated by the fact, that in spite of the immense amount of free nitrogen, combined nitrogen,, under existing commercial conditions, costs about 16 cents per pound. The nitrogen of the soil. — The store of nitrogen in the soil is in the humus, the residue of the organic matter that has undergone decomposition in the soil. The content of average arable soils in nitrogen is from 0.1 to 0.2 per cent but the nitrogen in humus is not available to the plant since it. is insoluble. It is a very fortunate provision of nature that a portion of the nitrogen that has been added to the soil in the organic matter has been thus stored. If it were all immediately available that which was not used by the growing plant would be quickly leached from the soil in the drainage water. In the cultivation of the soil which is necessary in modern agriculture where the crop is removed, the nitrogen needs of growing crops are in excess of the natural soil content, and therefore where farming is to be made economically profitable, it is necessary to increase the nitrogen stores of soils by the artificial addition of this -element. This addition may be made in a variety of ways. Besides the plant residues, animal material may be purchased, such as blood and bone meal, fish scrap, wool waste, etc. Guano, the excrement of birds, originally formed an important source of nitrogenous fertilizers. At the present time besides natural plant and animal material both ammonium 56 Agricultural Bacteriology sulphate and sodium nitrate are used as fertilizers. It is probable that plants can make use of nitrogen in a number of different compounds varying with the kind of plant. Some plants, as potatoes and rice, can use ammoniacal nitrogen; oats and barley make use of either ammoniacal or nitrate nitrogen showing no preference for either, while corn and beets must have their nitrogen needs supplied in the form of nitrates. Most of our cultivated plants demand all or a portion of their nitrogen in the form of nitrates and can make a normal growth only in their presence. It is thus essential that the nitrogen added to the soil be changed from protein nitrogen to nitrate nitrogen before it can be generally avail- able. This process can only be effected by the action of the microorganisms of the soil. Ammoniiication. — The process of producing ammonia from organic nitrogen is a complicated one both chemically and biologically. It is impossible to follow in detail the transformation, but the main steps are known, as are the groups of organisms concerned. The nitrogen in the plant and animal cell is largely in the form of protein, a group of very complex substances that, for the most part, are in- soluble. Under the influence of microorganisms, these complex substances are broken up with the formation of more soluble and simpler compounds. There is a constant tendency for the nitrogen to appear as ammonia, just as the carbon in a decom- posing carbohydrate is broken off as carbon dioxide. Ultimately all of the nitrogen appears as ammonia, and the name ammonification has been given to this step in the cycle of nitrogen. This change is a necessary process in nitrate formation as nitrate can The Cycle of Nitrogen 57 be formed only by the action of certain definite microorganisms on ammonia. Before protein can be made use of by micro- organisms, it must be changed into soluble form. This is accomplished by the action of protein dis- solving (proteolytic) enzymes which are elaborated by the bacterial cells. They act in a manner quite similar to such enzymes in the digestive juices of animals, as trypsin, the pancreatic ferment. In the decomposition of materials in which carbo- hydrates predominate, such as milk, the reaction of the substance after decomposition is acid, while in the case of the materials high in protein the resulting reaction is alkaline. The carbon of the protein appears in the form of carbon dioxide, as in the case of the decomposition of carbohydrates, the hydrogen as water, and the sulphur as hydrogen sulphide. Organic acids are also formed. A portion of these are in combination with radicles containing nitrogen in the form of amino acids. The simplest of these is glycocoll or amino-acetic acid. It will be seen later that the production of disease by microorganisms is often due to the formation of poisonous nitrogenous compounds in the body when some constituent is decomposed by these organisms. A great number of molds and bacteria, both aerobic and anaerobic organisms, are able to decompose nitrogenous plant and animal matter. This phase of the nitrogen cycle can thus be carried on in any type of soil whether well aerated or not, in con- tradistinction to the steps that follow in the forma- tion of nitrates which can go on only in the presence of air. The protein-decomposing organisms belong in the main to the type that can liquefy or digest such protein matter as gelatine and casein, two sub- 58 Agricultural Bacteriology stances that are constantly used in laboratory practice in the detailed study of bacteria. The rate at which the process goes on in the soil is determined by the kind of material that is being acted on, and by the kinds of organisms concerned. Certain nitrogenous substances are resistant to decomposi- tion while others are easily attacked. When dried blood, ground fish, tankage, and the liquid excrement of animals are applied to the land, the nitrogen is quickly changed to ammonia, while if. cottonseed meal or the solid excrement of animals is used the formation of ammonia will go on much slower. The availability of nitrogenous fertilizers is measured largely by the ease with which ammonification is accomplished by the soil organisms. Since the process of ammonification is an essential step in the cycle of nitrogen, it is necessary that it go on at such a rate that the plant crop will not be limited by the lack of nitrogen in an available form. All of the conditions that favor the development of bacteria in the soil are also favorable to the formation of ammonia. The establishment of a neutral re- action by the addition of lime, the aeration of the soil by the removal of water by drainage and by cultivation are conditions that favor the process of ammonification. One of the theories of diminished soil fertility seeks to account for reduced yields by the destruction of the ammonifying bacteria by the protozoa in the soil. The partial sterilization of soil by heat or by volatile antiseptics, such as carbon disulphide, often results in an increased plant growth. It has been believed by some that in such treatment of the soil, the protozoa were destroyed while the development of the ammonifying bacteria The Cycle of Nitrogen 59 went on unrestrained; hence a greater amount of nitrogen became available for the crop. The nitrogen that has been built into the tissues of the animal body is broken down into simpler com- pounds as a result of the metabolic activities of animals. These by-products often are not acted on by the ordinary bacteria that are able to decompose the more complex nitrogenous bodies. The nitrogen waste from animals is eliminated in the urine in the form of urea, hippuric acid and uric acid. The amount and relative proportions of these three com- pounds varies in the different animals. It is esti- mated that 60 per cent of the nitrogen of the food is eliminated by the horse in the urine, 42 per cent in the case of the sheep, and 31 per cent in cattle. In all cases the nitrogen in the three compounds men- tioned is changed into ammonia by means of a group of bacteria that are most often termed the urea- fermenting organisms. They differ widely in their form and structure, but all have the common property of forming an enzyme called urease which changes the urea to ammonium carbonate. This group is found in the soil and in the sohd excrement of animals. The mixing of the solid and liquid excre- ment results in the rapid change of the urea and allied compounds to ammonia. The strong odor of am- monia often noticed in the horse stalls is due to the fermentation of the urea. The urea-fermenting bacteria require oxygen for their growth. They are also favored by a strong alkaline reaction and are able to continue growth in the presence of quantities of ammonia that would quickly stop the growth of the common putrefactive bacteria. The urine of herbiverous animals is alkaline in reaction, while that of man and the car- 60 Agricultural Bacteriology niverous animals is acid. The former is a better medium for the growth of the urea-fermenting organisms. Uric acid is changed directly to ammon- ium carbonate or to urea and then to ammonia. Hippuric acid is likewise fermented with the for- mation of ammonium carbonate or first may be changed to benzoic acid and glycocoll. The later is then ammonified. The ease with which the ammoni- fication of the nitrogenous bodies in urines takes place accounts for their high availability. Nitrification. — While some of the plants having economic value can make use of ammonia, others can not, and it is necessary to have the ammonia oxidized to nitric acid. This process can be accom- plished by purely chemical means. Many porous substances have the property of occluding gases or of condensing them. The modification of platinum known as spongy platinum has this property in a high degree. If ammonia is brought in contact with spongy platinum, it will be oxidized to nitric acid. Before anything was known of the activity of bacteria in such processes, it was thought that the soil rep- resented such a porous medium. This view was strengthened by the manner in which nitrates were made which were to be used in the manufacture of gun powder. A mixture of organic matter of any kind and of earth was prepared. This was placed in a pile, usually the brush of trees was placed in the same, which was kept moist. An alkaline reaction was established by the addition of lime. The organic matter was decomposed and the leaching of the pile with water resulted in the removal of the soluble nitrates that had been formed in some manner in the saltpeter plantations as they were called. The calcium or sodium nitrate thus obtained could be The Cycle of Nitrogen 61 changed to potassium nitrate by treatment with the lye leached from wood ashes. Until the discovery of the great deposits of sodium nitrate in Chili, the larger part of the nitrates used in the industries were thus prepared. The Chinese are still using the process. Nitrates can also be obtained by the leach- ing of the soil to which large quantities of organic matter have been added and which has been pro- tected from leaching. During the civil war the con- federate states were forced to leach the soil beneath the old tobacco barns. The Chinese remove the dirt floors from their houses and dissolve the nitrate that has there accumulated. It is now known that the formation of nitrates in the soil and in the saltpeter plantation is traceable to the oxidation of ammonia by two groups of bac- teria, the first forming nitrous acid from the ammonia, while the second oxidizes the nitrous acid to nitric acid, which combines with some basic substance in the soil to form nitrates. These bacteria are specific in their action, the first group developing only when ammonia is present; the second making use of no other source of nitrogen than the salt of nitrous acid. These types of organisms use these simple substances as sources of energy and of nitrogen needed for cell formation, just as the putrefactive bacteria obtain energy and their nitrogen supply from the complex protein substances. These organisms were discovered and isolated in pure culture in 1889. They have some very charac- teristic properties. For example they are unable to grow in the presence of any considerable amounts of soluble, organic matter. Such compounds act on the nitrifying bacteria in a manner comparable to that in which an antiseptic acts on the common forms of 62 Agricultural Bacteriology bacteria. This peculiarity of the nitrifying bacteria makes it impossible to isolate them on the ordinary culture media used in the case of the other bacteria. Solid culture media made with silica jelly is used in place of gelatin or agar. Before the discovery of the nitrifying bacteria, it was thought that the presence of chlorophyll was an essential condition for the use of carbon dioxide as a source of carbon, and that no other type of life than the green plant was able to utilize so stable a substance as carbon dioxide. It was shown that the nitrifying , bacteria can decompose carbon dioxide in a manner similar to that of the green plant, ob- taining the energy needed for the process from the oxidation of ammonia and nitrous acids. Since the process is one of oxidation, these organ- isms can grow only in the presence of air. The ammonia in the soil or in the nutrient solutions used in the laboratory will be in the form of a neutral salt such as ammonium sulphate. If the nitrogen in this compound is changed to nitrous acid, the reaction of the medium will become more and more acid as the process continues. The process would soon cease in the soil if some neutralizing agent, such as lime- stone, were not added. The conversion of nitrous acid to nitric acid is retarded by the presence of ammonia. Nitrificalion in the soil. — If nitrification is to go on rapidly in the soil, conditions that will permit the rapid growth of the causal bacteria must be established. One of the most important conditions is a well aerated soil. If the pores are filled with water, anaerobic conditions prevail and no nitrifi- cation can go on. In those soils which naturally are well drained, as sandy soils, or in which the excess The Cycle of Nitrogen 63 of water is removed by tile drainage or otherwise, the air supply will be greater, and other conditions being equal, the oxidation of the ammonia formed by the ammonifying bacteria will go on quickly. It is difficult to establish the most favorable condi- tions with reference to oxygen in our soils. Only by frequent cultivation can the nitrifying process be kept at its maximum. The formation of nitrates is more rapid in the soil on which a cultivated crop is growing such as corn or roots. This accounts, in part at least, for the large amount of dry matter these crops will produce per acre. In the making of composts the frequent stirring of the pile favors the process of ammonification and of nitrification espe- cially. Nitrification goes on very slowly in acid soils such as marsh or peat soils. If these are treated with lime in such quantities as to establish an alkaline reaction, the formation of nitrates will be greatly increased. In water-logged soils the decomposition of the organic. matter is incomplete, and acid pro- duced accumulates. The removal of the water by drainage permits the air to enter and thus gives opportunity for the growth of aerobic microorgan- isms, such as molds, that will decompose the acids making the soil a better home for the nitrifying bacteria. The nitrification process goes on slowly at low temperatures. It is probable that it continues as long as the soil is not frozen. The optimum tem- perature for the organism is about 90° F. Conservation of nitrogen. — When moisture and temperature conditions are most favorable for crop production, the yield is often limited by the lack of sufficient quantities of some one element. Generally 64 Agricultural Bacteriology speaking this limiting element is most often nitrogen. It is again probable that the ammonia is oxidized as rapidly as it is formed, but not suflTicient ammonia is formed for the needs of the crop. As indicated be- fore, the process of ammonification is favored by the same conditions as are known to favor the process of nitrification. The plant leads a sort of a hand to mouth existence as far as the supp'y of" nitrates is concerned, since during the growing season the nitrates are used as fast as formed. After the crop is removed the process of decomposition continues and the nitrates accumulate in the soil to be removed during the wet periods of the fall, winter and spring. Since nitrogen is most frequently the element limiting the growth of the crop, and since the store of nitrogen in the soil is none too large, it is essential that the farmer use every means to conserve the nitrogen supply of the soil. Much can be done in this regard by keeping a crop on the land constantly. For example after the removal of corn the land may be planted to rye which will use up the nitrates in the soil. If this crop is plowed under in the spring, the organic matter will decom- pose, and the nitrogen be made available for the coming crop. It has been determined that four times as much nitrogen is lost in the drainage water as is removed in the crop. This loss is particularly heavy in the South where the long exposure of the soil to the winter rains gives a most favorable op- portunity for leaching. The fallow method of handling the soil results in the establishment of favorable conditions for de- composition, because of the well aerated condition of the soil and the retention of the moisture during the summer months. Plant food thus accumulates The Cycle of Nitrogen 65 in the form of nitrates so that when a crop of winter wheat or rye is sown in the fall, rapid growth occurs. Nitrate deposits. — Almost all of the nitrate used in the industries and as a fertilizer is obtained from natural deposits in Chili. It is believed that the de- posits are due to the accumulation of large amounts of organic matter in some arm of the sea. This was raised above the sea level and underwent decompo- sition in a region in which the rainfall was not suffi- cient to leach the nitrate into the deeper levels of the soil, so it accumulated in some such manner as it is now accumulating in certain parts of the West. In some sections of Colorado the nitrate content of orchards and of fields has become so high as to de- stroy all vegetation. Denitrification. — Nitrogen is removed from the reach of green plants by both chemical and biologi- cal processes. For example in the discharge of ex- plosives of all kinds, which contain as a rule great amounts of nitrogen, this gas is set free. The nitrates in the soil may be destroyed by bacteria. These processes are termed denitrification. There are still other processes by which the nitrogen is not lost from combination, but is changed into forms in which it is not available to the plant. In the absence of air and in the presence of organic matter many bacteria can use the oxygen contained in nitrates for their respiratory processes as the ordinary anaero- bic bacteria use the oxygen of sugar. This abiUty of reducing nitrates to nitrites and to ammonia is a very common property of bacteria and is made use of in the detailed study of organisms. When aerobic conditions are restored in the soil, the ammonia and nitrites will be reoxidized by the nitrifying bac- teria. There is no loss of nitrogen in the process ex- 5a 66 Agricultural Bacteriology cept such as may occur in a secondary reaction that may take place between the nitrite and ammonia in which the nitrogen of both compounds is set free. It is not certain this secondary reaction is of any importance in the soil, although it may be elsewhere, as in certain methods of sewage disposal. A much smaller number of bacteria are able to re- duce nitrates to free nitrogen. The conditions nec- essary for the process are first the presence of nitrate, second a supply of organic matter, and third an ab- sence of free oxygen. The organic matter is essential to furnish the energy needed to decompose the nitrate. It was seen that the nitrifying bacteria obtain energy from the oxidation of ammonia to nitrites and nitrates. If energy is set free in a chemi- cal reaction, energy will need to be absorbed to carry on the reverse operation. In the presence of air these organisms use the free oxygen and leave the nitrate untouched. The denitrifying bacteria are found in the soil and in manures, especially in horse manure. It is not believed that the process is of great economic im- portance, since conditions in the soil essential for the process do not obtain for any length of time. If nitrates were added to a rich soil or were applied simultaneously with barnyard manure, a portion of the nitrogen might be lost, but in ordinary soils no great loss of nitrogen can occur because of this process. Many soil bacteria can obtain the nitrogen needed to build their cells from nitrates. If any consider- able amount of growth of the organisms takes place at the same time the demand of the crop for ni-. trate is greatest, the crop may be limited in its growth, since nitrogen is most frequently the limit- The Cycle of Nitrogen 67 ing element. There would be the same objection to these bacteria as to weeds among a cultivated crop, namely, the removal of food that might otherwise be used by the crop. These bacteria ultimately die and the nitrogen is ammonified so they do not per- manently remove the nitrogen from the soil, but take it only temporarily from the reach of the plant. CHAPTER VII BARNYARD MANURES AND SEWAGE DISPOSAL Manures. — One of the most important by-prod- ucts of the farm as far as the fertility of the soil is concerned is the excrement of farm animals and the litter that is used as an absorbent in the stalls. Manure contains the four elements necessary for the maintenance of the fertility of the soil, nitrogen, phosphorus, potassium, and sulphur. None of these is available for the plant in the form in which it is excreted by the animal, but must undergo decompo- sition by microorganisms. Fresh manure is harmful to plants rather than helpful. The elements must pass through the cycle of changes that have been previously detailed. The farmer desires to conserve the value of the manure as far as possible and should handle it in such a manner that he may return to the soil the maximum amount of the fertilizing elements. Loss of the elements may occur by leaching of the piles. This is true for all the elements. Nitrogen may be lost by being converted into volatile substances. The farmer should also remember that organic matter is of value to the soil as a source of humus and to fur- nish energy for classes of bacteria yet to be described. From the standpoint of the kinds of microorgan- isms that grow in manures, this animal refuse may be divided into two classes, the basis of division being the amount of water they contain. Horse and sheep manure contain a smaller amount of water Manures and Sewage Disposal 69 than cow and hog manures. They are also more porous and lose water more rapidly. The solid ex- crement of the cow dries slowly. The lack of moist- ure and the porosity of manures from the horse and sheep permit the introduction of air, and hence favor the growth of aerobic organisms, especially molds. The respiration of the aerobic forms results in the production of heat which is not readily radiated on account of the non-conductivity of the organic mat- ter. As the temperature increases the more rapid growth of the organisms is made possible. This growth continues until the decomposition of the manure is very complete, and the loss of nitrogen and organic matter is marked. These so-called hot manures are subject to fire fanging. The loss of or- ganic matter can be prevented by the close packing of the piles to exclude air or by the addition of water. In the absence of the air the decomposition will be due to anaerobic forms and while the rotting will be complete in that the vegetable matter loses its iden- tity there is not as great a loss as under aerobic conditions. Cow and hog manure are cold manures on ac- count of their high moisture content and their close texture, giving no opportunity for air to penetrate. These manures do not overheat or fire-fang. In two piles of manure of the same composition one of which was piled loosely while the other was closely packed the following losses were noted. The loss of nitrogen from the loose pile amounted to 34 per cent and the loss of organic matter to 53 per cent, while from the closely packed pile, the loss of nitrogen was 28 per cent and of organic matter the same. The direct application of the manure from the stable to the field is the best way to conserve its fer- 70 Agriculturat. Bacteriology tilizing value. The use of deep stalls in which the litter is sufficient to absorb all the liquid excrement is highly desirable. The packing of the manure by the tramping of stock excludes the air and prevents the loss due to the growth of aerobic bacteria and molds. Sewage disposal. — The disposal of the wastes of human life is accomplished through the agency of microorganisms which will change the organic matter in it to simple and harmless compounds. When great numbers of people are brought together, as is the case in the large cities, the difficulty of the problem is greatly increased. The decomposition must be carried out under such conditions that it shall not become a nuisance or a danger to public health. It is of course desirable to return as much of this ma- terial as possible to the land because of its content in valuable, fertilizing ingredients. This method of disposing of the night soil is used in some of the large oriental cities, such as Tokyo, from which all the material is gathered at frequent intervals, and placed on the fields. In American and European cities water carriage is employed for the disposal of sewage. In some instances the sewage is distributed over the land in much the same manner as water is applied in the irrigated districts. It is necessary to have sandy land for this purpose so that the water will drain away quickly and admit the air to the soil so that the decomposition processes may be com- plete. If the soil is heavy as with clays, it will be constantly water-logged and the humus will accu- mulate to such an extent as to make the soil totally unfit for the purpose of decomposing large amounts of organic matter. As the water leaves the sandy soil, the solid matter of the sewage is filtered out Manures and Sewac.e Disposal 71 and a portion of the complex, organic matter that is in solution is held by the soil particles. The decom- position processes go on rapidly, and the organic matter is transformed into simple inorganic sub- stances which are leached out of the soil when sewage is next applied. The cities of Berlin and Paris make use of this method of sewage disposal. The farm home can use this same method with success if it is provided with a modern water supply so that water can be employed to carry the sewage onto the land. In most cities the sewage is turned into a body of water and the decomposition processes occur in the liquid rather than in the soil. If the body of water is large in proportion to the volume of sewage, the decomposition will take place without the produc- tion of objectionable odors or without injury to the water life. If the amount of sewage is too great, the stream will become a nuisance especially in the sum- mer and fish and other forms of life may be destroyed. The application of sewage to the land increases its fertility. On the sewage farms great crops of grasses and roots may be raised. The addition of sewage to a stream will, of course, produce an increase in the number of bacteria. This will cause an increase in the forms that live on bacteria such as the protozoa. The greater number of these low, animal forms will cause an increase in the crustaceae which serve as food for fish. Hence the addition of not too large quantities of sewage to a body of water usually re- sults in an increase in the number of fish. If these are consumed as food, some portion of the organic mat- ter is again made use of by man, and is not wholly lost. The farm home can make use of this method for the disposal of its sewage if a body of water of some size is available. In case any city draws its 72 Agricultural Bacteriology water supply from this source, it should not be used for the disposal of household sewage, because of the danger of spreading typhoid fever as will be ex- plained later. If the se-v^age is allowed to undergo partial or quite complete decomposition before it is discharged into a body of water, a much greater amount of sewage can be added to the water without injuring it in any way. Since it is not possible to establish conditions so that the decomposition can be carried out by aerobic organisms, as in the soil, the larger part of the decomposition is allowed to take place under anaerobic conditions in large tanks, called septic tanks because the processes are carried on by bacteria. The tanks are so arranged that the sew- age flows slowly through them; the solid matter settles to the bottom and forms the sludge. There soon accumulates on the surface a scum in which a portion of the gases formed in the decomposition is held. This excludes the air in a very perfect manner. If the sewage is allowed to remain in the tank 24 to 48 hours, the solid matter will be liquefied, the pro- tein changed to ammonia, the carbohydrates to acids and gases. The tanks are frequently called di- gestion tanks because of the nature of the changes which go on in them. The effluent from the tank is turbid and has a disagreeable odor. This partially decomposed material is often turned into a body of water, or it may be so treated that the decomposi- tion will be more complete. The more complete changes, which include such processes as the oxidiza- tion of ammonia to nitrates, can go on only in the presence of air. The sewage may be conducted onto filter beds of porous material such as cinders through which it is allowed to trickle slowly. The final stages Manures and Sewage Disposal 73 of decomposition here take place, and the effluent from the filter beds should be as clear as water and be harmless when added to water in any amount as far as the water life is concerned. The farm home will find it most convenient to use the septic tank for the disposal of the sewage and to apply the effluent of the tank either to the sur- face of the soil' or beneath the surface by means of tile. It is necessary to establish conditions that will FIG. 9 — A SEPTIC TANK A. drain from the house; B. baffle board to Iceep the scum (C) from passing into the second compartment; D. plug closing the entrance to the drain. The second compartment is emptied by withdrawing the plug before the sewage reaches the overflow. be favorable to the growth of the essential classes of bacteria. The installation of such a plant is a sepa- rate problem for each home. All that can here be done is to point out the necessary conditions that must be established. The tank must be of such a size that it will hold at least twice the amount of sewage produced daily. Before the sewage passes into the septic tank, it is desirable to have it flow through a grease trap to remove the fat that is found in the 74 Agricultural Bacteriology kitchen sewage for the grease is quite resistant to decomposition and may clog the drain tile. The tank must be so arranged that the sewage will enter without disturbing the sediment or the layer of scum. The inlet must be below the surface, and cross partitions should be placed in the tank to prevent currents and to keep all portions of the sewage in the tank for the same time. A second chamber is provided into which sewage enters from the first tank. The septic tank proper is thus kept continu- ally full. If the digested sewage is applied to the surface of the soil or in underdrains, it is necessary to discharge it at intervals rather than constantly, so as to give the water time to drain away and the air to enter in order that the decomposition may be completed. The second tank should hold from one- third to one-quarter of the daily volume of sewage. It is called a dosing chamber, and some means must be provided by which it can be emptied when full. This can be accomplished by installing a gate valve at the bottom which can be opened and closed from the surface. A more convenient arrangement is the automatic syphon by which the tank is emptied whenever the sewage reaches a certain depth. The drains into which the sewage is discharged are placed from eight to ten inches below the sur- face and are laid to grade with open joints as in the case of ordinary tile drains. When the tank is emptied the sewage flows into the tile and fills the entire length of the drain. It passes out of the open joints and percolates into the soil where the last steps in the decomposition of the organic matter take place, just as in the filter beds used in the disposal of municipal sewage. If the sewage was allowed to flow constantly from the tank in a small stream, it Manures and Sewage Disposal 75 would find its way into the soil through the first few joints of the tile; the soil in the immediate neighborhood would be kept water-logged, and the oxidation processes could not go on. The soil would soon become clogged with the undecomposed, or- ganic matter. The tiles should be laid near the sur- face of the soil (twelve to fifteen inches) so that ^f^^^S^ FIG. 10 — SEWAGE DISPOSAL The sewage is discharged from the septic tank into tile drains laid in cinders or gravel near the surface of the ground. The sewage passes out at the open joints; the water leaches away, and the organic matter is completely decomposed by the bacteria in the soil. oxygen shall be available for the aerobic bacteria. The tile may be laid in cinders or gravel if the soil is heavy in character. A sandy soil is best adapted for such a disposal system, but the method can be used in most types of soil. Since but little solid matter other than organic enters the septic tank, the accu- mulation of sludge is slow, and the tank will not have to be cleaned for months or years. The drains may have to be dug up and the tiles cleaned once in 76 Agricultural Bacteriology three or four years as earth worms bring in a good deal of soil. The system will work with httle atten- tion and furnishes a means by which the farm home can dispose of the household sewage in a convenient and harmless manner. This together with an abun- dant and well arranged water supply adds much to the comfort and healthfulness of the farm home. It is thus possible for the country dweller to have all the conveniences which the city home possesses in this respect and at practically no greater cost. CHAPTER IX THE FIXATION OF NITROGEN As has been seen the supply of nitrogen in the soil is the result of processes that have been going on from time immemorial under natural conditions. When human activities enter into consideration, the equilibrium of natural forces is upset. The de- composition of the organic matter goes on more rapidly and more completely in cultivated soil than in the virgin forest or prairie. The more aerobic con- ditions favor the more rapid decomposition of the humus that has accumulated, and unless care is taken to add increased quantities of organic matter to the soil, it soon becomes so depleted that profit- able crops can no longer be grown. The depletion of organic matter is of especial im- portance from the standpoint of the nitrogen supply because the humus is the chief source of nitrogen for the soil organisms. Some nitrogen is lost in the drainage water and also in the crop removed from the land. It is probable that some is also lost in the decomposition of nitrogenous matter and certainly in processes of denitrification. The depletion of the nitrogen content of the soil has been considered by some observers to be a most serious problem. Some have maintained that the population of the world will be limited because of the constant loss of nitro- gen from the soil. It is probable that such fears are unfounded, for many factors are at work tending to maintain the nitrogen content of the soil. 78 Agricultural Bacteriology Chemical processes have been devised by which the nitrogen and the oxygen of the air can be brought into combination. The most successful of these is the fixation of nitrogen by electric discharges. Where cheap electric power can be had, nitrates can be made at prices that enable them to compete with the natural product from Chili. Large quantities of such nitrates are made in the Scandinavian coun- tries where water power is abundant, and conditions are not such as to enable the power to be used for other purposes. Constant progress is being made in the development of methods by which the nitrogen of the air is made to combine with other elements, and it is highly probable that the world has little to fear from an insufficient supply of combined nitro- gen in the years to come. Nitrogen added to soil by i^ains. — Every elec- tric discharge occurring in the atmosphere results in the production of oxides of nitrogen. These and the ammonia in the air are returned to the soil in the rain water. It has been determined that from three to six pounds of nitrogen aje thus added to each acre per year. About 70 per cent of this is in the form of ammonia, the remainder in the form of oxides of nitrogen. Nitrogen fixation in soil. — The same crop can be grown on the land for thousands of years, the yield will soon reach a level below which it will not fall. This level is usually established by the rate at which some one element is made available. The limiting factor most frequently is nitrogen. The yield of the crop is usually larger than could be ac- counted for by the amount of nitrogen added to the soil in the rain water. It would thus seem that there must be factors at work in the soil that tend to main- Fixation of Nitrogen 79 tain the nitrogen content. During the latter part of the last century, Berthelot, a French chemist, studied the increase of nitrogen in soils on which no crop was growing, and which were protected from the rain. He found that a constant increase in nitro- gen took place. He estimated that the nitrogen thus added to the soil in the fields amounted to about 50 to 75 pounds per acre per year. When the soil was heated no increase in nitrogen went on. This indi- cated a biological process. It is now known that there are at least two groups of bacteria in the soil that are able to fix nitrogen. The first of these is an anaerobic group. The first organism studied was given the name of Clostridium Pasteur ianum. When a nutrient solution is made which contains all the necessary elements in an avail- able form with the exception of nitrogen, and also a source of energy in the form of a sugar, the organ- ism will grow. In the process of growth nitrogenous organic matter must be formed, and, since the only source of nitrogen is the free nitrogen of the air, the organism must in some way bring it into combina- tion. It was found that for every gram of sugar fer- mented about two milligrams of nitrogen were com- bined. Another group of nitrogen-fixing bacteria in the soil belonging to the aerobic type is known as the Azotobader group. Most of them have the ability to form a black pigment; the name Azotobader chro- ococcum has been given to this type. This type is more efficient in fixing nitrogen than the previous group in that for every gram of sugar fermented from 10 to 20 milligrams of nitrogen are fixed. Mem- bers of this group are found in nearly all soils, being more abundant in the more fertile soils. They are 80 Agricultural Bacteriology also found in water, frequently in combination with green algae. It is supposed that they live in a sym- biotic relationship, the algae furnishing the carbo- hydrate to the bacteria, and the latter nitrogen in an available form to the cells of the algae. Not only can sugar be used as a source of energy but also organic acids. It is thought that cellulose is also made available to the nitrogen-fixing bacteria by the cellulose-fermenting bacteria through the for- mation of sugars. It has been maintained that the nitrogen-fixing power of a soil could be increased by inoculation with cultures of these organisms. Such a hope has not yet been realized, although these types have been found widely distributed in all soils. Their ac- tion in the soil must be favored by the establishment of a suitable environment. A sufficient supply of both phosphorus and potassium is essential as is organic matter from the fermentation of which they may obtain the energy necessary for the combina- tion of the nitrogen. As noted in the discussion on manures, it is desirable to return to the soil as much of the organic matter as possible, irrespective of whether it contains any of the four elements that are known to be most important from the stand- point of the soil. One important role of this organic matter is to favor the growth of these nitrogen-fixing organisms. There is good reason to believe that these two groups of bacteria are important factors in the maintenance of the nitrogen content of the soil rather than simply scientific curiosities as some have considered them. Leguminous plants. — It has long been recog- nized that the leguminous plants have different properties from the grains and grasses in that they Fixation of Nitrogen 81 are able to produce luxuriant crops on lands on which the nonlegumes will make but a meager growth. They also seem to enrich the soil so as to increase the crop that follows them on the same land. This property has led to the inclusion of some type of leguminous plant in most systems of crop rotation. It was found by Liebig, the father of agricultural chemistry, that in some unknown manner the leguminous plants were able to increase the content of the soil in nitrogen and that they seemed to have sources of nitrogen that were not open to other classes of plants. / It had also long been known that there were commonly on the roots of the leguminous plants nodules or tubercles which were usually looked upon as galls similar to those produced on many plants by the stings of insects, and by other causes which stimulate the growth of the plant cells in the im- mediate vicinity to which the stimulus is applied. The tubercles were thought to be injurious or at least not of service to the plant. It had been found that the tubercles contained bacteria. Hellriegel and Wilfarth, German investigators, found in their study of the ability of different plants to grow in the absence of some one element that the nonleguminous plants were able to make but a slight growth in the absence of combined nitrogen. Some growth would always take place because of the content of the seed in nitrogen but when this was exhausted, the plant would die of nitrogen starvation. When legumes were studied, the results in some instances were identical with those obtained with the non-legumes, while at other times the legumes showed an ability to grow in the absence of combined nitrogen in the soil. No prediction could be made a*s to the outcome 6a 82 Agricultural Bacteriology of any experimcnl when legumes were used, as could be done with the other kinds of plants. These investigators found that the ability of the leguminous plants to grow in the absence of combined FIG. 11^— ^NOUULES ON SOY BEANS The noduU's on this legume arc among the largest known. nitrogen was correlated with tlie presence of tubercles oil the roots of tlic plant. If the soil was sterilized, 110 liii)crcles ai>pcarcd, and tlie plant was unal)le to glow except when nilrogeiioiis fertilizers had ])een added lo the nitrogen-free soil. A few drops of the Fixation of Nitrogen 83 teachings from the soil on which the legume in ques- tion had been grown was sufficient to induce tubercle formation and consequently growth, ff the leach- ings were heated, no effect was noted. It was thus evident that the causal factor was a biological one. Soon after the discovery of the relation of the nodule to the nitrogen needs of the plant, the nodule-forming FIG. 12— INFLUENCE OF INOCULATION Clover growing in a soil containing no-combinedinitrogcn. In jar No. 6 Ihc soil was devoid of nodule-forming bacteria; in jar No. .^) the soil con- tained an abundance of the bacteria. The meager growth in the absence of bacteria is due to the nitrogen in the seed. bacteria were isolated by the Dulch baclcriologisl, Beyjerinck. When leguminous plants are spoken of, the cultivated legumes come to mind such as the clovers, alfalfa, the peas and beans, the vetches, lupines and serradella. The native flora of all soils and of all parts of the world is made up in large part of legumi- nous plants. It has been determined that 20 per cent of the flora of the western prairies consists of wild or native legumes while slill higher figures have been oblained in the caslern slalcs. In size they 84 Agricultural Bacteriology vary from the smallest clovers to full sized trees, such as the honey locust. All, so far as known, have the same relation to the nodule-forming bacteria, and possess the ability to use the free nitrogen of the air. Leguminous plants are found growing on every type of soil. Some are adapted to acid soils while others grow best on alkaline soils. Many are adapted to high land, and a few are water plants. The legumes are distinguished from the grains and grasses by the high nitrogen content of the seed and the plant. Corn and oats contain about two per cent of nitrogen in the dry grain, while peas and beans contain over four per cent, and soybeans over six per cent. The leguminous seeds are thus valuable foods for both man and animals. Inoculation of the soil. — It is of course evident that if the soil does not contain the bacteria that are able to form the nodules and bring the free nitrogen of the air to the service of the plant, the legume must draw all of its supply from the soil. If the crop is removed, the soil will be more rapidly depleted of its nitrogen content than with a grain crop, and if the crop is turned under, no increase in the nitrogen content of the soil will have taken place. If the legumes are to be used to enrich the soil, the fields must contain the bacteria. The recognition of this fact led to the inoculation of the soil. At first soil from a field on which the legume in question had been grown was employed. Later the use of pure cultures of the bacteria was attempted. For many years it met with little success, due, apparently, to the fact that the bacteria on artificial cultivation lost their ability to form the nodules. More recently improved methods of growing the bacteria in the laboratory have been devised, and at present the Fixation of NiTROGElsr 85 use of artificial cultures for the inoculation of legumes is fairly successful. At times the results are excellent, again absolute failures are the result. The pure cultures do not give the same proportion of successes as does the inoculation with the soil from a field on which the legume in question has grown, and es- pecially when the plants have shown an abundance of nodules on the roots. The bacteria that will produce the nodules on the roots of one legume will not necessarily do so on a different legume. In the actual practice of inoculation it is the custom to employ the culture that was isolated from the same legume or to use the soil from a field on which it has been grown. There are some instances in which the same bacteria will function on different plants. The organism found in the nodule of the sweet clover will produce nodules on alfalfa and vice versa. The bacteria enter the plant through the root hairs. They stimulate the growth of the cells at the point of entrance and the nodule is produced. If this nodule is examined under the microscope, the plant cells will be found filled with myriads of motile bacteria which, in the young nodules, are rod-shaped but in the older ones assume abnormal shapes known as baderoids. In some not well- un- derstood manner, the bacteria are able to obtain their nitrogen from the air and to make it available to the plant. The bacteria derive from the plant the fermentable material necessary to secure the energy demanded for the fixation of the nitrogen. The relation is thus a mutually helpful or a symbiotic one. The plant can thus obtain a sufficient supply of nitrogen to make a good growth, but when grow- ing under natural conditions the plant derives a 86 Agricultural Bacteriology greater or less amount of its nitrogen from the soil in the same way as do other plants. No one can state the proportion of nitrogen taken from the air or from the soil under any given set of conditions. All that can be said is that the plant is unable to use the free nitrogen of the air, unless the nodules are present on the roots. If the nodules are few, a ORGANiq MATTER PLANTS FIG. 13 — THE CYCLE OF NITROGEN The green plants, the animals and the microorganisms arc concerned in the cycle of nitrogen and of the other elements. (After Wright.) small part of the nitrogen may come from the air, while if the roots are well covered with nodules, the plant will undoubtedly take the major part of its nitrogen from the air. Every farmer should make an effort to have all the legumes he may grow well inoculated. Other conditions must be made as favorable for the legume as possible. There should be an ade- quate supply of potash and phosphorus in the soil, and the reaction should be favorable for the par- ticular legume. When these conditions are met and Fixation of Nitrogen 87 the appropriate bacteria are present or have been added, nodule development should be abundant. It must be remembered that the only way in which the legume can increase the fertility of the soil is with reference to the single element, nitrogen. A leguminous crop may be grown on a field and be re- moved, and the soil remain as high in nitrogen as before the crop was grown, but this can never be true for potassium, phosphorous, and sulphur. The legume bacteria are motile. There is, how- ever, no reason to believe that they can pass through the soil in a horizontal direction for any distance. The plant root must come in contact with the bac- teria before infection can take place. Since the plant roots do not fill all the spaces of the soil, it is essential that the soil contain great numbers of these organisms in order that an abundance of nodules may be formed. If artificial inoculation is to be re- sorted to, it is important to bring the organisms in intimate contact with the roots of the young plant. This is best accomplished by the inoculation of the seed rather than by the inoculation of the soil. In the case of inoculation with soil, the seed may be moistened slightly and the fine soil thoroughly mixed with it. The seed should be treated shortly before it is to be sown. The pure cultures are usually added to water which is sprinkled over the seed. It must be remembered that unfavorable conditions such as drying and sunlight may destroy the organisms on the seed. If soil containing the organisms is avail- able in unlimited amounts, it may be broadcasted over the field, or it may be applied with a drill and well harrowed in, so as to mij^ it as intimately as pos- sible with the soil. 88 Agricultural Bacteriology Another practical method of securing optimum conditions favorable to the growth of a different kind of legume is to sow a small quantity of the seed in question with the regular crops. Thus, if it is de- sired to secure a catch of alfalfa on soil that has not grown this crop, instead of inoculation with a pure culture or infected soil, some farmers follow the practice of sowing a small quantity of alfalfa seed with all of their grain crops, even if the land is seeded down to red clover. In a few years this preliminary inoculation sufTices to infect the soil suflTiciently so that inoculation of the crop can be readily secured later. The same is true of soy beans. This Japanese bean is not native to this country and always re- quires inoculation at the outset, but if soy beans are grown on the same soil for three or four years, nodule development will readily occur. In some way the legume and the soil organisms become acclimated to each other. The matter of inoculation is especially important when a new legume is to be grown or when a legume is to be sown on a field on which it has not been grown for a number of years. The bacteria are able to grow in the soil itself. Experience has shown that they gradually decrease in number and after five or six years will be so diminished that inoculation is advisable if the legume is to be grown again. No very definite direction can be given as to the amount of soil that should be used in the inocula- tion since this will be determined by the number of bacteria in it. In case it is broadcasted over the land, several hundred pounds per acre should be added. It has been found in experimental work in the green house that the bacterial content of the Fixation of Nitrogen 89 soil to which sugar has been added may reach a point where one half pound per acre will produce a good inoculation. It has been shown that the composition of the plant is changed by the presence of the nodules in that the nitrogen content of the aerial parts of the plants bearing nodules is higher than plants on which nodules are not present. The composition of non-legumes growing with legumes is also changed in the same manner. A few non-leguminous plants may bear nodules on the roots and apparently have the same relation to free nitrogen as do the legumes. The most im- portant of these are the alders. The development of the nodules is very sparse as a rule. The legume furnishes the cheapest way of in- creasing the nitrogen content of the soil and at the same time of increasing its content in organic mat- ter. Where sandy lands are to be reclaimed and worn out soils restored, the legume is to be classed as a most important factor. The nitrogen thus added to the soil is estimated to cost from one-half to five cents per pound as opposed to 15 or 16 cents when purchased. PART III THE RELATION OF MICROORGANISMS TO FOODS CHAPTER X THE CONTAMINATION OF FOODS WITH MICROORGANISMS The decomposition of organic matter is due to the action of microorganisms that utihze the various compounds as food, and leave, as a result of their life processes, more simple substances or by-products. As most of these changes affect the quality of foods that are used by man, or even the domestic animals, it is desirable to protect food supplies in general, so far as practicable, from the action of such micro- organisms. Especially in the temperate zone is this question of food preservation of great importance, for the season during which plant growth takes place is short, and vegetable matter must be stored for use during the colder period of the year. Under the complex conditions in which we now live, the question of protection of food during the process of distribu- tion is likewise of great importance. While the action of most microorganisms in food supplies does not enhance the nutritive properties of foods, certain types are used to advantage in the preparation of some foods, as in the fermentation industries, in which the raw materials are trans- formed by the action of living organisms, Some by- Contamination of Foods 91 products are used as food or they may be of service in the preparation of food as is the case with carbon dioxide, formed by the action of yeast on sugar, which serves as a leaven to "raise" or lighten the dough in bread making. Milk. — In the following pages the discussion is limited chiefly to milk and dairy products, as practi- cally all phases of the relation of microorganisms to foods are well illustrated with milk and its products. There are especial reasons why a detailed discussion of the action of microorganisms on this food product is desirable. Milk is one of the most important foods in the dietary of the American and European people. It forms about one-sixth of the food of the popula- tion of this country and, for children, a much greater proportion of their nutriment. One milch-cow is kept for each 4.5 persons. A large portion of the milk consumed is used as raw milk, and hence its contamination with disease-producing bacteria is of great importance. Again its preservation is a problem that is presented to the producer and to the consumer daily, for, in the production and handling of milk, it becomes seeded with great numbers of bacteria which find in it a most favorable place for growth. The manufacture of butter and cheese was originally carried out on the farm ; their prepara- tion has now been largely removed therefrom, but the farmer is still the producer of the raw material, the quality of which determines the quality of the product. Factors governing decomposition. — In the de- composition of any substance the rapidity of the changes involved are determined by the number of organisms from foreign sources that are brought in contact with the material, and by the rapidity with 92 Agricultural Bacteriology which growth occurs, since decomposition processes can not occur without growth, no matter how great the initial contamination of the food. The question of food preservation, therefore, may be divided into two divisions: first, the contamination of the food; second, the destruction of the microorganisms con- tained in the food or the inhibition of their growth. The first is especially important with liquid foods, as milk, because the organisms can be uniformly in- corporated with the liquid, and their growth will not be limited to any one point, as in the case of solid foods. Again when once introduced, they can not be removed therefrom as from the surface of a solid. The source of contamination of foods in general is readily traced to contact with matter from the soil, water, or the contents of the alimentary tract of animal life. These materials harbor the bacterial life which is the cause of the changes involved, and if foods can be kept from direct contact with such organic wastes, it is comparatively easy to prevent in large measure the decomposition changes that will otherwise occur. In the protection and care of food products, it is desirable to do only those things that are of real necessity and value, rather than to waste time and effort in carrying out a mode of procedure that is unnecessarily refined. So much exaggeration is frequently found in the public prints relative to germ life and its dangers, that not infrequently un- necessary alarm is engendered in the minds of many people. This makes it important that consideration be given to the various sources of contamination from which milk becomes seeded with bacteria. Contamination of milk from the interior of the udder. — No other food is so subject to con- Contamination of Foods 93 lamination with materials rich in bacteria as is milk. It is impossible to produce it under conditions of cleanliness that are comparable to those demanded in the kitchen, the bakery or in the meat shop. It FIG. 14 — SECTIONAL VIEW OF UDDKR The milk formed by the tissues of the upper part of the gland flows tlirough the milk sinuses to the milk cistern and from this into tlie duct of the teat. (After Moore and Ward.) is not possible to produce milk that is wholly free from bacteria. When milk leaves the milk-producing cells of the udder of a healthy animal, it is probably free from these organisms, but this condition does not long obtain, for before it is drawn from the animal 94 Agricultural Bacteriology •it comes in contact with the bacteria which have in- vaded the udder through the opening of the teat and have estabhshed themselves in all portions of the spaces or channels which ramify through this organ. The greater number of organisms are found in the lower portion of the udder, in the milk cistern and in the large milk ducts. The opening of the teat comes in contact with material that contains the most varied kinds of bacteria, and it is probable that the milk ducts are invaded by many kinds. Only certain types however are actually able to grow in the udder and these only to a limited extent. As will be seen later, all body fluids have a germicidal action, and this is probably why the growth of the bacteria in the udder is not as rapid as would be thought possible under the favorable conditions with reference to the food and temperature. This germi- cidal action of milk is shown for some time after it is drawn from the udder, but it is of small importance when the question of milk preservation is considered. The kinds of bacteria that are able to grow in the udder are not those that are actively concerned in the spoiling of milk, hence, this source of contamina- tion of milk, although one which can not be avoided, is of small commercial importance. At times the udder may be invaded by bacteria upon which the milk has no germicidal action. Growth will then be unchecked and serious trouble may result. The milk at the time of withdrawal generally contains a few hundred bacteria per cubic centimeter. Great differences in individual animals are to be noted. In the same herd two animals were found that showed an average bacterial content of over 30,000 per cubic centimeter during a period of over one year. Another animal kept under the same con- Contamination of Foods 95 ,i di lions gave milk in which the average germ content was l)ul 800 per cubic centimeter. Since the greater number of bacteria are found in the lower part of the udder and hence in the first FIG. 15 — CONTAMINATION FROM TIIK All! 'I'his culture plate, ttiree inches in diameter, was exposed Icir thirty seconds in the barn during the feeding of dry fodder. A twclye inch pai! exposes over eighteen times the surface of this plate. milk drawn from each teat, it has often been the custom to discard the fore-milk or the first few streams from each teat. This will reduce the number of bacteria found in the milk, but will have little, if any, influence on the keeping properties of the milk, since the organisms found in the udder grow very slo^\■l>■ at ordinary temperatures. 96 Agricultural Bacteriology Contamination from the air. — It is impossible to keep the stable floor free from soil and manure which, when it dries, is ground to dust by the move- ments of animals and attendants. The dry feed that is used is covered with fine dust particles that are easily dislodged during the processes of feeding. FIG. ir, — DIRT FROM MILK The dirt adhfrfnl to each of these filters was removed from one T>inL of milk. 'I'he milks tested were produced on different farms. The coal of the animal is also covered with dust. All operations of bedding the animals, brushing them, sweeping Ihe floor, and feeding dry feed, such as hay and corn fodder, throw large quantities of dust into the air, and thus increase its bacterial content. This dust settles rapidly, and if the milk is handled in such an atmosphere, contamination will result. This source of contamination can be avoided by taking care that no operations by whicli Contamination of Foods 97 dust is produced are carried out shortly before the milking process. Conlamination from ihc animal. — The amount of foreign matter added to milk with the dust from Ihe air is small in comparison with that which may be inlroduced from the animal herself in case Ihe coal is soiled with mud or manure, as FIG. 17 — THE MODEL STALL Aslall oi Ihis type keeps the animals clean and thus aids greatly in pro- ducing good milk. is often the case. It is impossible to withdraw milk from a dirty animal without gross contamination. Every effort should be made to keep milch animals under such conditions as to avoid their becoming soiled with mud. Yards should be well drained or covered with some material that will not become muddy, such as gravel or cinders. The cows should be kept out of mud holes, ponds, and muddy streams, so that the udders will not become soiled. The barn should be constructed with due refer- ence to one of the most important operations that is 7a 98 Agricultural Bacteriology to be carried on in it, i. e., millving. The stalls should be so arranged as to prevent the animal from becoming coated with manure. They should be of a proper length for each animal, and the gutters should be deep and wide. An arrangement that forces FIG. 18— BACTERIA ON HAIRS The hairs from a clean cow were placed on the surface of a gelatine plate. liach colony represents one or more bacteria that were adherent to the hair. Ihe animal to the rear when she is standing and lends to draw her forward on lying down will aid in keeping her clean. Such a stall is shown in Fig. 17. The essential feature of this stall is the placing of a two by three inch piece of timber across the floor of the stall. This piece is so placed that when the animal is standing with her head against the slalted manger, Contamination of Foods 99 the cross piece will be just in front of her hind feet. Upon lying down the animal finds it uncomfortable to lie on the strip, and thus crowds to the front. An ample supply of clean bedding should be used, not moldy straw or litter from the horse stalls, but fresh straw, shredded corn stover, saw dust, or shavings. Every effort should be made to keep the animals in a clean condition since the cleaning of an animal FIG. 19— SANITARY MILK PAILS The small opening Is very efficient in keeping tlie dirt out of milk. covered with mud and manure will not usually be done. Prevention of contamination from the ani- mal, — Even under the best of conditions in both summer and winter, the coat of the animal will become dusty, and it is advisable to remove this dust as well as the loose hair before milking. This can best be done by wiping the udder and flanks with a clean, damp cloth. This process prevents the ready dislodgment of dust particles in the same way that dust cannot be readily raised from a moist floor. Sometimes it is customary in a high grade milk trade for the udders to be washed outright. If 100 Agricultural Bacteriology the udder and flanks are clipped, the cleaning process will be rendered easier and more effective. The exclusion of dirt from the animal may also be attained by the use of a small-topped milk pail, in which the opening of the top is restricted in some way. Such pails (Fig. 19) are very effective in the exclusion of dirt,, and are nearly as convenient to I i FIG. 20 — USE OF SANITARY MILK PAILS The open pail is fully exposed to the falling dust while the hooded pail excludes mueh of the dust and dirt coming from the animal. use as is the common open pail. The larger part of the dirt comes from the flank of the animal rather than from the udder. The milking machine is also an effective way of preventing the introduction of mud and dirt into the milk, since the milk passes from the teat directly into tubes that lead to covered pails. The effect which such factors have in producing clean milk is dependent on the condition of the coat of the animal. If the latter is very clean, the use of the covered pail will have httle, if any, influence in Contamination of Foods 101 improving Lhc quality of Lhc miliv, Inil if tlie animal is dirty, the influence will be great. The extent to which any producer can apply methods for the pre- vention of the contamination of milk is to be de- termined by the results produced, and whether he FIG. 21 — PRODUCING HUMAN FOOD In such a stal>li^ and from such cows iL is impossible to produce good milk. can obtain compensation from the consumer for the additional expense incurred. Common decency, however, demands that the introduction of visible cjuantiLies of mud and manure be avoided. Preven- tion of contamination should begin with those opera- tions that will have the maximum effect. Influence of the milker. — The milker should ap- preciate the sources of contamination and the means 102 Agricultural Bacteriology of preventing the introduction of microorganisms. The dress and hands of the milker should be clean, and the methods of milking such as to avoid con- tamination. Milking should not be done with wet hands. If the conditions demand something to soften the teats, vaseline may be used. The whole hand FIG. 22 — PRODUCING HUMAN FOOD A clean, well lif;!hLed, well ventilated stable and clean cows arc necessary conditions lor the production ol good milk. should be used in milking rather than stripping with the thumb and fore finger. Contamination from the utensils. — It is dif- ficult to clean dirty utensils with sufTicient thorough- ness as to remove all traces of organic matter to the extent that bacterial growth will not take place in case the temperature and moisture conditions per- mit. The thoroughness with which the utensil can Contamination of Foods 103 be cleaned is dependent on the material of which it is made. Wooden ware can be cleaned less easily and less thoroughly than can metal vessels. Again, if the utensil is so constructed that joints and angles exist that can not be readily reached in the cleaning by the cloth or brush, it will be difficult to remove accumulations of organic matter. The square angles encountered in milk cans, and the open joints that are found in the cheaper grades of tin ware easily permit this to obtain. The condition of the utensil is another factor which determines the ease with which the cleaning process is carried out. The smooth surface of a new tin vessel is much easier to clean than old rusted cans. Such complicated utensils as milking machines and cream separators are difficult to keep in a sani- tary condition. For example the rubber tubes used to conduct the milk from the teat to the holding can of the milking machine can not be entirely freed of milk. Likewise it is impossible to dry them suffi- ciently to prevent bacterial growth. In order to avoid a large amount of contamination from such sources it is necessary to immerse these tubes in an antiseptic solution such as lime water or dilute form- alin. Care should be taken to fill the tubes com- pletely with the solution and not to leave them par- tially filled with air. Cleaning of milk utensils. — Milk utensils should be washed as soon as possible after using, for if the milk is allowed to dry on the surface of the utensils, it is difficult to remove. They should be rinsed with cold water, then washed with a hot solu- tion of a washing powder (not soap powder), for soaps and soap powders are difficult to remove by rinsing, and are not as effective in the removal of 104 Agricultural Bacteriology milk and grease. A stiff brush should be used for scrubbing, for much of the dirt can be removed only by mechanical force. Finally the vessel should be rinsed with boiling water, using it in such quantities that the vessel will be heated sufficiently, so that rapid and complete drying will take place. The growth of microorganisms can not occur in the ab- sence of water. Imperfect washing will not be so serious when the utensil is perfectly dried, as will a more thorough washing with imperfect drying. If steam is available, the utensils can be thoroughly heated so as to destroy all organisms that are likely to injure the keeping quahty of the milk. Further- more, steaming facilitates the drying process. In the washing rooms of city milk depots, can driers form an important part of the equipment. All utensils should be washed after each period of use. This is especially true of cream separators. It is impossible to remove the accumulation of organic matter that collects on the wall of the bowl by run- ning water through the machine. It is essential that the machine be taken apart, well washed and dried. Strainer cloths should be washed as free from milk as possible and placed where they will dry quickly, so that no growth can occur in them. Contamination from factory by-products. — The cans in which milk is transported to the cream- ery and cheese factory are also used to carry the whey and skim milk back to the farm. This custom would be of no disadvantage if the cans were thor- oughly washed before being used again. This, how- ever, is the exception rather than the rule, and hence bacteria find their way from the whey tank to the cheese vat. The great opportunity for the whey tank to become seeded with harmful types of bacteria or Contamination of Foods 105 ycasls makes Ihis source of eonlaminalion of much importance in the manufacture of cheese. Such trouble can be avoided by heating the whey to a temperature of 140° to 155° F. as it passes from the cheese vat to the whey tanlv where it is stored until the following day. If the volume of whey is large, it will require considerable time for the temperature to fall to a point where any bacterial growth can take FIG. 23- WIIEY DISPOSAL It is impossible to clean such whey barrels. The whey may contain harmful Itinds of bacteria which (ind their way from the barrels to Die cheese kettle because of the poorly washed milk can in which tiic whey has been carried. place. Heating to the above temperature is sufTicient to destroy all non-spore-forming bacteria. Whey so treated will be sweet when returned to the farm and will be of higher feeding value. It will also be free from disease-producing organisms that may thus be carried from one farm to another. It has been found that the enhanced value of the butter and cheese is more than sufficient to pay for all cost of treatment. 106 Agricultural Bacteriology Factors determining number of bacteria. — Milk is subject to contamination at every stage in its handling, even after it has reached the consumer. The high bacterial content of market milk is due to two factors: first, the arriount of initial contamina- tion; second, the growth of the bacteria in the milk. A high bacterial content does not necessarily mean a milk produced under undesirable conditions with reference to cleanliness. Straining and clarifying milk. — It might be thought that the foreign matter introduced into milk could be removed, thus reducing, the bacterial content as well. For the removal of dirt, straining the milk is generally resorted to. This practice may be carried out so as to remove the insoluble material that has found its way into milk, but it will have little, if any influence, on the reduction of bacteria, since they are readily washed off from the surface of solid matter, and are able to pass the pores of the finest strainer that may be used. All processes of straining and filtering can serve only to improve the appearance of the milk, but have little if any influ- ence on its keeping quality or healthfulness. When milk is passed through a separator, there is an accumulation of more or less slimy material on the wall of the separator bowl. This material, known as separator slime, ,is made up of cellular elements from the interior of the udder, dirt, and other in- soluble matter. With perfectly clean milk, this slime is white, but when more or less mixed with dirt and other foreign matter, it is gray in color. Bacteria can not be removed from milk as from water by filtration, because the other solids in the milk, such as the fat globules, which are larger than the bacteria, would likewise be removed. The bac- Contamination of Foods 107 terial content of the slime removed from the milk in processes of clarification is very high, much greater than that of milk. Due to the small amount of slime removed from the milk, the actual reduction of the bacterial content is so. small as to be of no practical importance. A modification of the cream separator is used for clarifying the milk. While this process removes all dirt, it does not essentially change the keeping quality. Influence of food on contamination of milk. — The bacterial content of the feed or water consumed by the cow can not have a direct influence on the kinds or number of bacteria that are introduced into the milk, as these organisms do not pass from the intestine through the blood and appear in the milk directly. They may, however, have an indirect in- fluence by changing the type of the bacterial flora in the manure, some of which nearly always finds its way into the milk from the dirty flanks of the ani- mal. If the feed is such as to make the manure more liquid than usual, it is more difficult to keep the ani- mals clean. The use of improper feed may also alter the taste of the milk or its value as food. When such feed as cabbage, turnips, rape, wild onions, and some weeds that may be eaten in the pasture are consumed, cer- tain volatile principles are absorbed directly into the circulation and may then appear in the milk, just as the odor of onions appears in the exhaled breath. Where such substances are eliminated in the milk, the normal taste is changed. This of course does not injure the healthfulness of the milk but decreases its commercial value. Certain drugs, as mercury, arsenic, or strychnine, if given to cows may be eliminated with the milk. The milk of an animal 108 Agricultural Bacteriology receiving medicine should not be used for human food, and especially for the feeding of children. Fats readily absorb any odors with which they come in contact, and milk, by reason of its cream content, thus absorbs some odors, such as bananas, with especial avidity. In order not to injure the flavor of the milk it should be kept and handled in an at- mosphere that is free from odors of all kinds. These absorbed odors are often difficult to differentiate from those due to the growth of bacteria in the milk. Coiitamination of other food. — Oysters and other shell fish are in many respects comparable to milk in that they are subject to contamination and in that they can not be cleaned as can fruits, vege- tables and meats. Chopped meats are also quite comparable to milk in that they cannot be cleaned and in that bacterial growth takes place throughout the entire mass, the bacteria becoming incorporated with the meat during the grinding. It is essential that all foods that must be used without previous cleaning, and especially those that are eaten without cooking, be protected from con- tamination, both for aesthetic and sanitary reasons. Bakery goods, candies, etc., should be handled with due regard to cleanliness. Dust contamination and pollution incident to handling food products are especially to be considered. CHAPTER XI THE CONTAMINATION OF FOODS WITH PATHOGENIC BACTERIA Diseases of man and domestic animals, that are due to the growth of bacteria in the living body are often spread from one individual to another by dis- charges from the body of the diseased individual which in some way find their way into the body of another susceptible individual. The actual mode of transmission from one to the other can take place in a number of ways, as will be discussed in a later chapter. The contamination of foods with patho- genic organisms is one of the chief ways of distribu- tion. Milk is one of the most important foods in this respect, for it is subject to contamination, not only by the bacteria that produce disease in man, but by those that cause disease in the milk-producing ani- mals. Man is susceptible to a number of diseases that affect cattle, so milk may serve to transmit dis- ease from man to man, from cattle to man, and from cattle to cattle. The diseases most often spread by milk are tuberculosis, typhoid fever, diphtheria, and scarlet fever. Some idea of the importance of milk as an agent in the distribution of disease is shown in the following summary of epidemics that have been traced to milk in Boston for the years 1909-1911. 1907 Diphtheria 72 cases 1907 Scarlet fever 717 cases 1908 Typhoid fever 400 cases 1910 vScarlet fever 842 cases 1911 Tonsilitis 2,064 cases 110 Agricultural Bacteriology Tuberculosis. — Tuberculosis is a disease that af- fects many portions of the body. It is characterized by the formation of nodules or tubercles that increase in size, forming abscesses in the contents of which the tubercle bacillus is found. When these abscesses are in organs that have an opening to the exterior such as the lungs, the organisms may be thrown out from the body if these abscesses discharge their con- tents into the air passages. From the standpoint of the contamination of the milk, such abscesses in the lungs and udder are of the greatest importance. The infectious material is coughed up from the lungs; a portion is ejected from the mouth, while the re- mainder, which is swallowed, passes unharmed through the alimentary tract and appears in the feces. Since some fecal matter inevitably finds its way into milk, the opportunity is offered for the contamination of milk with the tubercle bacilli. Similar abscesses may also form in the udder, the contents of which will be discharged into the milk ducts. The extent of contamination is much greater in the case of tuberculosis of the udder, and hence is more important, although possibly it is less fre- quent than contamination from the lungs, either by means of manure or the dust from the stable. It is estimated that one to two per cent of tuber- cular cows have the udder involved. The per cent of animals having tuberculosis varies widely in differ- ent parts of the world. Generally in factory practice milk containing tubercle bacilli is diluted with the product of healthy animals/ to a considerable extent. It is probable that this dilution serves to lessen some- what the danger of the spread of tuberculosis by means of milk, but usually when the udder is af- fected, the danger of infection is quite marked. The Contamination of Foods 111 following figures give some idea of the frequency with which mixed market milk sold in the cities con- tains tubercle baciUi. Of 144 samples examined in Chicago in 1910, 10.5 per cent were found to con- tain tubercle baciUi. In New York 107 samples were examined in 1909, 16 per cent of which contained the organisms. In 1906, 233 samples were examined in Washington, of which 6.7 per cent contained the tubercle bacillus. It is impossible to examine market milk in any effective manner for the presence of tubercle bacilli in order to determine whether an animal is eliminat- ing the organisms; hence, under practical condi- tions, it is necessary to consider any animal that has the disease as a potential source of danger, although she may not be giving off the organisms. The usual method of preventing the contamination of the milk with bovine tubercle bacilli is to apply the tuberculin test to the animals, and to remove all animals which react to the test. The legality of the test as a means of thus protecting the milk supply has been passed on favorably by the United States supreme court. The danger of contracting tuberculosis from the ingestion of contaminated milk is very slight in the case of the adult on account of his relative insus- ceptibility, but with young children it is much greater. From present available data, it is estimated that about seven per cent of human tuberculosis is due to infection from bovine sources. Almost all of this is found among children. About 25 per cent of the disease in children is thought to be due to the use of contaminated milk. The organisms are able to penetrate the tissues of the throat, especially the toncils, from which they pass to the lymph glands of the neck, causing them to enlarge, or the bacilli may 112 Agricultural Bacteriology pass through the intestinal wall, producing tuber- culosis of the abdominal cavity. The importance of bovine tuberculosis as a factor in the occurrence of the disease in man has been established only within the last few years. Through detailed studies made on organisms isolated from fatal cases of the disease in people of all ages, it has been possible to ascertain whether the organism in question belonged to the human or bovine type. The organism from cattle is more virulent for most experimental animals than is the organism from man; this together with the differences in growth on culture media enables the bacteriologist to tell whether the organism originally came from cattle or from man. Foot and Mouth Disease. — FooL and moulh disease is another disease that affects both man and cattle. The organism is contained in the blisters that develop on the mouth and feet, and at times on the teats, where they are easily ruptured during milking. Under such conditions the organism, the nature of which is unknown, is thus introduced into the milk. The ingestion of infected milk may serve to produce the disease, especially in children where the same symptoms appear as in cattle. Normally this disease does not exist in this country, but from time to time more or less serious outbreaks have occurred, due to transmission in various ways from Europe and other infected regions. Garget. — Inflammation of the udder is often due to its invasion by streptococci. It is believed by some that the organisms from this source are the cause of epidemics of septic sore throat in man. It has been definitely proven that milk is an agent in the spread of this trouble, but it is by no means so Contamination of Foods 113 certain that the organism from inflamed udders is the cause of the trouble, for opportunity is constantly offered for the contamination of the milk from people suffering from the disease. In the case of certain diseases of milk-producing animals the organism is present in the milk at the time of its withdrawal from the udder, not only from animals that have the disease but from animals that are apparently in normal health. Malta fever, a disease of goats, and contagious abortion of cattle, are examples. In the case of the latter it is known that an animal may continue to excrete the bacilli for years. It is not known that the organism is of any sanitary importance, as far as man in concerned, but, undoubtedly, such animals are the cause of the spread of the disease to other individuals. It is in this way that the disease is spread by purchase of affected stock. In a general way it may be said that the milk of an animal that is suffering from any disease what- ever should not be used for human food. It may not be true that the milk would be distinctly harmful, but it is always well to err on the safe side. Such troubles as abscesses on any part of the body, in- flammation of the intestines, or any abnormal con- dition after calving should cause the rejection of the milk. Typhoid fever. — Of those diseases that do not affect cattle, but are spread by means of milk, ty- phoid fever is the most important. The organisms producing the disease enter the body with the food or drink. They establish themselves in the intes- tines, and from there penetrate to other parts of the body. They are eliminated in the feces and the urine. From these infectious materials, they are 8a 114 Agricultural Bacteriology brought in contact with food and drink by a num- ber of agents. Not only milk, but many other foods and water are concerned in the spread of typhoid fever. The methods by which food products may become con- taminated are so similar that all may be discussed together. Milk has one pecuHarity that is not com- J.HBI Af m m m t\ \M FIG. 24 — TYPHOID FEVER SPREAD BY MILK A village with the milli producers and dealers represented by small squares and the milk routes by lines. Three hundred and sixty-eight cases of typhoid fever occurred on the route of one milk man and but eighteen on the routes of the other nine men. Contamination of Foods 115 mon to most other foods in that the typhoid bacilli find in it an excellent culture medium, and since growth can take place at temperatures far below that of the human body, indeed at temperatures at which milk is often stored, the shght contamination that might be of small importance in other foods may be the starting point of a great epidemic when milk is concerned. The typhoid organism is unable to grow in water and does not persist for a long time. ;CESS POOL FIG. 25 — WELL CONTAMINATION The well water may be polluted from a cess pool or vault if the well is close to them by the percolation of the water into the well. Contamination of water supplies. — The most frequent manner in which water is contaminated is by its pollution with sewage containing the organism. The methods of disposal of both municipal and farm sewage often are such as to permit of its introduction into the water. Municipalities often draw their water supplies from a body of water which is con- taminated with sewage. In individual supplies, the farm well is often located in close proximity to the outhouse, and if the well is not protected from the entrance of surface water, or seepage from the upper soil layers, infectious material may be carried into 116 Agricultural Bacteriology the well by the drainage water. If the well is a drilled one, the iron casing should extend to an im- pervious layer of soil or rock, and the curb should be constructed so that no waste water can find its way FIG. 26— SAFEGUARDING THE WATER SUPPLY The ground water is sterile; surface water contains many bacteria and may contain harmful ones. The well should be protected from sur- face seepage into the well. If the well is dug instead of drilled, the upper portion of the protecting wall should be laid in concrete, and the surface properly protected by a concrete curb. No definite sLalemenls can be made as to the distance infectious material may be Contamination of Foods 117 carried by the drainage water, as it depends so much on the porosity of the soil. If the soil is clay, gravel, or sand, the movement of infectious matter will be only for a short distance, but if the soil is underlaid with limestone, underground channels may develop by the solution of the limestone that may carry the organisms for considerable distances. The well should be not less than 100 feet from any possible source of pollution. The chief protection is to be found in the filtering effect of the soil. If no water can enter the well until it has first passed through at least fifteen to twenty feet of soil, there will be little danger of infection. There are many ways in which contaminated water may come in contact with milk, as in the case of rinsing the milk utensils with cold water, and the accidental or intentional addition of water to milk. The protection of the farm water supply against contamination with typhoid bacilli is important, not only from the standpoint of the health of the farm home itself, but also from the point of view of the homes to which the products of the farm find their way. Milk is the most important product in this regard, since the typhoid organism can grow so luxuriantly in it, and since so large a proportion is used without previous heating. Springs are the outlets of underground streams. Spring water is usually free from bacteria as it issues from the ground, but it immediately becomes seeded with organisms from contact with the organic matter in the soil, unless special precautions are taken to guard it. Contamination from typhoid patients. — Di- rect infection of milk sometimes occurs where a person in contact with the typhoid case, as a nurse. 118 Agricultural Bacteriology also is concerned with the preparation of food or the handling of milk. Such infection can occur only when carelessness obtains with reference to the cleansing of the hands after handling the patient. In these days physicians give strict directions that all the discharges of a typhoid patient shall be treated with a disinfectant that will destroy the typhoid bacilli, but if care is not taken by those coming in contact with the patient, they may not only acquire the disease themselves, but may serve to spread it to others. The recognized case of typhoid is not so dangerous as those that are not clinically apparent, such as the mild cases for the treatment of which no physician is consulted. The attack may be so slight that the individual may not be aware of any appreciable illness. In these cases the virulent organism is eliminated to the same extent as in pronounced cases. Typhoid carriers. — When recovery from the disease takes place, the bacilli usually disappear from the body, but in about four per cent of cases, the organisms persist for a period of time. Excep- tional cases have been recorded in which the or- ganisms were eliminated for many years after re- covery. Such people are known as typhoid carriers. It is estimated that about one in each thousand of the population is to be classed as a typhoid carrier. Whenever such a carrier is engaged in the prepara- tion or handling of food, an epidemic of typhoid may result. An outbreak of 400 cases in New York city was traced Lo the person that had the disease forty-seven years before. As has been stated the contamination of milk is important, due to the op- portunity for the growth of the bacilli, but any food may become contaminated and thus become the Contamination of Foods 119 cause of trouble. Since no one recognizes the typhoid carrier as such,, and since generally he does not even know his own condition, the difficulty of protecting the public against such source of danger seems well nigh insurmountable. Oysters and typhoid fever. — Shell fish repre- sent another food that is not infrequently concerned in the spread of typhoid fever. When oysters are removed from salt water in which they have grown, they are placed in fresh water for a short period in order that they may be "fattened" or "plumped," due to the absorption of water. If the water in which they are placed is polluted with sewage, the oyster will be contaminated, and since they are often con- sumed raw, opportunity is presented for the trans- mission of the organism. The house-fly. — Another agency in the distri- bution of typhoid fever is the house fly. If infectious material is deposited where flies have access to it, they may carry the organisms to the food with which they come in contact. All privy vaults should be so constructed that flies can not or wfll not enter them; all homes and all places in which food is prepared or sold should be screened for protection. Diphtheria and scarlet fever. — Diphtheria and scarlet fever are also spread by means of milk. The opportunities for the contamination of foods with the causal organisms of these disease are not so varied as in the case of typhoid fever. It is probable that the infection is largely from mild cases that are not recognized or through the agency of one acting in the dual capacity of nurse and milker. Infant mortality. — It is thought that the milk supply has much to do with the high death rate of 120 Agricultural Bacteriology young children. In some American cities over 40 per cent of the children die before they reach one year of age. The greater number of these are fed on some substitute for mother's milk, cows' milk being most frequently used. It is claimed that if the milk were of better quality, if it contained less bacteria, and had undergone less decomposition, a great de- crease in death rate of artificially fed children would be noted. It is certain that the most powerful factor concerned in the great improvement of market milk that has taken place in recent years has been the effort to reduce the high rate of infant mortality. While it has undoubtedly been a great factor, many others have also functioned. Poisonous foods. — Foods may become poison- ous because of the decomposition induced by bac- terial growth. The foods most often, at fault are cheese, ice cream, fish, and canned goods. Any canned material that shows any abnormal appear- ance on opening, either in color or appearance, should not be used as food. Fish and shell fish in which any evidence of decomposition is at all ap- parent should be discarded. The decay of fruits is of small sanitary importance, for the portions af- fected may be discarded, and the remaining portion used. It must not be thought that all processes of decomposition make a food product harmful. As will be seen later, many food products are rendered more appetizing or more digestible as a result of decomposition processes of various kinds. CHAPTER XII THE PRESERVATION OF FOODS It is impossible to handle foods so as not to allow microorganisms to come in contact with them. With greater or less rapidity, depending on whether the organisms find in or on the food favorable condi- tions for growth, decomposition changes will ensue and the food is rendered worthless. In a previous chapter the ways in which foods become seeded with microorganisms were studied. The more cleanly the conditions under which foods are handled, the smaller will be the number of organisms brought in contact with them, and the less rapidly will the changes take place. The greater the number of or- ganisms initially present, the more rapidly the de- composition changes occur, other conditions remain- ing constant. Hence, one of the chief ways of pre- serving foods is to prevent its contamination with germ life. But mere prevention of contamination is not sufTicient and must be supplemented by other ways, which roughly may be divided into three classes, the removal of microorganisms from foods, their destruction, or the establishment of conditions that will retard or prevent their growth. These methods may be used singly, or in combination. The microorganisms that may adhere to the sur- face of solid foods may often be removed in large part by washing. Microorganisms are practically always contained in dirt and other matter foreign to the food. Consequently, the removal of this dirt 122 Agricultural Bacteriology will tend to reduce such contamination. Washing or wiping of fruits such as apples tends to remove the molds that are concerned in rotting processes, and the washing of meats and of many other foods will have some influence in retarding decomposition changes. Filtration of water. — Water may well be classed as a food, and while it is not exposed to decomposi- tion by bacteria, it may contain disease-producing organisms and thus serve to spread disease. The processes which are "sometimes used to preserve foods also destroy the pathogenic organisms which may be found in water. The bacteria may be en- tirely removed from water by passing it through a properly constructed filter. For this purpose, un- glazed porcelain filters are used for household and laboratory purposes. The pores of these filters are very fine and tortuous, so that the bacteria are held back even though the pore spaces are of greater average diameter than the organisms. If, however, the filters are used for a considerable period of time without cleaning, some forms of bacteria will mul- tiply in the pores and the filtrate will no longer be sterile. In actual use the filters must be frequently cleaned and sterilized, if they are to function in an efficient manner. The purification of water in per- colating through the soil is also due to the same principle. Many cities purify their water supplies by filter- ing surface waters through filters composed of layers of gravel and sand, arranged so that the finer ma- terial is on the surface. The upper surface of the filter soon becomes coated with a gelatinous layer of bacteria and sediment which is so coherent that it prevents the organisms in the water from passing Preservation of Foods 123 through the sand. As this sediment layer increases in thickness, it becomes less permeable, thus reduc- ing the amount of water which will pass the filter. In time this filtering surface must be removed. For the first few days after this disturbance, the effi- ciency of the filtration process is reduced, until the gelatinous layer is re-established. By means of this process, it is possible to reduce the bacterial content of surface waters from 95 to 99 per cent and to eliminate practically all danger from, typhoid and other water-borne diseases. Another method of purifying water supplies con- sists of producing gelatinous precipitates by the ad- dition of chemical agents such as salts of iron or aluminum. When these are added to water con- taining hme in solution, insoluble compounds are formed of a jelly-hke nature. All fine particles, in- cluding bacteria, are enmeshed in the gelatinous material, and thus can be removed readily by sedi- mentation or filtration. When water so treated is passed through coarse filters, these gelatinous pre- cipitates are readily removed and the water thereby not only clarified but its germ content materially reduced. The fine turbidity present in wines is removed in a similar way. When small quantities of gelatin are added, the tannin naturally present in wines causes an insoluble gelatinous precipitate to be produced which is readily removed by filtration. Preservatives. — While all foods contain more or less microorganisms, no appreciable decomposition can take place without the actual growth of such organisms; hence, any treatment that will inhibit or prevent the growth of bacteria and allied organ- isms will favor the preservation of food supplies. 124 Agricultural Bacteriology Cell-multiplication can be prevented by a great va- riety of chemical and physical agents. Many chemi- cal substances exert a deleterious effect on the growth of microorganisms, and when they are used in foods, are called preservatives. Among those most com- monly employed have been boric acid and borates, benzoic acid and its salts, salicylic acid, and formalin. Boric acid is used in butter, especially in that made in New Zealand and Australia which is to be shipped to the English^markets. Benzoic acid is used in catsups and ciders, while salicylic acid is contained in the canning powders that have been widely sold in the past. Formalin has often been used in milk. One part of formalin to 10,000 of milk will have a marked inhibiting effect on bacterial growth. The use of such chemicals in foods is prohibited by law in most states and by the national government in its control of the interstate commerce in foods. The use of benzoates is allowed in certain foods, but the label must bear the statement of the amount used. The question of the effect of such preservatives on health is one on which different views are held by the various authorities. The prohibition of their use is a wise one, since the foods in which they would be used can be preserved by other means concerning which there is no question as to their effect on the health of the consumers. Certain chemicals sometimes added to foods unite with definite decomposition products, thus masking the effect of the changes produced. If sodium bi- carbonate is added to milk, it combines the lactic acid, and thus reduces the acidity of the product. Sulphites are used with meats, particularly chopped meats, to impart a bright red color to the same and to neutralize the odors produced in putrefactive Preservation of Foods 125 changes. Potassium nitrate is especially used in corned beef. Some of the nitrate is reduced to nitrite which is an active agent. It also imparts a bright red color to the meat. The use of this latter sub- stance is not regarded as dangerous. Generally speaking, such chemicals can be used in sufficient amounts to accomplish the desired result without being evident to the taste. If their use was san- tioned by law, materials unfit for food would be sold. Essential oils. — In some cases, preservative ma- terials are added which impart a flavor to the food. Such substances as cloves, cinnamon, and mustard inhibit to a marked degree bacterial and especially mold activity, due to the action of the essential oils which they contain. They are used especially in pickles, catsups, mince meat and fruit cake. Creosote compounds. — In the smoking of meats, chemical compounds of the creosote type are pro- duced by the slow or imperfect combustion of wood and are deposited on the surface of the meats. Cer- tain woods, such as beechwood, yield a special flavor that is much prized. Of later years the so-called liquid smoke, which is a by-product of wood distilla- tion, is often used as a surface application. Its value as a preservative depends on the disinfecting action of the creosote. Organic acids. — Organic acids are widely used in the preservation of foods. The acid may be formed in the foods by decomposition processes or may have been added as in the case of the addition of vinegar to pickles. Sauerkraut is prepared by cutting cab- bage and packing it tightly in vessels with two per cent of common salt. The pressure and Ihe action of the salt extracts the juices from the plant tissue. 126 Agricultural Bacteriology This liquid, which contains sugar, protein material and various salts, makes an excellent medium for the growth of lactic acid-forming bacteria. The amount of acidity thus produced is suflTicient to in- hibit entirely all development of putrefactive bac- teria. As long as the acid reaction is maintained, the kraut remains edible. The acid may, however, be destroyed by the growth of molds and yeasts on the surface of the liquid, but if the sauerkraut is placed in kegs and thus kept from the air, no mold growth can take place. The action of acid-forming bacteria is also important in the preservation of certain pickles, especially cucumber pickles made in brine. The preservation of green fodder, especially corn, by placing in silos has become of great economic im- portance. Before placing the corn in the silo it is cut into short pieces, one-half to one inch long, so that it may be closely packed. The living cells soon use all the free oxygen that is in the silo for their respira- tory processes, and the air of the silo will contain only nitrogen and carbon dioxide. The silo must be so constructed that no air can penetrate into the silage except from the surface. Since one of the es- sential conditions for the development of molds is the presence of free oxygen, no growth of this type of microorganism can take place except in the upper layers of the silage into which the air has penetrated for a few inches. Such quantities of lactic and acetic acids are formed in the silage as to prevent the growth of putrefactive bacteria. It is beheved that the acids are formed as a result of the effort of the plant cells to obtain oxygen after the free oxygen has been ex- hausted. It has been seen that the anaerobic bac- teria obtain oxygen from sugars. In a similar man- Preservation of Foods 127 ner the cells of the ensiled material obtain oxygen from some of their constituents; acids, alcohol and carbon dioxide are produced as a result of the pro- cess. So long as the silo contains no free oxygen, mold growth is impossible; the acid reaction will persist, and putrefaction can not take place. In those areas in which molds grow, as near the surface, the acid will be destroyed and conditions thus established that will permit the growth of putrefactive bacteria. The silage in these places will be dark in color, will have an offensive odor, and the tissues will have disintegrated, while that in which the growth of molds and putrefactive bac- teria has been prevented will show none of these changes. Preservation by concentration. — Many foods that in time would ordinarily undergo fermentation changes are preserved by increasing the degree of concentration of their soluble solids. In bringing about such changes heat is often employed so that, the process of preservation is due, in part, to partial destruction of the bacteria by heat. Such methods of preservation may be produced by the addition of soluble substances, such as sugar in the case of jellies and preserves, or salt in the case of fish and meats, or the same effect may be caused by the evaporation of the water, as with some dried fruits which are rich in sugar. Syrups are preserved by the evaporation of the water in the sap of the sugar cane, beet,, or maple, as the case may be. If these sugary liquids are in- sufficiently concentrated by boiling, they ferment or ■'work." Condensed milk is another food product that well illustrates the application of both processes. It is preserved by the addition of cane sugar and by 128 Agricultural Bacteriology evaporation in vacuum pans until the concentration is such as to prohibit the development of bacteria. The bacteria are least resistant to the effect of concentration, while the molds are most resistant. In some cases it is not practical to prevent mold growth by concentration, and other means must be used such as the exclusion of the air by placing in closed cans or by covering the surface with a layer of parafTine, as in the preservation of jellies. Preservation by drying. — While some dried fruits, as raisins, currants, and prunes, are preserved by evaporation of the water and the consequent increased concentration of the sugar solution, other fruits, like apples, are kept by the reduced water content of the fruit. Bakery goods, such as crackers, are preserved in the same way. A good illustration of preserving through drying is seen in the case of hay. If it is placed in the mow too green, mold and bacterial growth go on apace, causing abnormal changes which render it unpalatable. Preservation by low temperature. — The use of low temperatures to restrain or prevent the growth of microorganisms is of the greatest importance. The methods involved in artificial refrigeration and their application to the cold storage industry are largely the outcome of the relation of cold to the preserva- tion of food supplies. The temperature zone within which bacterial growth can take place extends from a few degrees below the freezing point of water to about 176° F. It is true that no one organism possesses the ability of growing throughout this entire range of tempera- ture. Most kinds are able to develop from tempera- tures in the neighborhood of 50° F. to somewhat above blood heat. Also there are groups capable of Preservation of Foods 129 multiplying at or near the freezing point, and like- wise at temperatures of 120-140° F. Those types habituated to low temperatures are of much prac- tical significance in the storage of foods. The greatest importance of low temperatures in the preservation of foods is not found in the extreme temperatures that are employed in cold storage warehouses, but in the range which occurs in daily life under the conditions which obtain in the ordinary household. The great majority of bacteria grow most rapidly from 60° to 100° F. If the temperature is reduced to 50°, the rate of bacterial multiplication is much retarded, as is to be noted from the following table in which is given the time required for the division of a single bacterial cell into two completely grown daughter cells at different temperatures. The generation time of B. coli 45 °C. 113 "F. 20 minutes 40 " 104 " 17.2 35 " 95 " 22 30 " 86 " 29 25 " 77 " 40 20 " 68 " 95 16 " 60 " 120 10 " 50 " 14 hours, 25 min. As ordinary refrigerators maintain a temperature varying from 45° F. to 50° F., it is evident that bacterial growth is greatly retarded by maintenance of food material under readily available low tem- perature conditions. The zone of bacterial existence is far wider than the zone of growth. This fact is of importance in the storage of foods, for when they are removed from storage, the microorganisms that were present when the foods went into storage begin to grow, and the decomposition changes rapidly occur. Indeed, with some foods, especially meats, these changes develop Oa 130 Agricultural Bacteriology after freezing with even increased rapidity, owing to the fact that the muscle fibers are forced apart by the freezing process, allowing the cell juices to exude from the cells themselves. This permits the bacteria to act more readily on the meat than when confined exclusively to the surface. Even when stored at extremely low temperatures, below 0° F., foods spoil V <- i-~'-i /-I ' s PROGENY OF A SINGLE GERM ® IN twelve: hours FIG. 27;— COOLING THE MILK The rate of growth of bacteriajdecreases rapidly as the temperature is lowered. rapidly when removed, for while alternate freezing and thawing is very injurious to bacterial life, micro- organisms are able to withstand exposure to low temperatures for prolonged periods of time. In the storage of foods at low temperatures, the effect of freezing on the physical properties of food must be considered. For example, eggs, fruits, and vegetables can not be stored without injury at temperatures at which freezing will occur. If milk is frozen and allowed to remain in this condition for any considerable period, the fat is altered physically so that on thawing it does not mix thoroughly with Preservation of Foods 131 the serum. Longer exposure causes the casein to separate. In the case of foods that can be stored with im- punity at temperatures below freezing, the period of storage may be greatly extended, but with such foods as milk and eggs, where storage must be above freezing, the holding period is hmited because of the fact that bacterial development can occur at tem- peratures slightly above freezing. In milk the development of the acid-forming bacteria is stopped at these temperatures; consequently the milk may not undergo the customary souring change, but other bacterial types which act on the casein can grow slowly at these low temperatures, rendering the milk unfit for use within a few weeks. It is believed that many of the cases of poisoning due to ice cream have been caused as a result of the storage of cream, a practice that has now been largely abandoned. Even in the case of foods that are not contaminated with microorganisms, changes may go on that limit the time that the food can be held in storage. These changes are due to the enzymes that are normally present in many uncooked foods, and are usually termed autolytic changes. The ripening of meats is an example. In the case of eggs the white becomes less viscous, and loses to some extent its beating properties. Water also passes from the white into the yolk, the membrane surrounding the yolk weakens so that when the egg is broken, it is difficult to avoid mixing the yolk with the white. Preservation of eggs. — In preserving eggs it is necessary to prevent the invasion by bacteria and to limit the loss of water from the egg. Eggs are practically sterile when laid, but, owing to the porous nature of the shell, bacteria are able to penetrate it 132 Agricultural Bacteriology readily if tlie surface of the egg is moist. These organisms penetrate the shell in the same manner that they are able to grow through a porcelain filter. If the eggs are left in a dirty nest, or placed in a damp cellar, the adhering moisture may be sufficient to enable the bacteria on the shell to multiply and so penetrate the shell. In cold storage rooms it is essential that the temperature be kept quite constant, so that moisture shall not condense on the surface of the eggs. For home use, eggs may be preserved by placing them in some liquid in which bacterial growth can not take place, but the liquid must be of such a nature that it will not be absorbed by the eggs. For this purpose sodium silicate or water glass is most frequently employed. The collodial nature of this silicate prevents its passage into the egg, and the alkalinity of the solution stops all bacterial growth. No desiccation can of course take place. If the eggs have been invaded by bacteria before placing them in the water glass, the growth of the bacteria will not be inhibited. In such cases, gaseous by-products may be formed in the egg to such an extent that rupture of the shell occurs, in which case the ill- smelling decomposition products will be absorbed by the remaining eggs to such a degree as to injure their commercial value. The best practice is to place the eggs in water glass the'same day they are laid. Preservation by heat. — Practically the only way by which the microorganisms present in any food can be completely destroyed is by the application of heat. The vegetative growth of bacteria, yeasts and molds is easily destroyed, when subjected to temperatures approximating 140° to 150° F. The spore stages of Preservation of Foods 133 all types are more resistant, but particularly so with the bacteria which require a temperature exceeding that of the boiling point before they can be com- pletely destroyed. It is therefore easy to free any food substance from all organisms except the spores of bacteria. In practice two processes are used; first, the appli- cation of a temperature slightly exceeding the scalding point of water, from 140° to 165° F. This process, known as pasteurization, from the fact that it was first employed by Louis Pasteur in the treat- ment of wines to prevent abnormal changes, does not destroy all microorganisms, but only those in the vegetative or growing stage. The more stringent process, known as sterilization, utilizes temperatures equalling or exceeding the boiling point. The former method is employed when it is not desired to keep the material for long periods, as in the commercial handling of milk. It is also used when the presence of bacterial spores is of no importance due to the fact that their germination is prevented by the reaction of the material. The higher temperature is employed in the preparation of canned foods where complete destruction of bacterial life is necessary to preserve such material for relatively long periods of time. Pasteurization of milk. — The organisms con- cerned in the spoiling of milk are non-spore-forming bacteria, and are easily destroyed. Milk may also contain pathogenic organisms which it is necessary to destroy. Fortunately those disease germs that are likely to be spread through the agency of milk are non-spore-forming, so that protection of milk supplies may be secured through pasteurization. 134 Agricultural Bacteriology In the treatment of any food with heat, the physical effect on the material must be considered. In many cases the chemical and physical changes are such as to injure the commercial value of the food, although rarely do they injure its nutritive properties. If milk is heated above 145° F. for any length of time, the process of creaming is much retarded. The FIG. 28 — A PASTEURIZER FOR USE IN THE HOME A milk bottle with a tumbler for a cover. The bottle should be placed on a perforated pie tin so that it will not be broken by the heat. The water should be at nearly the san:ic level as the milk. A thermometer should always be used. fat globules in milk are not uniformly distributed throughout the entire milk, but are grouped in masses that present a relatively smaller surface in proportion to their volume than do the separate globules. The viscosity of the serum is such that it offers a certain amount of resistance to the rising of the fat. The larger the mass of fat, which is of less specific gravity Preservation of Foods 135 than the milk scrum, the more readily does it rise to the surface. In the commercial handling of milk in bottles, it is desirable that the creaming take place rapidly, since an indistinct or thin cream line is considered by the consumer as indicating a milk low in fat content. The cooked taste that is imparted to milk by too high degrees of heat is objectionable to many consumers. It has often been stated that heated milk is less digestible and likely to cause digestive troubles in children. Cases of malnutrition and abnormal development of the bones, rickets, have likewise been ascribed to heated or pasteurized milk. There is no reason to believe that milk as now treated is more likely to be the cause of such troubles than raw milk. In fact milk heated to the boiling point is successfully used with children. In the treatment of any food the degree of heat necessary to destroy the organisms is dependent on the time the material is exposed to its action. Low temperatures for long periods of time may be as effective as higher temperatures for shorter exposures. In the pasteurization of milk, the exposure may be at 145° F. for 20 to 30 minutes or 160° F. for a moment. Either of these methods will insure the freedom of the milk from pathogenic bacteria, and will destroy such a proportion of the acid-forming bacteria as to improve the keeping qualities of milk. In actual practice no method has been devised by which milk can be heated momentarily with perfect success. In machines designed for this so-called flash method of heating, the milk is allowed to flow through the heating chamber in a continuous stream. In such a device the rate of flow is not uniform in all parts of the machine. The milk in contact with the walls 130 Agricui.turai, Bacteriology Hows less rapidly than thai in the middle of the machine; consequently, while some of it may be heated sufficiently long to permit of the thorough destruction of all disease-producing bacteria, if they may perchance be present, other portions may be insufhciently heated. When the possibility exists that such organisms as the typhoid and tubercle bacteria may be present, it is apparent that no method can be regarded as wholly satisfactory that will not insure the destruction of these types. While this flash method of pasteurization was earlier adopted by many milk dealers for the treat- ment of milk supplies, it has not been with the full approval of health authorities, and gradually it has been displaced by the more efTicient holding method. The fundamental principle of the holding process is that a given quantity of milk can be held at any desired temperature for any given length of time. This makes it possible to treat the milk in such a way as to insure perfect safety, through the complete destruction of disease organisms. In the methods described the total destruction of all spore-forming bacteria is not attained. To pre- vent the subsequent germination and rapid develop- ment of these resistant spores, it is essential that the pasteurized milk be cooled quickly and stored at a low temperature. As milk is commercially handled, not all of the acid-forming bacteria are killed; hence, pasteurized milk sours as does raw milk, but the process is materially delayed. If heated at the higher pasteurizing limits, all but the spore-forming bacteria will be destroyed. Such milk will not sour, but decomposition changes will occur in which the casein and albumen will undergo a change. Preservation of Foods 137 Pasteurization of other liquid products is fre- quently employed. Beer is heated to low tempera- tures after it is placed in the bottles to prevent the appearance of fermentations that might impart an undesirable taste and appearance. The presence of alcohol and some of the extractives from the hops tend to prevent the growth of the bacteria that have not been killed by the heating. Wines are also heated in a similar way to overcome the turbidity that sometimes results from bacterial changes. Such acid products as tomatoes, rhubarb, and grape juice can be preserved by a short exposure at the boiling point. This insures the destruction of all but the spores of bacteria, the development of which is prevented by the acid reaction. To obviate sub- sequent infection, it is advisable to treat such material after it has been placed in containers. Such a method has recently been introduced with milk, but the attendant expense is such that it has not been generally adopted by milk dealers, even though it would insure complete immunity from disease. Preservation by sterilization. — The indefinite preservation of foods in closed containers has been rendered possible through the application of the process of sterilization. On this principle rests the great development of the canning industry which has assumed such tremendous proportions of late years. While it is impossible to raise the temperature of boiling water above 212° F., the temperature of steam when confined increases rapidly above that point. Under a steam pressure of fifteen pounds to the square inch, a temperature of 248° F. is attained, which is sufficient to destroy all forms of bacterial life, even the most resistant spores. 138 Agricultural Bacteriology In the treatment of many foods, the heating process can not be continued for too long a period without injury to the physical properties of the product, as for instance with milk, heating at excessive temperatures causes the casein to curdle. In the treatment of peas, if heated at too high a temperature, or for a prolonged exposure, some of the peas crack open, allowing the contents to escape. This results in a turbidity which is unattractive and gives the impression of an abnormal fermentation. To prevent this the cans should be exposed for a longer time at a lower temperature. In the earlier days of the canning industry much loss was oc- casioned from the development of abnormal fer- mentations, due to the growth of gas-producing bacteria, the spores of which were not destroyed by insufficient sterilization. Even under the anaerobic conditions which prevailed in the closed container luxuriant germ growth could occur. With milk, peas, and vegetables, the reaction of the liquid is sufficiently neutral to permit of the ready germination of any spores which may have escaped destruction in the heating process. As gas is generally produced as a result of such fermentation, spoiled food products preserved in tin containers can readily be detected by the bulging of ends of the can. Not infrequently may this pressure develop to the point where the cans actually explode. In the treatment of meat products, there is no practical danger of over-heating, so the losses which occur are relatively small In household preservation of vegetables, sterility can be secured by heating the product to the boiling point on three successive days, as previously des- cribed (page 30). Small steam pressure cookers Preservation of Foods 139 have also been recently introduced for household use. In the canning of fruits, as is done in the home, the sugar which is added for flavor aids in the preserving process, through the fact that it increases the con- centration of the liquid and also raises the boiling point of the solution so that the spores are subjected to a temperature above 212° F. All food materials must be thoroughly protected from contamination after they have once been heated. In commercial work the cans are often sealed while they are partially evacuated of air, and then heated after seaUng. CHAPTER XIII THE FERMENTATIONS OCCURRING IN FOOD PRODUCTS In the preparation and handling of foods, they inevitably become seeded with a great variety of organisms. This contamination is a more or less constant factor, in that representatives of the three great groups of microorganisms, the bacteria, yeasts, and molds, are always introduced. The relation between the groups will vary, depending on the nature of the food material. Whether one group or another is to be most active in the decomposition changes will depend on the composition and con- centration of the material. Such foods as meats, which are high in protein and low in carbohydrates, undergo putrefactive changes, due to bacteria which act primarily on the protein. If the material contains a large amount of sugar in proportion to the protein, and the reaction is not acid, the type of fermentation will be similar to that noted in milk in which the sugar is fermented with the production of acid, and the protein is not attacked to any degree. If the material contains sugar and has an acid re- action, a condition found in many fruit juices, yeasts are likely to be the dominant type of organism concerned in its decomposition. Alcohol and carbon dioxide are the chief products of yeast action. The fermentations of milk. — The souring of milk is so common that it is looked upon as a normal change; in fact, its absence is more likely to be Fermentations in Food Products 141 regarded as abnormal. If milk could be secured without microorganisms, it would remain unchanged for many days, but, in the normal course of events, it invariably becomes seeded with a variety of organ- isms, which in a short course of time are able to develop and produce the characteristic fermentative by-products that bring about the usual changes noted. These changes are not produced by a specific organism, but by a group of widely dis- similar species so far as form is concerned, which however are able to produce varying amounts of acids, particularly lactic acid. While this fermen- tation product is produced in such quantities as to characterize the change involved, yet other by- products are likewise formed, as other acids, gases, and various substances. Some of these flavor- forming products may be of value, as in the case of those producing agreeable flavors in butter. The curdling of milk. — The casein of milk is not in actual solution, but exists in a semi-solid condition in combination with calcium. As the bacteria multiply, a portion of the sugar which they use is changed to acid, which combines with the calcium, leaving the casein free, in which condition it is precipitated in the form of curd. If, as is the case with the true lactic bacteria, most of the sugar fermented is changed to lactic acid, and no gas is produced, the curd wiH assume a jelly-like consistency and the odor will be agreeable. Milk soured by the action of the normal lactic types forms an appetizing and healthful food. If the fermentative change is produced by organisms belonging to the colon group, gas will be formed in varying amounts, and^the curd will assume a more or less spongy structure, due to the gas imprisoned in' the curd. Acetic acid is 142 Agricultural Bacteriology produced in larger amounts than is lactic, and com- pounds of unknown composition are formed which make the milk wholly unappetizing. If the milk has been produced with due regard to cleanliness, the lactic fermentation will generally predominate, but if quantities of manure and dirt find their way into the milk, the colon group of bacteria will gain the ascendency. It is often asserted that the milk fermented by the colon group of bacteria is unhealthful. Whether this is true may be questioned, but every consideration, both aes- thetic and economic, demands that the milk be produced under conditions which shall render con- tamination as small as possible. Only a portion of the sugar of the milk is fermented by these acid-forming bacteria. The casein and the ash constituents of the milk are able to unite with a certain amount of acid, but as soon as free acid appears in the milk the growth of these bacteria is checked. The amount of acid normally formed ranges from 0.8 to 1 per cent. The acid-forming bacteria of milk are non-spore bearing, a fortunate provision as it permits of the use of the pasteurization process as an aid in the preservation of milk. Each of the groups is able to grow both in the absence and presence of air, again a fortunate provision, otherwise the use of either group in dairy manufacturing would be impossible. Milk forms an excellent example of the preservative action of acid against putrefaction. The increase of acid due to the lactic bacteria quickly inhibits the development of types capable of attacking the casein and albumen. As long as the milk remains sour, it is not subject to the action of putrefactive bacteria. But usually there appears on the surface of the sour, Fermentations in Food Products 143 raw milk in the course of time a white mold, called Oidium ladis, which uses the acid by-products as food, gradually changing the reaction from acid to alkaline. Under these conditions the putrefactive bacteria that are always present are able to develop, and soon the milk is changed to a vile smelling mass. Milk forms an excellent example of the sequence of decomposition processes in nature where one type of life is dependent upon the by-products formed by another group of organisms. In milk there is found a third group of acid- forming bacteria, which, on account of their relatively slow growth at ordinary temperatures, has httle or nothing to do with the common souring change. This type is not inhibited by the presence of free acid, but continues to develop after the ordinary lactic groups have ceased to grow, fermenting entirely in some cases the sugar of the milk. This group of bacteria is generally referred to as the Bulgaricus type, because of the causal relation which obtains between the Bulgarian sour milk and the organism causing this change. Bacillus Bulgaricus. This type of organism is associated with fecal matter and is able to grow especially well at high temperatures. Certain types of this group are widely used in the preparation of fermented milks, which have gained considerable vogue because of their supposed therapeutic value in the case of digestive troubles. It is not at all certain that this particular type of organism is of special value; the favorable dietetic action of fermented milk may be due more largely to the acid which it contains. Milk soured by the ordinary lactic bacteria may be as desirable as that fermented by Bacillus Bulgaricus. In fact most of the fermented milks, commercially prepared, 144 Agricultural Bacteriology are made by employing the two groups of organisms, the Bulgaricus group being used because it imparts a better texture to the milk, since it increases the viscosity of the liquid, thereby preventing the separation of Ihe curd from the whey. Butter milk and cottage cheese are to be classed as types of fermented milk. Both form excellent examples of a material that has undergone fermentative changes, and yet is a desirable and a healthful food. Milk in the fermented form is widely used in the southern states and in certain tropical regions where it would be difTicult to keep it in the unfermented condition. Sweet curdling of milk. — Sometimes milk be- comes seeded with other organisms than the usual lactic type and other fermentative changes arc produced. A common type of change that is especially likely to occur in the absence of the lactic fermentation is the sweet curdling change in which the casein and albumen are acted upon, the former being precipitated in a manner similar to the action of rennet. Since this change is caused by many spore-forming organisms, heated milks (sterilized or boiled) are peculiarly prone to undergo this change. In addition to the curdling enzymes analogous to rennet, digestive enzymes are also produced which are similar in their action to trypsin, which is found in the intestinal juices of all animals and which changes protein materials to soluble products. The bacterial trypsin gradually digests the curdled casein, so that the change is frequently called the digestive fermentation of milk. The digestive changes produced are very similar to those which take place in the decomposition of all proteins. Fermentations in Food Products 145 Butyric Fermentation. — Those organisms that are able to ferment sugars forming the volatile butyric acid are also found in milk and at times in the absence of the lactic acid bacteria produce their characteristic fermentation which is marked by the peculiar odor of the acid. Gas is also produced and at first the fermentation might be mistaken for one due to the colon group of bacteria. Slimy fermentations. — In sugar solutions fermentations are often noted that change the solu- tion into a slimy or ropy licjuid. In the manufacture of sugar the syrup may be changed into a mass, almost jelly-like in consist- ency, due lo the growth of bac- teria thai possess a gelatinous capsule aijout the cell. In maple sap a ropy change is often noted and the same is true of milk. This abnormal change seems to be produced most frequently at low temperatures. In milk two types of organisms may be con- cerned. The Norwegians have long used a fermented milk of this type as a drink. This prepared milk, known as "taettemjolk," has the taste of ordinary sour milk but the texture is more or less slimy, depending on how long the fermentation has been allowed to [irocccd. A slight increase in viscosity is desirable in any fermented milk that is to be used as food, lOa FIG. 2il SLIMY MILK It does not mix with water when poured into it. 146 Agricultural Bacteriology since such change prevents the settling of the curd and the appearance of the free whey which imparts to the milk an unappetizing appearance. This slimy fermentation produces so marked a physical change in milk that it is usually considered unfit for use but there is no reason to believe that such milk is at all harmful. Various other abnormal fermentations are some- times noted in milk when the lactic bacteria are replaced by other groups. Many bacteria produce colored by-products, and when such organisms grow in milk to any extent a color may be imparted to the same. The appearance of red and blue milk is thus explained. Bitter milk may be due to the ingestion of feeds which contain a bitter principle, or to the production of soluble decomposition products by the putrefactive bacteria. Some acid-forming bacteria are able to produce bitter flavors. Alcoholic fermentation. — The fermentation of sugar with the production of alcohol and carbon dioxide is due to yeasts. Bacteria may produce alcohol but never in appreciable amounts. The growth of yeasts is favored by the presence of cane sugar or glucose, and by an acid reaction, conditions that are found in fruit juices. Yeasts are found in limited numbers in the soil. They are carried by dust and insects onto the surface of fruits, and with any rupture of the skin which allows the juice to exude, favorable food conditions are at once estab- hshed. When sugar-containing fruit is crushed, as in the making of cider from apples and wine from grapes, the juice is abundantly seeded with yeast cells. Rapid development then ensues but the growth of yeasts is never so abundant as that of Fermentations in Food Products 147 bacteria. The fermentation is marked by the pro- duction of carbon dioxide and alcohol. In the production of fermented liquors, such as beer, it is necessary to seed the material to be fer- mented with yeast, since the mash does not naturally contain sufficient numbers to induce rapid action. Furthermore, the beer wort is heated to temperatures that will destroy any yeast it may contain in the process of extracting the soluble elements of grains. For this purpose pure cultures of yeasts are used by the brewer so that he can control the quality of the product, something that would be impossible if impure yeasts were employed since the character of the flavors produced would vary from time to time. The yeasts vary widely in the amount of sugar they will ferment before the action is stopped by the accumulation of by-products. The alcoholic fermentation does not occur nor- mally in milk, because lactose, the sugar in milk, is not readily susceptible to fermentation. Also, the extent of seeding with yeasts is not sufficient to enable fermentative changes to develop rapidly. As a consequence the activity of the yeasts is usually overshadowed by bacterial changes. While most yeasts are unable to act on milk sugar, yet lactose- splitting yeasts do occur not infrequently, and are often to be noted in milk if it is held for some lime until thoroughly soured. Yeasty fermentation occurs in gathered cream, especially where it is held for some days as that supplied to the large centralized creameries. It is also noted in cheese factories, especially in the whey tanks on account of the favorable opportunity for contamination and growth. When such fermented whey is returned to the farm in the milk cans and these are imperfectly washed, 148 Agricultural Bacteriology the fresh milk may become seeded to such an extent as to cause abnormal fermentations in the cheese, injuring its commercial value. This is particularly true with Swiss cheese, in which the development of acid in the process of making is not carried sufficiently far to transform all of the sugar. . Certain kinds of fermented milks are also prepared by the use of lactose-fermenting yeasts. When raw milk is used, the milk will be subject to both the acid and the alcoholic fermentations, and the taste of the acid milk will be modified by that of the alcohol. Two of these fermented milks are widely used in eastern Europe and western Asia. Koumiss is prepared from mare's milk by the nomadic peoples of the Caucasus. The fresh milk is inoculated with a little of the previously fermented milk which may be dried when it is desired to keep it for long periods. The drink contains about two per cent of alcohol and one per cent of acid. It has been introduced into western Europe and America because of its supposed therapeutic value in the treatment of tuberculosis, and for typhoid fever convalescents. An artificial koumiss is sometimes prepared by adding cane sugar to cow's milk and seeding it with ordinary yeast. Kefir is another drink prepared from milk by inoculating the milk with kefir grains which consist of masses of yeasts and bacteria. The grains are dried and may be bought as articles of commerce. The composition of kefir is quite similar to that of koumiss except that the content of alcohol is less. These drinks have been largely supplanted in this country by Yoghurt which is prepared by the use of the Bacillus Bulgaricus. Manufacture of vinegar. -Ethyl or grain alco- hol, as it is often called to distinguish it from methyl Fermentations in Food Products 149 or wood alcohol, furnishes a source of food and energy for a class of bacteria that are known as the acetic acid bacteria, since they oxidize alcohol to acetic acid. If cider, wine, or any similar organic liquid containing alcohol, is exposed to the air it soon becomes covered with a whitish or gray film, or membrane, and the alcohol is gradually changed to acetic acid. If the film, which consists of masses of bacterial growth is allowed to remain undisturbed, the liquid remains clear. If it is disturbed, the particles of membrane sink to the bottom, and the surface soon becomes covered with a fresh film. The film which is known as mother of vinegar may in- crease in thickness, and, finally present the appear- ance of a leathery mass. The acetic acid formation can go on only under aerobic conditions, since the organisms concerned in it must have access to the free oxygen of the air to convert the alcohol into vinegar. If the alcoholic liquid is kept in closed or completely filled barrels, the process does not occur. In the presence of too large quantities of alcohol, above 14 per cent, the acetic bacteria can not grow, and the alcohol is not attacked. In the fermentation of lactose by the lactic bacteria, not only is lactic acid formed but other substances that impart a cnaracteristic taste and odor to the fermented milk. So in the fermentation of alcohol by the acetic bacteria, not only is the acid formed, but also com- pounds having an agreeable odor and taste. The fermentation of cider and wine produce a vinegar that is valued much higher than that obtained by the oxidation of distilled alcohol, which is the great source of commercial vinegar. In the manufacture of vinegar on the farm the barrel should be filled from one-half to two-thirds 150 Agricultural Bacteriology full and then placed on its side. The circulation of air through the barrel can be facilitated if openings are made in the barrel heads just above the level of the liquid. To prevent entrance of flies these holes should be screened with wire netting which has been varnished so that the iron will 'not be attacked by the acetic acid. The addition at the start of a small quantity of vinegar, or better some of the mother of vinegar, serves to seed the liquid with the necessary acetic bacteria. The fermentative process progresses rather slowly at first but in a few weeks the vinegar will be ready for use. If it is desired to continue the process in the same barrel, fresh alcohol can be added through the bung hole by means of a glass funnel to which a rubber tube is attached. This enables the alcohol to be added without disturbing the bacterial film on the surface. In the manufacture of vinegar on a large scale, the provisions made for the aeration of the liquid are much more perfect, and the process goes on rapidly. Large wooden casks are filled with beech shavings, a wood that imparts no flavor to the product. The casks are provided with numerous holes near the bottom. The alcoholic liquid is sprinkled on the surface in such a way that it will be evenly distributed over the shavings, which soon become covered with bacterial growth. The fermentative process produces heat, and hence there is a constant current of air through the holes at the bottom of the cask. The air passes upward through the shavings and out at the top. In this manner the bacteria are provided with an abundant supply of air facilitating the oxi- dation process. As the by-products of the fermen- tation are also constantly removed, the change goes on continuously and with rapidity. Fermentations in Food Products 151 The vinegar may be invaded by bacteria that will destroy the acid, and expose the product to the action of putrefactive bacteria. It often becomes seeded with the vinegar eel, a minute worm that injures the quality and the appearance of the vinegar. Bread. — The making of bread represents another fermentation industry that is carried out in every home. If flour, water, a small amount of salt, and fat, such as lard or butter, are mixed and baked, a dense hard mass is obtained, such as a cracker or unleavened bread. If however, yeast is added to the mixture, and the dough placed under conditions which permits the yeast to grow, the sugar is changed into alcohol and carbon dioxide. The dough made from such flours as wheat contains glutin which imparts a plasticity to the mass. As the gas is formed, it is unable to escape readily from the dough, and the whole mass "rises," producing the "sponge" which characterizes all leavened breads. When the gas is heated in the baking process, farther expansion occurs, and the loaf becomes light and porous. The yeast may be obtained by saving a piece of the dough from the previous baking, by the addition of dried or compressed yeast, or by giving conditions favorable to the natural seeding of the materials. In the baking industry large quantities of compressed yeast are added to the dough and the rising takes place rapidly. If smaller amounts of yeast are used, as is usual in the home, a longer time must be allowed for the growth of the yeast and the formation of sufficient gas to produce the desired porosity in the bread. In this case the yeast is added to a thin mixture of flour and water, called the batter. After standing for a few hours in a warm place, ad- ditional flour is added and the yeast uniformly 152 Agricultural Bacteriology incorporated with the dough, by the kneading pro- cess. More or less bacterial growth takes place in the dough, depending on the length of time it is allowed to stand before baking. The bacteria have much to do with the flavors of the bread. If the development of the bacteria has been too great, the bread is likely to have an acid or sour taste. If no yeast is added, but the mixture of flour and water is allowed to stand, gas will be produced by the gas-forming bacteria that are normally in the flour. The yeasts that are naturally present will also func- tion. Such bread has a quite different flavor from the ordinary product, and is called salt-rising bread. The flavor is far from constant, since there is no control over the kind of organisms that grow in the mass. Studies made during recent years point to the use of pure cultures of bacteria in the manufacture of salt-rising bread and to a better control of the flavor of the product. Bread and other bakery good? are not likely to undergo decomposition changes to any great extent, as ordinarily they are consumed in a fresh state. If kept in a damp place, mold soon appears on the surface. In the baking process the vegetative bacteria are readily destroyed, but the spores resist. At times, especially in warm weather, bread and cake may undergo a slimy fermentation, due to the growth of the spores of certain kinds of bacteria that may be present either in the flour or the yeast. CHAPTER XIV THE RELATION OF BACTERIA TO BUTTER AND CHEESE The preparation of condensed milk and milk powder represents a method of conserving the milk in a less bulky form than the original product, a great advantage when it is to be transported for long distances. The manufacture of butter repre- sents a method of concentrating the butter fat of the milk, while in cheese making, the fat and the casein, together with a small part of the milk serum are concentrated. Importance of flavor. — The flavor of foods is very important, an agreeable odor and pleasant taste in any food making it much more appetizing, if not influencing its nutritive value. The use of condiments and flavoring substances of all kinds in many foods are evidence of the value of flavor in foods. If cream is separated from sweet, fresh milk, and the remaining part of the milk serum is eliminated by the churning process, the butter thus obtained will be quite devoid of flavor, and to those ac- customed to the butter made from cream that has been allowed to sour before being churned, it is not at all attractive. Sweet-cream butter is made in all the countries of southern Europe, while that from fermented cream is made in northern Europe, America, Australia, Asia and South America. Sour or acid cream butter therefore represents the great mass of the butter supply of the world. 154 Agricultural Bacteriology Acid-fermentation and flavor of butter. — As earlier set forth, two groups of acid-forming bacteria are concerned in the souring of milk. The lactic group tends to predominate in milk that is produced under clean conditions, since it grows more rapidly at ordinary temperatures than does the colon group, which, by reason of the products formed, injures the taste and odor of the milk, and hence the flavor of the butter. In the lactic fermentation not only is lactic acid produced, but also other acids, alcohols and esters that impart to the sour milk and cream an agreeable odor and taste. Butter fat has the power of absorbing some of these volatile sub- stances, just as it will absorb the odors of fruits when kept in the same container with them. The dif- ference in flavor between the sweet and ripened cream butter is, therefore, due to the absorption of some of these products during the ripening of the cream and the churning process. Cream that is allowed to sour spontaneously will usually contain many more lactic than colon organisms, and the butter made from it may be of very excellent quality. As long as butter was made on the individual farm, no great need of control was felt, but as its manu- facture became centrahzed in creameries, it became necessary to control not only the kind of organisms growing in the cream, but also the rate at which the fermentation should go on. The bacterial content of cream both qualitatively and quantitatively will depend on the method by which the milk has been creamed. If it has been allowed to stand in shallow vessels at the fluctuating air temperatures and exposed to contamination from the air, it will contain greater numbers and more varied kinds of bacteria, than if the creaming had Relation of Bacteria to Butter 155 been carried out at low temperatures and in covered containers. If the centrifugal separator is employed, the bacterial content of the cream will be similar in kind to that of milk. Control of flavor.— The modern methods of butter making are designed to give the maker control over the kinds of bacteria that are concerned in the fermentation of the cream. The first step in this direction was to add to the cream, selected pure cultures of lactic bacteria that had been isolated from varied sources, and tested as to their favorable flavor-forming properties and their ability to grow at the ripening temperatures. The organisms are propagated in milk that has been heated sufficiently to destroy all of the non-spore forming bacteria. This is then inoculated with the pure culture of lactic bacteria. If the butter maker exercises care in the prevention of contamination and in the control of temperature at which it is kept, this pure culture starter can be maintained for a long period of time. He is thus in position to grow the desirable organism in any quantity for addition to the cream which is to be fermented or ripened. The lactic bacteria are quite resistant to desic- cation. The commercial laboratories make use of this property and market the pure cultures of the organism not only in milk, but in a dried condition. These are usually prepared by adding some of the fermented milk to some inert substance, such as milk sugar or milk powder, and drying the mass at a low temperature. The organisms remain alive in this condition much longer than where they are exposed to their by-products as in milk. Pasteurization of cream. — If the starter is added to raw cream, its effect will depend on the 156 AoRicuLTunAL Bacteriology number of bacteria present in the cream. If the cream^is sweet and clean, the number of bacteria added£in the starter will greatly exceed those nat- urally found in the cream, and the|_fermentation will be largely due to the added bacteria. If, however, the* cream is quite sour from the bacteria already present, the fermentation may not be much influenced by the starter. In order to obtain the maximum effect and to give the organisms added in the starter a clear field, it is advisable to pasteurize the cream, and then add the starter. In the pasteurizing process the acid-forming bacteria will be destroyed, and the entire fermentation will be controlled by the added bacteria. By the use of this method, the butter maker has entire control over the flavor of the product, in case he is furnished with a good quaUty of raw material, i. e., sweet, fi'csh, clean cream. The ripening of the cream also aids in the churning process, causing the cream to churn more quickly, and diminishing the loss of butter fat in the milk. The ripening of the cream also improves the keeping quality of the butter made from raw cream. It is probable that if the butter is made from pasteurized cream, the sweet cream butter has better keeping qualities. The pasteurization also- destroys any pathogenic bacteria that may be present in the cream. The control of the ripening of the cream is desirable from the standpoint of the rapidity with which the process goes on. The flavor of the product will depend on the degree of acidity developed in the cream, or in other words on the relative amounts of flavoring substances and butter fat. If the cream is low in fat, the same amount of flavoring substances Relation of Bacteria to Butter 157 will impart to the butter a higher degree of flavor than if there had been twice as much fat in the cream. It is, therefore, desirable to stop the develop- ment of acid when it has reached the proper point. Also from the standpoint of convenience it is desir- able to have the cream ripened at that rate which will make possible the churning at about the same hour each day. This can be accomplished by varying the amount of starter, and by the control of the temperature at which the cream is kept. Starters are rarely used on the farm. It is certain that a great improvement in farm butter could be made by a more perfect control of the ripening process, which could be easily attained by the use of starters. Small quantities of starters can be made in such vessels as fruit jars or milk bottles. Flavor of artificial buller. — Oleomargerine is made from fats that are quite devoid of butter flavor. If this product is to be sold as a butter substitute, the butter flavor must be developed. This is imparted to the product by churning the fats with sour milk. The flavor of the oleomargerine is thus identical in origin and nature with the flavor of butter. Renovated butter is made from a poor quality of butter, the flavor of which is of low grade or decidedly below standard. The fat used in its manufacture is melted; the obnoxious flavors re- moved by passing air through it, and by washing the fat. The desirable flavor is then imparted by churning the fat with some milk soured by pure cultures of bacteria. In the manufacture of oleo- margerine and renovated butter, the most approved scientific methods are employed to impart to the otherwise neutral fats, the characlcristic flavor which is so much in demand on the butter market. 158 Agricultural Bacteriology Decomposition of butter. — Butter deteriorates more or less rapidly, depending on the kinds of bacteria present in the cream, and on the tempera- ture at which it is stored. The best keeping butter is that made from sweet cream that has been pas- teurized; the poorest keeping butter is that from a raw, sweet cream or from a sour cream that contains not only great numbers of undesirable flavor-forming bacteria but yeasts and molds. If the butter is stored at ordinary temperatures, the development of undesirable flavors is rapid, while at the temperatures maintained in butter storage rooms, below zero F., the changes are very slow. If the butter is kept in small packages so that a considerable surface is exposed to the air, the. spoiling will be more rapid than if the package is larger, or if the butter is hermetically sealed. Abnormal flavors in butler. — Various abnor- mal flavors are noted in butter. These may be due to feed, to absorption of odors by the milk or cream or bacterial by-products. The action of the bacteria may be indirect as in the case of the "fishy" flavor in butter which is due to the presence of small quantities of iron or copper dissolved from the utensils by the acid of the cream. All vessels should be well tinned so that no solvent action can be exerted by the acid. Cheese making. — In the making of cheese it is necessary for the casein and the fat to be separated from the milk serum. This separation is accom- plished by aflowing the milk to sour, or by the addi- tion of rennet to it. The latter is the method used in the making of all the varieties of cheese that aic of much commercial importance. Cottage or sour milk cheese is made by allowing the milk to Relation of Bacteria to Cheese 159 undergo an acid fermentation. It is ready for use as soon as the whey has been drained from it, and represents a form of fermented milk. The rennet cheese has no particular flavor at the time it is made, and moreover is rather indigestible. It is essential that the green cheese be allowed to undergo a ripening process in which is formed the peculiar flavors which characterize the various types. This process also renders the cheese more easily digested. Bacteria and other microorganisms func- tion in the ripening of the different varieties of cheese which vary greatly in texture and in flavor. The important commercial varieties of cheese are all made from the same raw materials, cow's milk, rennet and salt, but variations in the methods of manufacture, and in the conditions under which the ripening takes place, permit the growth of differ- ent classes of bacteria in the different types of cheese. The amount of whey allowed to remain in the cheese will cause a difference not only in moisture, but also in sugar, and hence in the acidity of the product, since all of the sugar will be fermented by the acid-forming bacteria of the milk which in large part are retained in the curd. The type of cheese in which a large amount of whey is left is called soft cheese, such as the Camembertand Brie; those from which the whey is more completely expressed are called hard cheese, as represented by the Ched- dar and Swiss types. The latter are usually made in considerably larger sizes, and in the case of the American or cheddar cheese may be made of any desired size. In the case of the soft cheese, the ripening is, in part, due to the action of organisms LhaL grow on Lhe surface of the cheese, and act on 160 Agricultural Bacteriology the curd by the secretion of soluble enzymes that diffuse into the cheese. If the cheese is too large, the outer layers will be ripe before the inner portions and the softness makes the handhng of large cheese impossible. Cheddar Cheese. — In the making of chcddar cheese, the most common variety made in America, it is essential that the milk contain great numbers of lactic bacteria, since it is necessai-y that a relatively large amount of acid be formed during the making of the cheese. If the milk is too sweet, or in other words, too low in bacteria, the maker adds a starter prepared from pure cultures of lactic bacteria, just as the butter maker does for the control of the cream ripening process. The action of the rennet which is used to curdle the casein is also facilitated by the acid reaction of the milk. Within a few minutes from the time the rennet is added the milk curdles and as soon as it is properly set, it is cut into small pieces for the purpose of facilitating the expulsion of the whey. With the development of acid, a certain action is exerted on the casein, which causes the pieces of curd to adhere to each other, and to fuse into one mass under the influence of the pressure exerted in the press in which the curd is placed after the whey has been largely removed. Bacterial growth in the curd occurs rapidly, the colonies developing in a manner similar to those obtained in the plate cultures of the bacteriologist. The rennet used also contains the digestive enzyme of the stomach juice, pepsin, which can exert its digestive action only in the presence of an acid. In the stomach of the animal, the hydrochloric acid secreted by the stomach wall is the activating agent, w liile in the cheese the lactic acid formed in the acid Relation of Bacteria to Cheese 161 fermentation of the sugar gives favorable conditions for action. The protein of the cheese is changed into soluble decomposition products, similar to those formed in the stomach digestion of nitrogenous com- pounds. The acid reaction established by the lactic bacteria serves to protect the cheese from the action of the putrefactive bacteria that are always present, in the same manner as the acid of silage protects it against putrefaction. It is evident that without the lactic bacteria the cheese will either remain in the same condition in which it is when removed from the press, or else it will undergo putrefaction. It is also evident that without the bacteria the manufacture of cheese would be impossible. Gassy cheese. — If the milk is handled in a way so as to allow the colon or gas-forming type of bacteria to overcome the usual lactic type, the quality of the cheese will be injured, due not only to the impair- ment of the flavor by the by-products of these organ- isms, but the texture of the cheese is rendered open or porous by the development of gas which permeates the mass of the cheese. The growth of mold which occurs readily on the surface of the cheese and is objectionable on account of discoloring the surface can be easily prevented by dipping the cheese in melted paraffin. The impervi- ous layer thus formed excludes the air, thereby pre- venting the growth of the mold spores. The cheese maker can not of course, test the milk of the different patrons of his factory for gas-forming bacteria by the methods employed by the bacteriolo- gist. He can, however, gain an idea of the quality of the raw product, by making what is known as a curd test, in which a small portion of each patron's supply is curdled with rennet, after which the curd lla 162 AdHICULTUKAL BaC-.TKHIOLOGV is broken up so as to expel the whey which is then turned off. The small mass of curd is kept for several hours at temperatures which favor the growth of the gas-forming bacteria, 100° to 104° F. The quahty of the milk is then determined by the appearance of the curd with reference to presence of gas holes, also the flavor and odor. This rough (pialitative test is JatH « r'jf^ ^ - . ^ r'j FIG. .30— .\ GASSY CHEESE Such u chveso h.is liUlc if any commercial value due to the objccLionahle flavors present. of great service to the cheese maker in detecting the c{uality of the raw material. All of the operations must be carried oul with due regard to contamination from outside sources, since it is essential that the changes noted in Ihe curd be caused only by the bacteria in the milk, and not by those introduced by the use of unclean utensils. Swiss cheese. — Swiss cheese is made from s-weet milk vvhich contains only small numbers of lactic bacteria. Tlic i-enncl used to curdle the milk is oblaiiu'd fiotu iKilui';d "I'cnnels," i. c, portions of Relation of Bacteria to Cheese 163 dried calves' stomachs which are soaked in whey and kept at a temperature of 80° to 95° F. for 24 to 36 hours. The whey contains numerous lactic bacteria and on the dried rennets, there are always organisms of the Bacillus Bulgaricus type. This serves as a starter to inoculate the milk with bacteria capable of fermenting the sugar and with others that produce propionic acid, and carbon dioxide. The milk is heated to about 135° F. and the curd removed from the whey in one mass, so as not to allow it to become cool, which checks the growth of the Bacillus Bul- garicus in the curd. At every step in the making of this and other kinds of cheese, the process is conducted in a manner which influences the growth of certain groups of organisms. Of course, these methods were primarily worked out -entirely from the standpoint of experience, but more recently their relation to the action of certain bacterial groups has been more definitely traced. In the making of cheddar cheese the salt is added to the finely cut curd before it is placed in the press, but in the case of Swiss cheese the salt is applied to the surface of the cheese. The most marked charac- teristic of Swiss cheese is the presence of gas holes, ranging from the size of a cherry to a walnut, which are scattered quite uniformly through the interior of the cheese. These so called eyes are formed by the fermentation of the lactates with the formation of propionic acid and carbon dioxide, the latter causing the holes in the plastic curd while the acid influences the flavor of the cheese. These organisms can not grow in the presence of salt, and it is therefore essential that an opportunity first be given for their growth. Later the application of salt to the outside checks the development of these gas-forming organ- 164 Agricultural Bacteriology isms as the salt gradually penetrates the substance of the cheese. Mold-ripened cheese. — Roquefort, a French cheese made from sheep's milk, Gorgonzola, an Itahan cheese, and Stilton cheese made in England are illustrations of hard, firm cheese which contain molds. Not only does the presence of these molds confer a peculiar flavor and appearance on the cheese, but undoubtedly the ripening or digestive changes are influenced by these types of organisms. With the Roquefort type a green-spored mold, quite simi- lar to the ordinary bread mold, is grown on rye bread, which, after drying, is powdered and the powder sprinkled over the curd before it is placed in the press. In order that the mold may have the necessary supply of air for the maturing of the spores the cheese is pierced with many small holes. The green color of the mold imparts to the cheese a marbled appearance, and the peculiar flavor is due, at least in part, to the same factor. In the other varieties mentioned, the mold is not added inten- tionally, reliance being placed on the contamination in the factory during the process of making. Soft cheese. — In Camembert, a French cheese, the curd produced by rennet is not cut but is placed in small molds to allow the whey to drain off. After removal from the press the cheeses are placed in a very moist room. The lactic fermentation goes on rapidly in the cheese, changing the curd to an acid mass that is favorable for the growth of molds. The characteristic mold of milk, Oidium lactis, and a white-spored mold related to the mold that grows in Roquefort cheese are essential to the ripening and the development of the characteristic flavor. It is essential that a certain balance be maintained be- Relation of Bacteria to Cheese 165 Lween the two types of molds, which can only be accomplished by proper temperature and moisture conditions. The inability of the maker to control these conditions makes the ripening a difficult problem, and a large portion of the cheese is of low value because of the non-development of the typical flavor. Brie, Limburger and brick cheese are other va- rieties of soft cheese that are made and ripened in a manner similar to Camembert. The manufacture of cheese is an industry that is closely connected with the farm, and is an example of the value which microorganisms exert in the preparation of a valuable food product. It indicates how valuable the products of decomposition may be in imparting a desirable flavor to what would other- wise be a tasteless product. CHAPTER XV THE CONTROL OF FOODS The various governmental units, national, state, and municipal, are all expending much effort in attempting to control the quality of food supplies. While national activity is confined to foods embraced in interstate commerce, and in the main is concerned with those which are preserved, yet the interstate control of fresh meats is a large factor in govern- mental enterprise. So far as municipal control is concerned, the regulatory service includes in the main only fresh food products, and of these milk is of the most importance. With the recognition of the fact that milk is the chief food product in its relation to health, especially of children, much more attention has been given of late years to the formulation of sanitary rules, than ever before. Even small cities and towns are now dealing in a direct way with dairy- men, so that by far the larger part of milk supplies used for direct consumption now comes under some kind of supervision. In order to have the milk reach the consumer in the city in an unchanged condition, the greatest care must be observed by all who handle it. The regulations which the modern city imposes on the milk dealer and on the producer are complex and cover every phase of its prodnction and handling that can in any way affect the value of the milk as hu- man food. The city can enforce its regulations by not allowing the sale of milk that has not been produced in conformity therewith. In order to determine whether the regulations are observed, two types of The Control of Foods 167 inspection are maintained: first, the examination of the milk in the city as to the number and kind of bacteria it contains; and second, an inspection of the dairy farms as to the methods there used and to the health of the animals. A summary of the rules imposed by the city of New York is given. It will be noted that the rules are intended to force the production of a clean and healthful milk. The Cows 1. The cows must be kept clean, and manure must not be permitted to collect upon the tail, sides, udder and belly of any milch cow. 2. The cows snould be groomed daily, and all collections of manure, mud or other filth must not be allowed to remain upon their flanks, udders or bellies during milking. 3. The clipping of long hairs from the udder and flanks of the cows is of assistance in preventing the collection of filth which may drop into the milk. The hair on the tails should be cut, so that the brush will be well above the ground. 4. The udders and teats of the cow should be thoroughly cleaned before milking; this to be done by thorough brushing and the use of a cloth and warm water. 5. To prevent the cows from lying down and getting dirty between cleaning and milking, a throat latch of rope or chain should be fastened across the stanchions under the cow's neck. 6. Only feed which is of good quality and only grain and coarse fodders which are free from dirt and mold should be used. Distillery waste or any 168 Agricultural Bacteriology substance in a state of fermentation or putrefaction must not be fed. 7. Cows which are not in good flesh and condition should be immediately removed and their milk kept separate until their health has been passed upon by a veterinarian. . 8. An examination by a veterinary surgeon should be made at least once a year. The Stable 9. No stagnant water, hog-pen, privy or uncov- ered cess-pool or manure pit should be maintained within 100 feet of the cow stable. 10. The cow stable should be provided with some adequate means of ventilation, either by the con- struction of sufficient air chutes extending from the room in which the cows are kept to the outside air, or by the installation of muslin stretched over the window openings. 11. Windows should be installed in the cow barn to provide sufficient light (2 sq. feet of window hght to each 600 cubic feet of air space the minimum) and the window panes be washed and kept clean. 12. There should be at least 600 cubic feet of air space for each cow. 13. Milch cows should be kept in a place which is used for no other purpose. 14. Stable floors should be made water-tight, be properly graded and well drained, and be of some non-absorbent material. ' Cement or brick floors are the best, as they can be more easily kept clean than those of wood or earth. 15. The feeding troughs and platforms should be well lighted and kept clean at all times. The Control of Faons 169 16. The ceiling should be thoroughly swept down and kept free from hanging straw, dirt and cobwebs. 17. The ceiling must be so constructed that dust and dirt therefrom shall not readily fall to the floor or into the milk. If the space over the cows is used for storage of hay, the ceiling should be made tight to prevent chaff and dust from falling through. 18. The walls and ledges should be thoroughly swept down and kept free from dust, dirt, manure or cobwebs, and the floors and premises be kept free from dirt, rubbish and decaying animal or vege- table matter at all times. 19. The cow beds should be so graded and kept that they will be clean and sanitary at all times. 20. Stables should be whitewashed at least twice a year unless the walls are painted or are of smooth cement. 21. Manure must be removed from the stalls and gutters at least twice daily. This must not be done during milking, nor within one hour prior there- to. 22. Manure should be taken from the barn, pre- ferably drawn to the field. When the weather is such that this cannot be done, it should be stored not nearer than 200 ft. from the stable and the manure pile should be so located that the cows cannot get at it. 23. The liquid matter should be absorbed and removed daily and at no time be allowed to overflow or saturate the ground under or around the cow barn. 24. Manure gutters should be from six to eight inches deep, and constructed of concrete, stone or some non-absorbent material. 170 Agricultural Bacteriology 25. The use of land plaster or lime is recommended upon the floors and gutters. 26. Only bedding which is clean, dry and absor- bent should be used, preferably sawdust, shavings, dried leaves or straw. No horse manure should be used as bedding. 27. The flooring where the cows stand should be so constructed that afl manure may drop into the gutter and not upon the floor itself. 28. The floor should be swept daily. This must not be done within one hour prior to milking time. 29. If individual drinking basins are used for the cows, they should be frequently drained and cleaned. 30. All live stock other than cows should be ex- cluded from the room in which the milch cows are kept. (Calf or bull pens may be allowed in the same room if kept in the same clean and sanitary manner as the cow beds.) 31. The barnyard should be well drained and dry, and should be as much sheltered as possible from the wind and cold. Manure should not be allowed to collect therein. 32. A suitable place in some separate building should be provided for the use of the cows when sick, and separate quarters must be provided for the cows when calving. 33. There should be no direct opening from any silo or grain pit into the room in which the milch cows are kept. The Milk House 34. A mflk house must be provided which is sepa- rated from the stable and dwelling. It should be located on elevated ground, with no hog-pen, privy or manure pile within 100 feet. The Control of Foods 1 71 35. It must be kept clean and not used for any purpose except the handling of milk. 36. The milk house should be provided with sufTicient light and ventilation, with floors properly graded and made water-tight. 37. It should be provided with adjustable sashes to furnish sufficient light, and some proper method of ventilation should be installed. 38. The milk house should be provided with an ample supply of clean water for cooling the milk, and if it is not a running supply, the water should be changed twice daily. Also a supply of clean ice should be provided to be used for cooling the milk to 50° F. within two hours after milking. 39. Suitable means should be provided within the milk house, to expose the milk pails, cans and utensils to the sun or to live steam. 40. Facilities consisting of wash basins, soap and towel should be provided for the use of milkers before and during milking. During the summer months the milk house should be properly screened to exclude flies. The Milkers and Milking 41. Any person having any communicable or infectious disease, or one caring for persons having such disease, must not be allowed to handle the milk or milk utensils. 42. The hands of the milkers must be thoroughly washed with soap and water, and carefully dried on a clean towel before milking. 43. Clean overalls and jumpers should be worn during the milking of the cows. They should be 172 Ar.RicuLTURAi. Bacteriology used for no other purpose, and when not in use should be kept in a clean place protected from dust. 44. The hands and teats should be kept dry during milking. The practice of moistening the hands with milk is to be condemned. 45. The milking stools should be at all times kept clean, and iron stools are recommended. 46. The first streams from each teat should be rejected, as this foremilk contains more bacteria than the rest of the milk. 47. All milk drawn from the cows 15 days before, or 5 days after parturition should be rejected. 48. The pails in which the milk is drawn should have as small an opening at the top as can be used in milking; top opening preferably not to exceed 8 inches in diameter. This lessens the contamination by dust and dirt during milking. 49. The milking should be done rapidly and quietly, and the cows should be treated kindly. 50. Dry fodder should not be fed to the cows during or just before milking, as dust therefrom may fall into the milk. 51. All milk utensils, including pails, cans, strainers, and dippers must be kept thoroughly clean and must be washed and scalded after each using, and all seams in these utensils should be cleaned, scraped and soldered flush. The Milk 52. Milk from diseased cows must not be shipped. 53. The milk must not be in any way adulterated. 54. The milk as soon as drawn should be removed to the milk house and immediately strained and cooled to the proper temperature. ' The Control of Foods 173 55. All milk must be cooled to a temperature below 50° F., within two hours after being drawn, and kept thereafter below that until delivered to the creamery. 56. The milk should be strained into cans which are standing in. ice water which reaches the neck of the can. The more rapidly the milk is cooled, the safer it is, and longer it will keep sweet. Ice should be used in cooling milk, as very few springs are cold enough for the purpose. 57. If separators are used, they should stand where the air is free from dust or odors, and on no account should they be used in the stable, or out of doors. 58. Milk strainers should be kept clean; scalded a second time just before using, and if cloth strainers are used, several of them should be provided, in order that they may be frequently changed during the straining of the milk. 59. The use of any preservative or coloring matter is adulteration, and its use by a producer or shipper will be a sufTicient cause for the exclusion of his product from the city of New York. Water Supply 50. The water supply used in the dairy and for washing utensils should be absolutely free from any contamination, sufficiently abundant for all purposes, and easy of access. 61. This supply should be protected against flood or surface drainage. 62. The privy should be located not nedrer than 100 feet of the source of the water supply, or else be provided with a water-tight box that can be readily 174 Agricultural Bacteriology removed and cleaned, and so constructed that at no time will the contents overflow or saturate the surrounding ground. 63. The source of the water supply should be rendered safe against contamination by having no stable, barnyard, pile of manure or other source of contamination located within 200 feet of it. In order that the farm inspection shall be as effective as possible, and to make the work of the several inspectors as uniform as may be, the dairies are scored. A copy of the score card follows. DEPARTMENT OF HEALTH The Cily of New York division of Gnieriil Sanilary Inspeclion Dairy Report Inspection No Time A. P. M.. Dale 191 1 Dairyman Owner 2 P. O. Address P. O. Address State 3 County State Party Interviewed 4 Milk delivered to Creamery at Formerly at 5 Operated by Address 6 Distance of farm from Creamery Occupied farm since 7 No. Cows No. Milking No. Qts. Produced 8 All persons in the households of those engaged in producing or hand- ling milk are free from all infectious disease. Weekly re- ports are being filed 9 Date and nature of last case on farm 10 WATER SUPPLY for utensils is from a located feet deep and apparently is pure and wholesome State any possible contamination lo- cated within 200 feet of source of water supply or if water supply is not protected against surface drainage 11 Water supply on this farm analyzed 191 Result 12 style of Cow Barn Length ft. Width ft. Height of ceiling ft. 13 Dairy Rules of the Department of Health are posted 14 Dairy Herd examined by on 191 Report The Control of Foods 175 Perfect Allow EQUIPMENT 15 COW STABLE is located on elevated ground with no stagnant water, hiog-pen: privy, uncovered cesspool or manure pit within 100 feet ^ 16 FLOORS, other than cow beds, are of concrete or some non-absorbent material 17 Floors are properly graded and water- tight 18 Cow beds are of concrete or planks laid on concrete 19 DROPS are constructed of concrete, stone or some non-absorbent material 1_ 20 Drops are water-tight and space beneath is clean and dry 21 CEILING is constructed of and is tight and dust proof 22 WINDOWS No total square feet there is 2 square feet of window light for each 600 cu. ft. air space (1 sq. ft. per each 600 cu. ft. — 1) 23 VENTILATION consists of sq. ft. muslin covered openings or sq. ft. open chutes in cj^iling or which is sufficient 3, fair 2, poor 1, insufficient 24 AIR SPACE is cu. ft. per cow (600 and over— 3) (500 to 600—2) (400 to 500—1) (un- der 400— 0) 25 LIVE STOCK, other than cows, are ex- cluded from rooms in which milch cows are kept - 26 There is-_ direct opening from stable into silo or grain pit 27 Separate quarters are provided for cows when calving or sick 28 COW YARD is properly graded and drained 29 WATER SUPPLY for cows is unpol- luted and plentiful 30 MILK HOUSE has direct opening into cow barn or other building 31 Milk house has sufficient light and ventila- tion 32 Floor is properly graded and water- tight _ 33 Milk house is properly screened to ex- clude flies 34 Milk pails are of smoothly tinned metal in good repair.;. 35 MILK PAILS have all seams soldered flush 36 Milk pails are of the small mouthed d^esign, top opening not exceeding 8 inches in diameter. Diameter 37 Racks are provided to hold milk pails and cans when not in use 38 Special milking, suits are provided 40 176 Agricultural Bacteriology 39 41 43 44 45 47 48 49 50 51 52 53 54 56 57 58 59 60 62 63 64 65 66 METHODS STABLE INTERIOR painted or white washed on which is satisfactory 3, fair 2, unsatisfactory 1, never FEEDING TROUGHS, platforms or cribs are well lighted and clean Ceiling is free from hanging straw, dirt or cobwebs Window panes are washed and kept W A L LS ~ A N b~ LED g'eS ~ areW.V " "f ree ~f ro" dirt, dust, manure or cobwebs FLOORS AND PREMISES are free from dirt, rubbish or decayed animal or vegetable matter COW BEDS are clean, dry and no horse manure used thereon Manure is removed to field daily 4, to at least 100 feet from barn 2, stored less than 100 feet or where cows can get at it Liquid Matter is allowed to saturate ground under or around cow barn Milking Stools are clean Cow Yard is clean and free from manure COWS have been tuberculin tested and all tuberculous cows removed Cows are all in good flesh and condi- tion at time of inspection Cows are all free from clinging ma- nure and dirt. (No. dirty) LONG HAIRS are kept short on belly, flanks, udder and tail UDDER AND TEATS of cow are thoroughly brushed and wiped with a clean damp cloth before milkin.g ALL FEED is of good quality and distil- lery waste or any substance in a state of putre- faction is fed MILKING is done with dry hands FORE MILK or first few streams from each teat is discarded , Clothing of milkers is clean Facilities for washing hands of milkers are provided in cow barn or milk house Milk is strained at and clean atm ©sphere Milk is cooled within two hours after milking to 50 degrees F. 3, to 55 degrees F. 2 to 60 degrees F. 1 Ice is used for cooling milk MILK HOUSE is free from dirt, rub- bish and all material not used in the handling and storing of milk Milk Utensils are rinsed with cold water immediately after using and washed clean with hot water and washing solution Utensils are sterilized by steam or boiling water after each using Privy is in sanitary condition, with vault and seats covered and pro- tected Perfect Allow 60 Remarks. Equipment 10 per cent. Methods 60 per cei Perfect Dairy 100 i Score per cent The Control of Foods 177 Grades of milk. — Recognizing the fact that milk is utilized for a, number of different purposes, ranging from infant feeding to its use as an ingredient in the preparation of food, many cities allow the sale of different grades of milk. The following grades are sold in New York: Guaranteed milk is usually that produced by animals that have been shown to be free from tuberculosis by the tuberculin test; which also does not contain over 30,000 bacteria per cubic centimeter, and is delivered to the consumer in bottles within 30 hours after withdrawal from the animal. Inspected milk is from animals free from tuber- culosis. It must not contain over 60,000 bacteria per cubic centimeter when delivered to the consumer. The farm must score at least 25 points on equip- ment and 50 points on methods. Selected milk must be produced on farms that score at least 20 points on equipment and 40 points on methods. Since animals may not have been shown to be free from tuberculosis, the milk must be pasteurized. The milk may not contain over 50,000 bacteria per cubic centimeter when delivered. The above requirements refer to the first grade. A second grade is provided for in which selected milk is that produced by cows that have been physi- cally examined by a veterinarian and pronounced healthy and free from tuberculosis. The farm must score at least 25 points for equipment and 43 for methods. No definite bacterial standard is estab- lished. If this grade of milk is pasteurized before sale no examination of the animals is required. A third grade that can be used only for cooking is often pi-ovided for. 12a 178 Agricultural Bacteriology Certified milk is that produced under the regula- tions and control of a medical milk conmmission appointed by the county medical society. The in- spections of the farm and of the milk are made by experts. The rules are usually very rigid and the number of bacteria can not exceed 10,000 per cubic centimeter. On some of the farms the milk is pro- duced with the most extreme care to avoid the intro- duction of dirt. The expense connected with its production is great, and this grade of milk must of necessity sell at a price that places it out of the reach of the ordinary consumer. Pasteurization of market milk. — The inability of cities to obtain milk from animals that are known to be free from tuberculosis, and the impossibility of protecting the milk from contamination with typhoid bacilli and other pathogenic organisms, has led to the adoption of pasteurization as a means of obtaining a safe supply. The temperatures recom- mended for the process are as follows: 158° F. for 3 minutes 155° F. for 5 minutes 152° F. for 10 minuLes 148° F. for 15 minutes 145° F. for 18 minutes 140° F. for 20 minutes These temperatures are sufTicient to destroy all pathogenic organisms. In actual practice the milk is heated to 145° F. for 25 to 35 minutes. PART IV TRANSMISSIBLE DISEASES CHAPTER XVI THE RELATION OF MICROORGANISMS TO DISEASES OF ANIMALS Communicable diseases. — The diseases of ani- mals may be divided to two classes: the organic or constitutional that are due to the faulty operation of some organ, and the communicable which are caused by the invasion of the body by some organism and the growth thereof with the formation of sub- stances that have a harmful action on the body. The latter are termed communicable diseases, be- cause the passage of the causal organism from the diseased to the healthy animal is sufficient to spread the trouble. The organic diseases can not be thus transmitted for they are not due to the presence of a living organism in the body. The communicable diseases are also termed infectious, contagious, and preventable, since the prevention of their spread can be accomplished by stopping the transmission of the organism. In some instances the knowledge of the manner of transmission is so complete that if stock- men could be induced to put that knowledge into practice, the diseases would soon disappear, while in other cases, the information is not yet sufficiently complete to enable their spread to be prevented. 180 Agricultural Bacteriology Such transmissible diseases as tuberculosis, conta- gious abortion, hog cholera, Texas fever and glanders entail an enocmous tax on the live stock industry of the world, as will appear in the discussion of the speci- fic diseases. Since the prevention of disease is a prob- lem that must always rest in the hands of the farmer himself, rather than in professional aid he may employ, it is desirable that every stockman be ac- quainted with the salient facts concerning the more important of the transmissible diseases of domestic animals, just as everyone should know something of the important transmissible diseases of man so that he may intelhgently protect himself from them. Infection. — In order to produce disease the organism must invade the body, must grow therein, and its by-products must exert an injurious effect on the body tissues. This sequence of events is known as infection. The severity of the attack of any communicable disease may vary from one ani- mal to another, due to the difference in resistance of the host and to a difference in the virulence of the organism, which may be defined as the power of the organism to multiply within the body and produce disease. Little is known of the conditions that increase or diminish the virulence of organisms in nature. The resistance of the host may be im- paired by any condition that tends to weaken the body, such as fatigue, exposure to cold, heat or dampness, improper diet, thirst, age, wounds, and other diseases. Neither the invading organism nor the host are to be considered as passive agents; the relation between them is a true struggle, a fight to the finish. In the struggle the host seeks to over- come the parasite by means that will be discussed later, and the organism proLecls itself that growth MiCROOUGANISMS AND DISEASE 181 may take place and perpetuation of the species be accomplished. The portals of entry into the body are the broken skin, or an injured mucous membrane, the alimentary tract, the respiratory tract, the genital tract and the conjunctiva, or the mucous membrane of the eye. Many organisms have specific, definite methods by which they enter the body of the host, as for example, the hog cholera organism enters by way of the ahmentary tract, while the tubercle bacillus enters in a variety of ways, as through wounds, by inhalation, or by ingestion. The tetanus or lock jaw bacillus is always introduced through wounds of the skin or of the mucous membranes. The original or initial infection is called the pri- mary infection, and may be followed by a second invasion with another kind of organism which may have been present in the body, but was unable to multiply until the resistance was first lowered by the primary infection. Infection is usually due to a single specific organism, but some troubles are due to a mixed infection with two or more organisms. When the body has been weakened , by organic diseases, it is sometimes more susceptible to invasion by certain disease-producing organisms, or results of invasion are more likely to be serious. The original trouble might have resulted in death, but the end is hastened by the terminal infection, as it is called. The invading organisms injure the tissues by the production of poisonous substances, known as toxins. In some cases the organisms grow in a limited area, and do not cause any great destruc- tion of the tissue at the point of growth, but the toxin is so active that a minute quantity is sufficient to cause death. Such a disease is known as toxemia, 182 Agricultural Bacteriology in contradistinction to the bacteremia or septicaemia, in which the entire body is invaded by the organism, as in the case of anthrax. Examples of toxemias are lock jaw and diphtheria. In still other instances the invasive powers of the organisms are not great, but the tissue is destroyed at the point of growth, as in the case of the pus-producing organisms. It is evident that the symptoms of any disease can not appear until the organism has had time to multi- ply and to form suflTicient toxin to have a visible effect on the body of the animal. This period which may vary from a few days to several months is called the period of incubation of the disease. The changes in the various tissues due to the action of the organism are called the lesions of the disease. With some diseases they are very characteristic, in others not. The external defenses of the body. — There are .many means by which nature has sought to protect the body against the invasion of microorganisms. The surface of the body is covered by the skin, and all of the cavities of the body that are in contact with the exterior are provided with a mucous mem- brane. The bacteria normally gain entrance with but few exceptions through these protective mem- branes, only as they are injured. The mucous membranes are always bathed with the products of glandular activity, which possesses a more or less marked germicidal or antiseptic action. By reason of this many of the organisms that come in contact with these fluids are thus destroyed. Wounds in the mouth and in the intestine must of necessity frequently occur, especially with animals that feed on coarse, dry fodder. Yet a harmful effect from such a source is rarely noted. The lungs and air Microorganisms and Disease 183 passages are constantly exposed to dust laden with adherent bacteria. These foreign bodies are re- moved by the action of the cilia of the cells lining the air passages. The hair-like appendages are con- stantly in motion and tend to move any foreign particle outward. The internal defenses. — After the microorgan- isms have invaded the tissues, their development can not go on unhampered, for the body has a number of internal defenses that must be overcome before growth and disease production can occur. An ani- mal is said to be immune to a disease when it resists the development of the organism, or is not injured by the poison which the organism produces. Various explanations have been offered to explain the im- munity of animals. The white blood corpuscles possess ameboid properties, or are able to ingest solid bodies like the bacteria and digest them. This process is known as phagocytosis, and such devouring cells are called phagocytes. Whenever any portion of the body is invaded by a foreign agent, or when any abnormal condition arises the phagocytes are at- tracted to this point as a result of chemical stimulus. This causes them to accumulate at or near the point of invasion, where they soon engulf and destroy many of the invading organisms. If these white blood corpuscles are able to overcome the harmful bacteria, the initial infection may be rendered of no importance. Again the organism may multiply and form poisonous products which may injure the body to the extent that, death is caused or under the stimulus of these harmful products, the body cells may react and form substances that neutralize or nullify in some way the poisonous effects of the products of the organism. These antagonistic body 184 Agricultural Bacteriology products are diffused through the liquids of the body, especially in the blood serum, and form the basis of the anti-serums that are used for protective purposes. A specific disease may occur only in a single host species, as in hog cholera, or it may be capable of spreading throughout a variety of different animals. Blackleg affects only cattle and sheep, while the anthrax bacillus produces a characteristic disease in man as well as in many of the lower animals. Numerous diseases, affecting man, such as typhoid fever, diphtheria, yellow fever, and cholera are limited to this host alone. Other warm-blooded animals are naturally insusceptible to these maladies ; they possess a natural immunity. Immunity is a condition of the body in which the disease organism is not able to develop, al- though in a closely related species, or by reason of some change in condition, development may be- come possible. Thus, sheep are highly susceptible to anthrax, but the Algerian variety is naturally immune to this organism. Sometimes immunity is racial in character, as is noted especially in malaria with the black race; sometimes it is influenced by age, as in the case of children's diseases like measles, chicken pox, and whooping cough. Well-condi- tioned young cattle are more prone to fall victims to blackleg than aged animals. Immunity may be either natural, as in the above cases, or acquired. If acquired, it may have been conferred by the operation of natural causes, such as a previous attack of the disease, or artificially pro- duced by inoculation or vaccination. Southern caltle acquire a natural immunity toward Texas fever by reason of the fact that from birth, they are Microorganisms and Disease 185 continually exposed to tick infection and have the disease in the mild form. With many diseases immunity is conferred by virtue of a first attack, as with smallpox, measles, and hog cholera. With some diseases such as pneu- monia and influenza in man, an initial attack seems to predispose the system to a second infection. An immunity that is due to recovery from a natural attack of a disease is called active, since the indi- vidual gains protection through the unaided action of its own tissues. The body has manufactured sub- stances that in some way counteract the poisons produced by the organism, the toxins. These bodies are called antitoxins on anti-bodies since their action is not always directed against the poison but may be directed against the organism itself. Thus in the body of an individual rendered immune by an at- tack the disease-producing organism may not be able to grow even • though it succeeds in invading the tissues. In the case of acquired immunity it may be active, as when the body is forced to manufacture its own protective substances or anti-bodies, or the im- munity may be passive as when the anti-bodies are transferred from the body of the individual that has made them to the body of another animal that it is desired to protect. Active immunity, whether natural or acquired, is always produced by the ac- tion of the organism that causes the disease. As has been seen a natural attack protects against a subse- quent one in the case of many diseases, as small pox for example. It is a well known fact that some outbreaks of a disease are very severe in that many of the infected die while in another outbreak of the same disease 186 Agricultural Bacteriology practically all the infected individuals recover. This can not be explained on the greater resistance of the second group over the first but rather the explana- tion is to be sought in the diminished virulence of the organism. As was stated the causes that induce such changes in nature are unknown. The first effort to impart immunity by artificial means was by the intentional inoculation of people with material taken from mild cases of small pox. The mild attack thus induced afforded protection to the individual against the more severe form of the disease. Later it was noted by Jenner, an English surgeon, that those individuals that had acquired cow pox by milking a cow suffering from this trouble were thereby protected against small pox. Due to this observation the inoculation of human virus against small pox was superseded by vaccination with material taken from the pustules of the animal disease, cow pox, or vaccinia, as it is called. It is now known that the organism causing cow pox is a modified form of the small pox virus. In some man- ner its residence in the body of cattle has so changed its properties that it is no longer able to produce a dangerous form of the disease in man, but it is able to stimulate the tissues to manufacture sufficient anti-bodies to protect the body for a number of years against a natural attack. The .vaccine used for inoculation contains the virus of small pox, the nature of which is unknown. All vaccines that are used as a protective measure against any contagious disease contain the virus of the disease against which protection is sought. The virus may be virulent; it may be attenuated or weakened; or it may be dead. The degree of protection afforded by the vaccina- tion process will depend on the extent to which the Microorganisms and Disease 187 organism has been attenuated. Thus the protection afforded by "killed" organisms is not as great as when the weakened organisms are used. In the case of human vaccination "killed" cultures are usually employed because of the possibility that the viru- lence of the weakened organism may accidentally be regained. The manner in which the different vac- cines are made will be discussed in the treatment of the specific diseases. Vaccines are used not only to prevent but also to cure disease. In the production of passive immunity the anti- bodies are transferred from the body of the animal in which they have been actively formed to the animal to be protected. This transfer is accom- plished by withdrawing a portion of the blood from the immune animal, and injecting it into the animal that it is sought to protect. Since the blood serum is used, the term protective serum or antiserum is often used. Because of the content of the serum in anti-bodies the term anti-toxin is also employed, as diphtheria anti-toxin. The blood of a hog that has recovered from hog cholera will contain sufficient anti-bodies to protect the individual against a subsequent attack, but not a sufficient amount so that the blood would bestow any marked degree of protection on another animal when inoculated with an amount that would be practicable to use. In order to make the method of practical value, the immune animal is forced to manufacture a larger amount of the anti-bodies than would normally be produced. An animal so treated is said to be hyperimmunized. In preparing hog- cholera serum, this is accompUshed by injecting into the body of the immune hog a large quantity of blood from a hog that is already sick with 188 Ar.RicuLTURAT. Bacteriology hog cholera. The specific organism causing hog cholera is yet unknown, although it can be trans- ferred by the use of blood from a sick hog. The in- troduction of a large quantity of the virus into the body of the immune hog causes the formation of an increased amount of the protective bodies. The immunizing process is thus repeated, until the blood contains such a quantity of protective substances that when transferred in practicable amounts, it imparts a considerable degree of immunity. The disease virus is obtained from a sick hog by bleeding from the throat. This virulent blood is usually in- troduced into the blood vessels of the animal to be hyperimmunized, which, when its blood is sufTi- ciently high in the protective bodies, is bled for the anti-serum by cutting off a piece of the tail. The animal can be bled several times in this way, a fresh cut being made each time until this appendage is too short for further use. The final bleeding is then made from the throat. In the preparation of diphtheria antitoxin the horse is used to produce the anti-bodies. This ani- mal is not susceptible to diphtheria; hence, the or- ganisms themselves can not be employed to stimulate the production of anti-bodies. The horse is, how- ever, susceptible to the toxin of the diphtheria organism. The organism is grown in the laboratory in beef broth, which is filtered through porcelain to remove all the bacteria, and gradually increasing doses of this filtrate are then injected into the body of the horse. At first very small doses can only be administered without killing the animal, but after recovery from the first injection, repeated doses of increasing amounts are applied, Lhe effect of which is to produce Lhe protective anti-bodies in the blood Microorganisms and Disease 189 of the animal. The blood is then drawn from the jugular vein; it is allowed to coagulate in order to remove the clot, and the blood serum is used. Not only does this serum protect the child from acquir- ing diphtheria by rendering him artificially immune but it acts as a curative agent in neutralizing the poison of the disease, if applied in the earlier stages of the disease. The blood serum of animals hyperimmunized against hog cholera or diphtheria varies greatly in the amount of anti-bodies formed. Before it is used it is necessary to know something of the strength or potency of the serum, so that the proper quantity to be used in the animal to be protected may be determined. This is accomplished in the case of hog-cholera, by inoculating a number of young pigs with a definite quantity of the disease virus, and a varying amount of the protective serum, noting the amount which is required to protect the animal against the artificial inoculation. In the case of the diphtheria antitoxin the determination of the dosage is made by using guinea pigs as an index of the strength of the antitoxin. Persistence of immunity. — Passive immunity produced by the transfer of the anti-bodies is always of short duration as compared with the active im- munity produced by artificial means, while the ac- tive immunity produced as a result of the natural cause of the disease persists for a still longer period. The variation in time during which protection per- sists must be taken into account in the practical employment of serums and vaccines in the preven- tion of animal diseases. Exit of organisms from body. — Almost with- out exception the pathogenic organisms grow only 190 Agricultural Bacteriology in the bodies of susceptible animals. Their contin- ued existence in nature is therefore dependent upon their expulsion from the diseased body, and the op- portunity for introduction into a new susceptible host. The exits from the host by which organisms find their way to new hosts vary in the different diseases. With intestinal diseases as hog cholera, the excreta serves as a mode of exit. In tuberculosis the secretions, as milk and saliva, function as car- riers of contagion. With some diseases the blood from wounds caused by biting insects or the dis- charges from abscesses on the surface of the body serve as channels of transmission. The transfer of the causal organisms from one animal to another may take place in a multitude of ways. In the same herd the healthy animal easily comes in direct contact with the infectious material from the diseased animal. The spread of the disease to other herds may take place through the transfer of an infected animal or infectious material, such as milk or contaminated objects. The direct methods of transfer can be quite readily guarded against, but the more indirect modes of transmission are much more difficult to detect. Thus hog cholera virus can be readily transferred by dogs, crows, or persons carrying the virus on their feet. The opportunity for the transfer of organisms for any considerable distance is dependent on the re- sistance of the organism, which is largely determined by the fact as to whether it produces spores or not. Most of the non-spore-forming organisms can not persist for any long period outside the animal, as they succumb quite readily to such unfavorable en- vironmental influences as drying, sunlight, and the action of saprophytic bacteria. The spore-forming Microorganisms and Disease 191 organisms, on the other hand, can persist for long periods due to the resistance of the spores to all or- dinary environmental conditions. Necessity for correct diagnosis. — It is very es- sential that a correct diagnosis of any of the trans- missible diseases be made, for the methods that will prove effective against one may have no effect against another. Especially is this true when the serums or vaccines are to be used in preventing fur- ther spread, for these substances are specific in their action. The farmer must usually rely on the expe- rienced veterinarian for a proper diagnosis of any transmissible disease and the veterinarian is fre- quently forced to call to his aid the facilities of a bacteriological laboratory. The use of drugs in the treatment of the trans- missible diseases is usually without any curative effect. The farmer must exert his efforts to prevent the disease, and especially to prevent their intro- duction onto his farm. CHAPTER XVII ANTHRAX, BLACK LEG, HEMORRHAGIC SEPTI- CAEMIA, AND CORN STALK DISEASE From a geographical and zoological point of view, anthrax is said to be the most widespread of all of the communicable diseases. In this country it is not of so much economic importance as some other dis- eases, but in other parts of the world, it is to be classed as one of the most important diseases affect- ing domestic animals. Because of the ease with which man acquires it from the animal, it is also in- vested with considerable sanitary significance. On account of the highly resistant properties of the or- ganism, any locality that becomes the seat of an outbreak is likely to remain contaminated for a num- ber of years. Before the communicable nature of Lhe disease was known, it had spread widely over Europe and Asia, and in France and Russia had caused great losses. Outbreaks have occurred in twenty-five of the Ameri- can states, and in certain regions, as in the delta lands of the Mississippi, it makes an annual appear- ance. By reason of the resistance of the spores, it is possible for infectious material to be transferred long distances. Many of the outbreaks in this coun- try have been due to contaminated hides which were removed from anthrax cadavers in China and South America. The disease in one that has played a great role in the development of bacteriology. It was the first disease that was shown to be due to the infection of Anthrax and Black Leg 193 the body with a specific organism. In 1876, Robert Koch showed that the organism that had been noted by Pollender in 1840 in the blood of animals suffer- ing from anthrax could be grown outside the body for long periods of time, and that its reintroduction into the body of susceptible animals gave rise to the disease with symptoms and lesions identical with those noted in naturally occurring cases. It was like- wise the first disease for which methods of preven- tion by vaccination were elaborated by the French bacteriologist Pasteur in 1881, and thus formed the starting point for all the discoveries of prevention and cure of communicable diseases by the use of biological products, vaccines and serums. The disease affects chiefly cattle, sheep, goats, horses, and less frequently hogs. Cats and dogs as well as birds are quite resistant. The disease is va- riously known in its several forms; splenic fever re- fers to the enlarged condition of the spleen, while malignant carbuncle refers to the development of large swellings on the surface of the body, a common manifestation of the disease in man when the organ- ism has been introduced through wounds. Infection. — In the case of cattle, sheep, and horses, the portal of entry is most frequently the ahmentary tract. It seems probable that in many cases the organism can pass through the uninjured wall of the intestine, but it is certain that its en- trance is made more easy by the presence of wounds in any part of the tract. It is probable that wounds are almost constantly present in the mouths of cattle that are grazing or that are fed dry feed. The trans- fer of the organism from infectious material, and its entry into the body is often accomplished by the agency of biting flies. It is thought that this is the 13a 194 Agricultural Bacteriology chief way in which the horses on the plantations in Mississippi become infected. The infection may oc- cur by grazing on infected pastures or by the use of infected food in the stable. It is not probable that the organism can grow to any extent outside the body, but on account of the formation of spores it can persist in the fields, yards and stables for years. The spores are formed only in the presence of free oxygen, a property that becomes of great importance in limiting the spread of the disease. The rapidity with which the disease progresses to a fatal termination varies greatly. Usually the first animals lost in an outbreak die without any apparent symptoms being noted. An animal may give the usual quantity of milk in the evening and be found dead in the morning. Such rapid progress of the disease leads to the suspicion of poisoning, or death by lightning in case a thunder storm has occurred. These conclusions may lead to the careless disposal of the carcass, endangering human life as well as the rest of the animals on the farm. It is well to con- sider all cases of sudden death in animals, especially when no cause can be given for the same, as due to dangerous causes, and act accordingly in the disposal of the carcass. More often animals show signs of illness for a longer or shorter period of time, a fever of 105°-108° F. being present. They may become restless, paw the ground or show violent movements and develop convulsions which are soon followed by death. Swellings on the surface of the body are some- times noted. In these cases the duration of the dis- ease is likely to be longer than where the infection has been more general. The mortality from the disease is from 70 to 80 per cent. Anthrax and Black Leg 195 Lesions. — One of the most marked changes to be noted in the post-mortem examination of typical cases is the dark blood which may appear almost tar-like. The blood does not coagulate like normal blood. The spleen or milt is usually greatly increased in size; is dark in color and has lost its structure as is shown by the semi-liquid consistency of its in- terior. Blood often issues from the rectum after death, and upon section, hemorrhages or bloody areas are found in which the blood has oozed from the blood vessels into the tissues. If the infection has taken place through wounds, carbuncles may develop on the surface of the body. The swellings are at first hard, hot, and painful, but later, due to the death of the tissue at the center of the swelling, the fever subsides, and no pain is evidenced by the animal when the carbuncle is opened. The exudate will be tar-like in consistency. Gangrene, due to a secondary invasion of the abscess with putrefactive bacteria, is often noted. Sudden death with black, non-coagulated blood and an enlarged spleen should always lead one to suspect anthrax. The disease is a true septicemia in that the organ- isms are to be found in great numbers in every por- tion of the body at the time of death. The absolute diagnosis of the disease is made by finding the an- thrax bacillus in the tissues, and in some cases in which the post mortem changes are not typical this is the only way in which the diagnosis can be made with certainty. Whenever an animal has died and anthrax is suspected, the remaining portion of the herd should be examined immediately, the temperature of each animal should be taken, and all that show any fever, 104° F. or above, should be allowed to remain in 196 Agricultural Bacteriology the infected quarters, while the remainder of the herd should be removed. Prompt separation of the animals and due care in the .disposal of carcasses and disinfection will do much to prevent continued loss. Vaccination. — Healthy animals should be vac- cinated. The anthrax vaccine is made by growing the organism at a temperature above the optimum. At these high temperatures the virulence is ma- terially reduced. The longer the organism is thus grown, the more its virulence is decreased. For successful protection, two applications of vaccine are made. The vaccine first administered is so at- tenuated that it has no effect on the large domestic animals, nor even on a guinea pig, but will kill a white mouse. The second vaccine, applied ten days later, is of sufTicient potency to kill a guinea pig but not a rabbit. The application of the second vaccine protects the animal from the natural course of the disease. It is, of course, impossible to use too strong a vaccine since the less resistant animals would be killed thereby. By using a weak vaccine first, and then a stronger, protection can be given with little loss due to the vaccination. In the practical ap- plication of the vaccine about one per cent of the animals are lost. The immunity thus conferred is active and persists for about one year. In some sections where pastures are permanently contam- inated, a single application of strong vaccine is employed. The losses under these conditions amount to several per cent, but the survivors are protected from a natural infection. With the recognition of the importance of care in the handhng of the car- casses and the use of vaccine, the ravages of anthrax have greatly decreased. Anthrax and Black Leg 197 Disposal of carcasses. — In the unopened carcass the organism can not form spores, due to the lack of oxygen, but in the discharges from the nose and rectum, and in any blood that may be brought in contact with the air in making a post-mortem examination spore formation readily occurs. In the vegetative form, the organisms are easily killed, but the spores are exceedingly resistant, so that every precaution should be taken to prevent the blood from coming in contact with the air. The carcass should not be skinned nor opened, unless it it neces- sary for the purpose of establishing the cause of death. If it is not possible to destroy the carcass at the place where death occured, it should be re- moved by placing it on a stone boat instead of dragging it on the ground. The opening of the carcass should take place only at the point where its ultimate disposal is to be carried out, which, if possible, should be by burning rather than burying. If the carcass is buried, it should be covered with quick lime and the spot so chosen that there will be no danger of the carcass being washed out. It is well to fence the spot so that dogs or stock can not have access. In the unopened carcass the vege- tative organisms rapidly die due to putrefactive processes. The great danger in anthrax lies in the non-recognition of the first case and in the careless disposal of the carcass. Anthrax of man. — Man is less suspectible to anthrax than are cattle and sheep. In the disposal of anthrax cadavers, especially when the disease is not recognized and the hide is removed, there is great danger of infection through wounds on the hands or those inflicted during the process of skin- ning. In case infection occurs, the trouble is likely 198 Agricultural Bacteriology Lo be localized in the form of a carbuncle at the point of entry. In the handling of infected material such as hides, wool, and bristles, the workers are hkely to become infected, especially when the material is handled in a dry condition so that the spores of the anthrax bacilli may be taken into the lungs or into the alimentary tract. Both respiration and ingestion anthrax are usually fatal. Importance of correct diagnosis. — There are a number of diseases that are frequently mistaken for anthrax, and since the advent of vaccines for the prevention of some of these diseases, it is much more important than formerly that a correct diag- nosis be made. In cases that show typical lesions there is httle danger of confusion, but cases occur that are more or less atypical, and frequently a bacteriological examination of the tissues is necessary to determine the cause of the trouble with accuracy. For this purpose samples of the tissues must be for- warded to some laboratory for examination. Such laboratories are maintained by many states in con- nection with the colleges of agriculture. The tissues should be so packed that they will not be a source of danger to those who must handle them in trans- portation. The tissue should be placed in a jar that is closed so tightly that no liquid can escape. The jar should be packed in a mixture of saw dust and cracked ice and sent at once to the laboratory. No disinfectant should be added if a bacteriological ex- amination is desired; the putrefactive processes must be delayed as much as possible by the maintenance of low temperatures. Every animal that dies from an unknown cause, should be subjected to a post-mortem examination by a competent person. In making such examina- Anthrax ane Black Leg 199 tion, it is well to proceed on the supposition that the cause of death is due to an organism dangerous to man. The hands should be examined for wounds, and in all cases it is well to coat the hands with some grease before beginning the examination. It should also be remembered that the cause of the death may be traceable to something which can be com- municated to other animals of the herd, and the examination should not be made, until the carcass has been brought to the place of final disposal. Such simple precautions may be cheap insurance against future trouble. Black leg. — The diseases which are most fre- quently confounded with anthrax are hemorrhagic septicaemia and black leg. The latter was classed as a special form of anthrax until the last half of the previous century, when it was found that the causal organism was very different. The bacillus of black leg or symptomatic anthrax, as it is often called, is an anaerobic, spore-forming organism. The spore can be produced in the tissues of the un- opened body, a factor which distinguishes it sharply from anthrax. Black leg is found in all parts of the world from the tropical regions to the northernmost limits at which cattle are kept. In the United States it is most common in the great grazing states of the southwest. In these sections of the country, black leg has been until recently the most important disease of cattle but the means of prevention have been so effectively applied that it has decreased greatly in amount. It is a disease to which young cattle between the ages of six months and two years are particularly prone. It is also a disease that is most likely to 200 Agricultural Bacteriology appear in the best conditioned stock and in those that are growing rapidly. It is supposed that this greater susceptibility of the animal is due to the accumulation of lactic acid in the tissues. The acid has a peculiar effect on the organism, greatly aiding its development in the tissues. The penetration into the tissues is through deep puncture wounds on the surface of the body. It is a disease primarily of the pasture, and hence ap- pears in the northern states only in the summer, but farther south it occurs during the entire year. It affects sheep and goats less frequently than cattle. The symptoms. — The symptoms of the disease are fever and a darkening of the mucous membranes which later may change to a purplish color. The tumors that develop on the heavy muscles of the breast and legs are the most prominent manifesta- tion of the disease. They may appear on any part of the body except below the knee and hock joints. The tumor is at first small and painful, but increases rapidly in size; soon the tissue dies, and no pain is shown by the animal when the tumor is opened. The exudate will be dark red and frothy, with the odor of acetone. The frothy condition is due to the production of gas from the fermentation of the sugar in the muscles. The gas can be noted in the unopened tiimors by pressure from the hand when a crackhng sound is heard, as the muscle fibers are forced apart by the movement of the gas. These tumors have led to the term black leg on account of the dark color of the affected tissue and the location. The gas formation continues after death, often greatly distending the carcass. The blood is normal in color and in coagulating properties. The spleen Anthrax and Black Leg 201 is also normal. In these two respects it is easily differentiated from anthrax. It is highly desirable to prevent the contamina- tion of the soil with the resistant spores that are formed in the affected tissue, and in the exudate from the wounds. In treating the disease the tumors are frequently opened, thus making it much more difficult to prevent the spread to other animals. The same care should be used in the disposal of the carcasses as in the case of anthrax. There is no danger of transmission to man. Vaccination. — The vaccine used for protective purposes is prepared by inoculating a young animal with the organism. After death has taken place the affected muscular tissue is removed and pounded to a pulp, which is then strained through a cloth and dried. In this form it may be stored. When it is desired to prepare it for use, some of the dried material is mixed with water and heated in an oven to 203° to 210° F. for six hours. The dried mass that remains will contain the spores of the organism in an attenuated condition. The powder is mixed with water and injected beneath the skin of the animal. The Bureau of Animal Industry of the U. S. Department of Agriculture has prepared large amounts that have been supplied to the cattle owners. Through the use of the vaccine the losses have been reduced from 10 per cent to one-half of one per cent. Hemorrhagic septicaemia. — This disease is characterized by the invasion of the blood current with the causal organism and by hemorrhages in various parts of the body, or by the passage of the blood vessels into the tissue, a phenomenon that is 202 Agricultural Bacteriology marked by the appearance of reddish areas of varying size. The organism causing the disease is one of a large group, producing a number of diseases in differ- ent animals. Among the most important are chicken cholera and the bubonic or Asiatic plague in man. In cattle, deer and related animals, outbreaks of varying intensity occur. The disease is found in all parts of the world. In our own country it has been most frequent in the upper Mississippi valley. The normal habitat of the organism is unknown. It has been thought by some to be a saprophytic organism, which, under unknown conditions, may suddenly become virulent, and thus cause an epi- demic that usually disappears as quickly and as mysteriously as it appeared. The rapidity of its appearance and the suddenness with which the ani- mals die, together with the helplessness of the owner to combat it, make it a disease much to be dreaded. Frequently the animals die without showing pre- vious symptoms of illness. This may lead to sus- picions of poisoning. In less acute types of the disease, weakness of the limbs may be noted. Re- covery is rare. The manner in which the organism enters the body is unknown, as is also the method of trans- mission from one animal to another. On post-mortem examination reddish spots vary- ing from a pin head to several inches in diameter are found beneath the skin. Hemorrhagic areas are usually present on the heart, stomach, and intes- tines. The blood is red and coagulates in a normal manner. The spleen is normal. It is frequently mis- taken for anthrax on account of the suddenness witn which death occurs. In atypical cases an examina- Anthrax and Black Leg 203 tion of the blood for the specific organism is neces- sary to confirm the diagnosis. No method of vacci- nation has been devised. The prevention of the disease must be accom- pUshed by isolation of the healthy animals from those that show any symptoms. Preferably the healthy animals should be removed to a fresh pasture or meadow and staked out so that no contact be- tween the animals can take place. The carcasses should be disposed of with the same care as in the case of anthrax. If animals die in the stable, the litter should be burned, the stable cleaned and dis- infected. The organisms are easily destroyed since they do not form spores; consequently there is no danger that they will persist in yards and stables as in the case of anthrax. Corn stalk disease. — In those sections of the country in which it is the custom to harvest the ear com in the row, and then turn the stock into the standing fodder, a disease known as corn stalk dis- ease is sometimes encountered. The trouble appears soon after the cattle or horses are turned into the field (4 to 10 days) and runs a rapid course. On account of the suddenness with which death occurs and the large losses which follow in a short time, it is often mistaken for a contagious disease, especially for anthrax, black leg, or hemorrhagic septicaemia. It is important, however, to differen- tiate the trouble which is probably physiological from those diseases that are caused by specific or- ganisms. There is no evidence that the disease is communicable. The differentiation can be made by the lack of abnormal changes in the tissues, and the relation of the appearance of the disease to the period of admission of the animals to the fields. CHAPTER XVIII TUBERCULOSIS Tuberculosis is one of the most important com- municable diseases of both man and the domestic animals. In the latter it is of both economic and sanitary importance, since as noted in the spread of disease by means of milk, a portion of the tubercu- losis occurring in man is due to the organism from bovine sources. Statistics show that one-seventh of all deaths of human beings are due to tuberculosis, and that one-third of the mortality occurring be- tween the ages of 20 and 45, the productive period of life, is caused by the tubercle bacillus. Animals affected. — All of the domestic animals may be affected but the disease is most prevalent among cattle, hogs, and fowls. The other domestic animals are rarely affected. Besides the domestic animals a large number of wild animals are also sus- ceptible. There is probably little or no opportunity for wild animals to come in contact with the organ- ism in nature, but when placed in captivity where there is opportunity for infection, the disease makes rapid strides. It is the chief cause of death of mon- keys, caged animals, and birds in zoological gardens. In the London zoological garden 30 per cent of the birds that died were found to have tuberculosis. A disease caused by an organism belonging to the same group as the tubercle bacillus produces what has been termed tuberculosis in some of the cold- blooded animals. Tuberculosis 205 Distribution. — Within very recent times the commerce in domestic animals and their products and in cultivated plants has increased in a most re- markable manner due to the development of meth- ods of transportation. From northwestern Europe the improved breeds of cattle, sheep, horses, and hogs have been shipped to all parts of the world, and with them have gone the diseases with which they were affected. Many countries into which such diseases were thus introduced were previously free from them, and could have been kept so, if there had been sufficient knowledge concerning the nature of these communicable diseases, their detection and mode of dissemination. This knowledge has been acquired only after the harm has been done in many instances. This country is still free from some of the communicable diseases that are a cause of great loss to the farmers of Europe, and it behooves us to use all possible precautions to prevent their introduc- tion into this country. The islands of Jersey and Guernsey are the only important breeding centers that are free from tuber- culosis, and this condition has resulted from a rigidly enforced rule that no live cattle should be imported onto these islands. The per cent of animals affected with the disease ranges from over fifty in some of the German states, as Saxony, to less than five in our western and southern states. The disease is not widespread in those sections in which cattle raising is not important and into which the improved breeds have not been introduced in considerable numbers. In England it is asserted that less than five per cent of the milking herds of Short Horn, Ayrshire, and Jersey cattle are free from tuberculosis while in Wis- consin, one of the most important dairy states in 206 Agricultural Bacteriology this country, not over one-third of the herds contain any tubercular animals. In the eastern states in which dairying has been longer established, the per- centage of affected herds is higher. The rapid spread of the disease within recent years is shown in the following figures which indicate the per cent of animals found to be tubercular on slaughter. Cattle % Calves % Sheep % Hogs % Horses /O 1898 1906 1898 1906 1898 1906 5.07 10.31 16.09 23.40 30.46 37.58 0.05 0.28 0.15 .33 0.24 0.50 0.35 1.40 2. .32 2.96 3.16 5.07 Bavaria 0.12 0.11 0.17 0.09 0.09 0.11 Prussia 0.12 0.16 Saxony : 0.16 0.30 The only figures available in regard to the spread of the disease in this country are those collected by the Bureau of Animal Industry in the meat in- spection service. In 1901, 0.12 per cent of the car- casses of cattle inspected were condemned; in 1911, 0.35 per cent were condemned. In 1901, 0.035 per cent of the hog carcasses were considered unfit for use on account of tuberculosis, in 1911 the per cent had increased to 0.11. The figures just given refer only to those carcasses in which the disease was so extensive that the entire carcass was condemned. No figures are available as to the per cent of cattle slaughtered that were found to be tubercular. The rules under which carcasses are condemned are changed from time to time, but the great in- crease in percentage of carcasses condemned on Tuberculosis 207 account of tuberculosis is evidence of the rapid spread of the disease in this country. The disease is most common in the pure bred herds ; not because these animals are more susceptible than grades, but because the opportunity for infec- tion in pure bred herds has been much greater by reason of the interchange through purchase. For the same reason large herds are more likely to be found to be tubercular than small herds. The disease is one that spreads among cattle kept out of doors by direct contact almost as rapidly as in the case of those that are stabled for a large part of the year. Different names are often applied to the .various manifestations of the disease in man; Consumption and phthisis refer to tuberculosis of the lungs; scrofula to tuberculosis of the glands of the neck; lupus, to that of the skin, and joint disease to that of the joints. In cattle, grapes and pearl disease, terms sometimes used by butchers, refer to tuber- culosis of the serous membranes. Infection. — The organisms enter the body through either the respiratory or the alimentary tract. The importance of these portals of entrance varies with different animals. It is probable that the bovine becomes easily infected through either of these ways, while in the case of the hog the in- fection under natural conditions is by way of the alimentary tract. The guinea pig, one of the most susceptible animals, acquires the disease with ease by the inhalation of the bacilli, while it is very resistant to infection through the alimentary tract. Distribution in the body. — ^The lymph glands which are widely distributed in the body are to be looked upon as filters, and tend to remove any foreign solid particles, such as tubercle bacilli, that have 208 AGRICULTURAL BACTERIOLOGY entered the lymph or blood circulation. The bacilli are most likely to lodge in those glands that are in close proximity to the point of invasion. Thus, the neck glands are most frequently diseased in the hog, and the glands near the lungs and intestines in the bovine. The lungs, liver, and spleen are also fre- FIG. 3t — TUBERCULAR OMENTUM One of the membranes from the abdominal cavity. The normal mem- brane is perfectly smooth. This form of the disease is frequently called "grapes." quently tubercular. The organs mentioned are those to which the greatest attention should be paid in making a post-mortem examination. Any organ may be tubercular, such as the heart, brain, udder and neighboring lymph glands, the joints, bones, and infrequently the lymph glands located in the muscles. Tuberculosis 209 The bacilli collect in some of these organs and begin to grow slowly. Their presence exerts a stimulus on the body cells in the immediate vicinity, causing the formation of the characteristic tubercles or nodules. The tubercle is most evident when lo- cated on one of the smooth serous membranes of the body, producing that type of disease known as pearl disease or grapes. The cells at the center of the tubercle are soon killed by the poison elaborated by the bacilli. As the area of the dead tissues con- tinues to increase, the tubercles may become conflu- ent, and form tubercular abscesses of varying sizes. Ultimately these abscesses break and discharge their contents into the air passages of the lungs, the milk ducts, the bile ducts, or into some other opening that will enable the bacilh to escape from the body. This condition is known as "open" tuberculosis as opposed to the "closed" form where the tubercles do not break down. The tubercular animal is of danger to others only as it is eliminat- ing the organisms from the body. If the disease has not reached the open stage, or the lesions are found in parts of the body that have no exterior opening, the animal can not be dangerous to others or to the human beings consuming the milk. It is im- possible to foretell when the closed form of the disease will change to the open as it is certain to do if the disease progresses. Hence every affected animal must be considered a potential source of danger to the herd and to public health. The tubercles vary in size from a pinhead to ab- scesses as large as the closed fist. The small tubercles are usually of a light pearly gray color throughout, or they may show a yellowish area at the center, composed of dead tissue. The larger tubercles and 14a 210 Ac.P.IGULTURAL BACTERIOLOGY abscesses may be filled with creamy pus, or with hard, gritty, yellow material due to the deposition of lime salts. The tubercle is then said to be calcified, and its contents have the appearance of corn meal. The lungs of a healthy animal are light pink in color and spongy in texture; in the tubercular organ [he firm, hard tubercles may be felt upon pressure, FIC. 32— TUBKHCIILAP. SPI.r'.KN [he hog from which Lhe organ came was fed milk conLaining Luberele iKicilii. Generalized tnbere\ilosis was produced in a few months as a rcsull of three such feedings. or they ma\' e\'en be raised above the surface of the lung. x\s previously stated, the disease is readily recognized in the liver and spleen by lhe sharp con- Li'ast ])eLween liie yellow arfecLed ai'cas and the sur- rounding heallhy tissue. Tubercular organs and glands arc usually in- cieased in size in comparison with the healthy tissue. lu lhe case of a tubercular iiddei' lhe disease is usu- all\ coiilincd lo a single (piarler and the alTeclcd Tuberculosis 211 part may be much enlarged. There is no fever nor pain in the tubercular udder as in the case of acute inflammations. Infection of the animal. — The organisms are eliminated in the sputum discharged from the mouth in the act of coughing, the feces, and to some extent in the milk. In the stable the material from the di- gestive tract becomes dry, and the dust therefrom with the adherent tubercle bacilli may be drawn into the air passages of healthy animals. The fodder or water may be contaminated with infectious material, or the transmission of the organism may be direct, through the diseased animal licking herself, and then being licked by a healthy animal. The milk becomes infected through the introduction of manure and dust, and also from tubercular udders. The feeding of such milk to calves and hogs readily serves to infect them. Hogs also acquire the disease from following cattle in the feed lot or from manure. The reproductive organs are rarely diseased, and calves from tubercular dams are usually healthy. The spread of the disease in the herd may be rapid or slow, the determining factor being the prevalence of cases of open tuberculosis. It is a popular view that the dis- ease will not spread among cattle kept out of doors or in stables well ventilated and lighted. Experience shows these views to be wrong, and that when open cases or spreaders, as they are called, which give out large numbers of the organisms are present the dis- ease will spread rapidly under conditions ideal in other respects. Infection of the herd. — ^The infection of Ihe herd takes place through the introduction of a tuber- cular animal, or through the products of a diseased animal, such as Ihe milk. In Ihe more acule com- 212 Agricultural Bacteriology municable diseases, the transmission of the organism from place to place may be accomplished by the transportation of infected objects, but in the case of tuberculosis, it is certain that this is of no impor- tance, and attention needs to be focused on the ani- mals purchased and the dairy products fed. The in- troduction of the disease in the latter manner is OOOOO OOOOO OOOO* OOOOOOOOOOO OQQQ99QQQQO •OOOOOO •©OOOOO •©OOOOO ooL oooo©i oooo©< oooo©i OOOOOO OOOOOO OOOOOO ••©©©©©©©••• •©© HERD 50LD ©©©• •©©©©©©©©©©• ©©oooo ©OOOOO OOOOOO ooe©^ Joo •oooo OOOOO OOOOO OOOOO OOOOO OOOOO FIG. 33 — BUYING TUBERCULOSIS Healthy animals (circles), tubercular animals (crossed circles) in the herds of thirteen farmers. The farmers purchased cattle at an auction. Tubercular animals purchased represented by black dots, healthy by half black dots. Twelve farmers bought tuberculosis, only two of whom had it in their herds previous to the purchase. easily guarded against by requiring that the by- products of creameries and cheese factories be prop- erly heated before being returned to the farm. The introduction of the farm separator has done much to lessen the spread of tuberculosis. The treatment of whey is to be recommended both from the standpoint of the spread of disease and the improvement of quality of the cheese. Tuberculosis 213 FIG. 34 — SPREADING TUBERCULOSIS BY SKIM MILK Two creamery districts in which tuberculosis was spread by mixed skim milk. Twenty-four and thirty-four per cent respectively of all cattle in these districts had the disease, while only eight per cent in herds in surrounding districts. (Healthy animals, circles; tubercular, black dots.) 214 Agricultural Bacteriology The prevention of the introduction of the in- cipiently diseased animal into the herd is a far more difiicult problem. The history of the disease in the individual animal usually shows a slow but not a continually progressive development. Periods of de- velopment are followed by periods of dormancy, in which neither the animal nor the parasite is active in its attack on the other. The organisms remain alive in the tissues and during periods of dimin- ished vitality in the animal may find opportunity for renewed action, causing a rapid progress of the dis- ease. In man recovery from infection is the rule rather than the exception, while in cattle recovery is probably so rare that it can not be considered as a factor in the fight against the disease. Fortunately the period of latency may cover several years, and so permit of its detection in the earlier stages of the disease before the infected animal becomes an ele- ment of danger. Detection of the disease. — In the early stages of the disease it is quite impossible to detect it by a physical examination alone. Not until the disease has made sufficient progress to affect in considerable degree the general health of the animal are the symp- toms apparent, even to an experienced person. In the last stages the animal becomes emaciated, the hair rough, the eyes sunken, and the head often ex- tended. The appetite may remain good, but the food seems to have no effect. If the lungs are in- volved, coughing may be noted, especially after the animal has been forced to violent exercise. In the case of glands that can be examined in the living animal, such as those of the neck and udder, an en- larged condition should always arouse suspicion. It is impossible for the average farmer or veterinarian TUBERCTILOSIS 215 to tell from physical examination alone whether an animal has tuberculosis or not, or predict the stage of the disease. An animal may l^e apparently in perfect health, and yet be dangerous to other animals because of the elimination of tubercle bacilli. Many of the open cases can be detected on physi- cal examination by veterinarians highly trained in FIG. 35 — A TUBERCULAIl ANIMAL 'I'lie animal was killed immediately after the photograph was taken. Extensive lesions were found in the lungs. physical diagnosis, especially when aided by a microscopic examination of the excretions. The physical diagnosis and detection of the open cases form the basis of the Ostertag method of eliminat- ing the disease, as practiced in Germany. This is applied especially where the per cent of diseased animals is so high that the removal of all tubercular animals becomes an economic impossibility. The method has not been used for a long enough period 216 Agricultural Bacteriology to give any indications as to its final success. In many portions of this country it would not place an impossible burden on the dairy industry, if all tuber- cular animals were removed from the herds. In order to detect the disease in the herd, and to deter- mine the real condition of animals to be introduced into the herd, a more refined method of detecting the disease than physical diagnosis must be employed. FIG. 36 — ADVANCED TUBERCULOSIS Extreme emaciation and lack of vitality arc symptoms of tuberculosis. The diagnosis of the disease can be made wiLh the greatest certainty by applying what is known as the tuberculin test. Tuberculin was originally devised by Robert Koch as a cure for tuberculosis. Its use in the case of cattle is of much value in indicating whether they are affected in any degree with the tubercle bacillus. If any tuberctilar tissue exists, the introduction of tuberculin causes a rise Tuberculosis 217 of temperature which can be easily determined by thermometer readings before and after the subcu- taneous injection of the tubercuhn. In making tubercuhn, the tubercle bacillus is grown in beef broth to which glycerine has been added. After the maximum growth has been ob- tained the cultures are heated to kill the bacilli and extract all of their soluble cell products. This extract is concentrated to a definite volume, and then filtered through porcelain filters to remove all the dead organisms. It is of course impossible for the tuberculin to produce tuberculosis, as has often been stated by those ignorant of its nature. When this material is brought in contact with the tissues of a healthy animal, no effect is to be noted, while in the case of a tubercular animal, an effect varying with the method of application is produced, because Lhe tissues of the diseased animal are supersensitive to the tuberculin. There are at present three methods of applying tuberculin: to the eye, when it is known as the ophthalmic test; into the skin, when it is called the intradermal test, and beneath the skin as it was originally used, when it is referred to as the subcutaneous test. In the former a drop of tuberculin is placed on the eyeball. A more or less marked inflammation results in a few hours in the case of a tubercular animal. If the tuberculin is introduced between the layers of the skin, a swell- ing results which is more extensive and persists for a longer period in a tubercular than in a healthy ani- mal. The subcutaneous test is the more reliable and used far more extensively than the others, although it takes considerably longer time to produce the desired reaction. In addition to the temporary fever produced in a reacting animal, a constitutional 218 Af;Rir.rLTiiRAL Bacti^riolocv reaction is frequently noted, as is indicated by loss of appetite, increased respiration, diarrhoea, and a local swelling at the point of injection. The thermal reaction is, however, most constant, and since it is FIG. 37 — INJECriNG TUBKTUUlLrN The tuberculin is injected by picking u]> a told of the skin and thrusting the needle of the syringe through the skin but not into the subcutaneous tissues. easily delected by taking the temperature, it is the characteristic on which the greatest reliance is placed. DelaiJs of ihe tuberculin test. — In testing an animal before tuberculin is injected, a series of tem- peratures should l)e taken to determine the normal Tuberculosis 219 range, and to note especially whether any abnormal condition obtains. The temperature is taken by means of a clinical thermometet inserted in the rectum. The temperature of the bovine varies con- siderably in different animals, and even in the same animal at different times of the day. The tempera- ture of healthy milch cows may range from 100° F. to over 102° F. The temperature of calves and fat stock is usually higher, while that of aged or weak animals is lower. The variations that may be noted in a well kept, healthy animal are illustrated in the following table, in which are also given the pulse and number of respirations per min- ute. Exercise, excitement, and hot weather tend to increase the temperature. The drinking of large quantities of cold water lowers the temperature for some hours. Temperature, Rate of Pulse and Respirations per Minute. Cow No. 1 Cow No. 2 Tem- Tem- perature Pulse Resp. perature Pulse Resp. 10 A. M... 99.5° F. 66 18 98.6° F. 60 15 12 M 100.8 54 15 99.4 54 15 2 P. M... 101.6 48 15 100.2 54 18 4 P. M... 103.0 66 24 102.7 72 24 6 P. M... 103.1 57 18 103.0 60 27 8 P. M... 103 56 16 102.0 60 14 10 P. M... 102 60 20 102.0 50 18 12 P. M... 102.5 56 16 101.6 54 20 2 A. M... 102.4 64 18 102.2 58 18 4 A. M... 102.2 54 24 101.5 60 24 6 A. M.„ 101.8 60 18 102.2 60 20 8 A. M... 102.5 56 16 103.2 60 18 220 Agricultural Bacteriology The veterinary thermometers of heavy glass are preferable to the ordinary physician's thermometer. Many are provided with a ring to which may be. attached a string bearing a small clamp at the other end. This clamp may be fastened to the long hairs at the base of the tail thus preventing loss or break- age if the animal ejects the instrument. The mer- cury column should always be shaken below 98° F. before the instrument is inserted. If the animal objects to the introduction of the thermometer, scratching her back with a curry comb will divert her attention, and aid in overcoming the difficulty. Lubrication of the bulb with vaseline makes the insertion easier. The temperature should be taken four times at two hour intervals before the injection of the tuber- culin. The injection is made by means of a hypo- dermic syringe, usually just back or in front of the shoulder. It may be injected wherever the skin is thin and loose. A syringe with a "slip-on" needle is preferable to one with a thread since the needle can be inserted, and the syringe then attached. A short, sharp needle of 15 or 17 wire gauge is used. This can be kept sharp by the use of an oil stone. The needle is inserted through a fold in the skin. Care should be taken in inserting the needle to see that it penetrates through the skin but not the deeper lying muscular tissue. Before the syringe is used it should be st-erilized by placing it in cold water and bringing the water to the boiling point. The dose of tuberculin varies, depending on the degree of concentration. The usual strength em- ployed requires 2cc. per 1,000 pounds live weight. It is desirable that the animals tested shall be in a normal condition; hence, the injection of the tuber- Tuberculosis 221 culin should be preceded by a careful examination of each animal. Any animals showing abnormal temperatures should not be injected. The normal physiological functions, such as calving, oestrum or "heat" may or may not affect the temperature. Complete notes should be made as to the condition of the animals so that these facts may be considered in the interpretation of the records. A negative reaction in the case of a cow in heat or that has recently calved is as reliable as on any animal, while a positive reaction may not be so reliable. As it takes a number of hours to produce the febrile reaction in the case of an affected animal, it is not necessary to take temperatures until eight to ten hours after the injection of the tuberculin. On account of the length of this period, it is most convenient to inject the tuberculin in the evening. Four or five temperature readings should be taken at two hour intervals until there is a marked and permanent decline toward the normal. In the case of a positive reaction, the temperature usually begins to rise 10 to 14 hours after injection, reaches a maximum in 12 to 16 hours and then declines rapidly. The maximum temperature may reach 105° to 107° F. or three to five degrees above the average normal temperature. The reaction is considered positive when the highest temperature after the injection is at least two degrees above the average normal before injection, or is one and five-tenths degrees above the highest temperature noted before injection. It is often a question of judgment as to whether a reaction is positive or not, especially in those cases in which the increase just reaches the standards assumed. In those cases, all circumstan- tial features, including especially the character of 222 Agricultural Bacteriology the temperature curve have to be taken into con- sideration. In no case should the decision of such doubtful cases be left in the hands of persons who are not thoroughly familiar with such work. The limitations of the test must be recognized and the results must always be interpreted with judgment. In making the test, conditions should be kept as nearly normal as possible. The herd should be' kept Hours After Injection. F 8 ID 12 14 16 18 20 107° ^ ^ 106° -.^^^ ^ --^^^ A -^^^ ^ "= ' —7^ — ^-=^ ^-.....^^^ 10 4° 3// /yz 10 3° / 102° 1 101° 10 0° FIG. 38 — TEMPERATURE CURVES 1. The temperature curve of" a healthy animal after the injeetioii of tuberculin; 2 and 3 the temperature curves of tubercular animals after the injection of tuberculin. (After Moore.) quiet, fed and watered as usual, unless the manner of watering would permit them to drink large quantities of cold water which might vitiate the results by excessive reduction of temperature. Pre- ferably water should be given in small quantities, if it is cold. Animals that show a doubtful reaction should be rclested after sixty days. If the retest is made at an earlier time, it is hkely to be less reliable, due to the effect of the previous dose of tuberculin. On the retest a triple dose of tuberculin should be used, and the leinpcralures after Ihe injection should Tuberculosis 223 be begun by the fourth hour, since the reaction is likely to appear earlier than on the original test. Animals recently infected but not yet containing diseased tissues, i.e., those in the incubation stage, or those in which the disease is dormant do not as a rule react to the test, while those in which the disease is far advanced often fail to react -because they are already saturated with the tuberculin naturally formed as a result of disease. The disease in the latter can be recognized by a physical examination; in the former only by a repe- tition of the test at intervals so as to determine its presence before it has made such headway as to make the animal a source of danger. The purely mechanical part of the test can be carried out by any intelligent farmer. He should learn how to read the thermometer accurately and acquaint himself with all the details of the test. No farmer need neglect the testing of his herd because of the inability to obtain a veterinarian, or on account of expense. The cost of the necessary instruments is small, and the tubercuhn can be purchased of reliable firms at a cost of from 10 to 25 cents per dose depending on the amount. Most experiment stations are in position to supply tuber- culin to bona-fide stock owners in their states. The advantage of being able to test one's own herd is great, since retests can be made on single animals as the occasion seems to warrant, and all aniinals purchased can be tested before they are placed with the herd. Freeing the herd from tuberculosis. — The methods to be followed depend on the value of the animals, and the extent of the disease in the herd. If buL few animals arc found Lo be infected, the 224 Agricultural Bacteriology cheapest and most effective way is to remove them. If a larger portion is diseased, and especially when the animals are valuable for breeding purposes, the herd may be separated into healthy and reacting sections which should be kept in separate quarters and pastures. The calves from tubercular animals should be removed at birth, placed with the healthy portion of the herd, and fed on the milk of healthy animals, or on that of the tubercular animals after proper sterihzation. In almost all cases, calves so treated can be raised to maturity in a healthy condition. As the herd is built up in this way, the old reacting animals can be discarded; by this means the valuable characteristics of the particular family can be transmitted through the progeny. This method of "weeding out" the disease instead of "stamping" it out by wholesale slaughter is known as the Bang system and has been widely used in Denmark where the percentage of affected animals is so high that immediate slaughter would be almost prohibitive. If fifty per cent or more of the animals react to the test, it is advisable to consider the entire herd as infected, and not attempt to eradicate by separa- tion and slaughter, but to build up the healthy herd by the progeny alone. This plan is recom- mended because it has been found by. experience that when so large a part of the herd reacts to the test, most of the remaining animals will react later. Many of the states require the removal of known tubercular animals from dairy herds. In most cases the state bears a portion of the loss by compensating the owner in part for diseased or reacting animals. The reacting cattle are usually slaughtered at some abattoir in which federal inspectors arc stationed. Tuberculosis 225 The meat of reacting animals may or may not be passed as fit for food, depending upon the extent of the lesions of the disease. It was formerly the custom to destroy all carcasses of reacting animals but this economic loss is no longer sanctioned. In the incipient stages of the disease, the affected parts are usually confined to definite organs, and if those are removed, no danger exists in the use of meat that has passed federal inspection. This salvage has reduced to a marked extent the cost of the eradication of tuberculosis. The basis for the compensation of the stockmen by the state is the sanitary importance of the disease, and the necessity of safeguarding the public welfare as well as the economic, relations. In determining this amount, the proportion paid by the state should not be too large or otherwise there is danger that the public treasury will be made the victim of unscrupu- lous design. Farmers do not appreciate the losses that are occasioned by this disease, as its course is so slow and insidious. If it ran as rapid a course as the more acute communicable diseases, its importance would be more appreciated. With the develop- ment of an unthrifty condition the farmer is apt to dispose of the animal to some one else, which simply perpetuates the disease in another herd. Many are sold as "canners." If the time ever comes when the losses due to the condemnation of carcasses because of tuberculosis is placed on the producer, in- stead of on the consumer of meat, farmers will be forced to the necessity of eradicating this plague to save themselves from such economic losses as now obtain. The rapid spread of the disease, especially in hogs makes this problem already a 15a 226 Agricultural Bacteriology factor of considerable importance in the price paid for swine. Vaccination. — Many attempts have been made to use vaccines in the prevention of tuberculosis but without success. Some of the methods employed have imparted a certain degree of immunity, but because of the fact that the protection persisted only for a few months, and it could be bestowed only on young animals, the methods have not been a practical success. In the present stage of develop- ment there is no use in the farmer expending money in attempting to use the methods now on the market. Prevention. — There is no doubt but that a breed- ing herd free from tuberculosis is a most valuable asset, and one that will increase in value as people become more awakened to the economic importance of the disease, and seek to purchase only sound cattle. At present it is difTicult, if not impossible, to avoid the introduction of tuberculosis into a herd where considerable numbers of cattle are pur- chased. The wide-spread distribution of the disease, even though sparingly present in any particular herd always raises the question of comparative cer- tainty that any animals purchased from such herds may not be in the incipient but non-reacting stages. The limitations of the tuberculin test are such that it will not enable one to recognize every case, no matter in what stage the disease may be. Individual animals may show a plainly negative reaction to the test at the time of purchase because of the dor- mant form of the disease, because the period of incubation has not been completed, or because of dishonest practices by the seller. These animals, if maintained in the herd, are quite certain in time Tuberculosis 227 to develop open tuberculosis, and be a source of loss if their condition is not detected. The only certain way to prevent the introduction of the disease is to purchase only from herds that are known to be absolutely free from tuberculosis. It is certain that loss can be largely avoided by the testing of all animals at the time of purchase, by keeping the animals separate from the herd for at least three months with a retest at the end of this period, and by the annual testing of the entire herd. In this way, the use of tuberculin serves as a cheap kind of stock insurance and should be maintained regularly as an annual duty in the herd. Where such vigilance is practiced, little danger from the disease need be feared. Tuberculosis of hogs. — As has been stated the infection of the hog takes place with the greatest ease by way of the alimentary tract, hence the lesions are most likely to be found in the glands of the throat, or in the mesenteric glands. The bacilli are almost entirely of bovine origin, the hog coming in contact with them in the manure, or in skim milk and whey. Due to the fact that hogs are not kept for long periods of time, there is little oppor- tunity for the disease to make such progress as to permit the animal to eliminate the bacilli, hence, there is probably little, if any, direct infection from one hog to another. The disease can be detected in the living animal by the subcutaneous tuberculin test. The tem-* perature of the hog is subject to great fluctuations. The animal must be kept in a normal and quiet condition during the test to prevent great fluctua- tions of temperature due to excitement. 228 Agricultural Bacteriology Avian tuberculosis. — It is certain that tuber- culosis has made rapid progress in barnyard fowl in this country during recent years, due undoubtedly to the commerce in breeding birds. In many sec- tions of the country it is becoming of great economic importance, causing in some instances, the death of 50 per cent of the flock in a year. The disease is characterized by the extreme emaciation of the bird in the last stages, and by paleness of the comb and wattles. Lameness is FIG. 3n — AVIAN TUBERCULOSIS Sections through the breast of a healthy chicken and of a tubercular one. Extreme emaciation is a symptom of avian tuberculosis. also an indication since the joints are often affected. These manifestations of the disease have given rise to the terms going light and rheumatism. Unlike mam- malian tuberculosis post-mortem changes of the di- sease are found chiefly in the abdominal cavity. The liver is often dotted with yellow necrotic areas which has caused the expression spotted liver to be commonly used. This organ is often much in- creased in size. The spleen is usually enlarged and often abnormal in shape. On section, the normal tissue may be found to be almost wholly destroyed. The intestinal wall may be studded with tubercles of varying size. In the more ad\'anccd cases the Tuberculosis 229 lesions will be found in other organs, such as the kidneys, lungs, and ovaries. The bacilli are ehminated in the excreta and infection is by way of the alimentary tract. The FIG. 40— AVIAN TUBERCULOSIS The liver is frequently involved. It is often studded with yellow areas and is usually enlarged. In this instance the liver weighed three-fourths of a pound. disease is spread from flock to flock by the sale of tubercular birds, and possibly by the sale of eggs, which, when the ovaries are tubercular, may con- lain the organisms. The chicks hatched from the eggs may develop the disease, a case of hereditary transmission. 230 Agricultural Bacteriology Tuberculosis can be detected with some degree of certainty by injecting a small amount of tuberculin prepared by the use of the avian tubercle bacillus. It is preferable to avoid the purchase of stock from suspected flocks and to get rid of the entire flock when the disease has made its appearance, rather than to attempt to eradicate it in other ways. There is no reason to believe the disease is of sanitary importance. It may have other economic aspects than those mentioned for it has been shown that the organism is capable of producing a non- progressive form of the disease in hogs which may cause the rejection of certain parts by the meat inspectors. To avoid such trouble and to prevent the spread of the disease in the flock, all dead birds should be buried so that they can not be eaten by hogs or birds. Chronic dysentery in cattle. — The tubercle organism belongs to a large group of bacteria that have certain properties in common, one being the relation to stains. All the members of the group are difTicult to stain, but when once the stain has pene- trated the cell, it is not easily removed, even by such reagents as mineral acids. This property has given the name acid-fast to organisms of the group. Other species than the tubercle baciUi are able to produce disease. Leprosy in man is caused by one of the group, as is chronic dysentery or Johne's disease in cattle. The disease is characterized by persistent chronic diarrhoea and marked emaciation and commonly terminates fatally. The walls of the small intestine are much thickened and thrown into folds. But httle is known of the manner of trans- mission of the disease or of its economic importance. Others of the acid-fast group are non-pathogenic. Tuberculosis 231 and are widely distributed in nature. They are present in milk and dairy products. The appear- ance of these under the microscope is so similar to that of the tubercle bacillus that a microscopic ex- amination of milk is of httle value, and resort must be had to animal inoculation to complete a proper diagnosis. Differential diagnosis. — There are a number of diseases that may be mistaken for tuberculosis in domestic animals. Actinomycosis may produce nodules in the udder that resemble tubercular nodules. Sheep are sometimes affected with an intestinal disease known as nodular disease, which to the uninitiated might be thought to be tuberculosis, but is really caused by a parasitic worm which burrows into the wall of the intestine, forming a greenish colored nodule about the size of a pea. CHAPTER XIX TEXAS FEVER, CONTAGIOUS ABORTION, AND FOOT AND MOUTH DISEASE Communicable diseases of both man and lower animals are caused not only by bacteria, but by microorganisms that" are classed as animals, the protozoa. Malaria and sleeping sickness are among the important human diseases caused by protozoa, and Texas fever is the most important protozoal animal disease. The pathogenic protozoa may be found in the intestines, and are then eliminated in the feces, from which there is opportunity for them to find their way into the bodies of healthy animals. Another class of protozoa are to be found in the blood and are trans- mitted from one animal to another by biting insects. The anopheles mosquito is responsible for the trans- mission of the malarial organism, and one of the cattle ticks for the transmission of the Texas fever organism. In the case of diseases transmitted by insects, there is opportunity for wide spread distri- bution, while in the case of the diseases caused by intestinal forms, the chance of the organism gaining entrance to the tissues of a susceptible host is much smaller. The fight against the spread of the insect- borne, protozoal diseases is a fight against the trans- mitting insect, the tick in the case of Texas fever. Transmission. — The parasite is found in the red blood cells which are destroyed by it, and the red coloring matter, the haemaglobin, is set free to be eliminated from the body in the urine, to which it Texas Fever 233 imparts a red or black color. The disease is often called tick fever because of the method of trans- mission, and red or black water from the dark urine. Of the ticks that are to be found on cattle, but one, Margaropus annulatus, is concerned in the trans- mission. The female tick that is infected with the parasitic organisms drops off the host animal to lay the eggs on the ground where they hatch in from thirteen days to six weeks, depending on the tem- perature. The young seed ticks soon fasten them- selves to an animal, if available, attaching them- selves to the inside of the thighs and flanks, along the belly and brisket, inside the fore legs, and around the root of the tail. If they are unable to do so, they starve. If they become attached to the host, they do not leave it, but exist there during the entire life history, living on the blood. The protozoa causing the disease pass from the female into the egg, and thence into the seed tick which infects the host animal. If the host is immune, no harm results, but if the animal is susceptible, a fatal form of the disease is usually produced. Young animals are not very susceptible to the disease. When raised in contact with the ticks, they gradually become infected with the protozoa during the time when the immunity is so high that a fatal form of the disease is not occasioned, and a permanent immunity is produced by the mild attack. The loss from the disease is not so much from death of animals, as from the fact that cattle can not be sent from in- fected regions to free areas. Thus southern cattle can not be shipped to the northern cattle markets, except under certain restrictions that add to the cost of marketing. In the markets the cattle from in- fected regions are sold at a lower price than the same 234 Agricultural Bacteriology grade from a noninfected territory. The ticks are also a constant drain on the vitality of the animals, diminishing the milk production, and rate of growth. The susceptibihty of cattle from free territory is so great that they can not be taken into infected terri- tory without immunization. This fact has limited the improvement of the southern cattle by the intro- duction of pure-bred animals from the north. FIG. 41 — TEXAS FEVER The dotted line represents the northern boundary of the infected terri- tory in 1906; the shaded areas represent the territory infected in 1914. Eradication. — It is, of course, impossible to al- low the free shipment of tick-infested cattle into the northern states where they would be brought in contact with susceptible animals. Before the nature of the disease was recognized such shipments made in the summer caused great losses in Illinois and In- diana. In 1891, the Texas fever line was established marking the boundary between the sections free from the transmitting tick and those in which it is still present. The line is not a fixed one but changes Texas Fever 235 from year lo year as the work in tick eradication progresses. The methods formerly used to free the cattle from ticks were based on the life history of the insect. Chemicals have now been found in which the animals can be dipped without injury to the animal but which will destroy the tick. The dip- ping solution used at the present time contains sodium arsenite. The total area rendered free from the tick since the beginning of the work in 1906 to July, 1914, is greater than the combined area of Mississippi, Ala- bama, Georgia and Florida. It seems reasonably certain that within a few years Texas fever will be eradicated from this country. The work of eradica- tion should progress rapidly when the stockmen realize the great economic advantages that will ac- crue to the industry from the eradication. Texas fever is a disease the eradication of which simply awaits the practical application of knowledge al- ready acquired, while in the case of other important communicable diseases, the information is not yet sufficiently complete, so that effective measures can be carried out, or else the nature of the diseases is such that there seems little hope of getting rid of them in the near future. Immuntzation. — The young animal is not very susceptible to Texas fever, and can be readily im- munized by introducing into the blood such a small number of the causal organisms that a fatal form of the disease will not be produced but which will cause a sufficient degree of immunity to protect against natural infection. The requisite number of organisms may be introduced by transferring blood from an infected animal to the animal to be im- munized. The amount of the blood to be thus trans- 236 Agricultural Bacteriology ferred depends on the susceptibility of the animal. If an old animal is to be immunized, one cubic centi- meter of the blood is used, but if a yearling is to be treated, three cubic centimeters of the blood may be employed without danger. Again, the tick itself may be allowed to infect the animal, the number of organisms introduced being governed by the num- ber of ticks placed on the animal. The method of immunization with blood is very successful, about three per cent being lost by vaccination and seven per cent by subsequent infection. The ten per cent loss when compared with the 90 per cent loss that was met with when non-immunized cattle were placed on tick-infested pastures is a measure of the success of the treatment. Certain cattle, as those of India, are resistant to the disease, and attempts have been made to breed strains that possess such an immunity. Contagious Abortion. — The term abortion sig- nifies the premature discharge of the fetus. If the abortion occurs late in the gestation period so that the young may live, it is often termed premature birth; there is, however, no essential difference as far as the cause is concerned between abortion early in the gestation period and that which occurs later. Causes of abortion. — The expulsion of the fetus may be due to slipping, injury by another animal, or other mechanical causes, and to feeds that have a specific action on the pregnant animal, such as grains and fodders that contain large amounts of smut or ergot. It is quite certain, however, that abortion as it is observed in cattle is almost wholly due to the invasion of the animal with a specific or- ganism which may pass from animal to animal, producing what is commonly known as contagious Contagious Abortion 237 abortion. The disease is of greatest economic impor- tance in cattle. A similar trouble, caused, however, by a different organism, is noted in mares, and a third organism is responsible for abortion in sheep. The disease as it appears in cattle has spread rap- idly in recent years, due to the great increase in the sale of breeding animals. It is now rare to find a herd of any considerable size that has an entirely clean record. The losses it occasions are felt espe- cially by the breeder who relies on the progeny of his herd for a large share of his returns, much more so than the farmer who is primarily interested in the production of milk. In the beef districts the disease of course becomes of major importance to the farmer. The loss is not wholly confined to that incurred from the death of the calf, for if the abortion occurs early in the gestation period, the animal will rarely prove a profitable producer of milk during that lactation period. If the abortion occurs late in the period, the flow of milk will be normal. In economic im- portance the disease ranks with tuberculosis, Texas fever, and hog cholera. Against these the farmer has much hope of making a successful fight with the knowledge that is already available, but against contagious abortion the case is far less hopeful. Cause. — The cause of bovine abortion is a small bacillus that does not form spores and is relatively non-resistant to disinfectants. It is called Bacillus abortus, or the Bang organism, after the Danish vet- erinarian who first discovered it. The organism has been found widely distributed in all parts of the world, and there is no doubt concerning its causal relation to the bovine form of the disease. Nature of the disease. — The disease is one that has a specific action on the pregnant animal, 238 Agricultural Bacteriology causing an inflammation of tlie uterus, with injury to the fetus or causing its death. The general health of the animal is not affected to any noticeable extent. The organism is eliminated at the time of abortion, and for an indefinite period thereafter in the discharges from the uterus. It has also been found in the milk. The organism may be present for months before abortion actually occurs, indicating that it is to be looked upon as a chronic disease. Again, an animal may continue to eliminate the abortion bacilli in the milk years after the last abortion occurred or when no abortion has been known to occur. The infection of an animal does not necessarily produce abortion, since this will turn on the extent of injury to the fetus. The in- fected animals that do not abort may be as danger- ous to healthy animals as those that have aborted. The fact that some unsuspected animals may thus act as bacillus carriers makes it an especially difficult disease to control in a herd. The natural manner of infection is not known with certainty. It has been shown by experimental methods that the infection may occur through the genital passages. It is probable that if infection occurs in this manner, it must take place before or very shortly after conception, as, after the uterus is closed, no invasion can occur through the blood. It is beheved by many that the male is one of the common agencies in the transmission of the organism from the infected to the healthy animal. It has also been shown that infection may occur through the alimentary tract by the ingestion of contam- inated food. By some it is thought that this is the most important, if not the sole way by which the organism enters the body under natural condi- Contagious Abortion 239 tions. Infection in this manner may occur after conception. It will be evident that if the disease is introduced into a herd, there will always be ample opportunity for. it to spread, whatever the method by which the organism invades the indi- vidual animal, since the stable and fodder is certain to be contaminated with the organism. The disease may be introduced into the herd by the purchase of an infected animal, and probably by the feeding of mixed creamery and cheese fac- tory by-products. While this last method of spread has not been proven, the presence of the organism in the milk of a considerable proportion of the in- fected animals and the fact that infection can occur by way of the alimentary tract would lead to the conclusion that this may be one method by which the disease is being distributed from farm to farm. This method of distribution can easily be avoided by pasteurizing the factory by-products, a method equally effective against both tuberculosis and foot and mouth disease. Detection of the diseased aniinal. — There is at present no method by which the infected animal can be detected with absolute certainty, and its introduction into a healthy herd avoided. Many infected animals can be detected by laboratory examination of the blood, but there is no test that can be apphed with the ease and surety of the tuber- culin test. The stockman must thus rely on what information he can secure concerning the presence of the disease in the herds from which he intends to purchase. Control of the disease in the herd. — It is not usual for an animal to abort more than once or twice, and such condition is frequently interpreted as an 240 Agricultural Bacteriology indication that a certain degree of immunity has developed as a result of the earlier attack. It seems probable that this is not the case, but that there is an age immunity, since the greatest number of abor- tions occur with heifers during the first and second pregnancies. It is not good practice to sell aborting animals with the hope of getting rid of the disease, since it will rarely if ever succeed. The replenish- ing of the herd by purchase will serve to continue the trouble, either by the addition of healthy animals to become infected, or by the introduction of new centers of infection. Prevention. — Many treatments have been de- vised and recommended for the prevention and cure of abortion. For example the internal administra- tion of carbolic acid, both with the feed, and by hypodermic injection has been widely used. There is little reason to believe that it has any favorable effect. The use of vaccines has been attempted but without success. It seems evident that the breeder and farmer must rely purely on sanitary precautions to prevent the spread of the disease. He must seek to destroy the organism in the infectious material discharged by the animal. The dead fetus and also the afterbirth should be buried; the contaminated litter should be destroyed; and the aborting animals should be flushed put with a 0.5 per cent solution of some of the soapy, coal-tar disinfectants, such as ly- sol, or with a 0.5 per cent Lugol's solution which consists of one part of iodine and two parts of potassium iodide dissolved in three hundred parts of water. The solution should be warmed to 100° F. The treatment should be continued daily until no discharge is to be noted. This procedure will not only serve lo limit the distribution of the organ- Foot and Mouth Disease 241 ism, but will be of some service in the prevention of sterility, which is a frequent sequence of abortion, and an important factor in its economic importance. It is also well to disinfect the bull after each service. A separate maternity stall should be provided to which all animals that are to calve at the normal time should be removed. This stall should be kept clean, and supplied with an abundance of clean bedding. Animals that show signs of aborting should not be placed in this stall, but removed from the stables to a separate building. Foot and mouth disease. — There are a number of transmissible diseases of domestic animals pre- valent in Europe that are not found in this country. Among them are contagious pleuro-pneumonia of cattle, hog erysipelas, and foot and mouth disease which affects the cloven-hoofed animals and man. All domestic animals imported from Europe must be kept in quarantine for a considerable period in order that there may be time for them to develop symptoms of any disease with which they may be infected, and to permit of a detailed examination as to the health. These precautions are taken primarily to prevent the introduction of diseases that are not known in this country. Even with all precaution there is always opportunity for some of these diseases to be introduced and to spread rapidly. Foot and mouth disease forms an excellent example. There have been six outbreaks of this disease as follows: 1870, 1880, 1884, 1902-3, 1908-9, 1914-15. The disease has, however, been eradicated at each ap- pearance by the federal officials. The method followed has been to slaughter not only the diseased animals, but all other susceptible animals on the farms on which the outbreaks occurred. In 1902-3 16a 242 Agricultural Bacteriology 4,461 animals were killed; in 1908-9 3,636 animals were slaughtered. The cost of the eradication of each of the outbreaks in 1902-3 and 1908-9 was approximately |300,000. Not a large amount to pay as insurance of the entire stock industry of this country against this disease for ten years. The disease is like other acute communicable diseases in that it has an ebb and flow. These waves occur at intervals of several years; the reason PENNSVLWkNI* riRM'B FIG. 42 — FOOT AND MOUTH DISEASE The outbreak of this disease in 1908 illustrates the rapidity with which a disease may spread under modern commercial conditions. for this variation in their severity is not known. In Germany after a period in which the disease was not especially important it began its ravages and in 1911, 3,000,000 cattle were affected, 1,000,000 sheep and 2,500,000 hogs. The loss from death of animals is not great, only about one per cent. The economic losses are due to the loss of flesh, to di- minished milk production, or to loss of reproductive power. II has been estimated for cattle that this loss ranges from $7.00 to $20.00 per head and pro- Foot and Mouth Disease 243 portionately less for smaller animals. It is thus clear that the disease imposes a great economic burden upon stockmen, and if by the expenditure of reason- able sums the country can be protected from it it is certainly wise to do so. The disease presents an excellent example of the influence of modern commercial conditions on the rate of spread. The infection of the great shipping yards is an especially important factor in the distri- bution. The outbreak of foot and mouth disease of 1908-09 was due to the importation of vaccine virus from Japan which was used in one of the vaccine establishments of Pennsylvania. Some of the virus was sent to a Detroit establishment which rented calves from a dealer for the manufacture of small pox vaccine. After the animals had been used for this purpose they were returned to the dealer and resold by him to farmers. Bovine animals inoculated with the mixed virus of cow pox and foot and mouth disease develop symptoms of cow pox alone, but when brought in contact with healthy animals the virus of foot and mouth disease may spread from the animals that show no symptoms. The calves in question were placed in pens in the Detroit stock yards, and from there distributed as shown in the diagram. Four days later a shipment of cattle was placed in the same yards, a portion of the shipment was reshipped to Buffalo, and from there a number of animals were sent to two towns in Pennsylvania in the neighborhood of which out- breaks of the disease occurred as illustrated in the diagram. In ten days the disease had traveled hundreds of miles. The rapid and effective work of the government oITicials was all that saved the 244 Agricultural Bacteriology country at that time from the permanent intro- duction of the disease. The outbreak of 1914-15 was possibly due to the importation of hides. In two weeks after the disease was first discovered in Michigan, it had spread through the infection of the Chicago stock yards from the Mississippi river to the Atlantic ocean, and had invaded fourteen states. Over 100,000 animals were slaughtered and buried on the infected farms. The cost of recompensing the farmers for animals killed and for disinfection was approxi- mately $4,000,000. Again not a large amount to pay to insure the stock industry when it is remein- bered that in England the losses in 1883 were esti- mated at $5,000,000. Nature of the disease. — The causal organism is an ultramicroscopic one, i. e., one so small that it can not be seen with the most powerful microscopes. In three to six days after the animal is exposed to the infection, the disease makes its appearance. .The onset of the trouble is marked by chills followed by fever which may cause the temperature to rise as high as 106° F. In one or two days blisters or vesicles about the size of a hemp seed or a pea are to be noted on the mucous membranes of the mouth, tongue, and gums. The vesicles are filled with a yellowish, watery liquid in which the causal organism is present. Similar eruptions appear on the feet between the digits and above the coronet. They may also appear on the udder and teats. The milking process rup- tures the vesicles and the organism finds its way into the milk by which it can be spread from one animal to another, and from farm to farm when mixed cheese factory or creamery products are fed. The vesicles increase in size until they reach the diameter Foot and Mouth Disease 245 of a dime or even larger. They rupture soon after their appearance, leaving reddened sensitive spots or erosions behind. Food is refused and the ropy saliva drools from the mouth. The soreness of the feet often renders it impossible for the animal to stand. The disease spreads with great rapidity in the herd. The chance that all of the herd will acquire it has led to the inoculation of the animals by the transfer of some of the saliva from the diseased to the healthy animals with the idea of shortening the period of trouble in the herd. In cases of doubt as to the nature of the disease the inoculation of a calf should give defmite information, since the inocu- lation should result in the characteristic vesicles in twenty-four to seventy-two hours. There are a number of diseases that somewhat resemble foot and mouth disease. Cow pox forms similar vesicles, but the inoculation does not result in symptoms of fever and eruption for at least ten days. In mycotic stomatitis or inflammation of the membranes of the mouth, the entire mouth cavity is inflamed and vesicles are rare and, if present, do not increase in size. The thin skin between the toes may be inflamed, but the vesicles do not appear nor is the udder affected. The disease does not spread and the inoculation of calves is not success- ful. In foot rot the inflammation of the foot is gene- ral, and the mouth remains unaffected. In ergotism or trouble due to the eating of too great quantities of smut, the mouth is not affected and the tissue changes are to be noted at the tips of the ears, end of the tail, and upon the lower part of the legs. 246 Agricultural Bacteriology Foot and mouth disease is transmitted to man by the use of infected milk. It causes eruptions in the mouth and on the fingers, but is seldom fatal, ex- cept in the case of weakened children. In the human it is known as aphthous fever. CHAPTER XX RABIES AND ACTINOMYCOSIS Rabies. — As has been shown the losses from Texas fever are needless, since ways are known by which it can be completely eradicated. Rabies is a disease that likewise could be made to disappear, if simple procedures that could easily be carried out were en- forced. The disease is primarily one of the flesh-eating animals, but is transmissible to all mammals through the bite of a rabid animal. In reality it is transmitted almost wholly by dogs, since the dog is the only ani- mal that is allowed to run about freely. It is found in most parts of the world; Australia and England are the only countries that are known to be free from it, and they are kept so through the rigid enforce- ment of wise quarantine regulations with reference to the importation of dogs. In this country it has spread rapidly within the last few years until at present it is found from the Atlantic to the Pacific. In 1908, it is known to have caused the death of 111 people. It is also of considerable economic impor- tance because of the loss of domestic animals. The sanitary and economic aspects of the disease are small when compared with some others, but all loss is so unnecessary that it seems advisable to discuss the disease in some detail, especially since there are so many misconceptions concerning it. The virus of unknown nature is known to be pres- ent in the saliva, the vitreous humor of the eye, lymph, milk, urine, and the peripheral nervous sys- 248 Agricultural Bacteriology tem. The presence of the virus in the saUva is the explanation of the transmission by the bite of an infected animal. Many times the saliva is so com- pletely removed from the teeth of the rabid animal that none of the organisms are introduced into the wound. Especially is this likely to be true when the wound is inflicted through the clothing; through the coat of a long-haired dog, or through the wool of a sheep. This is one of the reasons why only a small proportion of the human beings and of animals that are bitten by rabid animals develop the disease even though no protective treatment is employed. The virus is known to be present in the saliva from two to five days before the symptoms of the disease are evident. The wounds in which the in- fection is most likely to be of a serious nature are those inflicted on the head and face rather than on the extremities. The virus develops in the nerves and is more likely to establish itself in tissues that are rich in nerves than in those deficient in these structures. The extent of the bites is also an im- portant factor in determining whether infection is to occur, since the amount of virus introduced will be in proportion to the number of bites inflicted. The tissues seem to have the power of destroying a limited number of the organisms. Again if the wounds are such that bleeding is marked, there will be a tendency for the organisms to be washed out. The deep, puncture wounds are likely to be more serious than a tear in the flesh. It is commonly believed that there is a seasonal distribution of rabies, that it is more common dur- ing the so-called dog days of late summer. There is little or no basis of fact for this belief. There is, however, more opportunity for the rabid dog to Rabies and Actinomycosis 249 come in contact with human beings and with ani- mals in the summer than in the colder months when there is less out-of-door life. The regulations that require the muzzling of dogs during a few weeks in the summer have no justification unless the period is extended to include the entire year. The incubation period varies considerably in length, depending upon the location and extent of the bites. The symptoms are not evident until the central nervous system is involved. The rapidity with which this will occur is dependent on the dis- tance of the bite from the brain. The extent of the bite is also of importance in determining the rate at which the disease progresses. The average periods of incubation are as follows: Man 40 days Dog 21-48 days Horses 28-56 days Cats 14-28 days Pigs 14-21 days Sheep 21-28 days The virus may remain dormant after its introduc- tion into the tissues for a varying period of time, thus delaying the development of the symptoms for months and possibly years after the bite is inflicted. Symptoms. — The symptoms of the disease are increased nervousness and paralysis of the muscles. The groups of muscles most commonly involved are those of the throat, making it impossible for the ani- mal to swallow. There is a common idea that the rabid animal is afraid of water, from which originates the common term hydrophobia. When a rabid aniinal is thirsty and attempts to satisfy itself by drinking, a paroxysm of the muscles is produced. No food can be taken due to the inability of the animal to swal- low in the later stages of the disease. 250 Agricultural Bacteriology In certain kinds of animals, notably the dog, the nervous symptoms are more apparent. To this form of the disease the term furious rabies is applied, while with those animals in which the nervous symptoms are largely lacking and the paralysis of the muscles more evident the term dumb rabies is used. The rabid dog is extremely nervous and is unable to re- main quiet. It usually wanders long distances from home and during these journeys comes in contact with man and lower animals. The rabid dog does not go out of its way to attack other animals, but snaps at every thing that may come in its way, whether animate or inanimate. There is frothing at the mouth and the voice is more of a howl than bark, due to the affection of the muscles of the throat. The paralysis extends to other muscles, such as those of the limbs. The dumb or paralytic form of rabies being less common and less well known is often mis- taken for some other trouble such as choking. The dumb form is as dangerous as the furious, since the saliva contains the virus and may cause infection if brought in contact with an abrasion of the skin. The furious form is common in dogs, horses and other domestic animals and in man. The rabbit, so widely used in preparing the vaccine, is subject to the dumb type. Diagnosis. — It was stated earlier that only about 10 per cent of human beings bitten by known rabid animals develop the disease, due to the non-intro- duction of the organism into the tissues, or to the destruction of the organisms by the tissues. Death from rabies presents a series of horrible symptoms, and no chances should be taken in case a person is severely bitten, but the preventive treatment should be applied without delay. It is, however, Rabies and Actinomycosis 251 highly advantageous to know for a certainty whether the dog is actually rabid or not. The quickest way to determine with certainty the nature of the trouble is to confine the dog and note the symptoms. The disease is invariably fatal and death is commonly preceded by a definite sequence of symptoms so that there can be no mistake in the diagnosis. The desirability of making the diagnosis as quickly as possible is due to the fact that if preventive treat- ment is to be applied, it must be administered with- out delay as it is of no avail if not begun until the symptoms appear. If it is impossible to secure and confine the dog alive, the diagnosis can be made by a microscopic examination of the brain. The head should be removed, packed in ice and sawdust, and sent to the laboratory that is maintained by most states and large cities for such work. In killing the dog, care should be taken not to injure the brain or spinal cord. Preventive treatment. — The organism found in ordinary cases of rabies can be increased in viru- lence for rabbits by passing it through a series of animals. The so-called fixed virus will kill a rabbit in seven days, when introduced beneath the mem- branes of the brain. If the cord is removed from the animal immediately after death and allowed to dry over caustic potash, the virus becomes attenuated, the extent of the attenuation depending on the length of the drying process. The preventive treat- ment consists in giving hypodermic injections of a suspension of the dried cord. The first injections contain material that has been dried for about two weeks, while the subsequent injections are made with material that has been dried for a shorter period, until at last an injection of the fresh cord can 252 Agricultural Bacteriology be given without danger. Injections are made for a period of twenty-one days. Of all the persons, known to have been bitten by rabid animals, to which the preventive treatment has been administered at the Pasteur Institute in Paris, but one-half of one per cent have died of rabies while the mortality records of those that did not receive the treatment are ap- proximately ten per cent. All wounds that have been inflicted by a dog known to be rabid, or suspected of the disease, should be cauterized as soon as possible by the ap- plication of concentrated nitric acid, strong carbolic acid, or by a hot iron when the chemical agents are not available. This strenuous treatment will de- stroy the tissue about the wound together with the virus that has been introduced by the animal. The quicker the cauterization is carried out the more effective it will be. Eradication. — Since the disease is transmitted almost wholly by the dog, it could be prevented and eradicated by keeping all dogs that are allowed their freedom muzzled at all times. The effect of such measures is shown by the history of rabies in Eng- land. The following figures represent the number of reports of the disease. 1893 672 cases 1898 17 cases 1901 1 cases 1903-6 cases The reduction was due to the enforcement of muzzling laws after 1896. The muzzling regulations that have been passed by governing bodies in this country are rarely, if ever, enforced in an effective manner, due to the fact that many people believe it cruel to muzzle a dog. It is certain that the dis- Rabies and Actinomycosis 253 ease could be eradicated in a short time if its trans- mission could be prevented. It is also certain that muzzling of dogs for a short time is much more humane than to have hundreds of them as well as other animals and people dying from rabies each year. Actinomycosis. — Actinomycosis, or lumpy jaw as it is more commonly called, is primarily a disease of cattle, although horses, sheep, hogs, and dogs may be affected. Man is also subject to the disease. The causal organism is not one of the bacteria, but an organism much like a mold in its appearance under the microscope and in cultures. In this country the disease is not nearly so widespread as in some other sections of the world. It has been found in about one- .out of sixteen hundred animals killed in this country; Tuberculosis is many times more preva- l^itt and more important in every way, and yet in many places, because of the appearance of the dis- ease on the surface of the body, actinomycosis has made more of an impression on the popular mind than has tuberculosis. The disease is not one that spreads from animal to animal, and hence, like tetanus, can not be classed as a contagious disease. It is beheved that the organ- ism grows on certain plants as barley and is intro- duced into the tissues by the barley awns. It may also enter through a wound in the mouth or through a hollow tooth, and possibly may be inhaled. It is rarely fatal and when death is produced, it is due to mechanical causes as the interference with breath- ing or swallowing, or to the weakening of a blood vessel by the constantly growing fungus. Sympioms. — The first symptom when the disease is located in the throat, as is most common, is a 254 Agricultural Bacteriology slight swelling which gradually increases in size and is hard and dense. It undergoes disintegration at the center, and may discharge a thick yellow pus. The opening may heal over only to break out again. The opening by which the content of the abscess finds its way to the outside may be on the surface of the body or in the mouth or throat. The sore at the point of discharge may become very large and have the appearance of a head of cauliflower. FIG. 43 — ACTINOMYCOSIS The sponRy condition of the jaw bone was produced by the growth of the fungus. The growth of the tumor may continue for years. The tongue may be involved, in which case the disease is often given the name, wooden tongue. The organism may invade the bony part of the jaw, causing it to become spongy and enlarged. This permits the teeth to become loose so that some of them may fall out. The internal organs may be invaded by the organism. In the lungs, nodules that are ver>' similar lo the nodules found in tuber- culosis of the lungs may be formed under the stimulu- Rabies and Actinomycosis 255 of the fungus. These vary in size from mere specks to that of a pea. The spleen, liver and udder may contain actinomycotic nodules. The organism occurs in masses in the pus dis- charged from the nodules. Due to the color of the organism these masses of growth which can be seen by the unaided eye are often called sulphur granules. Treatment. — ^The disease is one of the few of those due to the invasion of the tissues by a parasitic organism that responds to treatment with drugs, the most successful of which is potassium iodide given in water as a drench. The dose is from one to two and one-half drams per day. The administra- tion of this amount of the drug can not be continued for any length of time without producing in the animal the effect which is known as iodism. This causes the eyes to run, the skin to become dry and rough, and a loss of appetite. When these evidences of the drug become apparent, its use should be dis- continued for a few days, and then begun later. In the case of milch cows, the milk should be dis- carded as the drug is excreted through this channel. The flow of milk is decreased and may stop entirely. The drug may also cause abortion. All animals do not react favorably to the drug. Where beneficial results are obtained, treatment should be success- ful in three to six weeks. Man does not acquire the disease from cattle, but becomes infected in the same manner as cattle, viz., through wounds in the mouth. The meat of affected animals can be used as food, if the disease is localized in the body. CHAPTER XXI GLANDERS AND TETANUS The most important transmissible disease affect- ing the horse and the closely related animals is glanders, or farcy, as it is often called. The disease affects primarily horses, mules, and asses, but dogs and cats may acquire it by feeding on the car- casses of glandered animals. The disease is also transmissible to man, and usually results fatally. Distribution. — Glanders is found in nearly all parts of the world; Australia is the only large area of any importance free from it. Great numbers of horses have been congregated from varied sources for war purposes, and have been transported to other lands, thus spreading the disease. It is asserted that glanders was introduced into Mexico at the time of the Mexican war by the American cavalry. During and after the civil war, the distri- bution was very rapid in this country, due to the sale of horses and mules by the government. At the present time glanders is most prevalent where large numbers of horses are brought together as in the lumber camps, on the ranges, and in the great stables maintained in the cities. Constant change is going on in such stables, and every horse purchased may serve to introduce the disease, unless precautions are taken to determine the health of the animal before it is allowed to come in contact with the healthy animals. The number of horses purchased by farmers is comparatively small, and unless the farmer buys range animals, or those that have been Glanders and Tetanus 257 in use by the large stables, little risk of acquiring glanders is encountered. Public stables and public watering troughs are undoubtedly agents in the spread of the trouble. It is considered a wise precau- tion by many not to make use of public watering troughs but to employ a pail. Symptoms. — In some respects the disease re- minds one of tuberculosis in that an animal may havp it for a long time, and yet remain in good flesh, and be able to stand a considerable amount of -v^ork. In other words many glandered horses havie an economic value, and yet are a constant source of danger to other animals with which they come in contact. It is thus considered wise that all glaijdered animals be destroyed, and that the owner be compensated by the state for the protec- tion of the industry in general. It is through the purchase of such chronic cases that the disease may be introduced onto the farm. The disease is primarily one of the membranes lining the nasal passages, and one of the most characteristic symptoms is the discharge of a sticky fluid, sometimes streaked with blood, from one or both nostrils. Small nodules may form on the upper part of the nasal septum. The nodules, which are translucent and grayish in color, may break and form ulcers, which destroy the surrounding tissue to a greater or less extent, and may even cause a perforation of the nasal septum. Similar nodules may be found in the lungs, and less often in the liver and kidneys. In glanders of the skin or farcy, nodules are found in the skin and the underlying tissues. These nodules are usually called farcy buds. They vary in size from a hemp seed to that of an egg. These 17a 258 Agricultural Bacteriology nodules break and form running sores on the sur- face of the body, the discharge being yellow and sticky. The sores thus formed often heal and leave marked scars on the head and legs, in which places they are most common. ^^^H WM /^l |5%£;aHH^H|V^ i* \ ^B m^^lrfW^^** • M.^. 1 ^^r .^^^^^H ^^H " '^^^^^^1 . ■ 1 ^m' " ^^^1 ^'^•n. ^M ^1 ^^m ^H9^l z^- ^M"^' ' |Bfl -'' ''.>r^*". ~^H ^H , ,. ^' Fg^^u . • * ^H H-" '^■MSI .^- "■_ '^.'Z ' ;*- '" K'r\-V' * " <* ^^^-K****. ;-5:oJ«j"^^'f-^P K^Ji-^ ^r? i^ *<- :',-'^ -'.^-^ !?^S^s.^ '■* ^ -v ** ■^^^^'.i^^^l^ - • >. ■ ~ ■-; v> ■ - *»t FIG. 41— GLANDERS Sores formed by the breaking ot the farcv buds. Note the swollen con- dition ot the leg. (After Reynolds.) The acute form of the disease is common in the mule and ass but is rare in the horse. Death often takes place in two lo four weeks, although the disease may become chronic and the animal live for a number of years. Treatment is of little avail. (Ireal pre- Glanders and Tetanus 259 caution should be exercised in the care of glandered animals, since if any of the infectious material is introduced into the eyes or nose, or comes in con- tact with a wound, infection of the human is likely to FIG. 45 — GLANDERS' The scars on the nose were formed by farcy sores- occur. The manifestations of glanders in man are quite similar to those noted in the case of the horse. Detection. — Glanders is often easily recognized by the characteristic lesions in the nasal passages or by the farcy buds. When the disease can not be recognized by physical examination, recourse must be had to some other method of diagnosis. The most common method is to apply the mallein test 260 Agricultural Bacteriology which is very similar to the tubercuUn test in nature and manner of application. Mallein is prepared by growing the glanders organism in glycerine broth. The culture is then killed by heating, and the dead cells removed by filtration. The mallein is injected beneath the skin, and a series of temperature read- ings is made both before and after the application of the mallein. A few hours after the introduction of the mallein there appears at the point of injection a swelling which, in the glandered horse, is hot and painful and continues to increase in size for twenty-four to thirty-six hours. The swelling persists for several days, disappearing in from eight to ten days. At the time the swelling is most prominent, the diseased animal appears dull, breathes rapidly, and has a poor appetite. In the case of the healthy horse, the swelling is small and disappears in twenty-four hours, and no signs of illness are to be noted, follow- ing the injection of the mallein. The constitutional reaction in the diseased animal is accompanied by an increase in the temperature ranging from two to two and five-tenths degrees. The increase begins about eight hours after the injection and reaches the maximum in ten to fifteen hours. The fever persists for twenty-four to forty-eight hours, instead of only a few hours as in the tuberculin test. In the healthy horse there is no appreciable rise in temperature. The test is not as accurate a method of diagnosing glanders as is tuberculin for tuberculosis, for some glandered horses do not react to the test, but a positive reaction is looked upon as proof of the diseased condition of the animal. Other tests are also employed, in which the blood is examined for certain of the antibodies that will be produced Glanders and Tetanus 2G1 under the stimulus of the glanders bacillus. These methods can only be carried out in the laboratory. The farmer must seek to protect himself by the purchase of animals from known healthy sources, and by care in preventing his animals from coming in contact with infectious material in public places. The organism does not form spores and hence is not especially resistant. Tetanus. — Horses and mules are often attacked by tetanus or lockjaw. The other domestic animals are also susceptible, but are not so often attacked. Man is likewise subject to the disease. Tetanus is not a contagious disease, since the organism is sup- posed to have its natural habitat in the soil, and, possibly, in the alimentary tract of the horse. The organisms do not spread from one animal to another. The organism is introduced into the tissues through a wound, most commonly a puncture wound into which dirt has been carried. Such wounds bleed little and therefore the foreign matter is not washed out by the blood, nor is it easy to re- move in the cleansing operations that may be em- ployed. Wounds caused by rusty, dirty nails often serve as a way in which the bacteria are introduced into the body. The infection may occur in opera- tions such as docking, castration of colts, and through the umbilical cord of the young foal. In man a large proportion of the disease is due to wounds produced by Fourth of July accidents. The filling of many forms of fire works is earth which may contain the extremely resistant spores of the tetanus bacillus. Some portion of the fiUing may be blown into the skin by a premature discharge of a fire cracker, or some other form of fire works. 262 Agricultural Bacteriology Symptoms. — The organism is a strict anaerobe. It grows only at the point at which it was intro- duced into the tissues and to a small extent even there. In fact so little evidence of its growth is shown by the tissues that it is sometimes impossible to determine the point of infection. The organism produces a most powerful poison, which is absorbed and carried by the blood to all parts of the body. The poison has a specific action on the nerves and causes characteristic spasms of certain groups of muscles. The muscles of the throat and jaw are often paralyzed, thus giving rise to the common name, lockjaw. The muscles of the neck may be involved, causing the head to be held in a stiff, out- stretched manner. The muscles of the tail and back are also often affected. The disease is usually fatal in sheep and in hogs, while about 75 per cent of the horses affected die. The duration of the disease in the horse may be a few days, or it may continue for several weeks. In man. the disease manifests itself in much the same manner as in the lower animals. Preventive measures. — ^A preventive, and to to some extent, a curative treatment has been de- veloped in the tetanus antitoxin. This antitoxin is comparable to the preventive serum used in hog cholera, and the diphtheria antitoxin used so widely in human medicine. In the preparation of the antitoxin, it is necessary to force a susceptible animal, like the horse, to pro- duce in its blood a quantity of the protective sub- stances so that the blood can be drawn and the serum obtained. In producing the serum, the animal is hyperimmunized by the addition of repeated doses of the toxin or poison produced by the organism, Glanders and Tetanus 203 beginning with very minute doses and gradually in- creasing. This treatment with constantly increas- ing doses of the toxin is continued until the body of the horse has produced a large quantity of the sub- stances that will neutralize the toxin. Fortunately the body can produce an amount of the protective substances in excess of that which is necessary to render harmless the toxin introduced. If some of the blood of the hyperimmunized ani- mal is carried to an animal that is just beginning to show symptoms of tetanus, the antitoxin will be ready to neutralize the poison as it is formed by the growth of the organisms. It will tide the body of the diseased animal over the period of danger and give it time to protect itself by the manufacture of its own antitoxin. The transfer of the protective substances is ac- complished by drawing a small portion of the blood, allowing it to clot, and using the clear serum, which formerly represented the commercial product. Ways have now been found by which the protective sub- stances can be concentrated by chemical means, a distinct advantage, since it avoids the introduc- tion of such large quantities of liquid into the ani- mal to be protected. The protective serum is expensive and hence is used only on valuable animals. Its widest use is in the prevention of the disease in man. The immunity thus produced is passive and persists for only a short time. CHAPTER XXII HOG CHOLERA The most important communicable disease of hogs is known as hog cholera, supposed to have been introduced from Europe in breeding animals. The first outbreak in this country of which record exists is that which occurred in Ohio in 1833. Since that time the disease has spread to all parts of the coun- try. In the corn growing states the losses occasioned by it are enormous. In the interval from 1894 to 1912 only eleven of the 92 counties of Indiana lost less than five per cent yearly of the annual hog crop, 38 lost between five and ten per cent, 30 between ten and fifteen per cent, 12 between fifteen and twenty per cent, and one county over twenty per cent. It is estimated that eighty-five per cent of the> losses incurred in the hog industry are due to this disease. From these figures it is evident that hog cholera places an enormous tax on the swine indus- try of the country. This disease, like foot and mouth disease, is one which presents high and low tides. A widespread outbreak occurred in 1886-7, another in 1894 and in the years thereafter, and still another began about 1911 and is still in progress. Its gradual spread from south to north is shown in the following figures which present the per cent of the annual hog crop lost through cholera. 1912 1913 Iowa 16 25.5 Minnesota 5.5 21.4 Nebraska 11 17.5 South Dakota 3.8 23.0 North Dakota 2 7.5 Hog Cholera 265 The disease is due to an ultramicroscopic organism that gains entrance to the body by the way of the digestive tract or through the broken skin. The causal organism is ehminated from the body in the feces and urine. All breeds of hogs are susceptible to the disease. It has been claimed by some that the mule- footed hogs would not acquire it, but experience has shown this statement to have no basis of fact. Symptoms. — The disease may appear as a typi- cal blood poisoning or septicaemia, as an intestinal infection, as a lung trouble, or in any combination of the three. It was earlier thought that there was more than one disease which affected hogs, but as methods of prevention have been devised, it has been found that all respond to the same treatment and hence must be caused by the same organism. The symptoms vary with the different manifesta- tions of the disease. The first hogs to die in any outbreak do so after having shown signs of illness but a short time. It will usually be observed that the sick hogs fail to eat, are affected with chills, and huddle together in the pens to keep warm. They stand with back arched and with the hind feet close together or crossed. They show stiffness of the muscles and joints, and may stagger and fall through weakness. The skin of ears, nose, abdomen, and inside the thighs may be reddened. The beginning stages are marked by a constipation, followed by a profuse diarrhea in which the feces have an offensive odor. If the lungs are affected, a hacking cough is noted and an increased respiration. The eyelids are often stuck together by a purulent discharge. The tem- perature is increased, reaching 104° to 109° F. 266 Agricultural Bacteriology If the attack is of longer duration as in the chronic form, there is more marked evidence of digestive disturbances. Animals with chronic cholera become emaciated, the hair may drop out, and even portions of the skin may die and slough off. As a rule they do not become profitable feeding animals even after recovery. It is sometimes diflTicult or impossible to determine from the symptoms alone whether hog cholera is present, in a herd. A careful post-mortem examina- tion of the dead animals is necessary in order to make a conclusive diagnosis. This examination should be made preferably on the carcass of a sick animal that has been killed, or on one that has just died. If the examination is delayed for a number of hours, it is hkely to be of little service in making a diagnosis because of post-mortem changes. Lesions. — The extent of the changes in the organs will depend on the length of the attack. If the animal has died of acute hog cholera, the lesions will not as a rule be so marked as in the more chronic form. The color of the skin should be noted. Red or purplish blotches are significant. The abdominal and lung cavities should be carefully opened and the following organs examined. The kidneys in the acute cases are likely to be darker than normal and to show small, red spots which impart to the organs a "turkey egg" appearance. The spleen or milt is usually enlarged, dark and soft; the liver is normal in appearance; and the membranes of the abdominal cavity, the stomach and the small intestine may show red areas as though blood had been spattered on them. It will be found impossible to remove the blood by washing, showing it to be in the tissues rather than on them. The hemorrhages are to be IToG Cholera 2f>7 found in many parts of the body and may vary in size from the pin point spots noted in the Ividneys to areas of considerable size. The lungs may or may not be affected. In case they are, the hemorrhages FIG. 46— HOG CHOLERA The turkey-egg appearance of the kidneys is an indication of cholera. are present and portions of the lung tissue may be consolidated instead of being soft and filled with air. The surface of the heart may show the red blotches. In the acute cases the inner hning of the large intestine is frequently found to be blood stained, 2f)8 Agricultural Bacteriology and the feces may be bloody. In the more chronic cases the most characteristic lesions of the disease are found in the large intestine, the so-called button ulcers which are round, hard, and yellowish with a dark center. They are distinctly raised above the surrounding, healthy surface of the intestine. In size they vary from a small point to the size of a 25 cent piece. The finding of such ulcers is to be considered as a positive indication of hog cholera, FIG. 47 HOG choleba Button ulcers on the inner wall ol the intestine in a case of chronic, hog cholera. (After Reynolds.) and it is the only lesion thaL can be regarded as absolutely diagnostic. The lymph glands in various parts of the carcass are found upon section to be enlarged and reddened. A number of causes may produce troubles that may be mistaken for cholera. Pneumonia due to exposure, dust or lung worms is sometimes con- founded with cholera. Improper feeding may cause intestinal disturbances. Slops containing alkalies, such as soap powders, are often a source of trouble in garbage fed hogs. Prevention.— Since but little can be done to cure the disease after it has made its appearance in the Hog Cholera 269 individual animal, the farmer must direct his efforts to the prevention of the disease. It should be re- membered that the organism is eliminated from the body of the affected animal in the urine and feces and that it is present in all the tissues of the body. An animal that has recovered from the disease may still harbor the organisms in its body and eliminate them. With these facts in mind the farmer can out- line his plan for the protection of his herd. No animal should be purchased from a herd in which cholera has been present during the previous year, nor from a herd that has been subjected to the pre- ventive treatment in which the virus has been em- ployed within six weeks. Animals purchased should be kept in quarantine for four weeks and then allowed to come in contact with a small number of the herd. If these exposed animals remain healthy after two or three weeks exposure, it is safe to place the purchased animals with the herd. It is not essen- tial that a rigid quarantine be established in the case of the purchased animals, for the prevention of in- timate contact will usually suffice. The method of keeping hogs in separate houses rather than in one large house has many advantages, one being that if cholera breaks out in one of the yards, it can often be prevented from spreading to the remaining sections of the herd. Hogs that have been shown at fairs are likely to be exposed to infection, not only at the place of exhibition but also during shipment. Care should be used in allowing such animals to mingle with the herd, until time has shown the animals to be free from infection. ,1 I The virus of hog cholera can be\arried on objects from one farm to another. It is probable that this 270 Agricultural Bacteriology is one of the chief ways in which the disease progresses in any locality into which it has been introduced. The farmer should remember that any object trans- ferred from an infected farm to his own may serve to carry the infection. The visiting of the hog yards in which an outbreak has made its appearance, the transfer of tools or wagons, animals such as dogs, or cows, are ways in which the disease spreads. It seems very probable that if the farmer takes care of those factors which he can control, he will have little trouble with those he can not control. The herd should be kept in as healthy a condition as possible by providing clean, well ventilated pens, clean feeding troughs, and proper feed, since any- thing that tends to weaken the animal makes it more likely to acquire cholera in case the organisms are taken into the body. It is generally believed that the feeding of new corn produces the disease. The feeding of large quantities of new corn may pro- duce digestive troubles and make the animal more susceptible to cholera, but naturally it can not cause hog cholera. If cholera makes its appearance in the herd, all the healthy animals should be removed at once to an- other field. One can not rely on the appearance of the animals as to whether they are infected or not. A much more reliable way is to take the tempera- ture of each animal, and to retain all showing any fever, in the yard with the diseased animals. The normal temperature of grown animals ranges from 101° to 103° F. In young animals the temperature will run somewhat higher, but in separating the herd all animals having a temperature of over 103.5° should be considered as infected. Hog Cholera 271 The carcasses of hogs that have died should be bui'ned, and also all litter from the pens. The care- less disposal of carcasses is one of the chief ways of spreading and perpetuating the disease. The hogs should be pastured in fields that do not border on the road and which are not traversed by streams, since infection may be introduced through either of these ways. The most certain way of protecting the herd against the disease is to apply the preventive treat- ment described later. Many cures for hog cholera have been proposed and are widely advertised. It is certain, however, that no treatment other than the administering of the protective serum is of any value. Protective treatment. — A hog that has recov- ered from a natural attack acquires an immunity to the disease, due to the presence in the blood of protective anti-bodies that have been formed under the stimulus of the disease organism. The amount of protective bodies that are thus produced as a re- sult of an attack of the disease is not sufficiently great so that the blood can be drawn and introduced into the body of another animal for the purpose of imparting immunity. If, however, such an immune animal is injected with large quantities of the blood of a hog that is very sick with the disease, the stimulus imparted by the introduction of the virus will cause the animal to form additional protective bodies, so that it will be practical to draw the blood and use it in protecting other animals. The immune hogs are said to be hyperimmunized. The protective serum is secured by bleeding the im- munized hog. This is done by cutting off the tip of the tail, about five or six cubic centimeters of blood 272 Agricultural Bacteriology per pound of body weight being drawn. This pro- cess is repeated three times, at weekly intervals. The animal is then given another injection of the viru- lent blood, is then bled twice from the tail and after the usual interval is bled from the throat. The blood is beaten with a wire as soon as it is drawn from the animal to remove the fibrin and prevent clotting. One-half per cent of carbolic acid is added as a pre- servative. Before the serum is used in the field it is necessary to determine its protective power. This is done by injecting varying amounts into susceptible pigs that are inoculated at the same time with some virulent blood. In this manner it can be determined how much must be used in actual work to protect an animal. It will be seen that the preparation of the serum is expensive because of the large number of hogs that must be used and the labor involved. Many of the states have established laboratories for the preparation of the serum. The price charged for it varies, the average being about one cent per cubic centimeter. The serum sold by the commer- cial companies is more expensive. By the use of the serum alone a passive immunity is produced that will protect the animal from a se- rious infection for six to ten weeks. If a small quan- tity of virulent blood is introduced into the animal at the same time the serum is injected, active im- munity will be produced which will generally pro- tect the animal for life. The serum can be applied, and in a week or ten days, a second dose of serum and also the virus. The first method is known as the serum-alone method, the second as the simultaneous method, and the last as the double or combination method. Each has its advantages and disadvantages Hog Cholera 273 which must be considered in determining which to apply. The serum-alone is safe but protects for only a short time, unless the animals come in contact with infectious material soon after treatment, in which case the results are substantially the same as are obtained in the simultaneous treatment. The method is of small value in the protection of breed- ing animals. It does allow the farmer to protect his herd for a short time when danger of infection is great. In the simultaneous method, some of the treated animals may die from cholera, because not sufficient serum was used to protect against the virus admin- istered. The animals in which acute cholera is thus produced may serve as centers of infection from which the disease may spread to other herds. This danger has led many to advise against the use of the simultaneous method. In herds in which the disease already exists, only the serum should be used. The combination method avoids the danger of the simultaneous treatment since rarely are any animals lost by cholera due to the treatment. It is more expensive since serum must be given twice. Breeding herds should be protected by the use of the combination method, even if cholera is not pres- ent in the vicinity, because it enables the breeder to send, without danger, breeding hogs into infected districts and to show at fairs. Application of the serum. — ^The serum may be applied by the farmer himself, but if the virus is to be used, as is the case in the simultaneous or com- bination methods, a veterinarian should be em- ployed, since the virus is dangerous material and if handled by those who do not appreciate its nature, trouble may result. The animals should receive a 274 Agricultural Bacteriology light laxative diet for a day or so before being treated, and should be kept in clean, dry quarters. Small hogs are usually injected in the arm pit. The animal may be held on its back between two round fence posts joined together by cleats. Larger animals may be snubbed to a post by a rope around the upper jaw, and the serum injected in the fold of loose skin at the side of the neck. The needle of the hypodermic syringe should be thrust deep into the tissue, not simply through the skin as when tuberculin is applied. If the infection of the animal with organisms that will cause inflammation and abscesses is to be avoided, it is necessary to see that the syringe is sterilized before use, by placing it in water, and bringing it to a boil. If the syringe has leather washers on the plunger, its sterilization must be accomplished by the use of chemical disin- fectants, since boiling would destroy the leather. The skin at the point where the injection is to be made should be scrubbed with a stiff brush, warm water and soap; then rinsed with some water that has been boiled and allowed to cool. The skin is then treated with a four per cent solution of carbolic acid, or tincture of iodine. Care should be used to keep everything clean during the process. The doses of serum are as follows: Weight of animal When virus is used No virus 0-20 lb s. 15 cc 10 cc 20-50 ' 25 " 20 " 50-75 ' 35 " 25 " 75-100 ' 40 " 30 " 100-150 ' 50 " 35 " 150-200 ' 55 " 40 " 200-300 ' 65 " 45 " 300-400 ' 85 " 65 " 400-600 ' 100 " 85 " Hog Cholera 275 The larger quantity of serum is used with the virus in order to protect the animal against the virus, which by itself would cause death. The virus is usually applied at some other point than the serum, as beneath the skin at the center of the space be- tween the fore legs when the serum is applied in the arm pit. For a few days after the serum is administered the feed should be reduced to about one-half the normal amount, gradually increasing until at the fourth week the full feed may be given. When only the serum is given, there should be little or no reaction. With the double or the simultaneous treatment in six to ten days after the injection, the reaction fever sets in and the temperature may rise to 106° F. The animals may lose appetite, have chills and present the symptoms of a mild case of hog cholera. The more susceptible animals may die from the effects of the virus. The hogs that show symptoms may eliminate the virus, and be the starting point of an outbreak of cholera in case they come in con- tact with susceptible animals. The results that have been obtained with the serum have been such as to recommend its use. When applied in herds in which the disease had already made its appearance, over 80 per cent of the animals have been saved, while the treatment applied before the infection of the herd took place has pro- tected over 90 per cent of the animals against in- fection. There seems to be little doubt but that any farmer or breeder can protect his herd against loss from cholera by the consistent and careful use of the pro- tective serum and the virus of the disease. It is a 276 Agricultural Bacteriology matter of some expense and the farmer must weigh the cost of the insurance against the probable loss from cholera before deciding whether to apply the treatment or not. CHAPTER XXIIl DISEASES OF FOWLS The transmissible diseases of fowls inflict a heavy tax on the poultry raiser and the general farmer. Present knowledge concerning many of these diseases is far from complete, and in many cases so frag- mentary, that no definite plan for the eradication and prevention can be devised other than the customary plan applicable in most cases of trans- missible diseases, viz., removal of affected indi- viduals, destruction of carcasses, and general cleanli- ness and disinfection. Chicken cholera. — Chickens like swine are sub- ject to dietary disorders which may often stimulate a true contagious disease in the rapidity, with which it appears in the flock and in its high mortality. Cholera is a term applied to many of such disorders that are not produced by a specific organism. The true chicken cholera is rare in this country and is due to the invasion of the body by a specific form of bacteria. Symptoms. — The urates, that part of the excre- ment excreted by the kidneys, in the case of healthy birds are pure white in color. In birds affected with cholera the urates are yellow, often a bright yellow, and sometimes a bright green. This change in color is not proof of the presence of cholera, but is a valuable indication of the disease. Diarrhea is usually present; the sick bird leaves the flock, be- comes weak and drowsy, acts dumpish, and the 278 Agricultural Bacteriology feathers are roughened. Intense thirst is noted, the appetite is poor, and the crop remains distended with food. There is a rapid loss of flesh. The disease makes rapid progress in the flock due to the short period of incubation, one to three days. Most of the affected birds die in a short time of an acute form of the disease; others may have a chronic type; recovery is rare. On post-mortem examination the digestive organs will be found to be inflamed, and the liver is usually enlarged and softened. The presence of cholera can, however, be established only by a bacteriological examination of the blood which will be found to contain great numbers of the causal organisms. The disease is a true septicaemia; the organism enters the body by the ingestion of con- taminated food or water which may become con- taminated with the excrement of the affected birds, or the material that drops from the beak. The extensive lesions in the intestine allow the excre- ment to become mixed with manure. The disease may be introduced into the flock by the purchase of a bird with a chronic form of the disease, or by doves and wild birds that fly from farm to farm. Prevention. — Nothing can be done for the birds that are infected. All efforts must be concentrated in preventing the spread of the disease. It should be remembered that every drop of blood contains great numbers of the causal organisms and that if any portion of the carcass is consumed by well birds, they are certain to become infected. It is advisable to kill the birds that show any symptoms of disease. This should be done in such a way that no blood is drawn. The dead fowls should be Diseases of Fowls 279 promptly disposed of; the feed and water troughs' should be thoroughly disinfected, as also the roost- ing houses. If possible the still healthy birds should be removed to fresh uncontaminated grounds. The causal organism does not produce spores and will not persist long outside the body of the bird. It is considered safe to bring new stock onto the place after the expiration of two weeks, in case the house and other contaminated objects have been thor- oughly disinfected. Infectious leukaemia. — This disease, which has also been called fowl typhoid, is often mistaken for true chicken cholera. It is, however, produced by a different organism. The disease is less rapid in its progress in the individual bird than is cholera. The diarrhea so characteristic of cholera is absent, and the intestines are pale instead of deep red as in cholera; the contents are normal in consistency while in cholera the intestinal contents are liquid and blood stained. The blood is quite free from the organisms. It is not especially important that a correct diagnosis be made as to which of these diseases is present in the flock, since identical methods of prevention should be employed with either. Cleanliness should be the chief reliance of the poultry man against these diseases. Roup. — Roup or diphtheria of fowls is considered to be the most important transmissible disease affecting the barnyard fowl of this country. It occurs in turkeys, ducks, pigeons, and pheasants, as well as chickens. The cause of roup has not been discovered and it is not certain whether chicken pox and canker are different diseases from roup or different manifestations of the same disease. 280 Agricultural Bacteriology It has sometimes been considered that this disease has some relation to diphtheria in man. There is no reason for such belief other than in certain forms of roup there may be formed a membrane similar to the membrane noted in diphtheria. The first symp- tom of roup is a watery discharge from the nostrils and often from the eyes. The bird becomes dumpish ; the breathing is often noisy, due to the obstruction of the air passages with the exudate. The fowl may be able to breathe only by opening the beak. Sneezing is frequent. The eyes may be covered with the dry discharge or they may be forced from the sockets, due to the accumulation of cheesy matter in the sockets. There may be found in the mouth and throat patches of grayish-yellow exudate or membranes. Death is often occasioned through suffocation due to the closing of the throat by the membrane. The swelling of the head caused by the accumulation of the exudate in the various cavities of the head has given rise to the term swell-head. It is considered that potassium permanganate is helpful both as a preventive and for the treatment of affected birds. It may be added to the drinking water in sufficient quantities to impart a pink color to the water. The heads of the affected birds may be dipped in one or two per cent solution of the same substance. Roup is to be differentiated from simple catarrh which closely resembles the human trouble known as cold-in-the-head. Simple catarrh is caused by exposure to dampness, cold winds, and by improper ventilation of the houses. It has been shown ex- perimentally that it is impossible to produce roup in these ways. There seems to be no doubt, however, Diseases of Fowls 281 that birds suffering from catarrh are much more susceptible to roup. Roup may be carried from flock to flock by the transfer of birds with a mild form of disease. Fowls should not be purchased from infected flocks and it is well to place in quarantine for some days new birds or those that have been at shows before plac- ing them with the flock. Any bird showing any dis- charge from the mouth or eyes should be removed at once from the flock. White diarrhea. — Of the diseases affecting young chicks white diarrhea is the most important. It is probable that more than one trouble has been classed under this name. The white diarrhea of young chicks caused hy Bacillus pullorum is certainly the most important. This disease offers an example of hereditary transmission- of disease. It has been shown that the ovaries of the hen may be affected, and that the ova contain the organism. The young chick becomes infected from the yolk sac. Some of the females that survive continue to harbor the germ and become bacillus carriers. The adult females may become infected by contact with other infected adults or by infected litter. They may then become bacillus carriers. The infection is in all probability acquired through the mouth. The economic importance of the disease is oc- casioned by its effect on young chicks. The greatest danger of infection is during the first forty-eight hours. The danger of infection is very slight after four days. The affected chicks appear stupid and remain under the hover or hen much of the time. The kfeiathers become rough and the wings droop. There 'is constant loss of weight. The chicks eat little 2(S2 Agricultural Bagtertology and appear unable to pick up their food. A whitish discharge from the vent soon makes its appearance. The discharge may be creamy or sometimes mixed with brown, and it is more or less sticky or glairy. In many cases it clings so closely to the down as to close up the vent. Many of the chicks peep con- stantly or utter a shrill cry, apparently of pain, when attempting to void the excrement. The ab- domen is enlarged and protrudes to the rear. The post-mortem examination shows no marked lesions. The organs are all pale; the alimentary tract is usually empty except for some slimy fluid. The prevention of the disease must rest on the non-introduction of bacillus carriers in the purchase of breeding stock, and by the purchase of eggs and young chicks from flocks that are known to be free from the disease. The wide spread infection of breeding birds is shown by the fact that in a flock in which the losses of the young chicks had been ex- cessive, over 80 per cent of the laying hens were shown to have diseased ovaries. The chicks that recover from the infection do not as a rule grow as rapidly as do noninfected birds. It has been shown quite conclusively that the feed- ing of sour milk to young chicks is of value in pre- venting the spread of the disease. The dishes in which the milk is kept should be cleaned daily and fresh supply of milk provided. The incubators and brooders should be thoroughly disinfected after each hatch and extreme cleanliness should be practiced in all regards in the handling of young chicks. CHAPTER XXIV BACTERIAL DISEASES OF PLANTS The most important transmissible diseases of ani- mals are those caused by bacteria rather than by the other groups of microorganisms such as molds and yeasts, while in the plant kingdom the reverse is true. The explanation of this is found in the fact that the bacteria prefer the alkaline reaction of the animal fluids, while the molds fmd more favorable conditions in the acid juices of the plant. The molds are also by nature better fitted to penetrate into the tissues of the plant than are the bacteria. Just as the increased commerce in animals has hastened and accentuated the spread of the trans- missible diseases of animals, the increased sale of seeds and plants of all kinds and their shipment from one part of the country to another has led to the rapid spread of both bacterial and fungus plant diseases. At the present time about thirty bacterial diseases of plants have been described. A few are wide spread and are certain to come to the notice of every one engaged in farming and are of great economic importance. The bacterial diseases of plants may be divided into four classes, depending on the manner in which they affect the plant: the blights, the rots, the wilts, and the galls. In the first the tissue is killed by the organism, but it is not decomposed as in the rots, in which the tissue is not only killed but decomposed; while in the wilts the passage of water to some por- tion of the plant is interfered with, and hence death of affected tissues soon ensues. 2R AcriTCUT.TCTRAl. PiACTF.P.IOT.Or.^' The complicalt'd quesLions LhaL arise in connection witii the immunity against bacterial diseases of animals do not occur in the bacterial diseases of plants. There is a difference in the susceptibility of different variclies of plants to the same organism. Efforts are being made to increase and extend this natural immunity. Much more can be done in an FIG. 48 — PEAR BLIGHT The bacteria causing the shriveling of the fruit enter through the blossoms. experimental way in the breeding of resistant varieties of plants than can be done with animals. Pear blight. — The most important of the bhghLs is that which affects the pear and apple, and to a lesser extent the quince, apricot, and plum. It was first observed in 1780 in the Hudson River valley, and as orcharding has spread westward the disease has developed, until it is now found in all parts of this country and Canada. In Colorado it found such favorable conditions that it has caused the abandonment of commercial pear growing. It has also caused great losses in (