MOLD COLONY, SHOWING MYCELIUM AND SPORE CLUSTERS COMMON MOLD (PENICILLIUM ), SHOWING MASSES OF SPORES [Both from micro-photographs made for this work] PRACTICAL DAIRY BACTERIOLOGY PREPARED FOR THE USE OF STUDENTS DAIRYMEN, AND ALL INTERESTED IN THE PROBLEMS OF THE RELATION OF MILK TO PUBLIC HEALTH : : : : By H. W. CONN, Ph.D. Professor of Biology of Wesleyan University and Bactenocogist of the State Board of Health of Connecticut ILLUSTRA TED NEW YORK ORANGE JUDD COMPANY LONDON Kegan Paul, Trench, Trubner & Co., Limited 1914 COPYRIGHT, 1907, BY ORANGE JUDD COMPANY All rights re serve a [entered AT STATIONERS' HALL, LONDON, englandJ DEDICATION In appreciation of the constant assistance received from her in this and other scientific works To My Wife PREFACE It is about twenty years since the serious study of the bacteria in milk began; for while, in an incidental way, certain problems were investigated earlier, not until about 1890 was it recognized that the subject had a large field of practical ap- plications. The development of Dairy Bacteriology has been due to the work of scientists on both sides of the Atlantic and this country has contributed no less than Europe to our present knowledge. As the study has progressed it has appeared that the bacteri- ology of milk products has an intimate relation to two different classes of problems. 1. The problems arising in the dairy proper and affecting many questions relating to milk, butter and cheese. It is no exaggeration to say that the development of the knowledge and application of dairy bacteriology has revolutionized all phases of dairying, until all processes, from the milking to the final consumption of the completed product, have been modified by bacteriological knowledge. The time has come when it is no longer possible to be a successful dairyman without a knowl- edge of milk bacteria. To-day all courses planned for the study of dairying devote more or less time to the subject, and the student who fails to acquire some practical knowledge of the relation of bacteria to milk products is not properly trained to make a practical dairyman. 2. The problems of the relation of milk bacteria to the public health. The increasing proof of the distribution of disease germs by milk, the accumulating evidence that the health of the public, especially of children, is closely related to the bacteria in milk, and the growing realization of the conditions VI PREFACE VII of filth and unwholesomeness under which milk has commonly been produced, have led to the active interest, on the part of health officials, in the nature of the public milk supply and primarily in bacteriological problems. A lack of proper in- formation on the part of health officials as to the conditions and possibilities of the dairy, and on the part of the dairymen as to the necessities and demands of the public and the method of meeting them, is to-day resulting in constant friction. It is to meet the demand from both of these sources that the present book is written. It is designed on the one hand for dairy students and on the other for all who are interested in dairy products from the standpoint of their production, their distribution, or their consumption. For this reason it combines the practical with the theoretical. In the first part is given a general discussion of the facts known concerning the relation of the bacteria in milk, and in the second part will be found practical directions for bacteriolog- ical analysis and methods of bacteriological study. It is hoped that thus the work may prove of use both as a manual for dairy students and as a reference book for dairymen and health officials who may be interested in all problems relative to the bacteria of milk. Middletown, Ct., October, 1907. H. W. Conn. CONTENTS PART I GENERAL DISCUSSION OF DAIRY BACTERIOLOGY CHAPTER PAGE I. What are Bacteria? i II. Types of Bacteria Found in Milk 21 III. Sources of Common Milk Bacteria 58 IV. The Growth of Bacteria in Milk 74 V. Disease Germs in Milk 86 VI. Dairy Methods 112 VIL Treatment of Milk for Market 144 VIII. Public Problems 168 IX. Bacteria and Butter-making . 193 X. Bacteria in Cheese 223-260 PART II LABORATORY DIRECTIONS FOR EXPERIMENTS IN DAIRY BACTERIOLOGY PAGE Laboratory Work in Dairy Bacteriology . . . 261-309 VIII ILLUSTRATIONS PAGE Mold Colony, Showing Mycelium and Spore Clusters- Common Mold (Penicillinm), Showing Masses of Spores Frontispiece Fine Needle, a Bit of Dust and Bacteria 2 Colonies of Bacteria Growing on Gelatin 3 Shapes of Bacteria 4 Flagella 5 Spores 7 Size of Yeasts, Mold Spores and Bacteria 8 Common Yeast 9 Yeast in Fermenting Cider 9 Common Mold, Penicillium 10 Common Mold, Aspergillus 11 Mold Showing Threads of the Mycelium 12 Mold Colony on a Gelatin Plate 13 A Sarcina Type 14 Varieties of Bacterium lactis acidi 27 Varieties of Bacterium lactis acidi 28 Colonies of Bacterium lactis acidi in Litmus Gelatin ... 30 Gelatin Stab Cultures of Bact. aerogenes and Bact. lactis acidi 31 Effect of Different Bacteria on Milk 33 Types of Bacterial Growth on Agar Slants 34 Production of Gas in Fermentation Tube Produced by Bact. lactis aerogenes 35 Liquefaction of Gelatin by Enzyme-forming Bacteria . . 39 Bacteria of Butyric Acid 43 Effect of Slimy Milk Bacteria upon Milk 45 Bacterium lactis viscosus, the Common Cause of Slimy Milk 46 Coccus, Producing Bitter Milk 48 Torula amari, Producing Bitter Milk and Cheese .... 49 Kefir Grains 51 Bacterium cyanogenes 53 Oidium lactis from a Surface Colony on Gelatin .... 56 Structure of Cow's Udder 59 Filthy Condition of Cow's Hindquarters 63 Proper Condition for Cow's Hindquarters 64 IX X PAGE Colonies of Bacteria Growing from Cow's Hair .... 65 Poorly-kept Cow Barn Sure to Give High Bacterial Con- tact to Milk 66 Relative Amount of Dirt Falling from the Udder During Milking under Different Conditions 72 Growth of Bacteria at Different Temperatures during 24 Hours 77 Tuberculosis Bacilli 89 Typhoid Bacilli 99 Typhoid Bacilli, Showing Flagella 99 Diphtheria Bacilli 103 The Model Dairy Farm 114 Poorly-kept Barnyard 115 Well-kept Dry Barnyard 116 Interior of a Well-equipped, Well-kept Cow Barn . . . 117 Colonies Developing from Bacteria Falling Upon a Plate Exposed During Milking in a Poorly-kept Barn . . .118 Colonies Developing from Bacteria Falling Upon a Plate Exposed During Milking in a Well-kept Barn . . . .118 Milking in a Milking Room, an Ideal Condition of Clean- liness 119 Milking in a Model Dairy Barn 120 An Ideal Locality for Milking 121 Method of Moistening the Cow's Udder During Milking . 125 Covered Milk Pail (North's) 127 Covered Milk Pails (Gurler's and Stadtmuller's) .... 128 Milking Machine 129 Use of a Milking Machine Out of Doors, Power Supplied by Animals in Treadmill 130 Results of a Rough Usage of Milk Cans on the Railroad . 146 Simple Improvised Methods of Pasteurizing without the Use of a Thermometer 155 Pasteurizer of the Discontinuous Type 157 Collecting Case for Milk Samples 175 Sediment from Centrifugalized Milk as It Appears Under the Microscope 182 The Use of Burettes for Determining the Amount of Acidity 202 Pieces of Milk Curdled with Gas-producing and Non-gas- producing Bacteria 239 Forms Used in Making and Draining Camembert Cheeses 244 Penicillium camcmberti 246 Common Mold on Soft Cheese, Penicillium camemberti . . 247 Camembert Cheese Cut Open to Show the Softening of the Curd 249 ILLUSTRATIONS ILLUSTRATIONS XI PAGE Worthless Camembert, Showing Pores Produced by Gas- forming Bacteria 252 Penicillium roqueforti 254 Test Tubes-Board of Health Pattern 264 Pipette and Glass Tube for Holding Same when Sterilized 266 Hot Air Sterilizer 266 Steam Sterilizer 269 Filtering Culture Media 270 Filling Test Tubes with a Measured Amount of a Culture Medium 271 Hardening Agar Slants 273 Vials for Milk Dilution 274 Autoclav for Sterilizing under Steam Pressure .... 275 Peri Dish 276 Counting-box 277 Platinum Needles 282 Forceps for Holding Cover-glasses 283 Centrifuge for Milk Testing 285 Making Gelatin Stabs 290 Cutter for Making Potato Plugs, Method of Cutting Them and Placing in Tubes 293 Types of Gelatin Stab Growths 294 Types of Gelatin Liquefaction ........ . 294 Types of Surface Elevation 296 PART I GENERAL DISCUSSION OF DAIRY BACTERIOLOGY CHAPTER I WHAT ARE BACTERIA? Bacteria were first seen two centuries ago by a Dutch micro- scopist, Leeuwenhoek, but were mostly forgotten for the century following. About 65 years ago it was first seriously suggested that these minute organisms might be of some significance in nature, and even at that time one unusual phenomenon of the dairy-blue milk-was attributed to the action of microscopic germs. About i860 Pasteur showed a close connection be- tween microscopic life and the souring of milk; but it was not until about 18 years ago that they were conceived to have a very intimate relation to dairying. Dairy Bacteriology has practically all developed in the last two decades. The advance has been extremely rapid, and it is not an exaggeration to say that in this brief time bacteriology has revolutionized all dairy methods, from the cow to the con- sumer's table. The dairyman of to-day, and even more the dairyman of to-morrow, must understand bacteriology both theoretically and practically. THE MICROSCOPIC APPEARANCE OF BACTERIA The dairyman who is trying to do any practical work with bacteria in his dairy business does not ordinarily need to use a microscope. The organisms are so small that they must al- ways be handled in masses, and for this purpose, though a microscope is extremely desirable, it is not absolutely necessary. The dairyman has rarely time to use a microscope properly. The student, however, who is learning of the bacteria in milk and their properties, must make a careful microscopic study. PRACTICAL DAIRY BACTERIOLOGY 2 Bacteria are all practically colorless, and as such are extremely difficult to see. It is almost impossible to study carefully a mi- nute, colorless object under the microscope. For the study of bacteria, therefore, it is common to color them with staining materials, which render them more visible. The method of staining and study will be found on page 283. Size.-Before we can enter into the study of their relations to dairying we must first learn what bacteria are and something also of their relations to conditions. The most striking fact about them is their extreme minuteness. (Fig. I.) When we FIG. I-COMPARATIVE SIZE OF POINT OF FINE NEEDLE (a), BIT OF DUST (b), BACTERIA (c) hear of 100,000,000 in a single drop of milk we are apt to be incredulous, feeling that there could be no room for them, quite failing to appreciate their minuteness. There can easily be 100,000.000 in a drop and still be room for more. A space the size of a pin-head may hold 8.000.000.000, and 100,000,- 000 will have plenty of room in a drop of milk. This extreme minuteness must always be kept in mind in attempts to under- stand them. This minuteness makes it impossible to handle them indi- vidually. The only way we can deal with them is to handle masses, and laboratory methods always begin with cultivating them, by which is meant, putting them into conditions where they will grow until large numbers may be obtained. Such masses or cultures we can easily deal with, and for this reason, in spite of the fact that they are the smallest known living WHAT ARE BACTERIA? 3 thing, most of the methods of bacteriological study do not even involve the use of a microscope. The unit with which the student commonly starts is not a bacterium, but a colony. By reference to experiment No. 6 it will be seen that a colony is a mass of bacteria in some solid jelly medium. (Fig. 2.) Such a colony is composed of thou- sands of bacteria, all of which are supposed to have risen from FIG. 2-VARIOUS TYPES OF COLONIES OF BACTERIA GROWING ON GELATIN a single bacterium by multiplication. The whole makes a cluster big enough to be seen without any lens, and since all the bacteria in it have come from a single one they are all alike. The colony may be picked out of the jelly, placed in some medium in which the bacteria will thrive, and then, after proper growth, the bacteria can be studied by the microscope. Such cultures form the basis of bacteriological study. Form of Bacteria.-Bacteria are of three quite different shapes, but all very simple, i. Simple spheres. (See Fig. 3, b, c, d.) Such spherical forms are called cocci, and in 4 PRACTICAL DAIRY BACTERIOLOGY common microscopical preparation no internal structure can be seen, the bacteria appearing as faint stained balls. The cocci, however, differ somewhat in their method of growth, thus enabling the microscopist to distinguish different kinds, as will be mentioned presently. 2. The rod formed bacteria. (Fig. 3, c, f> £•) These organisms are longer than they are broad, some- FIG. 3-SHAPES OF BACTERIA a. Spirillum. b. Micrococcus. c. Micrococcus. d. Streptococcus e-h. Rod-shaped bacteria. t and j. Divisions times only slightly so, but at other times very much longer, forming, indeed, long, slender threads. 3. The spiral formed bacteria. These are either a long coil spiral or a very short one with only a single turn. (Fig. 3, a.) The type is rare in milk, and it is not likely that the dairyman will ever run across them, even if he makes extended microscopic studies. Motility.-The next point of distinction among bacteria is based upon their motility. Some bacteria are capable of an active swimming motion, others are stationary. The motion is produced by minute, extremely delicate, vibrating hairs, called flagella. (Fig. 4.) The flagella are so delicate that they cannot ordinarily be seen in the living bacteria, and they do not stain by the ordinary method of staining. They are, there- WHAT ARE BACTERIA? 5 fore, never seen in the microscopic preparations usually pre- pared. They may be seen by special methods, but these are so difficult that the beginner cannot use them satisfactorily. The question of their motility is, however, usually determined with- out staining, by the study of the living bacteria. (Exper. No. 30.) These flagella are differently distributed upon different bacteria. Sometimes there is a single one on the end of a FIG. 4-FLAGELLA A. Peritrichic. B. Lophotrichic. C. Monotrichic. rod (Fig. 4, C)-monotrichic; sometimes a small tuft at one or both ends of a rod (Fig. 4, B)-lophotrichic; and sometimes there is a covering of flagella over the whole body of the bacterium (Fig. 4, A)-peritrichic. Multiplication of Bacteria.-The most unique property of bacteria is their almost inconceivable rapidity of multiplication, and it is this which makes them of importance in nature. The method of multiplication of bacteria is the simplest conceivable. The organisms elongate a little and then break in two in the middle, two individuals thus arising where there was formerly one. Each of these now elongates and in turn breaks, and thus by continued division the numbers increase. Figure 3, i and j, shows this reproduction both in the rod and cocci forms of 6 PRACTICAL DAIRY BACTERIOLOGY bacteria. The rapidity with which these bacteria can grow and divide is surprising. In some cases it has been found that under favorable conditions of food and temperature, they may elongate and divide every half hour, and if they should continue to reproduce at this rate for twenty-four hours it is easily proved that a single bacterium would have by that time about 17,000,000 descendants. If each of these should continue to grow at the same rate, each would have, in twenty-four hours more, 17,000,000 descendants, and then the numbers would develop beyond a possibility of appreciation by human mind. It will, of course, be readily recognized that the bacteria do not long continue to multiply at this rate, for, if they did, in a few days there would be no room in the world for anything but bacteria. Their multiplication is constantly being checked by adverse conditions-lack of food, lack of moisture, etc.- and thus they do not on the whole very materially increase in numbers. But this inconceivable power of multiplication they do possess, and whenever they are placed for a few hours under conditions where they can have plenty of food and mois- ture for growth, they will develop with enormous rapidity, thus producing most profound changes in the substance upon which they are feeding. If they chance to develop in milk, they will cause a variety of changes which it is our purpose to study, for they change its physical and chemical nature profoundly. These changes, due to this enormous possibility of multiplication, have made bacteria of great importance in dairying and elsewhere in nature. Production of Spores.-There is another method of producing new individuals, an understanding of which is of extreme im- portance in a knowledge of bacteria. It results in the produc- tion of new individuals, although it is hardly a multiplication since one individual only results from one to start with. The phenomenon in question is the production of spores, and it is illustrated in figure 5. The bacterium there figured consists of WHAT ARE BACTERIA? 7 rods. The contents of one of the rods collects itself in a spherical or oval body in the center of the rod. This later breaks out of the rod, the rest of the individual then dying and disappearing. The oval body itself is a spore, and is capable, when placed under proper conditions, of developing into a new rod. Inasmuch as only a single spore arises from a single bacterium, it is not a multiplication. Its purpose is not so much to increase the number of individuals as to enable the bacteria to endure adverse con- ditions without being killed. The ordinary bacteria are likely tp be killed by being dried and will readily succumb to moderate heat, a temperature of 1600 1 being suf- ficient to kill almost any of them. But these spores are covered with a hard case which enables them to resist the conditions which the active, growing and multiplying forms cannot resist. They may be dried for months, and even years, and still retain their vitality. They may be heated very much hotter than the active forms without injury; indeed, some of these spores may be boiled in boiling water for many minutes, for an hour even, or longer, without having their vitality destroyed, since, if the material is subse- quently cooled, the spores are capable of generating and growing into new bacteria. As the result of this it will follow that, while it is very easy to kill ordinary bacteria by heat, it is far more difficult to destroy spores. Some species of bacteria produce such spores while others do not, and hence some are much more easily killed by heat than others. Milk, for example, contains many kinds of bacteria. By the simple boiling or, FIG. 5-SPORE PRODUCTION 1 Temperatures used in this book always refer to the Fahrenheit scale. 8 PRACTICAL DAIRY BACTERIOLOGY indeed, the heating of the milk to a temperature of 1600, a vast majority of the bacteria are killed; but the few spores that may chance to be in the milk are not thus killed, and these will be able subsequently to develop. If milk contains spore bearing bacteria, it cannot be sterilized by boiling, and since it almost always does contain them, boiling is not suffi- cient to sterilize milk. This phenomenon of the high-resisting powers of spores must always be borne in mind in all problems of sterilizing. YEASTS Among the micro-organisms that one is likely to find in milk are some that do not belong to the bacteria proper, although closely related to them and of hardly less importance in dairy phenomena. The first type is yeast. Yeasts are miscroscopic cells of a round or oval shape, and may usually be distin- guished from any of the true bacteria by their somewhat larger size. The relative size of yeast cells and bacteria cells may be seen from figure 6. Occasionally we find small yeast cells and sometimes large bacteria, so that the matter of size is not in all cases sufficient to enable one to determine whether he is dealing with bacteria or yeasts. The primary difference between the two is in their method of multiplication. The multiplication of yeasts is different from that of bac- teria, and is known as the multiplication by budding. There appears on the side of the yeast cell a minute bud which con- tinues to increase in size (Figs. 7 and 8), until after a time it becomes as large as the cell from which it was produced. The FIG. 6-RELATIVE SIZE OF YEAST (&) MOLD SPORES (t) AND BACTERIA (a) WHAT ARE BACTERIA? 9 two may break apart at once, or each of them may in turn produce other buds before separating; but in either case there are now two cells where there was originally but one. Yeasts also multiply with considerable rapidity, but not, as a rule, as rapidly as many of the bacteria; nevertheless their great power of multiplication makes them of great importance among the forces of nature. The reproduction by budding is the primary characteristic which separates yeasts from bacteria, and the FIG. 8-YEAST IN FER- MENTING CIDER Sometimes called "wild yeast," coming directly from the air FIG. 7-COMMON YEAST Showing multiplication by budding determination of the method of reproduction will always en- able one to tell whether he has a yeast or a bacterium under study. When, however, one finds under his microscope rather large, round or oval cells, he is pretty safe in concluding that they are yeasts rather than bacteria, and if he can find small cells attached to the larger ones-buds-he may be absolutely sure that he is dealing with yeasts. A second distinction between yeasts and bacteria is in the type of fermentation they produce. Bacteria produce decay, putrefaction and lactic acid fermentation. Yeasts are more commonly associated with the alcoholic fermentation, by which some forms of sugar are easily converted into alcohol and carbon dioxide. The latter type of fermentation does not usually occur in milk. The yeasts do not play quite so important a part in dairying as bacteria, and yet, as we shall see later, in some phases of the subject they are of great importance. 10 PRACTICAL DAIRY BACTERIOLOGY These are plants of considerably larger size and much higher structure, and they do not logically belong to the consideration of bacteria in milk. Their relations to milk and milk products, however, are in many respects very much like those of bacteria, and in certain phenomena asso- ciated wtih the ripening of cheeses they are of as great importance as bacteria themselves. It is necessary, therefore, for the dairyman to know something about them. They are plants that in all cases are large enough to be seen with the naked eye, although the microscope is required to study them. They consist of a mass of branching threads of very minute size. (Figs. 9, io and n.) Sometimes the threads are large enough to be seen with- out a microscope and sometimes not. This mass of threads is almost always color- less, or of a pale white, and grows on the surface or in the substance of various materials upon which the plant feeds. On cheeses, for instance, it thrives in pro- fusion upon the surface, and sometimes extends into the cheese itself. There are a great many species of molds, but they are all very much alike so far as concerns the formation of this colorless thread {mycelium}, although there are some differences to be seen here. But molds have methods of mul- tiplication which commonly enable us to distinguish different species. After they have grown luxuriantly, they usually send minute branches into the air, the ends of which finally produce rounded spores, or reproductive bodies. These are sometimes produced in chains, sometimes in masses, and the method of MOLDS FIG. O-COMMON MOLD, PENICILLIUM Method Of forming spores WHAT ARE BACTERIA? 11 spore formation is the chief means by which the microscopist separates the different figures from each other. Figures 9 and 10 show the method by which some of the common molds produce their spores. The studying of molds for the purpose of dis- FIG. 10-COMMON MOLD, ASPERGILLUS Mycelium and method of forming spores tinguishing spores does not, however, belong to our immediate subject. Such types as are concerned in dairy problems will be mentioned later in the proper places. (See frontispiece.) NAMES APPLIED TO BACTERIA The detailed classification of bacteria is rather complicated and difficult, and need not detain the dairy student. A few terms, however, are so general that it is necessary to learn their meaning, especially since there is considerable confusion in 12 PRACTICAL DAIRY BACTERIOLOGY their use. The following brief classification will explain the meaning of the common terms in use: Large colorless plants forming delicate threads and frequently colored spores MOLDS 1 FIG. II-MOLD SHOWING THREADS OF THE MYCELIUM (MAGNIFIED) Single celled plants, multiplying by budding. (Fig. 7) YEASTS Single celled plants, multiplying by division. (Fig. 3) BACTERIA 1 Other fungi, like mushrooms, would belong here, but play no part in dairying, and may here be neglected. WHAT ARE BACTERIA? 13 Spherical Bacteria: Dividing in one plain so as to form chains. (Fig. 3, d) Streptococcus Dividing in two plains and not forming chains. (Fig. 3, b, c.) Micrococcus Dividing in three plains and forming cubical masses. (Fig. 13.) Sarcina BACTERIA FIG. 12-MOLD COLONY ON A GELATIN PLATE Rod Shaped Bacteria: With flagella and consequently motile. (Fig. 4, a, b.) Bacillus Without flagella and consequently non-motile. (Fig. 3, e, f, g.) n Bacterium 14 PRACTICAL DAIRY BACTERIOLOGY Spiral Bacteria. (Fig. 3, a.) Spirillum The genus bacillus is further divided as follows: Bacilli with only one flagellum (Fig. 4, C) are named Monotrichic Bacilli, or Pseudomonas Bacilli with a tuft of flagella at one end (Fig. 4, B) are called Lophotrichic Bacilli Bacilli with flagella over the whole body (Fig. 4, A) are called Peritrichic Bacilli It will thus be seen that the term Bacteria applies to the whole group of organisms that multiply by divis- ion, whose study constitutes the study of Bac- teriology, while the term Bacterium refers to a single division of the group, viz: the non-motile, rod forms. The term Bacillus should apply to motile forms only. The names Bacillus and Bac- terium are sometimes confused; for example, the tubercle Bacillus is really a Bacterium since it is non-motile, but the name Bacillus was given it years ago before the above distinctions were recognized, and it has not been changed. Some other bacteria named twenty years ago retain their earlier names in some books, but they are slowly having their names brought into harmony with the above distinctions. The term Coccus is applied to any spherical organism of the group bacteria. FIG. 13-A SARCINA TYPE The question whether bacteria are plants or animals has been much discussed and answered in both ways. The reason for the uncertainty is the fact that our biologists are unable to give any clear distinction separating animals from plants, and with any definition that they can give, bacteria show relations to both plants and animals. The method of forming spores allies them to plants, while their frequent motility, together with the fact that they live upon foods similar to animals, allies PLANTS OR ANIMALS WHAT ARE BACTERIA? 15 them to the animal kingdom. After long discussion it has been quite definitely concluded that they are closely allied to yeast, and these are in turn closely related to molds. Biologists agree that molds are plants, and, therefore, the conclusion to- day is that bacteria are also plants, but of an extremely low type. It is a matter of no special significance, excepting to the scientist, whether we consider them as belonging to one or the other. Their relations to the dairyman are the same whatever we call them. RELATIONS TO CONDITIONS Temperature.-The bacteria behave very differently at differ- ent temperatures. Their rapidity of multiplication is very de- cidedly modified by the different degrees of temperatures. In general, at low temperatures, multiplication is comparatively slow. At freezing they do not multiply at all; but when the temperature is raised above freezing they begin to grow, and their rate of multiplication increases with the increase in the temperature up to a certain point, an optimum temperature. If the temperature is raised higher, the rate becomes slower again, until finally they stop growing. If the temperature is raised still higher, they are injured; and finally by a certain amount of heat they are killed. Thus there is: i. A minimum temperature, the lowest at which they will all grow. 2. An optimum temperature, at which they will grow best. 3. A death temperature, which will kill them. Minimum Temperature.-Some species will grow, though slowly, at temperatures only just above freezing, multiplying at 33°, while other species will not grow at all at such tempera- tures. Some will hardly grow at temperatures lower than that of body heat, 98°. Others again seem to require a temperature much higher than this, even as high as 1200 to 1300. Between these two limits different species show all kinds of variations. Optimum Temperature.-This also differs very much for dif- ferent species of bacteria. Some will grow at 6o° to 65 , 16 PRACTICAL DAIRY BACTERIOLOGY others at 130°. But the majority of them grow most rapidly at temperatures somewhere between 8o° and ioo°, a fact that explains the rapidity of the souring of milk in hot weather, and the quickness with which any organic material decays during the summer. Above ioo° most bacteria are injured, and if the temperature is raised, they soon stop growing. There are, how- ever, a few species which grow at unexpectedly high tempera- tures; some types are found that grow most rapidly at a tem- perature as high as 1400 and some even higher. These peculiar bacteria are called thermophiles. Such bacteria may be found in milk by proper means, but it is needless to say that they have no important relations in dairy problems, for dairy products are never kept at a temperature as high as these. Death Temperature.-Most active bacteria are killed by a temperature of 1500 kept up for half an hour. This tempera- ture may, therefore, be satisfactorily adopted for pasteurizing. (See page 151.) At this temperature, however, they die slow- ly, while a temperature of 1600 destroys them much more quickly, and 1700 to 1800 more rapidly still. Hence, the higher temperatures are usually adopted for pasteurization. A total destruction of bacteria, including their spores, can be brought about only by temperatures above that of boiling water, and this is usually accomplished in the case of liquids in a closed chamber, in which the steam can be generated under consider- able pressure. If the steam is allowed to collect in such a chamber at a pressure of 115 lbs., the temperature, then, will be about 2400. This temperature kept up for half or three quarters of an hour destroys even the most resisting spores and may be relied upon to produce the perfect sterilizing of any liquid. Laboratories usually have a small apparatus designed for this purpose, called an autoclav (Fig. 76), and this is used constantly for sterilizing of liquids. Relation to Cold.-While heat will destroy all bacteria, cold will not do so. It is practically impossible to destroy the life WHAT ARE BACTERIA? 17 of bacteria by freezing, at least with any certainty, for no mat- ter how low the degree of temperature used, the life of some of these organisms seems to be totally resistant to cold. Experi- ments have shown that bacteria cooled to the temperature of liquid air, or even liquid hydrogen, are not killed, and after being warmed again still retain their life. Although these ex- tremes of temperature do not destroy all bacteria, the simple matter of freezing and thawing will kill a great number of in- dividuals. If water containing a large number of bacteria is frozen and subsequently thawed, the bacteria will be found much reduced in numbers, although they are not by any means all killed. When, therefore, water is contaminated by sewage containing typhoid bacteria, and ice is collected from it for domestic purposes, the typhoid bacteria may still be left alive in the ice. Such ice may still be a source of danger. But it must also be remembered that the freezing destroys a very large proportion of these germs, so that the danger from this use of ice is far less than the use of water from the same source. Relation to Air.-Nearly all living organisms require air, and it was formerly supposed that nothing could live without it. Certain types of bacteria, however, are able to live without air. Indeed, some species, while they grow readily if they have no contact with air, fail to grow at all when the slightest amount of air is present, growing only in the absence of oxygen. This type of bacteria is spoken of as anaerobic. At the other extreme there is a long list of bacteria which can grow only in the presence of air, failing to grow if they do not have oxygen at their command. This is the type of aerobic bacteria. Be- tween the two is an intermediate group capable of growing either in the air or out of contact with it, and this type is spoken of as facultative anaerobic. The most important of all dairy bacteria belongs to this class, the common lactic acid bacterium, Bact. lactis acidi. 18 PRACTICAL DAIRY BACTERIOLOGY Relation to Moisture.-Bacteria will grow only in the pres ence of considerable quantities of moisture; indeed, they de mand more moisture than most organisms. They will hardly grow at all unless there is 30 per cent, of moisture in the ma- terial in which they are living, and even then the growth is slow. On the other hand, they flourish most luxuriantly in localities where the water is from 90 to 100 per cent. Hence, as materials dry, bacteria will cease to grow in them, and any substance that can be dried can be thoroughly protected from their action. This explains why dried fish and dried meat, fruits, dried milk, etc., will keep indefinitely. The drying, however, does not actually kill the bacteria, for although they do not grow when the water is extracted from them, they may remain alive for weeks, months, or even years. In other words, it is impossible to depend upon drying as a means of destroying bacteria, for while many individuals will fail to live, many others do not seem to be injured at all by the drying, and are capable of resuming life again as soon as they find moisture. Relation to Food.-Bacteria are just as dependent upon food as are larger organisms, and they feed upon a greater variety of materials, perhaps, than any other organisms. There are some that appear to be capable of feeding upon what is called inorganic food; that is, upon mineral materials. The vast ma- jority of bacteria feed upon organic material, by which is meant substances that have been produced by other animals or plants. Their relation to food is very much like that of the animal kingdom, and in general they feed upon the same kinds of food that animals do: meats, gluten, milk, eggs, etc. It is of importance to note, however, that whereas they feed with great readiness upon proteid foods, like the lean part of meat, white of eggs, gluten of wheat, legumen of beans, peas, etc., they do not feed, as a rule, upon sugar or starches. Yeasts, on the other hand, are adapted to feed upon sugars. We find, there- WHAT ARE BACTERIA? 19 fore, that of the three great food products upon which animals feed, proteids, carbo-hydrates and fats, the bacteria feed espe- cially upon the proteid foods, and but rarely upon the carbo- hydrates. There is one conception of importance to the dairy- man. Certain types of bacteria act upon milk sugar (a car- bo-hydrate), converting it into lactic acid and causing the milk to sour. There are some kinds that are capable of acting on fats. The food which bacteria consume may be either living or dead when they attack it. In the case of most bacteria the organisms are unable to feed upon the material while it is alive. If bacteria, for example, are placed upon living muscle, they are unable to attack it and soon die; but if they are placed upon the same muscle after it is dead, they feed upon it readily and cause it to putrefy. These organisms which feed upon lifeless bodies include the vast majority of bacteria. There are, how- ever, other species of bacteria that are capable of living upon the bodies of animals and plants while these are still alive. In- asmuch as they can feed upon living organisms they are liable to produce disease and constitute in general the disease bacteria. Bacteria feeding upon living animals and plants are called parasites. Bacteria feeding upon dead animals and plants are called saprophytes. Both saprophytes and parasites are of great importance in dairy problems. In order to understand some of the topics discussed in the following pages it is necessary to recognize the distinction be- tween organised and unorganised ferments. The former term applies to bacteria and yeasts which produce their action by growth. They are actual living bodies which grow and multi- ply. When they produce a fermentation, like the alcoholic or the lactic acid fermentation, they increase in numbers during the process; that is, they act by growth. The unorganized fer- ENZYMES OR CHEMICAL FERMENTS 20 PRACTICAL DAIRY BACTERIOLOGY merits, enzymes or chemical ferments, are not alive. They ap- pear to be chemical substances which produce their fermenta- tions by their simple presence. They do not live, grow or multiply, but they have remarkable powers of bringing about changes in other bodies. While these enzymes are not alive, they are always produced by living bodies and from no other source. For example, the living cells in the glands in the stomach produce one of these enzymes, which we call pepsin, and which has the power of greatly changing the chemical nature of the swallowed food while it is in the stomach. Fre- quently similar enzymes are secreted by bacteria. In the study of bacteria we are, of course, dealing primarily with the living or organized ferments. But in some cases, as we shall see, they produce their action directly through the secreted enzymes, and thus in their study we have to consider both classes of action. It is always important to bear in mind that the organized ferments are ever increasing in numbers, when they have proper food, while the enzymes do not thus increase in amount, except as they are secreted by living cells. If we stop the life of bacteria by an antiseptic, we stop its fermentation, but such antiseptics will not stop the action of an enzyme, since an enzyme acts chemically and not by its life. CHAPTER II TYPES OF BACTERIA FOUND IN MILK In the study of the types of bacteria that may be found in milk, three different factors are to be considered: I. Their action on milk, and its relation to all who deal with this product. 2. Their relation to butter and cheese making. 3. Their rela- tion to the human organism and, therefore, their effect upon the persons using the milk as food. Certain bacteria that are troublesome to the dairyman are perfectly wholesome to the consumer, while, on the other hand, some bacteria that are very injurious and even fatal to the consumer are of no special signif- icance in dairying, so far as their effect upon milk is concerned. We shall also find that organisms which are of great nui- sance to a person dealing in milk are favorable and, indeed, indispensable to butter and cheese making. These problems must, therefore, be considered independently. It will be most convenient to study milk bacteria under the heads of normal and abnormal types. Since milk does not usually contain any bacteria at the time when it is secreted from the milk glands, there are really no such things as normal bacteria. Nevertheless, it is practically impossible to procure milk from any cow, no matter under how cleanly conditions it may be kept or how carefully we work, in such a manner that it will be sterile by the time it reaches the milk pail. We may, therefore, recognize bacteria as inevitable, and the distinction be- tween normal and abnormal bacteria will, therefore, be as follows: Normal bacteria are those that under all ordinary cir- cumstances are practically certain to be found in milk; their presence must always be expected, and it is practically im- possible to avoid them, Abnormal bacteria are those which are 21 22 PRACTICAL DAIRY BACTERIOLOGY less common, occurring in milk under certain conditions but which can generally, by proper means, be prevented from getting into the milk in quantities large enough to produce any effect. NORMAL MILK BACTERIA The Souring of Milk.-The most common change which oc- curs in milk is its sourhig. This has always been recognized and it has been supposed to be a natural change in the milk, the supposition being that milk would sour, just as blood will clot. The studies of bacteriologists have shown, however, that this is a mistake. The souring of milk is not a characteristic of the milk itself, for it will not occur in perfectly pure milk. It is always brought about by the growth of bacteria. Never- theless, it is of almost universal occurrence even in good milk. Indeed, if one obtains a lot of milk which will not sour when kept for a day or longer in a warm temperature, it at once arouses suspicion that some means have been adopted for keep- ing the milk sweet, either by the use of preservatives, or by pasteurization. This is not necessarily the c^se, however, for, under some conditions, pure milk may be obtained which will not undergo the ordinary souring; but the phenomenon is so common that it is to be expected in all samples of normal milk. The souring is the result of the production of lactic acid from milk sugar. Milk contains about 4 per cent, milk sugar and, under the action of certain bacteria that grow in milk, this milk sugar is changed into lactic acid. The change is frequently ex- pressed by the following formula: Milk sugar Lactic acid C.Hl2O« = 2C3H.OS There is no question, however, that this does not represent the change that actually takes place. A molecule of milk sugar contains a certain multiple of the formula above given, but the multiple is not known, and the change into lactic acid is a far more complicated one. than TYPES OF BACTERIA FOUND IN MILK 23 would be indicated by the simple equation above. Its exact nature is not understood even by chemists, and it is sufficient for the dairyman to know that milk sugar is changed to lactic acid. The lactic acid causes the milk, first, to turn sour, as in- dicated by chemical test, or by its taste; and, later, when the acid rises to .7 or .9 per cent., the milk curdles. This phenomenon is also a somewhat complicated one. Milk contains casein in a state of partial solution. As long as the liquid is slightly alkaline, this partial solution is maintained, but the casein will not remain dissolved in an acid solution. As soon as the liquid becomes sufficiently acid, the casein, by a well known chemical law, is precipitated and the milk curdles. Any other acid will produce exactly the same result, as may be proved easily by pouring into milk a little vinegar, or any other acid. The curdling of milk is produced by the appearance of an acid re- action. Curiously enough the three factors, bacteria growth, milk sugar destruction and acid formation, though causally connected, do not run parallel, sometimes one and sometimes another of the three running ahead of the others. In other words, it is impossible to determine the amount of acid from the number of bacteria present.1 The reasons for this, are not well understood. Souring of Milk and Thunder Storms.2-We have just no- ticed that the souring of milk is always the result of bacterial action. There has been a wide-spread belief that thunder storms bring about the souring and curdling of milk, and the general opinion has been that this is due in some way to the direct action of thunder or lightning. It frequently does happen that milk will sour and curdle during a thunder storm, but the con- clusion that there is any real connection between the two is a mistake. The suggestion that electricity has some direct effect upon milk was advanced some years ago and carefully tested • 1 Haacke. Arch. f. Hyg. xliv., p. 16, 1902. "Treadwell, Science, xvii., p. 178, 1894. 24 PRACTICAL DAIRY BACTERIOLOGY but the result demonstrated beyond a doubt that the electric current will not sour milk. It was then suggested that the electricity of the thunder storm produced ozone in the air, and this hastened the souring. This ozone theory, also, was carefully tested and proved to be without foundation, for ozone in itself has no power of affecting milk. Moreover, further experiments proved that, unless bacteria are in the milk, thunder storms have no effect. Milk that is deprived of bacteria may be kept for months during the whole season of thunder storms and never show the slightest trace of souring, showing, of course, that thunder storms have no direct action upon the milk, since they never affect milk from which the bacteria have been re- moved. Practical experience has also shown that if the milk is kept sufficiently cold in an ice chest, or by other means, the thunder storms have no effect upon it. Only milk that is not sufficiently cooled, or that is not cooled at all, is affected by thunder storms. From all these facts it becomes evident that if thunder storms have any effect upon milk it is only indirect. The reason that the souring of milk frequently occurs at the same time with thunder storms seems to be very simple. Bacteria will grow most readily at warm temperatures. Thun- der storms occur at seasons of the year when the tempera- ture is high; and the same atmospheric conditions which bring on thunder storms are likely, and, indeed, certain, to stimulate the growth of bacteria. If the temperature is high, and the milk is not sufficiently cool, it will sour very rapidly, whether any thunder storms are occurring or not. Hence it is not strange that, after a thunder storm, we often find milk which has not been kept cool, soured and curdled, and our natural inference is that the thunder storm has had some connec- tion with it. The whole phenomenon appears to be one of bacteria growth, stimulated by warm temperature. The souring of milk by thunder storms may be absolutely prevented by keeping the milk cool. TYPES OF BACTERIA FOUND IN MILK 25 Varieties of Bacteria Which Sour Milk.-The bacteria which sour milk are known, in general, as lactic acid bacteria. There are many varieties of them, however, differing more or less from each other. A very large number of different acid bac- teria have been separated from milk by bacteriologists, and have been described. Exactly how many of them there are it is im- possible to say. The reason for this impossibility is the fact that up to the present time we do not know what constitutes a type or a species of bacteria. We know that large numbers of lactic bacteria have been found which differ from each other in certain small points. Whether these differences are enough to separate them into different types or species, or whether they are only slight variations which appear in one and the same organism, is not yet known. We do not know, moreover, to what extent these slight differences may come and go under different conditions. For example, the various types of this class of organisms show great variation in the rapidity with which they can curdle milk. Some of them curdle it in a very few hours where the temperature is warm, others require several days for producing the same effect, while still others will not curdle the milk under any circumstances. Whether these should be recognized as different varieties, or simply as modified forms of the same type has been a subject of dispute. It is certain that this power of curdling milk may be increased in the same organisms by proper treatment in the laboratory, and the general opinion, to-day, is that some bacteria which curdle milk and others which sour it without curdling, cannot be regarded as separate species, but only as modifications of the same. Similarly in regard to other slight differences, our bacteriologists are not certain as to how wide variations may be found in the same organism. For this reason it is impossible to state how many species of lactic acid bacteria there are. Al- though this is true, it has become evident in the last few years that the lactic acid bacteria separate themselves into two some- 26 PRACTICAL DAIRY BACTERIOLOGY what distinct groups, more or less sharply separated from each other. The more bacteria are studied, the more clear it be- comes that these groups represent very distinct types, each one of which may have a large number of varieties differing in slight points. We shall, therefore, understand the lactic bac- teria best if we briefly consider the characteristics of these primary types. BACTERIUM LACTIS ACIDI-(Streptococcus lactarius) This organism is the lactic acid bacterium of milk, par ex- cellence, being the type that, in the vast majority of cases, pro- duces the souring and curdling of market milk. It is common, not only in milk from all sections of the United States, but also in all the markets of Europe, and must, without doubt, be looked upon as a common cause of the souring of milk. It is interesting to note that this species was not the first lactic acid bacterium found in milk, and described as the cause of milk souring. One of the other types mentioned below was found earlier, because it produces a large colony on gelatin. But when milk is studied by our more modern methods, this Bact. lactis acidi type is found to be by far the most common.1 It is not to be inferred from this statement, however, that it is al- ways the cause of sour milk, for, while it is the cause in the majority of cases, not infrequently samples of milk are found that have been soured by one of the other types mentioned later. It will be noticed that two names have been given above to this organism. These are not, by any means, the only names that have been applied to it, for it has been found by many bacteriologists, many of whom have given it distinct names, not knowing that they were studying an organism already named. The reason why we have given the above two names 1 Esten. Ann. Rep. Storrs. Exper. Sta., p. 44, 1896. Kozai. Zeit. f. Hyg., xxxviii., p. 386, 1901. Weigmann. Cent. f. Bact. II., v., p. 825, 1899. Gunther and Thierfelder. Arch. f. Hyg. xxv., p. 164, 1895. TYPES OF BACTERIA FOUND IN MILK 27 should, however, be explained. The organism in question ap- pears commonly in the form of a short, non-motile rod, which is not infrequently divided into two, as shown in figure 15, c, giv- ing the form known as the diplococcus. When it appears in this form, the question arises whether it is not more properly called one of the coccus type than a Bacterium. The longer forms, as shown in figures 14 and 15, may simply represent the cocci, elongating and about ready to divide. If this is the case, the organism should not be called a Bacterium, which term is, as We have seen, applied to rods, but rather Streptococcus, which is the name given to spherical forms having one division plane. (See p. 13.) If it is a coccus form it should be called Streptococ- cus; then the name Strepto- cocci lactarius is the cor- rect one to adopt.1 If, however, the organism is regarded as a short rod, then the term Bacterium should be applied, and the name Bact. lactis acidi should be used.2 It may seem strange that bacteriologists cannot agree as to whether this most com- mon milk organism is a Bacterium or a Streptococcus, but the fact is, that there is no sharp criterion by which the microscopist can tell whether he is dealing with an extremely short rod or a spherical form that is slightly elongated just be- fore division. The same culture of bacteria from which figure 14, d, was drawn was submitted to two microscopists, one of whom called it a Coccus and the other a Bacterium. Because of this difficulty in finding any sharp distinction between the two, it has been impossible to state with absolute certainty FIG -varieties of bact. lactis ACidi (conn) 1 Kruse. Cent. f. Bact. II., xxxiv., p. 737, 1903. a Leichmann. Cent. f. Bact., II., pp. 281 and 777, 1896; V., p. 344, 1899. 28 PRACTICAL DAIRY BACTERIOLOGY whether this organism is properly called a Bacterium or a Streptococcus. The term Bacterium, however, has been ap- plied to it for nearly twenty years, and only in the last year or FIG. 15-VARIETIES OF BACT. LACTIS ACIDI (HEINEMANN) two have some suggested that it ought to receive the name of Streptococcus. This recent suggestion has not as yet been very favorably received, and it is still known, and for some time, doubtless will be known, in literature, by the name of Bact. lactis acidi, although the name Streptococcus is taking its place.1 1 Heinemann. Jour, of Infectious Diseases, III., p. 173, 1906. Jour, of In- fectious Diseases, IV., p. 1» TYPES OF BACTERIA FOUND IN MILK 29 It is possible that this type should really be divided into two, one of which is a Streptococcus and the other a Bacterium. This seems to be the conclusion of recent works.1 But these two are in other respects almost alike and in our review here it is not wise to try to make this distinction.2 Isolation of Bacterium Lactic Acidi.-It is an extremely easy matter to obtain this organism from milk, as follows: A sample of milk that has become somewhat sour, but has not yet curdled, should be used for the purpose. This will contain millions of bacteria, and should be very highly diluted for the experiment. A dilution of one to a million is not too high for the purpose. Litmus gelatin plates should be made as de- scribed in experiment n. These are to be kept at about 700 for two to four days when they will be found filled with minute red points, or colonies. These are colonies of lactic acid bacteria. On such a plate will likely be found, however, several kinds of colonies, and perhaps two or three types of acid colonies. But the colonies of the Bact. lactis acidi can, when the plates are properly made, be distinguished from others by the use of a low magnifying power under the microscope. Each of the colonies is found to be quite red, very small, and growing zvholly underneath the surface of the gelatin. Examination with a microscope shows an appearance like figure 16, the most 1 Lbhnis. Cent. f. Bact., II., xviii., p. 97, 1907. 2 Besides the names given above the following names have been applied to lactic acid bacteria that are in essential respects like the one described and should at least be regarded as belonging to the same group: Bact. or Streptococcus Giintheri. Streptococcus acidi lactici. Bact. acidi paralactica. Bact. lactis acidi aromaticum. Bact. lactis acidi maltigeum. Bact. lactis acidi purum. Bact. lactis acidi acerbum. Staphylococcus lactis acidi. These are all by some regarded as identical with the common pathogenic form Strep, pyogenes. Heinemann. Jour. Infec. Dis., IV., p. 87, 1907. 30 PRACTICAL DAIRY BACTERIOLOGY distinctive feature of which is the rather dense red colony, with a series of very slight projections around the edge, so that the colony looks almost burr-like. These projections are very short and not al- ways found; but when they are found, it is practically certain that the colony represents the Bact. lactis acidi. It should also be borne in mind that no bacterium that grows on the surface of the plate is of this type, for this organism grows only under the sur- face. Characteristics of Bact. Lactis Acidi.-This organism is of so much importance to dairymen that it is necessary to study it in more detail, and the following description of the growth of this organism and the various culture media used in the laboratory will serve to identify this type. Its growth on the surface of any culture medium is extremely scanty. No growth can be seen on potato, and none on the surface of gelatin plates. When inoculated on agar slants there is an extremely thin growth, almost invisible, although it may be seen, by very careful examination, as a clear, transparent film. On the other hand, it grows fairly well in the depth of culture media away from contact with air. Inoculated by a needle, into a gelatin tube, it grows well below the surface along the line of inocula- tion (Fig. 17), but it stops at the surface and never spreads over it. When inoculated into ordinary bouillon, usually no visible signs of growth are seen, although the organism does grow slightly. Inoculated into fermentation tubes containing bouillon with milk sugar or dextrose, there is always a scanty growth giving rise to slight turbidity, and growth may extend up the closed arm of the tube, as well as in the open arm, in- dicating that the organism grows without contact with oxygen (Facultative anaerobic'). FIG. l6-COLONIES OF BACT. LACTIS ACIDI IN LITMUS GELATIN TYPES OF BACTERIA FOUND IN MILK 31 The most interesting characteristic is its effect upon milk. When inoculated into sterilized milk, it grows readily and, in a short time, begins to produce acid. The rapidity of the acid production is variable with different cultures. It is so rapid in some cases that, if the specimen be placed at body heat, it will curdle in six hours. With other cultures, the curdling under similar circumstances would not oc- cur for twelve hours, or three days, and with some cultures curdling never occurs. All specimens of milk, how- ever, become acid, although not al- ways sufficiently to precipitate the casein. Between these extremes every conceivable grade may be found in cultures that are, in other respects, identical; and they represent, doubt- less, one type, differing in its power of producing acid. The power of producing acid not only varies in the different samples of this type, but in different cultures from the same original source. It increases if the bacteria are cultivated for a while in milk. If, for example, a culture is taken directly from sweet milk, it will usually curdle the milk slowly; but if daily transferred from one culture of milk to another for a few days, and then taken out and tested, its power of producing the acid is found to have much increased. Since this power thus varies in successive cultures from the same original source, it would seem that the fact that FIG. 17-GELATIN STAB CULTURES a. Bad. ladis aerogenes b. Bad. ladis acidi 32 PRACTICAL DAIRY BACTERIOLOGY some of the cultures found in a sample of milk will sour and curdle it quickly, while others do not, only indicates that we are dealing with varieties of the same bacterium, and not with different species. To this type belong the largest number of bacteria known to cause the common souring of milk. Most of the butter starters (see page 203) are cultures of this class and the same is true of most of the cheese starters. Several different types of lactic acid bacteria have been described by Freudenreich as associated with the ripening of Swiss cheese. These, as he studied them, showed considerable difference in their action on cheese. Some appeared to benefit the cheese, while others did not. They showed other differences also, but most of them belong to this same general type of bacteria. This emphasizes the fact that the Bact. lactis acidi represents a group of varieties, and not a single organism. If the question is asked whether it represents a single species, or a collection of species, the answer is, that no one knows what is meant by the word species among bac- teria, and hence, this question cannot be answered. It cer- tainly represents a collection of forms related to each other, but showing some noticeable variations.1 The type of milk curdling produced by this organism is quite easily recognized. The milk becomes strongly acid, and turns into a hard curd, without any trace of gas bubbles, and without the separation of any whey. (Fig. 18, B.) The milk simply becomes one smooth, solid curd, with a clean, sharp, sour taste, and no odor. This type of curdling has been recog- nized as a desirable one by the dairyman, since it is most favor able for dairy processes, and is consistent with the production of the best grades of butter and cheese. This organism grow readily at temperatures from 6o° to ioo°, growing more rapid ly at higher temperatures. At a temperature of about 700 it 1 Muller. Cent. f. Bact., II., xvii., p. 468. Lohnis. Cent. f. Bact., II., xviii., p. 97, 1907. TYPES OF BACTERIA FOUND IN MILK 33 FIG. l8-EFFECT OF DIFFERENT BACTERIA ON MILK A. Bact. aerogenes. B. Bad. ladis acidi. C. & D. Liquefying bacteria grows with great rapidity, and at this temperature it seems to be more vigorotis than any other bacterium ordinarily found in milk. For this reason, as we shall presently see, milk kept at 700 becomes, in a short time, almost completely filled with this species of bacterium at the expense of all the others that might have been there originally. This type of organism is the dairyman's friend. 34 PRACTICAL DAIRY BACTERIOLOGY The second type of lactic acid bacterium differs in many re- spects from the first.1 It is not so common as the first type, but is, nevertheless, very widely distributed, and is very likely to be present in any sample of milk in some quantity. If it is present in large quantities, it becomes a very great nuisance; if only a few are present, it may do no injury. The organism in question can commonly be distinguished by a study of the BACTERIUM AEROGENES FIG. 19-TYPES OF BACTERIAL GROWTH ON AGAR SLANTS a. Filiform, b. Echinate. c. Beaded. d. Effuse, e. Arborescent gelatin plates described on page 288 by the fact that it produces a strongly acid colony which grows on the surface of the gela- tin as a round, more or less raised mass, spreading sometimes to the size of 2 or 3 mm. Bad. aerogenes, when examined with a microscope, is seen to be an organism somewhat larger than the other lactic type, and one which is, beyond question, a Bacterium, being con- siderably longer than it is broad. Its growth on various laboratory media is very easily distinguished from the first lactic acid type. When inoculated on the surface of agar tubes, or potato, it grows luxuriantly, forming a thick, whitish layer. In gelatin tubes it both grows along the needle inoculation and 1 Dominikiewicz. Milchztg., p. 817, 1903. Baginsky. Zeit. f. Phy. Chern., xiii., p. 434, 1889. TYPES OF BACTERIA FOUND IN MILK 35 spreads over the surface. (Fig. 19, b.) In bouillon it grows luxuriantly, producing a turbidity and sediment. Inoculated into fermentation tubes containing sugar, it grows both in the open and closed arm, and in all cases produces gas which collects at the top of the closed arm. (Fig. 20.) This lat- ter is the most important character of this type. When inoculated into milk, it causes the souring of the milk, which is rapidly followed by curdling, the rapidity of the curdling varying in dif- ferent specimens. The curd which is produced differs very much from that of the first type of lactic acid bacteria. It is always more or less filled with gas bubbles, and when care is taken to ob- tain a typical curd, it appears, as shown in figure 18, A, crowded with holes which represent the bubbles of gas formed by the organism. The whey commonly separates in a short time from the curd, and the final appearance is strikingly different from that of the curdled milk produced by the first type of lactic acid bacterium. There is also a difference in the type of lactic acid produced by the two organisms, the first producing what chemists refer to as right-handed lactic acid, and the second producing chiefly left- handed acid, two terms referring to the effect of the acid upon polarized light.1 This organism plays a great deal of mischief in dairying. FIG. 20-PRODUCTION OF GAS IN FERMENTATION TUBE, PRODUCED BY BACT. LACTIS AEROGENES 1 Utz. Cent. f. Bact. II., xi., p. 600, 1904. Thiele. Zeit. f. Hyg., xlvi., p. 395, 1904. Heinemann. Jour. Biol. Chern., II., p. 603,. 1907. 36 PRACTICAL DAIRY BACTERIOLOGY The production of gas is the cause of the ruin of vast quantities of cheese. If milk contains a considerable quantity of this bacterium, instead of the more common non-gas-producing type, when it is made into cheese, the bacteria grow and de- velop gas, the cheese becomes filled with the bubbles, swelling more and more, until it finally results in what is known as swelled cheese. At the same time that this swelling occurs, the flavor of the cheese becomes unsatisfactory, so that the swelled cheese is practically worthless, at least in a case of extreme swelling. These organisms have been the cause of the loss of enormous amounts of money on the part of cheese makers. In butter making they are not so disastrous, but here, too, their presence is undesirable, for they sometimes produce unpleasant flavors in the creams, resulting in an inferior grade of butter. This type of organism, therefore, is decidedly the dairyman's foe. Varieties of Gas-producing Bacteria.-Bad. aerogenes has been mentioned above as a single type, but there is a long list of bacteria capable of developing acid-producing gas, and grow- ing, generally, much like the one which we have described.1 These have been separated into several groups, with several names. The dairyman cannot expect to separate the different types from each other, and it is not appropriate in this work to at- tempt to describe them. They may all be considered together, therefore, as the type of gas-producing Bad. ladis aerogenes. One special type, however, which in most respects agrees with the one described above, must be specially noticed. This one also produces acid and gas from milk in the same method as the Bad. aerogenes, and its growth on potato, agar and else- where agrees in almost all respects with it. But it is found to be motile, and covered with a number of flagella. It is, there- fore, not a Bacterium, but a Bacillus. The organism in ques- 1 Harrison. Cent. f. Bact. II., xiv., p. 359, 1905. Conn. Ann. Rep. Storrs. Exper. Sta., 1906. TYPES OF BACTERIA FOUND IN MILK 37 tion, though not so common as the others, is yet not infre- quently found. It is of some importance, however, because it is generally recognized as indicating the presence of fecal contamination, and is known to bacteriologists by the name of B. coli communis. This is a type of Bacillus found living in the intestines of animals, and is not found in any abundance in nature, outside of the intestines. When, therefore, any water is found to contain this organism in any quantity, it is a fairly safe indication that the water is contaminated with sewage. If bacteriologists, in their study of drinking water, discover the presence of this organism they conclude that the water is contaminated with sewage and, hence, unsafe to drink; not because B. coli is in itself dangerous, but because it indicates sewage contamination. What about the presence of this organism in milk? In the first place, if the presence of B. coli in milk should result in its being pronounced unfit for drinking, a large pro- portion of the milk in our markets would be under ban, for, while it is not by any means universal in milk, still it is found with very great frequency. Indeed, it is quite evident that one would naturally find it in milk. Anyone who knows the conditions under which milk is produced, even in the best and most cleanly dairies, will easily recognize that there is always a possibility of the milk being contaminated with animal excreta. This excrement is almost sure to contain B. coli; hence, it will follow that the milk will very likely show its presence from such contamination. It may, therefore, be fairly safe to assume that the presence of this bacillus in milk indicates the con- tamination of the milk with animal faeces; but it by no means follows that this should be sufficient to condemn milk for drink- ing purposes. It is true that water contaminated by sewage is unsafe to drink, because it contains excreta from human bodies and is, therefore, quite likely to contain germs of human diseases, chief among them typhoid fever. Anything con- 38 PRACTICAL DAIRY BACTERIOLOGY taminated by the typhoid germ is dangerous. But the cattle which produce milk do not suffer from typhoid fever and, with the exception of tuberculosis, which will be especially mentioned later, no diseases found in the intestinal canals of animals are likely to be distributed to man. Hence, while a small quantity of animal faeces in milk may not be an appetizing matter to think of, it does not necessarily, or even probably, render the milk at all likely to distribute infectious diseases to those drink- ing it. Thus it follows that, although the presence of B. coli may render water unsafe, because it suggests sewage contam- ination, its presence in milk does not render the milk unsafe, but merely indicates that there may have been a certain amount of contamination with animal faeces. This fact is of universal occurrence, as any one knows who has ever visited a cow stall. B. coli is not harmful in itself, and animal faeces are by far less dangerous than human excreta. Clearly the presence of B. coli in milk has a very different meaning from its presence in water. Other Types of Lactic Bacteria.-The two types of bacteria described are by far the most important of the acid bacteria of milk. Each of these, as indicated, represents a type containing many varieties rather than a single species. In addition to these there are other bacteria that produce lactic acid not strict- ly belonging to either of these types. Some do not produce any gas, but grow quite abundantly upon the surface of culture media, sometimes producing small, and at other times quite large, colonies. It is impossible in this work to attempt any description of these forms. Although there are numerous va- rieties of lactic bacteria, in the vast majority of cases milk is soured by one of the two types above mentioned. Whether the one or the other of these types will be found to cause the spontaneous souring in any sample of milk cannot be predicted; for it is partly dependent upon temperature. If the milk has been kept at 700 or lower, it will, in most cases, be soured by TYPES OF BACTERIA FOUND IN MILK 39 Bact. lactis acidi, but if it has been kept at 80 or above, it is more likely to be soured by the Bact. aerogenes type. Milk soured at high temperatures there- fore commonly contains left-handed lactic acid, while that soured at low temperature contains right-handed acid.1 These facts are of special importance in cheese making as will be noted later. PEPTONIZING AND RENNET FORMING BACTERIA At times the dairyman is puzzled by a somewhat unusual phenomenon, for he finds his milk curdling, but not becoming sour, and he speaks of the phenomenon as "sweet curdling." It is apt to occur in the fall or spring, when the food of the cattle is being changed. It is due to a class of bacteria that have the power of secreting certain chemical fer- ments very similar to those produced by the digestive glands of the stom- ach and intestine. Upon almost any gelatin plate made from milk (see page 280) there will be found, in addition to the colonies of lactic acid bacteria, a number of pits in which the gelatin is being dissolved or liquefied. This effect is produced by bacteria, and if a platinum needle be dipped into one of these pits and then FIG. 21-LIQUEFACTION OF GELATIN BY ENZYME- FORMING BACTERIA 1 Ferguson. Die spontane Zersetzung der Milch. Gottingen, 1902. Conn. Ann. Rep. Storrs. Exper. Sta., 1906. 40 PRACTICAL DAIRY BACTERIOLOGY stabbed into a gelatin tube, in a day or two it will be found that the bacteria thus inoculated are liquefying the gelatin, as shown in figure 21. These bacteria do not liquefy agar, and can, therefore, be best studied in gelatin. If these bacteria be inoculated into milk, they will usually curdle it in the course of a day or two; but they do not produce any acid, hence they act differently from the lactic acid bacteria. The curdling is actually brought about by a chemical ferment secreted by the bacteria, which is very similar to rennet secreted by glands in the stomach of mammals, the material used by cheese makers to curdle their milk. Such chemical ferments are called enzymes; and it is an enzyme secreted by this class of bacteria which causes sweet curdling.1 But these bacteria produce a second action, since liquefaction of gelatin is also produced by an enzyme, though not by ren- net. The enzyme in question is quite similar to another di- gestive ferment in the stomach, and also somewhat like pepsin. It has the power of converting casein and other proteids into soluble products, prominent among which is peptone, which is a sort of soluble proteid. Hence, this class of bacteria is fre- quently called the peptonizing bacteria. They are also called liquefiers. Since many of them are putrefactive in their action, they may, in the end, fill the milk with putre- factive products. Thus, this class of bacteria actually secretes two different enzymes, or ferments. The first one curdles milk, much as rennet does, the second softens the casein and converts it into rennet-like bodies. Hence, when they are inoculated into sterilized milk, the milk first curdles, the curd being rather soft and usually not acid. After a few days the curd begins to show 1 Fermi. Cent. f. Bact. x., p. 401, 1891; xii., p. 713, 1892. Conn. Cent. f. Bact. xii., p. 223, 1892. Lubbert. Zeit. f. Hyg. xxii., p. 1, 1896. Duclaux. Le Lait., 1887, TYPES OF BACTERIA FOUND IN MILK 41 signs of softening, because it is being converted into peptones, or other soluble substances. As these are formed, they are dis- solved and the curd disappears. Since this is a process very similar to digestion of milk, in the stomach and intestine, it is customary to say that the curd is digesting. This may continue until the whole curd is dissolved into a watery liquid, or it may stop after it is only partly dissolved. (Fig. 18, C and D.) With some species of this class the digestion occurs without any previous curdling, the rennet action being slower than the digestion. In these cases the milk simply becomes more trans- parent, and analysis shows that it has digested much the same as in the other cases where the curdling first appears. The liquefying bacteria are very numerous and are almost always found in samples of fresh milk. They are of no great importance in the ordinary handling of milk since they are commonly overshadowed by the lactic acid bacteria. Though they sometimes produce a sweet curdling, they rarely develop sufficiently in milk to show signs of the digestion just de- scribed. Occasionally, however, they are of significance in cheese making. During the long ripening of cheese they have a better chance to grow than in milk. Whether they have much influence upon the hard cheeses is not fully known yet, but in the ripening of soft cheeses, they sometimes multiply so extensively as to produce very bad results, and to cause much loss to the cheese makers. While, therefore, they are of little importance to the one who handles milk, the y play a consider- able part in the making of cheese. Some instances have been described where this class of bacteria seems to be particularly abundant and develops instead of the lactic type. The trouble has been attributed to the bacteria in the udder, where they are thought to be especially abundant in the case of incomplete milking.1 1 Burri. Milchztg., p. 70S, 1903. 42 PRACTICAL DAIRY BACTERIOLOGY Bacteria Producing Both an Acid and a Ferment.-The peptonizing bacteria usually fail to produce acid and, indeed, make the milk slightly alkaline. Among the acid forming bac- teria are some which produce a peptonizing enzyme, like those just mentioned, but they produce acid at the same time. This type is thought by Gorini to have an important part to play in the ripening of some cheeses1; but there is no sufficient proof of this at present. One variety of this class has been found to be the cause of a bitterness in some types of cheese, bring- ing great losses upon the dairymen. They make the milk sour and are not, therefore, readily detected in normal milk. They can easily be found by the ordinary litmus gelatin plate methods. NEUTRAL TYPES A large number of milk bacteria apparently play no part in dairy problems, and, therefore, we need not consider them except by a passing word. Upon studying the litmus gelatin plates (page 289), we will find small colonies that are not acid, that do not liquefy the gelatin, but appear simply as whitish or greyish dots, some on the surface and others under the surface of the gelatin. If we will remove and study these by themselves, we will find most of them to be neutral organ- isms. They do not sour or curdle the milk, nor do they pro- duce any changes in it that are noticeable under any ordinary conditions. Neither do they seem to have any pathogenic effect upon persons drinking the milk, although they may be present in considerable quantities. Up to the present time, therefore, we have no reason to regard them of any special significance to dairymen. We need consider them no farther, beyond stating that they are frequently present in considerable numbers. 1 Gorini. Cent. f. Bact. II., viii., p. 137, 1902. TYPES OF BACTERIA FOUND IN MILK 43 The presence of butyric acid, accompanied by a rancid odor, is not uncommon in milk products. It is well known in samples of butter and, once in a while, is found in old samples of cheese. In milk, however, butyric acid fermentation rarely occurs. It BUTYRIC ACID FERMENTATION FIG. 22-BACTERIA OF BUTYRIC ACID (SHATTENFROH AND GRASSBERGER) is sometimes produced by certain types of bacteria (Fig. 22), which are practically always present in milk, although they rarely have an opportunity to develop.1 These organisms are commonly anaerobic, and will not develop in milk kept under ordinary conditions. Moreover, the rapid growth of lactic bacteria in milk, in almost all cases, prevents the butyric acid germs from developing at all. For these reasons, this fermenta- tion does not occur in ordinary milk, and in the common bac- teriological study of milk, butyric bacteria do not appear. They can, however, usually be found by a simple device, since the most common types of these organisms are spore producers and are, therefore, not killed by ordinary boiling temperatures. If milk be boiled for a few moments, and then plates be made from 1 Kedroski. Zeit. f. Hyg. xvi., p. 445, 1894. Schattenfroh and Grassberger. Arch. f. Hyg. xxxvii., p. 54, 1900; xlii., p. 219, 1902; lx., p. 40, 1907. 44 PRACTICAL DAIRY BACTERIOLOGY it, the spores will develop. (See page 299.) If these plates be cultivated under anaerobic conditions, the butyric bacteria will have a chance to develop and will produce colonies in the usual way, which subsequently may be isolated and studied. But the study of anaerobic bacteria is too difficult for the be- ginner. Moreover, these organisms are of so little importance in dairy problems that it is not worth while to consider them further here. The only conditions under which they become of dairy importance is in the samples of old butter that they have turned rancid, and this phenomenon is hardly important enough to deserve here our special attention. ABNORMAL MILK BACTERIA Under this head is included a variety of bacteria which pro- duces unusual fermentation in milk, and occasionally gives rise to important dairy troubles. They are by no means always found, and, by proper care, they may be completely avoided. Some of those listed below are very rare and are placed here for convenience' sake only; others, however, are quite common. Slimy Milk.1-This is one of the commonest of troublesome dairy infections. There are two different types. In some cases the milk is found to be slimy at the time it is drawn from the cow. Such a trouble is due to a diseased condition of the cow's udder, and is generally attributed to garget In such a case it is frequently accompanied by bloody milk from the dis- eased udder. Such milk should always be excluded from the dairy. In fact, no milk that is bloody or slimy when drawn 1 Adametz. Landw. Jahr., p. 18S, 1891. Marshall. Bui. 140, Mich. Exper. Sta., 1897. Ward. Bui. 165, Cornell Exper. Sta., 1899. Konig. Spieckermann and Tillman's Rev. Gen. d'Lait, ii., p. 115, 1902. Gruber. Rev. Gen. d'Lait, ii., p. 97, 1902. Harrison. Rev. Gen. d'Lait, v., p. 73, 1905. Burri. Cent. f. Bact., II., xii., p. 371, 1906. Weigmann. Milch. Zent., i., p. 5, 1905. TYPES OF BACTERIA FOUND IN MILK 45 from the cow should be used for food purposes. Gargetty milk is not caused by bacteria. The other type of slimy milk is due to the growth of bacteria. The sliminess in this case is not apparent when the milk is drawn, or for a number of hours afterward; but by the time the milk is from 12 to 24 hours old, depending upon the temperature, there begins to be evident an un- usual change; instead of souring in the normal way, the milk becomes more or less viscous and, finally, may be extremely slimy, so that it may be drawn out in long threads which, when dipped out with a dipper, will run down in long streams. (Fig. 23.) Such slimy milk is apt to be counted as a total loss, for it is usually regarded as absolutely worthless. But there is no reason to think that the milk is really un- wholesome. Indeed, in Norway the people are fond of such milk and frequently use artificial means to pro- duce it, preferring slimy milk to the ordinary kind. In this country slimy milk is thrown away, or fed only to pigs. Since the sliminess does not appear until the milk is several hours old, no loss ensues when the milk is used immediately. Only milk distributed to consumers or kept for a day or so will be thrown away because of sliminess. FIG. 23-EFFECT OF SLIMY MILK BAC- TERIA UPON MILK (RUSSELL) 46 PRACTICAL DAIRY BACTERIOLOGY The cause of such sliminess is, so far as known, the growth of certain types of bacteria. Several different kinds have been found capable of making milk slimy. The most common one of these seems to be Bad. ladis viscosus (Fig. 24), which was first described in Europe, and has later been found in a number of such infections in America. This organism is widely dis- tributed but, unless present in great numbers at the start, it does not, as a rule, develop in milk, since the growth of lactic bacteria prevents its becoming very abundant. Although sev- FIG. 24-BACT. LACTIS VISCOSUS, THE COMMON CAUSE OF SLIMY MILK (ward) eral other species of bacteria are able to produce slimy milk they are not commonly causes of dairy troubles. Some va- rieties of Bad. ladis acidi and Bad. acrogencs produce slimy milk. A slimy milk infection is sometimes persistent, remaining in dairies for weeks, and even months, and may be distributed from one dairy to another until a large community is more or less infected with the same trouble. One of the common methods by which a large community may become infected with such a trouble is through a central creamery or milk distributing station. The trouble begins in some one dairy, and the milk TYPES OF BACTERIA FOUND IN MILK 47 from this dairy is carried to a creamery or station; from here the cans which brought it are freely distributed to other dairies, and, in time, nearly every dairy in the community will receive these cans which may have been partially washed in the cream- ery, but never thoroughly sterilized. By these means the germs present in the cans are readily distributed and the sliminess begins to appear in one dairy after another. The remedy for such cases is clear. A more thorough washing and sterili- zing of the cans at the central distributing station must be adopted so that no farm will ever receive a can containing any of the undesirable bacteria. In this way the spread of such an infection may be checked. In all cases there must be some original source from which the trouble arises, and this source must be looked for carefully and, when found, eliminated if possible. Studies of such in- fection have shown, at least, three possible sources. I. Some- times the germs have been obtained from the water used in washing the cans or, more likely, from the water in which the cans were cooled. The presence of the slimy bacteria in the cooling water will surely, in time, infect the inside of the cans, when they will bring about the trouble. 2. Sometimes it has been found that the udder of a single cow has been infected with the germ, and this has affected the milk of the whole dairy. 3. The slimy milk germ has also been found in the dust of the food which has been fed to the animals. No other sources, as yet, are known. Hence, the dairyman who finds this trouble appearing in his dairy should look to his water supply, to the food of the cows, and should, for a few days, keep separate the milk from the individual cows to see whether the sliminess of the milk affects the entire herd, or only one or two cows. As soon as this is determined, he will know whether he needs to clean the udders of one or two cows, or to find some more general source of infection. It is impossible here to give directions how to discover and remove the cause 48 PRACTICAL DAIRY BACTERIOLOGY of such infection; for conditions vary with different dairies; but generally, when an intelligent dairyman has discovered the source of the infection, the remedy will suggest itself. BITTER MILK The appearance of a bitter taste in milk is a rare trouble for the milkman, though a common one for the cheese maker. The causes of the bitterness are, at least, three, i. Sometimes it happens that the milk has a bitter taste as soon as it is drawn from the cow. In such a case it must be attributed to some food which the animal has been eating. It is known that if the cattle feed upon lupine or ragweed, a certain amount of bitterness develops in the milk. The remedy is simply to change the food of the cow. 2. Sometimes it happens that milk tastes perfectly sweet when freshly drawn, but, in the course of a few hours-some- times not for a day or two-a bitter taste develops in the milk. Here the taste is due to the growth of bacteria in the milk (5. liguefaciens amari, M. casei amari), which produce certain bitter products.1 This type of dairy trouble is quite rare, and only two or three exam- ples of it have been carefully investigated. (Fig. 25.) But bitter tastes in cheese, due to a similar cause, are common. Two different bacteria have been found to give rise to the bitter tastes. The source of the organisms was traced, in one case, to the udders of certain cows, but in other cases the source was not found. One extensive series of bitter milk in- fection was found in a cheese factory in Canada, and produced a great deal of trouble. In this case, the cause of the bitterness fig. 25 coccus PRODUCING BITTER milk (conn) 1 Conn. Cent. f. Bact., ix., p. 653, 1891. Bleisch. Zeit. f. Hyg., xii., p. 81, 1893. Hugge. Rev. Gen. d'Lait., v., p. 470, 1906. TYPES OF BACTERIA FOUND IN MILK 49 was traced to a yeast which had made its way into the dairy supplying the factory, and which was producing the mischief? (Fig. 26.) This bitter yeast was found on the leaves of trees under which the open cans of milk were allowed to stand. In all such cases the only remedy is, to trace the trouble to its source, so as to be able to prevent a recurrence of the trouble, and then to clean thoroughly the udders of the cows and disinfect all milking utensils-cans, vats, etc. 3. The third type of bitter milk is of less importance to the dairyman. It occurs in milk which has been subjected to heat for the purpose of sterilizing it. Such action destroys most of the bacteria present, but sometimes leaves alive some of the spore-bearing organisms. When such milk is kept for some days, although it will not sour and usually will not curdle, it does undergo other changes, and frequenty becomes bitter. The bitterness may increase in strength with each day, making the milk very unpleasant to the taste. Inasmuch, however, as the use of sterilization is not a common process in dairying, and sterilized milk is rarely kept for any length of time, this type of bitter milk is of very little significance. There have been in- stances where factories that put up a sterile milk have experi- enced considerable loss from the development of bitter or- ganisms in their product. Their only remedy is found in more complete sterilization, by applying higher heat, which will kill all spores. Alcoholic Fermentation.-Although some forms of sugar FIG. 26-TORULA AMARI, PRODUCING BIT- TER MILK AND CHEESE (HARRISON) 1 Harrison. Ontario Agr. Coll., 120, 1902. 50 PRACTICAL DAIRY BACTERIOLOGY readily undergo alcoholic fermentation, this is not true of milk sugar. Fruit juices are quickly acted upon by yeasts, and con- verted into Carbon dioxide and alcohol. Milk sugar, although readily affected by bacteria, is not easily acted upon by yeasts, and does not normally undergo alcoholic fermentation. The reason for this has to do with the chemical nature of the sugar molecules, and does not concern us here. It is very im- portant to remember, however, that when solutions of fruit sugar are left exposed to the air, they ferment, giving rise to alcohol; but when milk sugar is exposed to similar conditions, lactic acid is produced, but never alcohol. It is, however, possible to produce an alcoholic fermentation in milk by adding thereto a little cane sugar and then inoculating it with yeasts. This fact has been learned by manufacturers of condensed milk to their sorrow'. The common brands of condensed milk are preserved by the addition of a large amount of cane sugar. Since bacteria cannot thrive upon cane sugar, it serves as a partial disinfectant for the milk. But although bacteria will not grow in condensed milk, yeasts are capable of growing there, and of fermenting the sugar. It sometimes happens that yeasts get into the milk, and the process of condensing does not kill them. When the milk is subsequently sealed in the cans as condensed milk, the living yeasts are inclosed. They find the sugar a medium favorable to growth, and an alcoholic fer- mentation occurs. Immense losses have at times occurred as the result of such growths. To prevent this loss, the process of condensing and final sealing must be carried on in such a way as to avoid yeast contamination, or the yeasts must be destroyed by heat. The possibility of making milk undergo an alcoholic fermen- tation by the addition of yeasts is made use of in the manu- facture of kummys.1 This beverage was originally prepared by 1 Schipin. Cent. f. Bact., II., vi., p. 775, 1900. TYPES OF BACTERIA FOUND IN MILK 51 the Arabs from mare's milk, which will undergo an alcoholic fermentation; but now an imitation product is widely made and used, prepared from cow's milk. A small quantity of sugar is added to milk, and some common baker's yeast. An alcoholic fermentation soon begins, and the fermented prod- uct is kummys. Various modifications of this gen- eral process are adopted by different makers, for kummys has become a commercial article. Several other alcoholic beverages, in different countries, are made by special methods from cow's milk. The first of these to which attention was given is kefir, a beverage long known in the Caucasian mountains, and produced from or- dinary milk by adding some bodies, called kefir grains. (Fig. 27.) These grains have been in the possession of the people from time immemorial. They are placed in milk, where they soon produce a fermentation. After the fermentation is over, they are removed and used in some more milk, or, per- haps, they are dried for preservation for future use. Kefir grains consist of a mixture of several kinds of bacteria and of yeasts.1 The nature of their action is essentially a double one. The bacteria have, first, a chemical action upon milk sugar, convert- ing it into a type which is easily fermentable by yeast, and the subsequent alcoholic fermentation which occurs, is due to the FIG. 27-KEFIR GRAINS 1 Freudenreich. Land. Sahr. d. Schw., 1896. 52 PRACTICAL DAIRY BACTERIOLOGY action of the yeast upon the modified milk sugar. The beverage that is produced contains alcohol, lactic acid, and certain other flavors, which are produced by the bacteria action on the milk. Other types of fermented milk beverages are found in va- rious countries. One called leben1 is common in Egypt. Mazun 2 is made in abundance in America. The Turkish people have one which they call yoghourt.3 In Sardinia an alcoholic milk beverage is called goiddu* In all of these cases the bever- age is made by ferments which the people prepare by special methods and keep on hand. In many places the ferment is simply a little of the old fermented milk inoculated into the new milk. Most of these forms of alcoholic milk have been studied by bacteriologists and have been found to be based, in general, upon the same principle. They all represent a combined action of bacteria and yeasts. The bacteria, in all cases prob- ably, first change the nature of the milk sugar, so that it is fermentable, and the yeast subsequently changes the sugar into alcohol and carbon dioxide. These beverages are frequently regarded as more easily digestible than ordinary milk, and they certainly have a more pleasant flavor. Perhaps their popularity is due simply to the fact that they contain alcohol. FERMENTATION CHANGING THE COLOR OF MILK The first type of fermentation ever studied in milk was blue milk which, even 60 years ago, was found to be a more or less common dairy trouble in certain sections of Europe. The phenomenon appears as follows: The milk is perfectly normal when drawn, and for several hours afterward, but at about a time when it begins to become sour, blue spots appear in it, which extend through the milk 1 Rist and Khoury. Ann. d. 1'Inst. Past., xvi., p. 65, 1902. - Duggeli. Cent. f. Bact., IL, xv., p. 577, 1905. s Zeit. f. Fleisch u. Milch Hyg., xv., p. 248, 1905. Grisconi. Milch. Zent., II., p. 425, 1906. TYPES OF BACTERIA FOUND IN MILK 53 until the whole finally becomes a deep blue color. The cause of the phenomenon was studied in 1840 and found to be a bac- terium, to which the name Bacillus cyano genes was given.1 This bacterium has been found many times since, and is one of the well known dairy organisms. (Fig. 28.) It is not very common, how- ever, and dairy infections due to blue milk are extremely rare. The organism has been found in Europe and Amer- ica, also, but dairy infection of blue milk has seldom been reported from American dairies. It is very easy, however, to produce blue milk by inoculating with the bacteria in question, but it does not seem to have been of common occurrence enough to have caused much trouble in this country. Red Milk.-A red color in milk is not an uncommon phe- nomenon. Often it is because the milk is bloody, through some wound in the udder. Sometimes cows give red milk after eating madder, or some other plants. In all these cases the trouble shows itself at the time of milking. But, in addition to such causes, red milk is sometimes due to bacteria, although as a dairy phenomenon, this is of very rare occurrence. When it does occur, the milk is generally red only on the surface. Several bacteria are known to make milk red, among which are: B. prodigiosus, Bad. erythrogenes and B. lacto rubifaciens and others. They are fairly common in milk, but rarely do they cause any trouble.3 FIG 28-B. CYANOGENES 1 Fuchs. Mag. f. d. ges. Thierh., 1841. 2 Baginsky. Cent. f. Bact., v., p. 448, 1889. Gruber. Cent. f. Bact., II., viii., p. 457, 1902. Gratz. Milch. Zent., i., p. 9, 1905. Menge. Cent, f, Bact., I., vi., p. 596, 1889, 54 PRACTICAL DAIRY BACTERIOLOGY Other Colors in Milk.-Several other bacteria are capable of producing color in milk. Some turn it yellow, some green, some brown and some black. By choosing proper species of bacteria, fermentations can be produced in milk, showing all the colors of the rainbow. But these phenomena, although found in the laboratory, and of great scientific interest, do not appear in practical dairying. Some of these colors occasionally appear on soft cheeses that are ripening, but they do not occur in milk. For these reasons we shall not here consider them further. MILK HAVING THE TASTE OF TURNIPS Milk is sometimes found to develop a turnipy taste, a fact which, until recently, has been attributed to cows eating turnips, or some similarly tasting food. In some cases this is probably the correct explanation; but a careful study of the phenomenon has recently shown that some of their troubles are due to the growth of bacteria in the milk. If the taste is directly due to the food eaten by the cows, it will appear in the milk when the milk is drawn. But in some cases this turnipy taste does not occur until several hours after the milk has been drawn: consequently it must be due to the growth of micro-organisms.' Bacteria producing this turnipy taste have been separated from milk and, by experiment, have been shown to produce this turnipy taste (B. lactis foctidus). The dairyman, therefore, must remember that this trouble may he due to the food, or there may be a bacteria infection. He can easily determine which it is by noticing whether the taste is present when the milk is drawn, or develops some hours afterward. In the latter case he may get rid of it by enforcing greater cleanliness, even though he still feeds turnips to his cows. 1 Gruber. Hyg. Rund., p. 626, 1903. Ritland. Zeit. f. Fleisch. u. Milch. Hyg., xiv., p. 276, 1904. TYPES OF BACTERIA FOUND IN MILK 55 A somewhat peculiar infection in milk has been discovered in one or two cases in Europe, and has been called soapy milk; but it has not, so far as is known, occurred in America. The milk in question develops a slight soapy taste which is decidedly unpleasant. Two different species of bacteria (Bact. sapolacti- cum and B. lactis saponacei} have been isolated from such milk, and have been proved to produce the trouble. The phenomenon is of no special significance, at least in American dairies.1 SOAPY MILK OIDIUM LACTIS This organism is very widely distributed and plays an im- portant part in certain dairy processes, although it has very little or no relation to milk used as milk. It differs from other bacteria in appearance and method of growth (Fig. 29), and seems to be about half way between a bacterium and a mold. Like molds, it grows generally upon the surface of liquids and solids, and does not, like bacteria, penetrate into their depths. When growing upon the surface it produces long, delicate threads, which radiate from a central point where the growth starts and extends over the surface of the medium for consider- able distances. At first it looks exactly like a mold, but, after growing for about two days, these threads break into a large number of very short segments, which serve as spores; for they will grow and generate into fresh threads, if planted upon favorable places. The organism grows with extreme rapidity, and when planted on the surface of hardened milk, like a soft cheese, it will spread over the surface so rapidly that, in the course of a very few hours, it may cover the whole surface. For this reason, it is found to be one of the obstacles to the 1 Eicholz. Cent. f. Bact., II., ix., p. 631, 1902. Weigmann and Zirn. Cent. f. Bact., xv., p. 463, 1894. Marshall. Bui. 146, Mich. Exper. Sta. 56 PRACTICAL DAIRY BACTERIOLOGY proper handling of soft cheeses, since it may choke out other organisms, whose growth is desired. (See page 250.) Oidium lactis has no appreciable action upon milk, neither souring nor curdling it, nor making any change in its flavor in the short period before the milk would ordinarily be consumed.1 Upon further growth, however, it gives rise to chemical decomposition, the result of which is the development of strong flavors, and FIG. 29-OIDIUM LACTIS, FROM A SURFACE COLONY ON GELATIN these are of significance in certain types of soft cheese and probably also in rancid butter. The organism is extremely abundant, and found practically in all dairies. It does not, however, ordinarily appear in milk or in the gelatin plates made from fresh milk, but it is almost certain to be found in old milk. It is almost always present in old butter, and may be found practically in all soft cheeses during the early process of their ripening. While, therefore, the organism has no important bearing upon milk, it is one of extreme importance in cheese making, as will be noticed later. There are several varieties of it, differing only slightly from each other. 1 Lang and Freudenreich. Land. Jahr. d. Schw., 1893. TYPES OF BACTERIA FOUND IN MILK 57 LIST OF MORE COMMON MILK BACTERIA The following list gives the bacteria that are most likely to be found in milk1 : Species almost sure to be found- Bact. lactis acidi (Strep, lactarius). M. lactis various (S. pyogenes aureus). M. lactis albus (S. pyogenes albus). M. lactis citreus (S. pyogenes citreus). Bact. aerogenes. B. coli communis. Oidium lactis. Species less sure to be found, but very common- B. lactis fiuorescens (B. pyocyaneus). B. prodigiosus. B. subtilis. B. aero genes capsulahis. Yeasts, both white and red. Miscellaneous molds. 1 Bergey. Bui. 125, Pa. Dept. Agri., 1904. For classification of Dairy Bacteria see Conn. Ann. Rep. Storrs Exper. Sta., 1906. Diiggeli. Cent. f. Bact., II., xviii., p. 37, 1907. CHAPTER HI SOURCES OF COMMON MILK BACTERIA Is Milk Sterile When Secreted?-The first question naturally arising is whether there are any bacteria in milk at the time it is secreted from the cow. This question has been subject to long dispute, and although many experiments have been made, there is not even yet absolute agreement. It is certain that if the udder of the cow is diseased, the milk may be contaminated, even when secreted. It has long been known that when a cow has tuberculosis in the udder her milk is practically certain to con- tain tuberculosis bacilli. It is equally sure that inflamed ud- ders infect the milk with bacteria. When the udder is the seat of the inflammation it co nmonly produces pus, and this naturally finds its way into the milk. With the pus there are practically sure to be bacteria that are associated with the inflammatory troubles. These are usually chain forming Streptococci. (Fig. 3, d.) Hence, the presence of large numbers of such bacteria, especially if accompanied by numerous pus cells (see page 182), indicates a diseased condition of the udder. Such are found in animals having mammitis or mastitis. These bacteria, if in considerable numbers, are certainly detrimental to the health of the persons drinking the milk. The practical con- clusion is that no animal suffering from any udder affection should be allowed to furnish milk for public consumption. Whether milk at the moment of secretion from the healthy udder ever contains bacteria is not so easy to settle. Some have thought that bacteria actually come from the blood of the healthy cow, so that milk contains them when secreted. Others have been even more positive that no bacteria come from such a source, if the cow is in health. The general opinion is. however, that, at least under ordinary conditions, if the cow be in health 58 SOURCES OF COMMON MILK BACTERIA 59 and her udder not injured in any way, the milk she secretes is at the moment of secretion free from bacteria. This question is of far less importance than might seem likely, for whatever may be the condition of the milk when secreted, it is never sterile at the time it leaves the udder. Bac- teria are known to be abundant in the udder. These bacteria probably come from without, entering through the teat into the milk ducts, feeding perhaps on the remains of the milk that clings to the passages between the different milkings.1 How far into the ducts these bacteria ex- tend is again a matter of differ- ence of opinion.2 Some have held that they are in all the ducts in the glands; others in- sist that the inner ducts are germ free in normal cows.3 (Fig. 30.) But these great dif- ferences of opinion arc of no great practical importance, for bacteria are certainly present in the milk ducts, so that the milk, even though sterile when secreted, is practically sure to be contaminated by bacteria be- fore it reaches the mouth of the milk duct. It is thus very dif- ficult to draw sterile milk from the cow in spite of the greatest FIG. 30-STRUCTURE OF COW'S UDDER Showing ducts in which bacteria grow 1 Ward. Bui. 178, Cornell Exper. Sta., 1898. 2 Simon. Hyg. Rund., X., p. 71, 1900. Harrison. Jour. Ap. Microscopy, V., pp. 20-29, 1903. 3 Freudenreich. Cent. f. Bact., II., x„ p. 401, 1903; xiii., p. 281, 1904. Uhlmann. Rev. Gen. d'Lait., iii., p. 163, 1904. Russell and Hastings. Ann. Rep. Wis. Exper. Sta., p. 164, 1904. 60 PRACTICAL DAIRY BACTERIOLOGY care. By extreme precautions in special tests small quantities of sterile milk have been thus obtained; but these precautions are not practical in ordinary milking, so that the dairyman must recognize that he cannot by any means of milking obtain sterile milk. Bacteria in the Milk Ducts.-The types of bacteria which come from the milk ducts are varied. They are mostly cocci and usually are not very numerous. Some of them liquefy gelatin and peptonize casein, while others do not; some produce small quantities of lactic acid while growing in milk, but others have no appreciable action on milk. These udder bacteria do not, as a rule, contain any representatives of the common lactic acid types, since neither Bad. acrogenes nor Bact. lactis acidi are common inhabitants of the glands or milk ducts. A very few cases have been found in which the latter organism is present in considerable abundance in the milk ducts. But all experimenters agree that this is an unusual occurrence, and that whereas it does occur in some cases, the general rule is that milk drawn directly from the milk ducts does not con- tain the lactic acid organisms, which in older milk are by far most common. In recent years the presence of Streptococci in milk has been sometimes regarded as rendering the milk sus- picious for reasons already noted. It is very important to notice, therefore, that the freshest milk from the best of cows is prac- tically sure to contain Streptococci coming directly from the udder. The cocci thus found appear to be similar to, if not identical with, some of the widely distributed forms that are commonly associated with inflammatory processes. {Strepto- coccus pyogenes, etc., Staph, aureus and Staph, albus, etc.) Whether they are identical with them it is difficult to say, but at all events their presence in milk cannot be a ground for condemning milk, unless it be to condemn all milk as an article of food; for otherwise nearly all fresh milk would be condemned. As a rule, the Streptococci in milk do not form SOURCES OF COMMON MILK BACTERIA 61 long chains, and it has been suggested that, whereas the pres- ence of these common forms may not render milk suspicious, the presence of the long chains may do so. Upon this matter there is as yet no information. It is quite certain that these bacteria get into the milk ducts from the outside. Some have tried to show that they may come from the tissues of the cow, being passed through the gland when the milk is secreted. But while this may be true in case the cow is suffering from some bacterial disease, such as tuberculosis or inflammation in the udder, it is practically certain that this does not occur in normal animals. If we recog- nize that they come from the exterior, it is natural to look for them in greatest abundance just inside of the openings of the teats. They are, indeed, found in the largest numbers just within the teats, where they enter between the successive milk- ings, and, in lesser numbers, further within the gland. They are in just the position to be largely washed out of the ducts by the first streams of milk drawn. This milk, called the fore-niilk, is quite sure to contain more bacteria than milk from other por- tions of the milking. During the milking the bacteria may be so thoroughly washed out by the milk stream that the last milk may be quite sterile. Only certain kinds of bacteria appear to be able to live thus permanently in the milk ducts. When the experiment is tried of injecting common bacteria into the ducts, it is found that they do not live long, but completely disappear in a few days. Even the most common of dairy bac- teria, Bact. lactis acidi, are only rarely found in the milk ducts.1 Aseptic Milk.-The term aseptic milk has been sometimes given to milk drawn from a cow with exceptional precautions, so as to remove as far as possible bacterial contamination.2 To 1 Burr. Cent. f. Bact., II., p. 236, 1902. 2 Backhaus and Appel. Ber. Landw. Inst. Univ. Konigsberg, p. 73, 1900. Willem and Minne. Rev. Gen. d'Lait., iv., p. 121, 1904. Lux. Cent. f. Bact., II., xi., p. 195, 1903. 62 PRACTICAL DAIRY' BACTERIOLOGY produce such milk the udder, flanks and under side of the cow are carefully washed; the teat washed with water or with a solution of io per cent, borax; sometimes a little of the borax solution is injected into the milk duct. The milker washes his hands and milking utensils, and puts on a clean milking gown. Previously a flask with a narrow neck is plugged with cotton and sterilized in the laboratory. This is taken to the milking room, and the cotton removed only at the moment of the milking. The first quarter of the milk is drawn in a sepa- rate vessel, and then the mouth of the flask is placed close to the teat, and the milk is drawn directly into it, after which it is immediately closed again with the cotton plug. In this way the chance of external contamination is practically elim- inated, and the milk which is obtained contains only the bac- teria from the udder itself. Such aseptic milk contains much fewer bacteria than ordinary milk, but even with these pre- cautions sterile milk is not obtainable. It usually contains, however, only the harmless cocci, which have little effect upon it, and it may be kept for a long time without souring or show- ing other changes due to milk bacteria. Such a method of milk- ing is, of course, impractical. A somewhat close approximation to it is obtained by modern milking machines, which will be referred to on a later page. EXTERNAL CONTAMINATION Most of the bacteria in ordinary milk come not from the udder, but from external sources. These sources are severa'.. and may be considered in succession. The Cow.-One of the chief sources of bacteria is the cow herself. Her skin, even when kept in fairly good condition, is never very clean and will always hold more or less dirt and dust laden with bacteria. The cow in many poorly kept dairies is, as is well known, rarely, if ever, cleaned: her flanks, tail and skin become covered with a coating of manure, until 63 SOURCES OF COMMON MILK BACTERIA FIG. 31-FILTHY CONDITION OF COW'S HINDQUARTERS TOO FREQUENT ON DAIRY FARMS the amount of filth attached thus to the animal is surprisingly great. All of this filth is laden with bacteria in large numbers. (Figs. 31 and 32.) It is impossible for the cow to move with- out more or less of it falling from her body, and during the whole process of the milking, by the movements of the flanks, 64 PRACTICAL DAIRY BACTERIOLOGY FIG. 32-PROPER CONDITION FOR COW'S HINDQUARTERS the rubbing of the skin by the milker, and by the switching of the tail, there is constantly being shed a shower of such particles from her body. This continues during the whole milking; and the milk pails, which are generally widely opened, are calculated to receive as large an amount of it as possible. The filth that thus finds its way into the milk pail consists of almost every conceivable kind of material. Besides excrement there are grass, straws, insect wings, hairs and almost every other kind of small particles. (Fig. 33.) The nature of this dirt and its SOURCES OF COMMON MILK BACTERIA 65 amount depend upon the condition of the cow. There will be less on cows during the pasturing season than during their life in the barn in the winter. It will vary much with different foods. Dry foods, like hay, appear to furnish more bacteria in the manure than moist foods, and the milk sub- sequently receives them. It frequently happens that a certain food fed to the cows injuriously affects the keeping of the milk because the bacteria, which the food furnishes, find their way into the milk from the filth on the cow; for ex- ample: the feeding of turnips. The Air.-In earlier studies of bacteriology, it was thought that the greatest source of bacterial contamination was the air. More careful study of later years has produced con- siderable change in this belief. Fresh air out-of-doors does not contain many bacteria, and if milking could take place in the open, free air, this source of contamination would be almost excluded. In a close barn, however, conditions are very different. The motions of the crowded cattle are dislodging bacteria from their skins, which float for some time in the air. Hay, dirt, cobwebs and other dry dust-producing materials are allowed to ac- cumulate. Dirty straw bedding is another source of air con- tamination. (Fig. 34.) The general manner of feeding the animals is even a larger source of contamination. If dry hay, or other dry food, is thrown down in front of the cattle, a large FIG. 33-NUMEROUS COLONIES OF BACTERIA GROWING FROM COW'S HAIR PLACED *N GELATIN 1 Backhaus. Milchztg., p. 357, 1897. 66 PRACTICAL DAIRY BACTERIOLOGY amount of dust will arise and spread through the air of the stable. Such dust is crowded with bacteria, many of which are alive and ready to grow. The common practice of keeping the cattle in the same room where they are milked is thus very productive of a large source of air bacterial contamination.1 The number of bacteria coming thus from the air varies im- FIG. 34-POORLY-KEPT COW BARN, SURE TO GIVE HIGH BACTERIAL CONTACT TO MILK mensely with the conditions of the stable. It is largest during and immediately after the feeding, especially when cows are fed upon dry food. The longer the time elapses after feeding, and the longer time the bacteria have to settle to the floor, the clearer the air becomes. But in the crowded conditions of the stables and the constant motion of the animals and their attendants, the bacteria never settle entirely out of the air and will always be a source 1 Barthel. Rev. Gen. d'Lait, V., p. 505, 1902. 67 SOURCES OF COMMON MILK BACTERIA of contamination. If milking takes place immediately after feeding, the number of bacteria from this source is very high. These facts are easily demonstrated by simple tests. The air of the dairy and the out-of-door air to which the milk may be exposed are also sources of some contamination, but these are usually slight. One curious instance has been recorded of milk being filled with troublesome organisms from the open cans standing under trees on the leaves of which the organism was growing. The bacteria derived from the air are, as a rule, harmless forms. Bact. lactis acidi is undoubtedly in many cases derived from this source. We do not positively know the orig- inal source of this germ, although Barthel has found it upon the leaves of growing plants, whence it might easily get into hay.1 But we do know that it gets into the milk from the dust and the air of the milking stable, and from the dirt attached to the skin of the cows, from faeces and indeed from almost all objects in the barn. Bact. aerogenes probably also comes from the air. Other bacteria from this source are representatives of the liquefying type and tend to produce liquefaction. Many neutral bacteria are also found. Milk Vessels.-Perhaps the most prolific source of milk con- tamination, so far as numbers are concerned, is found in the bacteria which remain alive in the milk utensils between one milking and the next. Bacteria are not usually killed by short drying. After each milking the milking pails and other utensils are commonly washed, sometimes, indeed, thoroughly washed with hot water and soap; but no such washing is sufficient to destroy the bacteria which may be in the pails. No simple washing will make a milk pail bacteriologically clean. After any ordinary washing there will be left clinging to such utensils many bacteria which are ready to develop as soon as fresh milk is added to furnish them with proper food and proper condi- 1 Barthel. Rev. Gen. d'Lait, V., p. 223, 1906. Burri. Cent. f. Bact., II., x., p. 756, 1903. 68 PRACTICAL DAIRY BACTERIOLOGY tions. Every different vessel or utensil with which the milk comes in contact, on its way from the cow to actual consump- tion, adds its quota of bacteria. Probably the various vessels and utensils supply the largest source of milk bacteria.1 The great difficulty of keeping these clean leaves them almost filled with bacteria. As the milk passes from the cow through one utensil to another it gathers more and more bacteria. For example, in a rather badly kept dairy the following figures were found by Bergy: Number of bacteria in milk from udder 400 Number of bacteria in milk in bucket 800 Number of bacteria after passing strainer .... 60,000 Number of bacteria after passing cooler 34,000 Number of bacteria in tank below cooler 173,000 Number of bacteria after passing bottler 84,000 While these numbers do not regularly increase they do show that there is a growing contamination from the various utensils with which the milk comes in contact. The irregularities in the results are doubtless due to the difficulty of getting average samples, always a great difficulty in such analyses. The Milker.-Of late years it has become more evident that the bacteria from the milker or other persons in the dairy are among the most serious. This is not so much because of the number of the bacteria that may enter the milk from this source but of their types. In ordinary dairies the milker rarely makes any special toilet before milking, and is liable to perform this task in old, soiled clothes, with no special attempt at cleaning his hands or face. Under these circumstances, while he is not so great a source of bacteria as the cow, some of these organ- isms are sure to fall from his hands or clothing into the milk vessels. The number of bacteria from such a source is, probably, 1 Erf. and Meleck. Bui. No. 131, Kan. Exper. Sta. 1905. SOURCES OF COMMON MILK BACTERIA 69 not great and does not add materially to the bacterial content. But in one respect these bacteria assume a more important significance. The bacteria which produce diseases in one an- imal do not necessarily produce diseases in other animals. Those which produce diseases in cattle, with some exceptions (tuberculosis), do not usually have the same effect on man. But it is evident that any disease germs that may be present in one man are just the kind that can develop in any other human being. Therefore, the bacteria contamination from human sources is more dangerous to other human beings than any infection from animals.. For this reason the bacteria which enter the milk from the milker are liable to be much more dangerous than those which come from any other source. This we shall consider in the next chapter. SOURCES OF COMMON MILK BACTERIA While at present no complete statement can be made as to the sources of all the milk bacteria, a few facts concerning some of the more common are of value. The common Streptococci and other coccus forms appear to come mostly from the udder directly. In some cases this may indicate a diseased condition of the udder, but even from the healthiest cow the milk will be sure to show these types at the outset. Bad. ladis acidi is derived, in most cases, not from the udder but from the dust of the air, or the hair of the cows, and pos- sibly in some cases from the faeces. But beyond doubt the largest source of these germs in the ordinary dairy is the milk vessels. This species grows abundantly in milk, and will always be left in large numbers in the milk vessels, even after thorough wash- ing. Hence, milk vessels are its chief source. Its original source has not been so easy to trace. It is sometimes found in the feed of the cattle, and Barthel has found it upon the 70 PRACTICAL DAIRY BACTERIOLOGY leaves of plants, where he thinks it multiplies naturally. Whether this may be regarded as its natural home cannot yet be stated. It is suggestive to note, however, that when found in fresh milk it does not appear to be in a vigorous condition, and grows much less rapidly than it will after it has been developing in milk for a number of hours. This suggests that whatever be its immediate source in the dairy it does not come from a location especially well adapted to its life. Bact. acrogenes and other gas-producing organisms are also from sources external to the cow. They practically never come from the cow's udder, but chiefly from the dirt of the cow or from the faeces, and they may, also, be derived from the air or other external filth contamination. Sometimes they, too, will be found in incompletely sterilized milk vessels. The practical conclusion to be drawn is that the dairyman's method of avoid- ing the troubles arising from gas-producing organisms is the extra care taken to prevent external contamination of the milk, or extra thoroughness in washing vessels. B. coli communis.-This organism, so far as we know, comes wholly from faecal contamination. It is quite common in milk, as would be expected from the well-known fact that it is a constant inhabitant of the intestine, and that almost all milk is contaminated with manure. Its presence in milk would prove a very serious contamination, if the faecal contamination were from human beings, but inasmuch as it comes from cows the contamination is less serious than it would be from a human source. The other types of bacteria found in milk need not especially detain us here. The udder itself furnishes merely harmless germs, at least in cases where the udder is free from all dis- ease. The kinds of bacteria which produce trouble in milk, slimy milk, bitter milk, etc., come from such a miscellaneous variety of places that no definite sources can be named. In cases of trouble each must be traced to its own source, which SOURCES OF COMMON MILK BACTERIA 71 is sometimes the udder, sometimes the food, sometimes the faeces, sometimes the water, sometimes infected cans from a creamery or cheese factory, etc. The dairyman must simply use his own ingenuity in detecting the source of the trouble in each individual case. SIGNIFICANCE OF DIRT IN MILK From the facts above outlined it is evident that the dirt which gets into milk is a very important source of organisms which are likely to produce trouble, since they may injure the milk or butter, and may produce even more trouble in cheese- making. The presence of dirt means infection with bacteria and, in many cases, with types of bacteria that are emphatically mischievous in the dairy; it always means filthy dairy condi- tions. The recognition of this fact has led to attempts to estimate the amount of dirt present in milk, as one of the means of determining its quality. Milk which contains a large amount of dirt must be far inferior in quality to a clean milk, since it will contain more bacteria. Clean milk will keep better, and it will be less liable to contain disease germs. Several methods of estimating the amount of dirt in milk have been devised, none of which are very accurate or very satisfactory. These are described in experiment 46. The methods there given will give the dry weight of the solid, insoluble particles of dirt which get into the milk, and which may be filtered out. But they do not give any estimation of such particles as are too small to separate out by sedimenting or centrifugalizing, and they give no indication of the portion of the dirt that may have been dissolved in the milk. The ab- solute amount of dirt found is of no special significance, but if numerous estimations are made by the same individual on a series of milk samples, the relative amounts will indicate the relative cleanliness of the different samples. Estimates made 72 PRACTICAL DAIRY BACTERIOLOGY by different observers do not agree very closely because of slight differences in technique. Figure 35 shows the amount of dirt collected under the udder of a cow during milking, both with, b, and without, a, previous washing of the udder. The amount is certainly sufficient to emphasize the importance of this source of bacterial contamination. The determination of the amount of dirt in milk certainly gives a gauge as to the care exercised in its production. But whether such dirt determination can be of any value in the FIG. 35-RELATIVE AMOUNT OF DIRT FALLING FROM THE UDDER DURING MILKING, UNDER DIFFERENT CONDITIONS A. Udder muddy. B. Udder slightly soiled. One-half natural size. (Fraser) municipal testing of milk is somewhat questionable. It is not yet possible to give any standard as to the amount of dirt that may be allowed in passable milk, and there seems to be no reason for believing that the dirt determination will be adopted as one of the methods of municipal examination of market milk. If milk is allowed to stand for several hours in a glass jar until the dirt settles at the bottom, an examination with the naked eye will give the relative amount of dirt, and the results will probably be as valuable as the longer laboratory methods of determining the quantity, especially when it is remembered that the amount of dirt thus obtained varies quite widely with dif- ferent experimenters and different methods. Special dirt filters SOURCES OF COMMON MILK BACTERIA 73 have been devised by which the amount of dirt can be quickly determined. These are intended to be used by milk inspectors in their ordinary routine examination of market milk. They have not as yet come into common use. A brief summary of the facts of this chapter may be useful. The bacteria that get into the milk from the udder are mostly cocci. Most of these are harmless, and they will always be found. Some appear to be identical with forms that are asso- ciated with various forms of inflammation, and if these be abundant they may make the milk unwholesome. None of them have any noticeable effect on the milk. An unusual number of Streptococci is usually accompanied with pus cells, and injures the milk. The bacteria that get into the milk from other sources are those common in air, soil and water, most of which are harmless. Among these are two types of lactic acid bacteria, various forms of putrefactive organisms and large numbers of miscellaneous species. From these sources, too, icome the bacteria that occasionally produce trouble, such as those causing slimy milk, blue milk, tainted milk, sweet curdling, etc. Some of these produce great losses in cheese-making by causing un- desirable flavors. The putrefactive bacteria are always unde- sirable but fairly common. From the milker or dairyman may come certain dangerous disease germs. Nearly all dairymen, to-day, have come to recognize the de- sirability of keeping the number of bacteria as small as possible in milk, and are looking for methods by which this can be done. In order to consider intelligently methods by which a dairy can accomplish this it will be necessary to learn certain important facts concerning bacteria growth. The practical application of the facts just given will therefore be reserved for a later chapter. CHAPTER IV THE GROWTH OF BACTERIA IN MILK The original bacterial contamination of milk accounts for the bacteria that may be found in fresh milk, but gives no idea as to what may be found at any subsequent time. The rapid growth of those which get in and the great differences in rapid- ity of growth of different species are such that the bacterial content of any sample is undergoing constant changes. To un- derstand dairy problems we must study the growth as well as the source of milk bacteria. Bacteria in Normal Milk.-The number of bacteria in fresh milk varies widely. It may be as low as too per c.c. in some of the best dairies, or it may be as high as half a million, or even higher, in the poorer type of dairy. Absolutely no average can be given that has any meaning, for, as will be pointed out in a later chapter, the number will depend upon the methods used in the dairy. The kinds of bacteria present in different samples of fresh milk will also vary widely. If, however, we take the milk of an average dairy kept in fairly good condition, we should find the bacteria in fresh milk somewhat as follows: There will be very few of the Bact. lactis acidi type. These are frequently so few that they cannot be detected by the com- mon methods of analysis. There may be one or two per cent., or sometimes more, but they are always in small numbers, rarely as high as io per cent. Their number is largely dependent upon the cleanness of the milk vessels. Gas-producing acid organisms are also liable to be present; but these, too, are few in numbers in good milk, and their presence in any considerable numbers is unfortunate, because they are liable later to produce 74 THE GROWTH OF BACTERIA IN MILK 75 erouble in the milk. The largest part of the bacteria will be cocci, some of which liquefy gelatin and belong to the pepton- izing group, and some of which do not. These organisms come directly from the udder and can practically never be avoided. Their presence in milk is normal, even in the best dairy con- ditions, and there is no reason for regarding them as injuring the milk, for most of them have no action upon it and are per- fectly harmless to man. In addition to these there will be a varying number of miscellaneous bacteria, liquefiers and non- liquefiers. Their number and types will depend upon the extent of the contamination of milk with manure, dust and other sources of bacteria. These miscellaneous bacteria are the ones most likely to produce trouble. EFFECT OF TEMPERATURE ON NUMBERS The number of bacteria in milk at any subsequent period is more largely dependent upon the temperature and age than upon the original contamination.1 Milk when fresh from the dairy, even from the slovenly, filthy dairy, will rarely contain more than half a million or so bacteria per c.c., and usually a much smaller number. By the time the milk is one or two days old the numbers may amount to 50,000,000, to 100,000,000, or even more. Milk from an ordinary cleanly, carefully kept dairy may have no more than from 10.000 to 50,000 per c.c. at the outset. But this milk, also, in the course of one or two days may have numbers as high as 100.000,000. The number of bacteria present at any time is, therefore, more dependent upon the age of the milk and the method of keeping it than upon the original amount of contamination. Milk which originally contained large numbers of bacteria, if kept cold, will in 24 hours show a much lower bacteria count than milk which originally had small numbers but was kept under warmer conditions. This 1 Conn. Bui. 26, Storrs. Exper. Sta., 1903. 76 PRACTICAL DAIRY BACTERIOLOGY should always be considered in drawing any conclusion as to the numbers of bacteria in milk. In ordinary municipal testing of milk this feature is too frequently lost sight of, or, at least, not properly considered. A single illustration will suffice to show the effect of the dif- ferent temperatures upon the numbers of bacteria. A sample of milk was examined for bacteria when fresh, and was found to contain 6,525 per c.c. It was then divided into two lots, one preserved at 500 and the other at 700. Fresh milk 6,525 per c.c. 25 hours old at 500 6,425 " " 25 hours old at 700 6,275,000 " " GROWTH OF BACTERIA AT 50° AND 70° The difference in this case is very striking. In these two samples of the same milk one showed no more bacteria at the end of 24 hours than at the beginning, while the other had increased 1,000 fold. It is not common to find quite such a marked difference, but in all cases the difference in the number of bacteria in two samples thus tested is very great. It is im- portant to note that these two samples of the same milk would receive very different verdicts from any municipal examina- tion. One would be passed as an exceptionally good milk, and the other condemned as a very bad milk. But if the bacteria in the fresh milk were the common harmless species, liable to be found in milk, the sample which at the end of 24 hours con- tained 6,000,000 would be just as wholesome as the one that contained 6,500. In other words, the ordinary examination of milk, which condemns milk containing more than a certain number, fails to reach the desired end. It does not distinguish between good milk, in which the harmless lactic bacteria have become quite abundant, because of a few extra degrees of tem- perature, and unwholesome samples of milk, in which the num- THE GROWTH OF BACTERIA IN MILK 77 bers of bacteria are due to the fact that the milk contains a large amount of filth, with its special and injurious types of FIG. 36-GROWTH OF BACTERIA AT DIFFERENT TEMPERATURES DURING 24 HOURS Each dot representing a single individual a. At 50° . 7 bacteria b. At 70° . . . 700 bacteria bacteria. The bearing of this upon the question of municipal examination will be considered later. The rapidity of growth of common bacteria at different tem- peratures is graphically shown by the accompanying figure 36. THE GROWTH OF DIFFERENT SPECIES When drawn from the cow milk is at a temperature that especially favors the development of most ordinary species of bacteria. While different species differ somewhat as to the temperature at which they grow best, a majority of them can multiply most rapidly at about body heat, between 900 and 1000. Some species, however, find a lower temperature especially favorable. Hence the kinds as well as the numbers of bacteria in milk are largely affected by the temperature.1 At 33°.-If milk is frozen, bacteria do not grow at all. If it is only a degree or two above freezing, they grow very slowly, 1 Conn and Esten. Ann. Rep. Storrs. Exper. Sta., 1901. Conn and Esten. Rev. Gen. d'Lait., i., p. 121, 1901. 78 PRACTICAL DAIRY BACTERIOLOGY so slowly indeed that for several days there is practically no increase in numbers. But even if milk is kept only one or two degrees above freezing, the bacteria will increase, and will in the course of time-in two or three weeks-be found to be extremely abundant. It is important to note that the bacteria which are found at these low temperatures do not produce the ordinary changes in milk. The milk does not sour or curdle and, as a rule, there is no visible sign in such milk that the bacteria have become abundant. Such milk may have a some- what oldish taste, but, as it is neither sour nor curdled, would usually be pronounced sweet and would naturally be used with perfect freedom. The milk, however, may contain bacteria in great numbers, and may become harmful because of them. At 40°.-If the milk is warmed to about 400, nearly the same statement can be made, excepting that the bacteria tend to grow a trifle faster and will become abundant somewhat earlier. It cannot be emphasized too strongly that milk or cream re- tained at these low temperatures, while it will remain sweet for many days, and even for some weeks, cannot after the first few days be looked upon as wholesome, since the bacteria have been growing, even though slowly, and may be present in enormous numbers. It may be possible that some cases of milk poisoning, especially such as come from ice cream, may be attributed to this cause. (See page 109.) The emphatic lesson to be drawn is that no normal milk that has been kept for many days is safe, even though it still remains sweet. At 50°.-If the temperature is raised to 500, the bacteria develop more rapidly, although very much more slowly than at higher temperatures. At this temperature the species which grow are commonly not lactic bacteria, but a large variety of miscellaneous forms. This is a matter of some importance, since while lactic bacteria are harmless and in some respects useful, the miscellaneous types which develop at 500, though many of them are harmless, are more likely to contain species THE GROWTH OF BACTERIA IN MILK 79 which give ptomaine poisoning and possibly summer complaint and cholera infantum. At 60°.-If the milk is warmed to from 6o° to 70°, the growth of bacteria is very much more rapid. It is extremely important to notice the type of bacteria specially favored by this temperature is the normal, harmless, useful lactic acid bacteria and Bact. lactis acidi. If milk is kept constantly at 6o°, the lactic acid bacteria multiply far more rapidly than any other species. Examinations of milk at intervals of an hour or two show a constantly increasing proportion of this type. At the outset the number of lactic acid bacteria is comparatively small, frequently less than 1 per cent, and seldom as high as 10 per cent.; but they constantly increase hour after hour, and when the milk is 24 hours old, the lactic acid bacteria are apt to com- prise 50 per cent, of the whole. In another day, just about the time the milk is ready to sour, the proportion of this type is commonly over 95 per cent, and sometimes too per cent. In other words, if milk is preserved in a temperature from 6o° to 700, the lactic acid bacteria far outrun other species and soon take their place. The acid which they develop seems to be injurious to the other species, and these rapidly disappear as the lactic organisms increase in numbers. As a rule, therefore, milk which sours at a temperature of 70 0 or thereabouts will be found to contain nearly a pure culture of the common lactic acid type, the other organisms that were so abundant at the outset having disappeared. For this reason we understand why lactic acid bacteria are regarded as a dairy bacteria par excellence. Although this species is not present in fresh milk in great numbers and con- stitutes only a small proportion of the whole, yet by the time the milk is 24 hours old, at ordinary temperatures, they have so outnumbered the others that they constitute the larger pro- portion of the bacteria present. 80 PRACTICAL DAIRY BACTERIOLOGY At 80° to 100°.-If the milk is kept at temperatures from 8oG to ioo°, the results are far more variable. The bacteria grow very much more rapidly, but we cannot tell so surely what species will develop in greatest abundance so as to become pre- dominant. In some cases here also the Bact. lactis acidi assumes predominance and sours the milk; but in a great many other cases, apparently the majority, some of the other types assume the upper hand in milk. It frequently happens that at these higher temperatures the gas-producing bacteria, Bact. aerogenes, be- come especially abundant, and by the time the milk has soured it will contain large proportions of these organisms that are so mischievous in various dairy products. For this reason it is extremely important that milk to be used for butter-making, and especially for cheese-making, should be kept at temperatures below 8o°, and after the curd- ling by rennet should be cooled to below 8o°. The presence of the gas-producing organisms is sure to produce mischief, whereas the proper growth of the other lactic bacteria is a necessary accompaniment to a good butter or cheese. From the facts here outlined it is evident that dairymen should endeavor to get milk quickly to a temperature below 700 in order to stimulate the development of the proper lactic acid bacteria. It should be remembered, however, that if the starter of the proper or- ganisms be added to milk or cream so that this organism is present in special abundance, a higher temperature of 8o° may frequently be safely used to hasten the souring of the milk. Under these circumstances, since the desirable organisms are added in great abundance, they succeed in producing their action on milk in spite of gas-producing organisms or other bacteria that may be present in smaller quantities. If, however, ordinary milk or cream, without a starter, be ripened at a tem- perature of 8o°, or thereabouts, the results will be very un- certain, and in some cases gas production will give rise to trouble and serious loss. 81 THE GROWTH OF BACTERIA IN MILK PHASES OF BACTERIA GROWTH IN MILK Germicidal Phase.-For some time after milk has been drawn from the cow bacteria fail to develop; indeed, many tests have shown that for the first few hours there is an actual reduction in the numbers of bacteria.1 The exact reason for this reduc- tion has been the subject of some discussion, but the different ways of explaining it need not occupy our attention. The general feeling has been that there is present in milk some sub- stance which is inimical to some kinds of bacteria and actually destroys them. For this reason this period has been called the germicidal phase. It lasts for a varying time, depending largely upon temperature. It commonly continues only a few hours, sometimes not more than two. At other times it may last for several hours, especially if the milk is kept cold. (See experi- ment on page 297.) The practical value of this fact is simply that for a period of a few hours after milk is drawn the bacteria do not develop, and the handler of the milk has, thus, a short period in which he need not fear much bacteria growth, and during which he can bring the milk into conditions for further preservation. First Period of Growth.-Following the germicidal phase the bacteria grow with a rapidity depending, as we have seen, on temperature.2 For a number of hours there is an increase of most species. Most of the kinds of bacteria present in the milk find it a favorable medium, and multiply with varying degrees of rapidity according to the nature of the species. But some multiply more rapidly than others, so that the proportion of the different types will constantly change as the hours pass. For a period of 12 hours, at ordinary temperatures, there is 1 Hunziker. Bui. 197, Cornell Agri. Exper. Sta., 1901. Koning. Milch. Zent., I., p. 99, 1905. Mayer. Rev. Gen. d'Lait, IL, p. 381, 1903. 2 Koning. Rev. Gen. d'Lait, 1904. Gissier and Gasching. Ann. d. 1'Inst. Past., xvii., p. 540, 1903. 82 PRACTICAL DAIRY BACTERIOLOGY a fairly regular increase of nearly all species, although the common lactic bacterium grows more rapidly than any other. The length of time of this period of increase depends upon the temperature of the milk. If kept cold, there may be a day, or even two days, during which time there will be shown a general increase in the numbers of nearly all kinds of bacteria. Second Phase.-After the numbers of bacteria have become fairly large, 10,000,000 to 20,000,000 per c.c., it is found that the Bact. lactis acidi has gained the upper hand of the others.1 This species, present in small numbers at the outset, has in- creased in proportion until at the close of the first period there may be from 20 to 40 per cent, of them. During the second period, which now follows, they rapidly increase until they finally come to be nearly 100 per cent. This type of organism has, in other words, almost or completely replaced the numerous species found at the outset. This second phase ends with the souring and the curdling of the milk. Third Phase.-After souring and curdling, the bacteria in the milk cease growing, the amount of acidity being so high that they cannot thrive. Many of the bacteria die, and there is a fairly rapid reduction in numbers until, after a few days, they are once more very few. The milk may now remain for a long time in this condition without further bacteria growth. Later, however, there are sure to develop some conditions which re- duce the acidity. This third phase, however, does not affect the milk handler, but chiefly the cheese producer; for it is only in milk products that have been kept many days, or several weeks, that the later changes occur. These later changes are variable, but, in general, they consist of a growth of molds over the surface of the milk or cheese, which reduces the acidity to a neutral, or slightly alkaline, condition. After the acid has disappeared, bacteria will again begin to grow. The bacteria which develop in the third phase are not lactic germs, 1 Bienstock. Arch. f. Hyg., xxxix., p. 390, 1901. THE GROWTH OF BACTERIA IN MILK 83 but belong more commonly to the type which liquefies gelatin, digests casien and produces general putrefaction and decomposi- tion. In the end, unless it dries, they cause the putrefac- tion and decay of the product, a change that would have oc- curred in the milk earlier if it had not been for the protecting action of the lactic bacteria. These further changes, however, need not be considered at this point. The phases above described occur normally in milk that is preserved at a temperature not above 700. If the temperature is higher, the changes will be quite different. The different stages will not only follow each other more rapidly, but the souring of the milk will be produced by the growth of the dif- ferent kinds of bacteria, and the resulting material will be of a totally different nature. It may be full of gas bubbles, it may have vile odors, but, in most cases, it will be a product which dairymen will recognize at once as abnormal and un- desirable. While the phases above outlined may be taken as illustrating the general changes in milk, they can only be looked upon as representing an average. Different samples will differ very much. Sometimes the milk will contain bacteria more vigorous than the lactic acid type, and these will gain the upper hand, as, for example, when the slimy bacteria are present. More- over, the various bacteria in a sample of milk have influence upon each other. Sometimes one species will hasten the growth of another species, as shown by Marshall,1 and sometimes one will delay the growth of others. The phenomena are so widely varied with different samples of milk that any description will be only approximate. The course as given above represents the usual series of events when milk contains the normal bac- teria and is kept at about 6o° to 700. 1 Marshall. Cent. f. Bact., II., p. 400, 1905. Bouska. Rev. Gen. d'Lait, III., p. 1, 1903. 84 PRACTICAL DAIRY BACTERIOLOGY PROTECTIVE ACTION OF THE LACTIC ACID BACTERIA The role which the lactic bacteria play in milk is one of con- siderable importance, since they protect the milk from putre- faction.1 Most proteid materials readily putrefy from the action of bacteria. Why does not milk undergo the same change? The other bacteria which are liable to be found here, especially the liquefying type, if left to develop unhindered, would produce a series of decomposition changes in the milk, as they do in meat or eggs, resulting in a variety of bad tastes and odors, ending in putrefaction, and in some cases would produce poisons, which would be extremely injurious. But ordinarily milk will not putrefy. The rapid development of the lactic acid bacteria prevents the growth of these miscellaneous organ- isms. As a result, although it contains a great quantity of easily putrefiable proteid, it is prevented from undergoing this putre- faction by the development of the lactic acid bacteria. One practical side of this question relates to the pasteurizing of milk. As we shall notice later, the ordinary pasteurizing of milk destroys a large number and sometimes all of the lactic bacteria, but leaves in an active condition many kinds of bac- teria, especially those producing spores. It frequently happens, therefore, that in pasteurized milk there are no lactic acid bac- teria present whose growth can subsequently prevent the milk from putrefaction by means of the other bacteria. Pasteurized milk will ordinarily not sour and seems to keep good for a con- siderable time; but it inevitably undergoes certain types of de- composition, and the products which appear after the bacteria have begun to grow are very much worse than those in sour milk. Pasteurizing, therefore, deprives the milk of the pro- tecting action of acid bacteria, so that after the other bacteria have a chance to grow, as they are sure to do in a few days, 1 Bouska. Land. Jahr, der Schweiz, 1903. THE GROWTH OF BACTERIA IN MILK 85 the milk becomes very vile and unfit for use. These facts are of much more importance in cheese-making than anywhere else. When the milk is used for drinking it is almost sure to be consumed before this protective action becomes noticeable; but cheese, the casein of milk, must be kept many weeks for ripening. To a less extent the facts are true of the ripening of cream in butter-making. We thus see the reason why lactic bacteria are necessary in butter and cheese-making, and why starters have become so widely used. It has even been seriously suggested that milk to be used for drinking should be inoculated with lactic bacteria to insure their presence and aid in the exclusion of other species. Here, too, we find an explanation of the fact that sour milk and butter milk are frequently recom- mended for drinking, these products being practically sure to contain large numbers of the lactic acid bacteria. From all of these facts it will be evident that the question of number in the bacterial content of any sample of milk is a very complicated one. The original contamination is only one factor and a small one. The age of the milk, the temperature at which it is kept, the relative abundance of the different species at the outset, the extent to which the different species are favored by the temperature, the influence of one species upon another,-all these, and other factors as well, combine to determine the bacterial content of any sample of milk. It is manifestly impossible to make any general statements as to what numbers or kinds of bacteria should be found in any sample of milk. CHAPTER V DISEASE GERMS IN MILK There is a wide-spread feeling that the bacteria in milk are closely associated with the distribution of diseases.1 This idea is undoubtedly well founded, but in the minds of most people it is too indefinite to have much meaning, and its very indef- initeness gives rise to erroneous impressions. The simple fact that milk contains bacteria does not render it dangerous to drink, nor that it contains large numbers of bacteria. It has been known for many years that sour milk is a perfectly health- ful product, and physicians, to-day. frequently recommend sour •milk or buttermilk as a diet for invalids or infants.2 Now, for reasons that we have already noticed, it is perfectly clear that buttermilk and sour milk will contain bacteria in enormous numbers. Buttermilk may frequently contain as many as 500,000,000 bacteria per c.c., and sour milk will contain them, sometimes, in equal quantity. The fact that these beverages, with such enormous numbers of bacteria, are perfectly healthful and wholesome, is a clear indication that the simple presence of bacteria in milk is not the fact which renders it suspicious, or in any degree less wholesome.3 Indeed, buttermilk is frequently recommended as a diet because it con- tains such large numbers of bacteria. It clearly follows that the impression which many people have that large numbers of bacteria render milk dangerous is a mistaken one. Any attempt to determine the healthfulness or the harmfulness of milk based upon the number of bacteria which it contains is, therefore, fallacious. 1 Busey and Kober. Rep. Health Off. of Dist. Col., 1895. 3 De Mattos. Jahrb. f. Kinderheilk., LV., p. 1 Salge. Jahrb. f. Kinderheilk., LV., p. 157. 3 Bienstock. Ann. 1'Inst, Past., XIV., p. 750, 1900. 86 DISEASE GERMS IN MILK 87 If the simple presence of bacteria in great numbers does not make milk dangerous, it must be that certain kinds of bacteria are harmful, while others are harmless. To understand the relation of milk bacteria to health, therefore, it is necessary to examine more closely the question of the kinds of disease germs liable to be in milk.1 The accumulated information of the last 20 years has shown us that four specific diseases are commonly thus distributed, together with a fifth type of dis- ease which is less definite. The specific diseases are tuber- culosis, typhoid fever, scarlet fever and diphtheria, and the less definite type includes the diarrheal troubles known commonly by the name of cholera infantum, summer complaint, infantile diarrhea, etc. These are all characterized by diarrheal dis- turbances, but with such varying symptoms that they are not recognized by physicians as sharply defined diseases, and they are moreover produced by more than one kind of a germ. DISEASES DIRECT FROM THE COW Diseases that come difectly from the cow are clearly more difficult to avoid than those which come from external sources. Fortunately, however, the diseases that attack cows do not usually attack man, and, with one exception, are not positively known to be distributed by milk except in the rarest of in- stances. The one disease thus distributed is tuberculosis. TUBERCULOSIS Extent of Tuberculosis.-The very great publicity that has been given in recent years to problems associated with tuber- culosis in cattle renders it unnecessary here to do more than summarize the chief conclusions. It is, to-day, thoroughly recognized that this disease is one of the most serious menaces 1 Klein. Jour, of Hyg., i., p. 78, 1901. Ernst. The Infectiousness of Milk, Boston, 1895. Lee. Ann. Pub. Health Assn., 1898. Wilckens. Zeit. f. Hyg., xxvii., p. 264, 1898. 88 PRACTICAL DAIRY BACTERIOLOGY to the dairy industry. It is known to be present and increasing in our dairy herds.1 It is known that in the more thickly settled parts of this country, as well as in Europe, the percentage of the disease among cattle is quite large, although it is difficult to give exact percentages. In Denmark and Germany 40 to 50 per cent, of the cattle are affected. In cold climates where the cattle are kept housed much of the time it is more common than in warm countries where they remain out of doors. In America it does not yet appear to be as common as in Europe; but still in the dairy districts of this country it is also very com- mon. It is apt to run through a whole herd after it once gets a foot-hold. Sometimes practically every animal in a herd will have the disease, while in another herd none will be found. With such wide differences averages mean little. The disease is frequently introduced into a herd by the purchase of stock that appears to be healthy but is really infected with the dis- ease. Hence, highly bred stock is most likely to be found infected. Statistics have also shown that the' disease has been rapidly increasing among cattle in the last 25 years. Such statistics, it is true, are open to some criticism. 25 or even 15 years ago the importance of tuberculosis in cattle was not recognized, and the method of detecting the presence of the disease was uncertain; careful records as to the percentage of tuberculosis among cattle were then scanty and unreliable. It is only about 15 years ago that any very strenuous attempt was made to determine the percentage of tuberculosis among cattle. As years have passed the importance of the matter has drawn more attention to it, and statistics have been collected with more and more thoroughness and completeness. Such in- 1 Salmon. 20th Ann. Rep. Bu. An. Ind., p. 89, 1903. Salmon. Bui. 53, Bu. An. Ind., 1904. Salmon. Bui. 38, Bu. An. Ind., 1906. Salmon. Bui. 33, Bu. An. Ind., 1901. DISEASE GERMS IN MILK 89 creasing completeness of inspection and examination would inevitably seem to show that the disease is on the increase. For this reason, and for others that we need not mention, the statistics are open to criti- cism. In spite of the crit- icism, however, no one questions the general belief that this disease has been on the increase among cat- tle, though how rapidly we do not know. It is now agreed by all that tubercu- losis is, perhaps, the most serious problem that the dairyman has to face and one that threatens to un- dermine the whole in- dustry.1 Tubercle Bacilli in Milk.-So far as concerns our problems, however, it is the relation of this tuberculosis to milk and the distribution of the disease from animals to man that interests us. The most important facts may be summarized, as follows: Animals suffering from tuberculosis may, under some con- ditions, produce milk that contains tubercle bacilli. This has been proved by studying the milk from such animals by two methods: I. The inoculation of such milk into susceptible animals, like guinea-pigs, results, in many cases, in the guinea- pigs rapidly acquiring tuberculosis.2 2. A microscopic study of such milk has demonstrated the presence of tuberculosis bacilli. (Fig. 37.) Although the microscopic examination of milk may FIG. 37-TUBERCULOSIS BACILLI (LEHMANN & NEUMANN) 1Markl. Milchztg., p. 232, 1901. Beck. Zeit. f. Fleisch u. Milch Hyg., xi., p. 245, 1901. 2 Mohler. Bui. 44, Bu. An. Ind., 1903. Schroeder and Cotton. Bui. 86, Bu. An. Ind., 1906. Schroeder and Cotton. Bui. 88, Bu. An. Ind., 1906. 90 PRACTICAL DAIRY BACTERIOLOGY detect the presence of tuberculosis, the method is too unreliable to be of practical value as a means of testing milk. Nowhere, as yet, has the examination of milk for the detection of the presence of this organism been undertaken as a method of municipal routine, and whether this will ever be done is doubt- ful. Such study, however, demonstrates beyond peradventure that the milk of tuberculosis animals may contain tuberculosis germs. It is then clearly important to know at what stage of the disease the milk may become thus affected, and upon this sub- ject there has been a large amount of experimenting with more or less contradictory results. If the disease is located in the udder, the milk is practically sure to be infected with tuber- culosis bacilli in greater or les's numbers. Perhaps 8 per cent, of the tuberculous cattle have the disease in the udder (Pear- son), although some place the number lower. If the disease occurs elsewhere in the body, but not in the udder, the results are less certain. It has be?n shown, however, that some animals that react to the tuberculin test without showing signs of the disease in the udder produce milk containing tuberculosis bacilli. Such reacting animals clearly have the disease, but it is apparently located elsewhere in the body than in the udder; their milk may produce tuberculosis when inoculated into guinea-pigs, indicating that under some conditions animals that have no visible signs of udder disease may produce infected milk.1 It is not of so much importance to settle the matter absolutely as it seems at first, because many animals are doubt- less infected with the disease in the udder without showing it by any visible signs. Moreover, an animal affected with the disease may at any moment have the disease attack the udder, 1 Rabinowitsch. Zeit. f. Hyg., xxxvii., p. 439, 1901. Mohler. Bui. 44, Bu. An. Ind., 1903. Obermuller. Hyg. Rund., p. 712, 1897. Pettersson. Cent. f. Bact., xxxii., p. 274, 1903. Beck. Hyg. Rund., p. 490, 1901. DISEASE GERMS IN MILK 91 after which for considerable time she may produce infected milk without there being any visible indication that the udder has been affected. The only logical conclusion to be drawn is that any animal suffering from the disease is a source of danger, inasmuch as we cannot tell when she may begin to produce infected milk. At the same time, we must recognize that only a small proportion of the animals actually suffering from tuberculosis produce infected milk, although we have no means of knowing how large the proportion is. It is a matter of more practical importance to learn whether market milk is actually infected. A microscopic study of market milk and market butter has demonstrated beyond peradventure that the milk sold to the public is more or less frequently infected with the tuberculosis bacillus from animals. Here, again, per- centages are quite impossible, because of the difficulty of the tests and the lack of a sufficient number of experiments. One very important phase of the relation of this disease to milk distribution should be emphasized and clearly understood. The tuberculosis bacillus does not multiply in milk. This germ will not grow at all, unless the temperature is kept close to body heat, and it does not develop outside of the human body by any known natural media. The germs which pass from the cow into the milk will remain alive therein for a considerable length of time and are, perhaps, capable of doing injury; but they do not increase in numbers. Hence, it follows that the general mixing of milk before it reaches market is quite sure to distribute the germs somewhat widely in the different por- tions of the milk, and this dilution greatly decreases the number in any particular sample of milk.1 Inasmuch as the germs do not grow, the more the infected milk is diluted with uninfected milk the more the germs will be reduced in numbers in each sample. A second factor of no less importance is that if these germs 1 Gebhardt. Virch. Arch., p. 119, 1890. 92 PRACTICAL DAIRY BACTERIOLOGY are capable of producing tuberculosis in man-a question to be noticed presently-they will do so only when present in con- siderable numbers. The human body has certain powers of resistance against them, and the danger in swallow'ing a few is comparatively small, whereas, if large numbers are taken into the body the danger is greater. Hence, milk that con- tains only a very small number of these germs, scattered somewhat indefinitely, is practically harmless, and would prob- ably never produce tuberculosis; whereas milk that contains them in larger numbers, so that a person who takes a drink of it would swallow considerable numbers of the germs, is more likely to be a source of danger. The Use of Milk in Tuberculous Cattle.-The possibility of the distribution of tuberculosis by means of milk has a number of very practical bearings upon dairying. Experiment has shown that such milk is liable to produce tuberculosis when fed to susceptible animals. The animals that most commonly acquire the disease from milk are calves and pigs. The milk of tuber- culous cows is, in many cases, the source of tuberculosis among calves feeding upon the milk. Whether calves from tuberculous mothers can be reared without danger of the disease is one that not unfrequently arises as a practical question. It has been demonstrated that, excepting in the very rarest of instances, this disease is not hereditary, and that calves when born, even though their mother is diseased, are free from it. Under these circumstances, if they can be kept later from infection, they may be raised with as good chances for health as any other calves. In order to do this, however, they must not drink their mother's milk, but must be removed from tuberculous mothers and fed only upon the milk of healthy animals, or upon milk that has been pasteurized. If this is done, there is no need of sacrificing calves of mothers that are suffering from tuberculosis. There is little doubt, however, that by means of the milk of such animals this disease has become common in our DISEASE GERMS IN MILK 93 herds and is becoming more and more wide-spread. Cream- eries, skimming stations and cheese factories constitute potent agents in such distribution. These central stations receive milk from a wide territory and, having passed the milk through the separator, return to the farmers the skim milk from the creameries, or the whey from cheese factories. The routine methods in running such stations never allow a farmer to receive back his own skim milk, but he receives the equiv- alent amount that chances to be ready for distribution at the time he is ready to take it. The result is that the milk of any tuberculous cattle in the district will in the course of time be distributed through the whole territory. This will be followed by the presence of tuberculosis among new herds, especially of calves, and also by the development of this disease among swine that are fed upon such products. These central stations are without doubt one of the chief agents in distributing tuber- culosis over the country. There is one remedy for this, and one only. The milk from such creameries should be pasteurized before it is returned to the farmer. In Denmark this is required by law, and the farmers never take back to their farm milk that has not been subjected to a heat supposedly sufficient to destroy tuberculosis bacilli. This procedure has rarely been adopted in this country, but until it is adopted no dairyman can be safe from the danger of tuberculosis among his calves and swine, so long as they are fed upon skim milk returned from the creamery or cheese factory. Relation to Mankind.-A phase of this subject of even more general interest is the relation of the milk from tuberculous cattle to man, and the very important question whether tuber- culosis among mankind is not attributable to the use of milk from tuberculous cattle. Over this question there has been in the last io years much dispute, many differences of opinion and a vast deal of experimenting. Out of the growing mass of facts that has been accumulating during this period have 94 PRACTICAL DAIRY BACTERIOLOGY gradually been emerging a few salient conclusions, which are now practically demonstrated: I. The bacterium which produces this disease in cattle and in man is essentially the same. After much experimenting it has been agreed that there are slight differences between bovine and human tuberculosis bacillus.1 These differences are in minor characteristics, which can be recognized by the bacte- riologists. One of the primary differences is that in experi- menting with animals the bovine bacillus proves to be, as a rule, of a more violent type than the human bacillus.2 In spite of the slight differences, however, it has been proved that the two types of organisms are so closely related that the germs from either man or cattle may produce the disease in the other. Many instances have been shown where the human bacillus is capable of producing tuberculosis among cattle, although, as a rule, of rather a mild type. The experiments of a reverse nature, by which the bovine bacillus is inoculated into man, are more difficult. A sufficient number of observations along the line of accidental inoculation have shown that in some cases tuberculosis has been produced in man by the bacillus that comes from cattle.3 The general conclusion, therefore, to-day, is that while there are differences in germs from the two sources, either may be a source of danger to mankind. 2. A second question over which a difference of opinion has arisen is whether pulmonary tuberculosis in man is likely to be due to the germs in milk, which are taken into the stomach. The stomach would not naturally be looked on as an entrance to the lungs. But it has been shown that the mode of entrance for pulmonary tuberculosis is not necessarily, as has been fre- quently assumed, through the respiratory organs, but that at least in many cases, germs entering the body through the 1 Behring, Romer and Ruppel. Beit. z. Exper. Thierapis, Marburg, 1902. De Schweinitz. 17th Ann. Rep. Bu. An. Ind., 1900. 2 Muller-Erfurt. Zeit. f. Fleisch u. Milch Hyg., xii., p. 12, 1902. 3 De Schweinitz and Schroeder. Bui. 52, Part II., Bu. An. Ind., 1905. DISEASE GERMS IN MILK 95 intestines find their way to the lungs and produce infection there? Hence, the fact that milk is swallowed and not taken into the lungs is no argument that it may not be a source of pulmonary consumption in mankind. Indeed, in most recent times it has been claimed that most cases of pulmonary tuber- culosis come from infection through the intestines rather than through the lungs.2 3. From these facts it has been finally agreed that the milk of tuberculous cattle is undoubtedly a source of danger to those drinking such milk. When the question of the extent of the danger is raised, however, there is still a wide difference of opinion. In the light of the facts in our possession to-day we cannot doubt that the danger exists, but whether it is a slight or serious danger we do not positively know? On the one hand, we find it claimed that a large part of the human tuberculosis, including pulmonary as well as intestinal, is attributable to the bacteria of the milk supply. Behring thinks that the larger part of pulmonary consumption is contracted in childhood from drinking milk, but that it may remain dormant for many years showing itself later in life? If this is the case, milk is the great source from which this scourge is contracted. On the other hand, it is held by Koch and others that this source of danger is a comparatively slight one. The great source of tuberculosis is, they hold, in breathing the germs directly into the lungs. Hence, it is the sputum of human patients rather than the milk of tuberculous cows that is the source of greatest danger. 1 Calmette and Guerin. Ann. d. 1'Inst. Past., XX., p. 354, 1906. Ipsen. Berl. Klin. Wochensch., p. 791, 1906. 2 Arloing. Cent. f. Bact., I., xxxi., p. 255, 1902. Zwick. Zeit. f. Fleisch. u. Milch. Hyg., xvii., p. 69, 1906. Tonzig. Arch. f. Hyg., xli., p. 46, 1901. Schroeder and Cotton. Bui. 95, Bu. An. In., 1906. 3 Speck. Zeit. f. Hyg., xlviii., p. 27, 1905. 4 Sobotta. Zeit. f. Fleisch u. Milch Hyg., xvi., p. 88, 1904. Behring. Berl. Klin. Wochensch., pp. 233, 337, 689, 1903. Behring. Tuberkuloseentstehung, Tuberkulosebekampfung und Sauglings- nahrung, Berlin, 1904. 96 PRACTICAL DAIRY BACTERIOLOGY This school recognizes that for young children, who live largely upon milk and whose resisting powers are slight, milk is a serious danger, but it does not look upon it as of much danger to adults. Between these two somewhat radically different views it is too early to decide definitely. It will be recognized that the adoption of one or the other attitude will make a great difference in the methods of fighting this disease. If Behring's idea is correct, tuberculosis among mankind is to be fought by getting rid of tuberculous cows, and to do this he has devised a method of preventive inoculation, which he thinks is efficient in protecting cattle from the disease. If the other view is cor- rect, the disease must be fought essentially as it is to-day, by protecting mankind from infection from other human patients, and chiefly from the sputum. One or two facts bearing upon this question it is necessary to mention. Tuberculosis is common among nations drinking cow's milk, but it is also common among some nations that do not use cow's milk as a food. Japan, Greenland and Turkey, for example, do not use cow's milk but suffer from this dis- ease.1 It is also important to note that although tuberculosis among cattle has been on the increase in the last 25 years, tuberculosis among men has been upon an equally constant decline. This decline in the disease among men has probably been rightly attributed to improved hygienic conditions. It is certainly not due to decreased chance of contagion from tuber- culous milk. It is interesting to note that the decrease in the disease has not affected the intestinal tuberculosis among chil- dren, a form which, more than any other, would naturally be attributed to milk. In the light of all these facts it must be admitted that the milk of tuberculous cows is a source of danger, although we may not yet agree as to the extent of the danger. However great or however small the danger may be the desirability of 1 Heyman. Zeit. f. Hyg., xlviii., p. 45, 1905. DISEASE GERMS IN MILK 97 guarding against it is evident enough. The milk of our markets certainly contains tubercle bacilli. In Berlin 30 per cent, of the market milk contains them, and it is not likely that our markets are in much better condition. Every dairyman should recognize the force of the question how he may best protect not only his own herds from this menace, but the health of his patrons from the menace of the disease through the milk of his cattle. The first safeguard to be taken is to use every means to free his herd from the disease. The methods of doing this are given in other places and cannot be dwelt upon here.1 The only way that a dairyman can be sure that no dangerous tuber- culous milk is furnished to his patrons is by having all his herds properly tested with tuberculin, and so treated as to be sure that none of them are suffering from the disease. The fact that such large numbers of animals suffer from tuberculosis has made it hitherto practically impossible to bring about an elimination of all tuberculous animals from the milk- producing herd of the public. The expense of destroying all tuberculous animals has been quite prohibitive. Under these circumstances milk derived from tuberculous animals has been and still is being used beyond doubt. Such milk can be rendered perfectly safe, however, by the process of pasteurization, which will be referred to later. It should, perhaps, be stated here that no bacteriological nor chemical analysis and no system of dairy inspection yet devised can thoroughly guard the public milk supply so far as concerns the distribution of tuberculosis. Until a law requires the universal testing of all cattle and the immediate slaughter of all reacting animals any public dairy inspection will be futile in this respect. 1 Harding, Smith and Moore. Bui. 277, N. Y. Exper. Sta., Geneva, 1906. 98 PRACTICAL DAIRY BACTERIOLOGY OTHER DISEASES DERIVED FROM THE COW There are no other diseases to which milch cows are subject that we know positively are liable to infect the milk to any considerable importance. Animals with foot or mouth disease or splenic fever will give infected milk, and man might take the disease, but these diseases are sudden and violent, and are, moreover, at least in this country, quite rare. Lockjaw and hydrophobia might sometimes be transmitted through milk. But the violence of the diseases and the ex- tremity of the sickness will hardly ever make it possible that the milk of such animals should reach the public for consump- tion. At all events we have at the present time no suspicion that the public is in any source of danger from the milk from such animals, and this subject may be passed with the simple caution that no animal suffering from any violent disease should be allowed to furnish milk for public consumption. After the milk is drawn from the cow there are, at least, three kinds of disease germs that may and sometimes do find their way into it and become a source of danger to the con- sumer. Typhoid Fever.-The first and most important of these is the typhoid fever germ.1 (Figs. 38 and 39.) Typhoid fever epi- demics attributable to milk are fairly common and, within a comparatively few years, at least fifty have been thus traced. The general character of such an epidemic is sharp and violent but short. There will usually appear within a few days num- erous cases of fever; but after a week or two further new cases become less common and soon disappear. Such a brief DISEASES DERIVED FROM EXTERNAL SOURCES 1 Freeman. Med. Record of N. Y. Mar., 1896. Fulton. Jour, of Hyg., i., p. 422, 1901. Rembold. Cent. f. Bact. I., xxxiii., p. 204, 1903. DISEASE GERMS IN MILK 99 epidemic gives every indication of being confined to a single instance of infection. They differ notably from typhoid fever epidemics due to the water supply in the fact that the latter are much more lasting, continuing for weeks and months. No milk epidemic has, as yet, been known to last long, and all seem due to one brief period of infection. The explanation of this is natural. The chance of infection of milk with typhoid fever bacteria is always a remote one, and it is never likely to continue for any considerable length of time. In other words, FIG. 38-TYPHOID BACILLI Ordinary culture Flagella not shown (Lehmann & Neumann) FIG. 39-TYPHOID BACILLI Showing flagella (Lehmann & Neumann) the infection is confined to one or two days only, after which the milk from this same source usually becomes normal again. A single day's infection will produce the short, sharp type of epidemic above referred to. We must next ask for the source by which milk becomes in- fected with typhoid fever germs. It must first be recognized that only one source of typhoid fever germs is known, viz: the intestinal contents of a typhoid fever patient. They pass out of the body in the faeces or from the mouth and are distributed by various means. The methods by which they get into the milk are, of course, not always the same, and they may be grouped under three heads: 100 PRACTICAL DAIRY BACTERIOLOGY i. Direct contact with a patient suffering from this disease Sometimes patients carry these germs for months and even years after recovery from the disease. Such "chronic bacilli carriers" are a constant menace when employed in the dairy.1 Typhoid fever, as is well known, does not always confine a patient to his bed, and sometimes it is of a mild enough character to allow him to continue for days, or even weeks, about his ordi- nary occupations. Such cases are called "walking typhoid." He frequently may not know that he has typhoid fever; but during all this period he is discharging typhoid germs from his body and is liable to infect anything he touches. If such a per- son is employed in a dairy and touches the milk cans or handles the various utensils, the chance of the germs from his hands or clothing entering the milk is very great. Some of the typhoid fever epidemics have been traced to such causes. 2. The patients who are sick enough to be confined in bed may, in an indirect way, be the cause of an infection of the milk. Discharges from such a patient always contain typhoid fever bacteria in abundance. These discharges may be care- lessly handled by persons who are employed in the dairy, or such persons may soil their own hands with the soiled clothing or bedding from typhoid patients. Subsequently they may milk the cows or handle milk cans or other utensils. Here is evi- dently an easy source of infection. The only safeguard against this chance of trouble is the absolute prohibition of anyone who has anything to do in the dairy having any connection with the sick room of a person suffering from intestinal fevers. Some health inspectors now forbid the sale of milk from any farm or dairy where there is a case of typhoid or other in- fectious disease. 3. Infected water is in some cases a source of trouble. The discharges of typhoid fever patients thrown upon the ground, or otherwise improperly disposed of, may percolate through 1 Lentz. Hyg. Rund., xvii., p. 377, 1907. DISEASE GERMS IN MILK 101 the soil with the drainage, or may run over the surface with rain-water and eventually find their way into wells, brooks or rivers. Sewage frequently drains directly into rivers. The water thus becomes infected with the typhoid bacilli, which may remain alive for some time; subsequently such water may infect the milk in several ways. If the milkman has the dishonest practice of watering his milk, the chance of contagion is evi- dent. But it is also a common practice for the dairyman to cool milk by submerging the milk cans in cold water nearly sufficient to cover them. Under these circumstances, the cans become covered with water containing typhoid fever germs, and there will occasionally be an opportunity for some of them to pass into the milk pails, so that the milk will thus be in- fected.1 It is even possible for such contaminated water to get into the milk cans in the course of washing the cans. If milk cans are washed in water that has not been boiled, or are rinsed in cool water, or if they are washed with cloths that have been rinsed in cold water, there is always a possibility of the introduction of typhoid germs into milk. One very severe typhoid epidemic at Stamford was caused by rinsing cans in cold water after washing them in hot water.2 Lastly, cows wading in such polluted water may get the typhoid germs on their udders and teats, from which entrance to the milk is easy. One typhoid epidemic8 of 43 cases was traced to the use of whey from a cheese factory. It may seem that the chance of infection from these sources is remote, and that under no conditions could many bacteria find their way into the milk. These are undoubtedly correct inferences, but in dealing with typhoid fever we have one source of danger that does not occur in the distribution of tuberculosis. Typhoid bacteria multiply in the milk. As a result, if from any one of these sources a very small number, perhaps even 1 Sedgwick and Chapin. Bost. Med. and Sur. Jour., p. 129, 1893. 2 Smith. Rep. of the Stamford Typhoid Epidemic, New Haven, 1895. 3 Dabney. Phila. Med. News, 63, 1893. 102 PRACTICAL DAIRY BACTERIOLOGY half-a-dozen, of these microscopic germs make their way into the milk, they find the milk such a favorable condition for growth that they multiply rapidly and, in the course of a few hours, become indefinitely numerous. By the time this milk has reached the consumer they may have become so abundant as to constitute a very considerable source of danger. It is beyond doubt this multiplication of bacteria in the milk that explains the violence of the milk typhoid fever epidemics. Wherever the germs are present at all they are likely to .be abundant from this growth, and under these circumstances a large proportion of those drinking the milk are sure to be infected. In this re- spect the conditions are very dififerent from the distribution of the typhoid by water. In water the multiplication of the germs is slight, and they rarely become very abundant. As a rule, water epidemics of typhoid, though wide-spread, affect only a small portion of those drinking the water, while a much larger proportion of those drinking milk are thus infected. It is, therefore, of the utmost importance for the dairyman to re- member that typhoid must be absolutely excluded, for the en- trance of a very few germs is a source of danger; a condition quite different from that of tuberculosis, where the presence of a small number of germs in the milk is not a source of danger to anyone. The milk has much less chance of becoming in- fected with typhoid bacteria than with tuberculosis germs, but when it does occur the danger to the consumer is very much greater. Since typhoid fever epidemics due to milk are frequent, health officials are sometimes called upon to trace their origin. The steps in such a problem are simple. In tracing the source of a typhoid epidemic the first step is to determine the limits of the disease, and if possible to find whether the cases occur irr such a way as to exclude the water supply as a source of contamination. If all of the cases occur rapidly, new cases failing to appear after two or three weeks, a single temporary DISEASE GERMS IN MILK 103 source of infection must be looked for. This will commonly exclude the water supply, since if a water supply is infected the trouble is apt to last a long time and will never be confined to one single period of infection. The next step will be to de- termine whether all persons suffering from the disease have had any one point of contact, as having attended a banquet, for example, at any particular time. If so, the disease may be traced to something used during the banquet. If no such com- mon point is found, the next point of suspicion would be the milk supply, and the ques- tion would be raised wheth- er all of the persons suffer- ing from the disease had obtained milk from a com- mon source. If this is found to be the case, the matter is practically demon- strated, and it is only nec- essary to trace the milk to its origin, and find the methods by which it is dis- tributed. There is usually little difficulty in detecting the source of contamina- tion, either from a typhoid fever patient in the dairyman's family or employ, or from an infected well or brook, from which water has come into contact with the milk cans. Diphtheria.-A few epidemics of this disease have been at- tributed beyond doubt to milk.1 In these cases the infection occurs after the milk has been drawn from the cow, although FIG. 40-DIPHTHERIA BACILLI (LEHMANN & NEUMANN) 1 Abbott. Jour. Path, and Bact., II., p. 35, 1894. Dean and Todd. Jour, of Hyg., II., 1902. Klein. Cent. f. Bact., vii., p. 489, 1890. Mackenzie. Med. News, p. 180, 1893. Marshall. Jour, of Hyg., vii., p. 32, 1907. 104 PRACTICAL DAIRY BACTERIOLOGY some have believed that the cow herself may suffer from the disease and affect the milk through the udder. No such in- stances are positively known, but it is known that secondary infection is possible. It is well known that convalescents from this disease carry active germs in their throats for some time after they appear perfectly recovered. (Fig. 40.) Such persons would naturally be allowed to return to work in the dairy, and the chances of infection in such instances are very great. Al- though such instances are not common they have been known; and the milk supply must be looked upon as one of the sources of the distribution of this disease.1 Scarlet Fever.-Scarlet fever has almost exactly the same relation to milk as diphtheria. We have as yet no proof that the disease may come from the cow, although one epidemic of a somewhat similar disease has been attributed to diseased cows in Herndon, England, and other cases have been reported of a similar nature. There is fairly good evidence that the milk may become infected by persons who have the disease or are re- covering from it. When such persons have direct or indirect contact with the dairy there is occasional opportunity for the milk to become infected. The cause of this disease is as yet unknown, but it is doubtless some living germ. At all events those who subsequently consume such infected milk do some- times contract the disease. A few epidemics have been traced sufficiently to convince us that here is a positive source of danger, though perhaps not a very great one. Asiatic Cholera is also transmitted by milk, but this is a matter of no special significance in the United States. 1 Davies. Jour, of Hyg., p. 338, 1901. Klein. Cent. f. Bact., ii., p. 219, 1887. Lee. Hyg. Rund., p. 227, 1900. Tingvall. Hyg. Rund., p. 109, 1904. DISEASE GERMS IN I:ILK 105 DETECTION OF DISEASE GERMS IN MILK There are within our reach at present no practical means by which we can examine milk and determine whether it contains the germs of any of these four diseases. It is true that proper study will sometimes detect the presence of tubercle bacilli in milk; but the methods are long, complicated and very difficult, and so uncertain that a negative result has no meaning. It would never be possible by any methods in our possession to- day to make a microscopic examination of milk and guarantee the milk as being free from tubercle bacilli. It is still more im- possible to detect the presence of the other three disease germs. The cause of scarlet fever is not known and cannot, of course, be recognized in milk. The typhoid bacillus very closely re- sembles B. coli, which we have seen is always to be expected in milk, and while the two bacteria can be distinguished from each other, the methods are too long and uncertain to make it possible to apply them in any practical way to the study of milk. The diphtheria bacillus, too, may be detected but only by methods of no practical value in the study of milk. Moreover, no sample of milk is likely to be thrown under sus- picion as distributing disease until long after the particular sample is consumed, when it is too late to make any study. It is important to notice that the numbers of'bacteria in milk have no relation whatsoever to its chance of distributing these four diseases. Milk that contains as many bacteria as 100,000.- 000 per c.c. is no more likely to contain these germs than milk that contains not more than 10,000, excepting on the general ground that the milk with the higher numbers has been perhaps more carelessly produced. In short, the plans of municipal in- spection which give us facts concerning the bacteria in milk give us not even a hint as to the germs that cause these four diseases. Furthermore, dairy inspection by any means as yet devised is not accurate enough to enable the inspector to de- 106 PRACTICAL DAIRY BACTERIOLOGY termine whether tuberculous cattle arc present among the herds furnishing milk to the public, or to guarantee the consumer against contagion from typhoid or diphtheria from all the sources above mentioned. The sources of infection from typhoid fever, diphtheria, scarlet fever or tuberculosis are varied and so rare that dairy inspection will give little protec- tion against them. A rigid dairy inspection, it is true, forces the dairyman to be more careful in the handling of his milk and more cleanly in his general methods; but this will not guarantee that the tuberculosis bacillus is not constantly finding its way into the milk, nor will it insure that typhoid fever germs are never present. On general principles the most care- fully kept dairy would be least likely to furnish milk infected with disease germs. So far, then, as dairy inspection can bring about this desired result, it will reduce the danger of contagion; but since even well kept dairies have in some cases been known to distribute milk contaminated with disease germs, there is no method at hand by which it is possible for the pub- lic to be guaranteed that the milk which comes from the dairies is free from the germs of these four specific diseases. In other words, as valuable as dairy inspection may be, it must be rec- ognized that it is inadequate at the present time as a guard against tuberculosis, typhoid fever, scarlet fever and diphtheria.1 DIARRHEAL DISEASES This type of disease has quite a different relation to our problems. Diarrheal diseases are somewhat indefinite, the term including such common troubles as summer complaint, cholera infantum and other miscellaneous types of intestinal troubles common in warm weather. The causes of these diseases are, as yet, unknown. They are not specific diseases like the four 1 Bienstock. Arch. f. Hyg., xxxix., p. 390, 1901. Eichert. Zeit. f. Fleisch u. Milch Hyg., viii., p. 86, 1898. Lubbert. Zeit. f. Fleisch u. Milch Hyg., vii., p. 1, 1896. Neumann. Zeit. f. Fleisch u. Milch Hyg., vi., p. 53, 1894. DISEASE GERMS IN MILK 107 already mentioned, and none of them can probably be attributed to any particular type of bacteria. Delepine has concluded that they are sometimes due to bacteria of the type of B. coli; found in the faecal matter1. The organism that he thinks is the cause differs from the ordinary B. coli in producing a small amount of acid and in not curdling the milk, and is frequently distributed by milk. Lameris has concluded that diarrhea may be produced by a Streptococcus that comes from the udders of cow's suffering from mastitis, and inflamed udders have cer- tainly been known to produce milk that gives rise to diarrhea.2 It has been shown that such milk may still produce the trouble? even after boiling, and this and other general facts have led to the belief that this type of disease is of a toxic rather than a bacterial nature.3 By this is meant that the direct cause is some substance of a poisonous nature that is swallowed with food or drink, and directly poisons the in^ dividual. This does not mean that bacteria are not the real agents, but that they act in an indirect way. In the case- of tuberculosis the germs enter the body and live within the living tissues as paiasites, but in the toxic diseases the bacteria grovy in the food or drink, and there produce poisons. When thes$ foods are subsequently swallowed they, with their poisons, will be absorbed from the digestive tract and cause a direct poisom ing. If this is true, it will follow that toxic poisoning would result from the use of food only after bacteria have had an opportunity to develop sufficiently to produce their toxins. It would follow, therefore, that their frequency would be pro- portionate with the number of certain species of bacteria in the foods. • ' With these facts in mind the relation of such troubles to milk becomes suggestive. While there are other sources for 1 Delepine. Jour, of Hyg., p. 68, 1903. 2 Lameris. Zeit. f. Fleisch u. Milch Hyg., xi., p. 114, 1900. 3 Groning. Inaug. dis., Bern, 1901. 108 PRACTICAL DAIRY BACTERIOLOGY them it appears that they may in part be attributed to milk bacteria.1 They seem to be associated with large numbers of bacteria in milk. They are especially common among children in warm weather, and the mortality from such diseases rises and falls with the number of bacteria in milk. This in itself does not prove much, however, for the number of bacteria in milk simply rises and falls with the temperature, and it may be that temperature itself is the primary factor. But it becomes more significant when we learn that these diarrheal troubles during the severe warm weather are far less abundant among children that are breast-fed, thus obtaining their milk from a source where the number of bacteria will be compara- tively small, than among those fed on cow's milk. Again ex- perience has shown that pasteurizing the milk, which reduces the number without destroying them all, causes a notable re- duction in the amount of intestinal disturbances and in the mortality of children from such sources. Lastly, among children fed upon certain improved forms of milk, which we shall notice later, the death rate from such causes is notably less than among children fed upon ordinary milk. In our large cities the children's death rate is startling in hot weather, and is always directly associated with a poor quality and a high bac- terial content of the milk. TOXIC POISONING FROM MILK PRODUCTS Closely related to the last topic are the occasional instances of toxic poisoning from milk, cream or cheese. These are charac- terized by violent cases of illness that follow the consumption of a single lot of poisonous food and come to a crisis and pass off rapidly. A few instances of such severe illness due to drink- ing a lot of milk2 have been recorded and several of similar 1 Rickards. Jour, of Hyg., 1903. 8 Zammet. Brit. Med. Jour., p. 2054, 1900. Vaughan. Arch. f. Hyg., xxvii., p. 308, 1896. DISEASE GERMS IN MILK 109 results from eating poisonous cheese.1 Cases of poisoning from eating ice cream are still more common. In all these instances certain kinds of bacteria have developed in the food until they have produced a considerable quantity of toxin, sufficient to bring about violent illness to the person swallowing it. The poison that causes the trouble has been separated from cheese and called tyrotoxicon by Vaughan, and he has also found in some samples of ice cream the species of bacterium that pro- duces the toxin. The only way of protecting milk from this trouble is to use it fresh, for it will never appear until the bacteria become very abundant. No means can be given for avoiding poisoning in cheese except that of using only good milk. We know so little as to the matter that we are as yet helpless in regard to it. Probably the use of lactic acid starters in cheese-making, which is becoming common, will do much toward eliminating such troubles which, fortunately, are very rare. Ice Cream Poisoning.-Instances of this nature are more com- mon and are probably more easily avoided. They occur almost always in hot summer weather and are apparently most liable to occur in the hot days that follow a period of cool weather. That they are attributable to certain poisons in the ice cream produced by the growth of bacteria is beyond question. It has been shown above that if the milk is kept cool for a number of days the lactic acid bacteria fail to grow; but at these low temperatures other kinds of bacteria do grow, even though somewhat slowly. If the cream collected from day to day is kept for a number of days in the cool temperature of an ice chest, there will be a continuous growth of these other types of germs which are not checked by the development of the lactic bacteria. During a period of cool days there is no de- mand for ice cream; the cream accumulates in the ice chest, 1 Vaughan. Med. and Sur. Reporter, ixiii., p. S84, 1890. Vaughan. Med. News, II., p. 644, 1887. 110 PRACTICAL DAIRY BACTERIOLOGY and these bacteria that love the cool temperatures are growing all the time, so that in the end they will be present in large numbers and will produce a quantity of toxic products. Now, if the cream is made into ice cream when the next hot days appear, the toxins which have been developing may cause among those eating the cream a violent intestinal disturbance. Little more is known concerning this trouble than the facts here stated. The cause is probably the development of toxic germs in cream kept at fairly low temperatures unchecked by the growth of lactic germs, which would at a higher temperature have prevented their development. It must be emphasized, however, that if these troubles do come from such a source, it is not the mere presence of numbers that produces trouble. As already mentioned, sour milk and buttermilk are wholesome products and have never been sus- pected of causing diarrheal troubles; yet they contain bacteria in prodigious quantities. From this it will follow, of course, that there are some kinds of bacteria whose presence in milk is deleterious and others whose presence is harmless. What types of bacteria are concerned in producing these toxins, we do not know at present. It is a growing belief that the bacteria concerned belong to the putrefying class. Putrefying bodies certainly do frequently show the presence of toxins, and it would be natural to assume that milk in "which such germs are growing would contain enough of the poisonous bodies to pro- duce a toxic poisoning in children. We have already learned that the putrefactive bacteria frequently liquefy gelatin and are consequently easily detected on common gelatin plates. Hence, we should be led to throw under suspicion any milk that shows large numbers of liquefiers. On the other hand, it is quite cer- tain that the lactic acid bacteria are not responsible for these troubles. Indeed, the presence of lactic acid bacteria prevents the growth of the putrefactive forms, and when the acid type becomes very abundant, as high as hundreds of millions per c.c., DISEASE GERMS IN MILK 111 the milk may for this very reason be perfectly wholesome, since the putrefactive bacteria have not been able to grow suffi- ciently to produce any considerable amount of toxin. On the other hand, another sample containing smaller numbers may be liable to produce intestinal disturbances, because the acid bacteria have not developed sufficiently to prevent the growth of the toxic forming species. Sour milk is practically sure to con- tain few putrefactive bacteria, if the lactic acid type is abund- ant. Evidently the toxic poisoning and the diarrheal troubles are closely related. In the former a large amount of poison is developed and swallowed at one time, producing violent results; in the latter the toxins are in smaller quantity but may be taken time after time in successive meals, producing a long continued effect. CHAPTER VI DAIRY METHODS We are now prepared to understand the meaning of various dairy methods which have been devised for improving the conditions of milk. To appreciate these it must be remembered that milk is an ideal medium for bacterial growth. It is a good food, is well supplied with water and is, when drawn from the animal, at a temperature best calculated for the most rapid growth of nearly all types of bacteria. It should also be borne in mind, that the bacterial contamination of milk is mostly external to the cow, and that although some of the bacteria of milk come directly from the udder and are unavoidable, the vast majority are from external sources, which can be more or less con- trolled. All reforms in the dairy are aimed, therefore, to ac- complish three ends: I. To insure the use of none but healthy cattle. 2. To protect the milk as far as possible from external contamination from bacteria after it leaves the cow's udder. 3. To produce conditions which so far as possible prevent the growth of these bacteria. In the outline which we will give of methods to be adopted for these purposes, we shall first ex- plain all of the improvements which have been proposed in dairy methods for improving the milk product; but since it is impossible for most dairies to adopt them all, we shall in the next chapter summarize briefly the more important of the prac- tical plans that may be adopted for producing a general im- provement in milk without involving very great expense.1 1 Fraser. Ill. Exper. Sta., 1903. Fraser. Bui. No. 92, Ill. Exper. Sta., 1903. Marshall. Bui. No. 28, Mich. Exper. Sta., 1905. Haecker and Melick. Bui. 87, Neb. Exper. Sta., 1905. Doane. Bu. 88, Md. Agri. Exper. Sta., 1903. 112 DAIRY METHODS 113 The dairyman may feel that some of the precautions are un- necessary, and that so many changes from old methods are needless. But he should remember that conditions are rapidly changing and that there is a much more emphatic demand for cleanness in milk than ever before. He should also remember that it is exactly these precautions that will enable him to guard the butter and cheese-maker from troubles which we shall notice later. The demand for greater care in the produc- tion of milk is a growing one, and one that will be more and more rigidly enforced. AT THE BARN The Health of the Cow.-This hardly belongs to our immediate subject, but it is so closely related to the wholesomeness of milk that it cannot be passed unnoticed. A veterinarian should periodically inspect the cattle, to determine their soundness; and no animal suffering from any udder disease should be allowed to contribute to the milk supply. The importance of excluding tuberculous cattle from those contributing to the public milk supply cannot be over-rated. The only method by which this can be done is not only to have an inspection of all the cattle, but to have at intervals a tuberculin test applied to the whole herd to detect this disease in any animal. Any animal that reacts must be separated at once from the rest of the herd, and her milk must not be sold. A universal tuberculin test of the cattle in the country is not possible under present condi- tions, but it must be emphatically insisted that so long as the cows that furnish milk to the public are not thus inspected and tested, the public is in a constant source of danger from tuber- culosis. Careful watch should be kept of the udder of the animals, and whenever any signs of udder disease appear in the form of inflammation or hardness, running sores or the appearance of bloody milk, the animal should be excluded at 114 PRACTICAL DAIRY BACTERIOLOGY once from the milk-producing herd until complete recovery has taken place. It is well also to remember that exercise is needed by the cow, both for its own health as well as for that of human beings, and that the best of results can never be obtained unless the cows are given a certain amount of outdoor exercise. The Stables.-The location of the cow stables should be such as to give the freest possible circulation of air. (Fig. 41.) It is much better to have them on high ground in which there is ready drainage than on low ground. Both air and light in the FIG. 41-MODEL DAIRY BARN stables are necessary for the best results. Each cow should have three to four square feet of window surface and 400 to 450 cubic feet of air space. If possible a separate place for calving should be provided. While the animals are in the yard, as they should be daily, the stables should be thoroughly aired. (Figs. 42 and 43.) The older plan of keeping the barns tightly closed for the purpose of saving warmth injures both the quality of the milk and the health of the cattle. A good system of ventilation is necessary. The cleanliness of the stable is a matter of utmost importance. The habits of the cow and the nature of the manure are such DAIRY METHODS 115 as render a high state of cleanliness very difficult. But the dairyman should understand that all accumulation of manure or other filth is a direct detriment to the quality of the milk. The cleaner the stable the better the result. (Fig. 44.) The removal of the manure from the stalls should be as frequent as possible. In the better kept dairies this is done several times a day, although upon the ordinary farm it is less frequently at- tended to; but it should never be less than twice a day. The more frequently it is removed, and consequently the cleaner the FIG. 42-POORLY-KEPT BARNYARD, WET AND FILLED WITH MANURE cows and stalls are kept, the better the results on the milk product. The manure, when removed, should be taken as far as possible from the barn and should not be heaped outside of the barn close by, nor be allowed to accumulate in the cellar. By far the best method is to distribute it daily upon the fields, where it is to be used as a fertilizer. If this daily spreading is impracticable, it is advisable to remove it to some distance from the barn as often as opportunity permits, always remembering that the greater the accumulation of manure the worse the re- sults on the milk. Attention should be given to the dust, cob- FIG. 43-WELL-KEPT, DRY BARNYARD 116 FIG. 44-INTERIOR OF A WELL-EQUIPPED, WELL-KEPT COW BARN 117 118 PRACTICAL DAIRY BACTERIOLOGY webs and hay that may be clinging to the ceilings of the barn, for all such are traps for accumulating dirt, as well as sources of bacteria, thus aiding in contaminating the milk. Plastered or sheathed walls and ceilings are very much to be preferred to a rough finish. The bedding of the cattle is a matter of some importance also, and clean shavings appear on the whole the best for this purpose. They serve better than straw or hay, FIG. 45-COLONIES Developing from bacteria falling upon a plate exposed during milking in a poorly-kept barn (Fraser) FIG. 46-COLONIES Developing from bacteria falling -upon a plate exposed during milking in a well-kept barn (Fraser) inasmuch as they give rise to less dust and dirt and are not so liable to be a source of bacteria contamination for the milk. A coat of whitewash should be applied with a spray pump at least once a year. It helps to disinfect and makes the stable light and clean. Flushing with water should be frequent. (Figs. 45 and 46.) DAIRY PERSONNEL There has been a strong tendency in recent years to give much greater attention to the habits of the persons employed on a dairy farm. It is not uncommon to find them dressed in DAIRY METHODS 119 white clothes, which are better than colored clothes because they show dirt when soiled. The milking clothes should be made of washable materials. These are worn during the milking or handling the milk, and some dairies insist that this clothing should be sterilized each day. Directions for thorough washing and drying the hands before milking, and for the care of the person are coming to be common in the better class of dairies. FIG. 47-MILKING IN A MILKING ROOM An ideal condition of cleanliness We have already learned that bacteria from the persons em- ployed in the dairy are more likely to be dangerous than those from any other source. For all of these reasons it is desirable that proper care should be given to persons employed in the dairy. No one should be allowed to handle any milk, to wash the milk cans, or to handle the milking utensils in any way, who is suffering from any contagious disease. Nor indeed should any farm furnish milk to the public if there is a case of typhoid, 120 PRACTICAL DAIRY BACTERIOLOGY scarlet fever or diphtheria among its employees, unless a health inspector pronounces the sanitary conditions satisfactory. THE MILKING ROOM A separate milking room is a rarity in our dairies, but from the standpoint of the purity of the milk such a room, separate from the stalls where the cows are kept, is eminently desirable. FIG. 48-MILKING IN A MODEL DAIRY BARN In the places where this has been tried, it has produced an im- proved quality of the milk. The reason is evident enough. Since the cows are only taken to the room for the few moments of the milking, it is possible to keep this room in a condition of far greater cleanliness than the cows' stalls can possibly be kept. (Figs. 47 and 48.) The cows soon adapt themselves to the new routine, yield their milk readily in the milking room, DAIRY METHODS 121 and dairymen who have used separate rooms for this purpose have been highly satisfied with their utility. There are, of course, objections. More space is required if the cows are to be milked in a separate room, and it is more trouble to re- move the cows from their stalls and take them to the milking room and subsequently lead them back again. The plan thus involves some expense; but the extra work certainly results in an extra purity of milk, and is one of the devices that should FIG. 49-AN IDEAL METHOD OF MILKING be adopted where one is attempting to obtain the highest grade of product. Where possible, milking out of doors is to be especially recommended. (Fig. 49.) During the milking process the cows should not be allowed to feed, nor should the feeding precede, but rather follow the milking. The feeding of the cows is almost always sure to fill the air with more or less dirt and dust from hay and dry grain. Experiment has shown that a great number of the bacteria in milk may come from the feed of the cows, if the feeding has occurred before or during the milking. If fed immediately after, the bacteria have a chance to settle to the floor before the next milking and do not produce so much trouble. The 122 PRACTICAL DAIRY BACTERIOLOGY effect of feeding cows upon dry corn stover before and after milking is shown by the following diagram: DIAGRAM SHOWING THE EFFECT UPON THE BACTERIA OF FEEDING CORN STOVER The milking room should, if possible, be flushed with water after each milking. There should also be flowing water, soap and towels to permit the milker to wash his hands after milking each cow, and whenever they are soiled.1 THE COW Emphasis must be placed upon the grooming of the cows. In earlier years it was almost unknown to pay any attention to the grooming and cleaning of the cattle. (Fig. 31.) For- tunately, there has been a decided change in this respect, and at the present time cattle in dairies are not infrequently groomed and brushed, and are sometimes kept in a condition as cleanly as that of the ordinary horse. If a proper milk supply is to be obtained, this is absolutely necessary. The habits of cows, and especially the close confinement in which they are kept in stalls, inevitably results in a large amount of manure adhering to the animal's flanks, tail and udder, and unless this is removed by curry comb and brush, with washing if necessary, the character of the milk is sure to suffer. The long hairs of the cow's body are especially liable to catch such filth, and the cutting off with 1 Stocking. Bui. 42, Storrs Exper. Sta., 1906. DAIRY METHODS 123 scissors or a clipper of the long hairs from flanks where manure is liable to collect, from the tip of the tail, and especially around the udder and on the under part of the body, is to be recommended. If this hair is occasionally cut, the contamina- tion of milk from the cow's body is lessened. The cow should not be allowed to wade through mud and manure such as is frequently found in the barnyard. This will mean the cleaning of the barnyard and, in many places, a thorough draining. A clean, dry barnyard for the cattle to use for exercise will con- tribute much to the cleanness of the cows and hence of the milk. (Fig. 43.) MILK VESSELS Perhaps the most important factor for producing a reduction of bacterial contamination is the proper cleaning of the milk vessels. From facts given in a previous chapter it is evident that all vessels which have held milk should receive a most thorough cleaning. This refers to milk pails, strainers, coolers, separators, milk cans, glass bottles and, in fact, to all dairy utensils. The washing of such utensils is no easy task, and it is not an exaggeration to say that upon an ordinary milk farm vessels are never washed clean. They are, it is true, frequently scrubbed with warm water and soap, but no such washing will result in removing all of the bacteria. After the most thorough washing and scrubbing there will still be left many of these organisms in the cracks and clinging to the milk vessels, ready to begin feeding upon the next lot of milk and to multiply. Cold water is practically useless for removing them. All milk vessels should be of metal, with a smooth surface. If the coating of tin is worn off, they should be discarded, for they cannot be kept clean. They should not be allowed to become dry before washing, for dried milk is difficult to remove. They should be first soaked in warm water to loosen the milk; then thoroughly washed in hot water, preferably containing soap or sal-soda, 124 PRACTICAL DAIRY BACTERIOLOGY and thoroughly scrubbed; after this a second rinsing in hot water is extremely desirable in order that the milk pail shall be cleaned in fair condition. After washing they should be placed inverted in the sun until used again. They should not be tightly closed. Such a washing as this is about all that they can receive upon an ordinary farm, but this is not enough to sterilize them. There is, indeed, no means by which the farmer can so thor- oughly wash his milk cans as to get rid of all bacteria. Every creamery, cheese factory or separating station should under- stand this and appreciate that a satisfactory washing can be accomplished only where there is a supply of steam. Hence, no creamery should depend upon the farmer to wash milk cans. Where a supply of steam is to be had a sterilization must follow the washing, but it should be different from that frequently seen. The common procedure is to place the can for a few seconds over a jet of exhaust steam, and after a short time to remove it that it may be replaced by a second. The steam used under these conditions hardly heats the can hot enough to kill any bacteria, and while cans thus steamed are certainly some- what more free from them than those not thus treated, the impression of the dairyman that he has thoroughly cleaned his cans of bacteria is incorrect. The only method by which milk vessels can be absolutely freed from bacteria is by thorough sterilization. This can be done by steam if the cans be placed in a steam chamber built for the purpose, and the steam turned on and left to act upon the cans for an hour or so. In no other way can absolute sterilization be ac- complished. Washing with hot water is better than with cold, washing with sal-soda is better than without it, subjecting them to a jet of exhaust steam is better than simple washing, and sterilizing in a steam chest is better than all. Each dairyman should adopt as thorough cleaning as is practicable. In washing dairy utensils special attention should be given DAIRY METHODS 125 to separators, which are a very important source of bacteria. These are frequently insufficiently washed, particularly hand separators. They are difficult to clean adequately, since it is commonly not easy to steam them. For this reason all the more care should be exercised in using plenty of boiling water and soap, and steaming all parts that can be steamed. Caution should be given that all pieces of cloth used around a milk room need frequent sterilization. A neglect of this precaution has sometimes caused seri- ous troubles in making starters in butter or cheese- making, and keeping unde- sirable bacteria away from them. FIG. 50-METHOD OF MOISTENING COW'S UDDER DURING MILKING (FRASER) THE MILKING Moistening the Udder.-The value of moistening the udder with a damp cloth or sponge before milking is greater than one would imagine. Its purpose is, of course, to prevent the fall of dirt into the pail. It may be done with vaseline or a borax solution, but plain water serves just about as well. The udder should not be left wet but simply damp. No simple procedure is more efficient than this in reducing bacterial contamination. (Fig. 50.) The value of such treatment in reducing the number of bacteria is shown in the following diagram: 126 PRACTICAL DAIRY BACTERIOLOGY DIAGRAM SHOWING THE EFFECT UPON THE BACTERIA OF MILK OF WIPING THE UDDER WITH A DAMP CLOTH Some dairies have the practice of brushing the cow just be- fore milking. This by no means serves the purpose of wiping with a damp cloth. On the contrary it simply stirs up the dirt and dust and leaves it floating in the air in a position to settle readily into the pail. The result is that there is an actual in- crease of bacteria rather than a decrease. The amount of increase, as shown by one series of tests, is indicated in the ac- companying diagram. DIAGRAM SHOWING THE EFFECT OF DRY BRUSHING OF THE UDDER BEFORE WIPING The Covered Pail.-In very recent years changes have been made in the methods of milking, designed for reducing the amount of dirt which falls into the milk during the milking process. The first and simplest of these is the change in the DAIRY METHODS 127 style of milk pail. The old fashioned pail had a flaring top, the purpose of which was to make the milking as easy as possible, but incidentally it resulted in exposing the milk to much con- tamination by dirt and bacteria. Various devices for protecting the milk from such exposure by the use of covered milk pails, are now used. Figures 51 and 52 show some of these devices. There is quite a great variety among them, but they all have the general plan of decreasing the size of the opening of the milk vessels so as to expose less surface for the entrance of FIG. 51-COVERED MILK PAIL (NORTHS) dirt, and they also have in the opening some kind of a cloth strainer for catching the larger particles of dirt and keeping them from the milk. The pail of Gurler uses a layer of ab- sorbent cotton for the purpose. Such milk pails produce a de- cided improvement in the milk. Careful testing has shown that by their use a large proportion of the dirt that gets into milk may be avoided. Their use has been extending rapidly, and the covered milk pails will soon doubtless take the place of the old flaring tops. Every dairyman should recognize this as one of the easiest, cheapest and most efficient means at his disposal for improving the character of his milk. It is necessary to use 128 PRACTICAL DAIRY BACTERIOLOGY them intelligently, however, for a careless milker can obtain as bad results from a covered pail as from any other. In their use special attention must be given to the cloths used for strainers. These must be sterilized daily by washing and boil- ing. It is sometimes customary to change the strainer cloth for a fresh one between the milking of each two cows. Strainers FIG. 52-COVERED MILK PAILS (GURLERS AND STADTMULLERS) made of bolting cloth, Irish linen, or flannel are the best, and these may be used over and over again with the proper washing. If absorbent cotton is used for a strainer, it must be thrown away after the milking. Milking Machines.-A still more recent means of reducing contamination is by machines designed to convey the milk directly from the cow's teat to closed sterilized milk cans, with- out allowing it to come into contact with the air, or exposing it to contamination with dirt.1 These milking machines consist of rubber tubes ending in special cups for attachment to the teats of the cow and connected at the other end with large cans, which can be sterilized. (Fig. 53.) The cans are connected with a vacuum system of tubes, and at the point where the rubber tubes are attached to the can there is a mechanical device 1 Lane and Stocking. Bui. 92, Bu. An. Ind., 1907. DAIRY METHODS 129 by which the vacuum is made to draw through the tubes inter- mittently, thus imitating natural milking. (Fig. 54.) This draws the milk through the tubes to the can. Details of these machines need not concern us here, but their efficiency in milk- FIG. 53-MILKING MACHINE (United States Department of Agriculture) ing is beyond question. Tt would seem that such a plan, which carries milk directly from the teat to a sterilized can, without contact with the air, would be almost ideal and would remove practically all dirt contamination. All ordinary sources of filth contamination are excluded by such milking machines, and 130 PRACTICAL DAIRY BACTERIOLOGY where they have been intelligently used they have been found to be efficient in producing a much cleaner quality of milk. On the other hand, they are themselves difficult to clean. Long rub- ber tubes which have been filled with milk are by no means easy to keep sweet, and when they are used by careless employees the bacteria become very abundant inside the tubes and the FIG. 54-USE OF A MILKING MACHINE OUT OF DOORS Power is supplied by animals in treadmill on right (Kansas Experiment Station) other parts of the somewhat complicated machine. In other words, it requires very great care in cleaning and sterilizing these milking machines in order to produce better results than are produced by hand milking, or even as good; but they can be cleaned, and, if care is taken to sterilize thoroughly all of the apparatus, better and more reliable milk can be obtained by the use of the milking machine. Directions for cleaning these ma- chines will always be given with the machines themselves and need not be given here. The general principle, however, that extreme care should be taken to disinfect thoroughly the rubber tubing after each milking cannot be too highly emphasized. The use of these milking machines certainly offers a great DAIRY METHODS 131 promise in the milk product. They are hardly practical, how- ever, at the present time upon the small farm, because of their expense. But in a dairy where there is a herd of fifty or more cows they become very economical because of the ease with which a single man can milk a number of cows at once by means of them. One serious objection has been raised against the use of these machines that has in some places caused their abandonment. There is no way yet devised by which the milker can see the milk as it passes into the collecting can. If he chances to milk a cow that has experienced some accident so as to produce bloody milk the milker will not discover it until he opens the can into which he has perhaps already drawn the milk of several cows. The result will be the spoiling of the whole milk in the can which would have been avoided if he had been able to detect the bloody milk at the time of the milking. Rejecting of Fore-milk.-For reasons already indicated the first milk drawn at each milking will contain more bacteria than the rest, inasmuch as it washes out the bacteria at the mouth of the milk ducts. It has become a practice in the better dairies to reject the fore-milk, either allowing it to waste upon the floor or collecting it in a separate dish. There is no doubt an advantage in this, but the extent of the advantage has been overdrawn. The extra number of bacteria obtained in a pail of milk from the entrance of the fore-milk is very small in pro- portion to the larger number that enter the milk from other sources, and while the rejection of the fore milk is to be recom- mended its value is not so great as is sometimes supposed. Its value is shown by the accompanying diagram. Stripping.-The practice of milking the cows thoroughly dry at each milking also tends to increase the purity of the next milk drawn. If a considerable quantity of milk is left in the udder, it has a tendency to hasten the development of the bacteria in the udder so that the next milk that is drawn con- 132 PRACTICAL DAIRY BACTERIOLOGY DIAGRAM SHOWING THE EFFECT UPON THE BACTERIA REJECTING THE FORE-MILK Fore-milk not re- jected, Fore-milk rejected, Decrease due to rejection of fore- milk, tains more than it otherwise would. The practice should, there- fore, be adopted of milking the cows as dry as possible. Its value is shown by the accompanying diagram. DIAGRAM SHOWING THE EFFECT UPON THE BACTERIA OF STRIPPING Milked dry, Not milked dry, Increase due to not milking dry, Removal from the Milking Room.-All milk should be re- moved from the milking room immediately after milking, and the quicker the better. The air of the room is sure to be filled with bacteria or dust, which will get into the milk. Moreover, the odor in the milking room is commonly quite noticeable, and it is well known that milk absorbs odors rapidly. Accordingly if the milk stands in the barn, it will collect an appreciable odor, and one that decidedly detracts from its value. Hence, the more quickly the milk is removed the better; and it is best to have a Collecting can outside of the milking room and to empty the DAIRY METHODS 133 milking pail between the milking of every two cows into the receptacle outside. The value and necessity of this procedure will, of course, vary with the cleanliness of the milking stall itself. The Value of the Trained Dairyman.-Apparatus without a proper man to use it is valueless. It makes no difference how many rules may be drawn concerning the dairy, how complicated the apparatus becomes, or how careful the directions given to the employees; it is quite impossible to expect satisfactory re- sults without properly educated and trained assistants. An un- trained man will only succeed in getting bad results, even with the best of apparatus. Special emphasis, therefore, should be placed upon the matter of the necessity of education. It must be recognized that the employees in our dairies at the present time are, in many cases, without any proper training. They do not know the character of the product they are producing; they do not know the dangers to which it is subject; they do not understand the universal presence of bacteria; they do not un- derstand, in general, the problems that are concerned with their business. They are quite likely to believe the whole subject of bacteria in milk to be foolishness and not worth their attention. Under these circumstances, no matter how many directions are given, or how much instruction there may be, satisfactory re- sults will never be obtained. We may look with more confi- dence upon the dairy conditions after the next few years. At the present time a large number of young people are being trained in the necessary knowledge to enable them successfully to handle the problems of dairy work. Only as more education is spread will it be possible to bring about the desired end in the dairy industry. The great significance of this matter of intelligent training is shown by the accompanying diagram. In a barn were employed a number of regular milkers, who had no special knowledge of bacteria, and one person, who had studied bacteria so that he understood the principles concerned. 134 PRACTICAL DAIRY BACTERIOLOGY DIAGRAM SHOWING THE VALUE OF TRAINING THE MILKER UPON THE NUMBER OF BACTERIA Milker No. i. Regular men, Difference in favor of No. i, Both were supplied with the same apparatus for milking, but the student used it more intelligently. The length of the lines shows the relative number of bacteria in the milk drawn by the regular milkers and the trained student. The result is striking and most emphatically shows the emphasis that is to be placed on the proper training for men employed in the dairy. AT THE DAIRY Location.-Thought and care should be given to the location and arrangement of the dairy. It should be in a light, airy location, unconnected with stables or living rooms. The floor should be of cement with good drainage. Windows should be abundant to let in plenty of light, covering a space equal at least to 15 per cent, of the floor space. There should be a satisfactory plumbing system both for furnishing an abundant supply of clean water and for carrying away wastes, the latter being as important as the former. That a dairy should be kept clean hardly needs emphasis, but it cannot be too strongly brought to the attention of all interested in the handling of milk. Constant cleaning of all utensils and vats, floors and walls, with hot water and soap, and steaming of all cans, bottles or pails in which milk is placed must be recognized as a necessary condition for a, properly kept dairy that shall furnish the best of milk. No DAIRY METHODS 135 milk spilled on the floor should be allowed to sour, but should be washed up at once. No dairy is properly equipped unless it has an ice supply. The amount of ice needed will vary with the climate, being, of course, less in colder than in warmer regions. For an ordinary climate at least 40 lbs. of ice are needed for every hundred pounds of milk produced. Cooling.-The importance of cooling the milk, and cooling it immediately, cannot be over-stated. As we have already seen, bacteria grow very readily at a temperature of the animal's body, very slowly at temperatures below 50 degrees, and scarcely at all at the temperature of freezing. When the milk is drawn from the animal it is, therefore, in a condition to stimulate the growth of bacteria to its utmost. It is true that for a while, because of the germicidal property of milk, the bacteria do not grow; but this condition lasts only a short time, after which, i f the milk is warm, they begin to develop with the greatest rapidity. If the milk, however, has been in the meantime cooled to a low temperature, the bacteria that have found their way into the milk will not grow rapidly, and the milk can be kept for a very much longer period. These facts are so simple as hardly to require statement, but, unfortunately, many a dairy- man, although he may theoretically understand them, fails to appreciate their importance. It not infrequently happens that a milkman finds that his morning's milk sours when delivered to his customers more quickly than milk of the night before, and he has been puzzled over the matter. The reason usually is that the night's milk, after milking, is placed at once in a cool place, an ice chest or a cold stream, and by morning it has acquired a fairly cool temperature. In the haste of the morning, however, the milkman does not cool his morning's milk but car- ries it to market in a warm condition. By the time it reaches the customer the bacteria have begun to develop. In the mean- time the milk of the night before has not been warmed up suf- ficiently for the bacteria to begin to grow rapidly. Thus, the 136 PRACTICAL DAIRY BACTERIOLOGY morning's milk soon contains many more bacteria than the milk that is twelve hours older, and hence it sours much more quickly. The lesson to be drawn from this is the necessity of immediately cooling the milk to a temperature as low as 400, if possible. It should be remembered, too, that it is just as necessary to cool good milk as it is to cool dirty milk. Unless this is done the good milk will soon contain as many bacteria as the dirtiest milk. It should be recognized, however, that the value of the cooling of the milk is only to decrease the rapidity of the multiplication Of the bacteria and does not necessarily affect its healthfulness. The milk, as we have seen, may distribute the germs of certain diseases. If these germs have found their way into the milk, the fact that the milk has been cooled will not destroy them and will have no value in rendering the milk wholesome. None of the disease germs known to be found in milk would be killed, or even injured, by the temperature of the ice chest, at least if continued only for a few hours, and therefore the practice of cooling the milk cannot be looked upon as a safeguard for protecting the public from the specific diseases liable to be dis- tributed by it. On the other hand, the development of the diarrheal diseases which, as we have learned, probably come from poisons secreted in the milk by certain kinds of bacteria may, perhaps, be checked by the use of cool temperatures that will prevent the growth of bacteria. Concerning this we know practically nothing at the present time. The value of the im- mediate cooling of the milk, therefore, is primarily in making the milk keep longer, and not in rendering it more wholesome. It decreases numbers only and does not remove disease germs. Any method that cools the milk rapidly is satisfactory. It may be done by immersing it, already bottled, into cold water. This has the advantage of cooling it rapidly in the jar so that no further disturbance of the milk is necessary. Cooling in cans is less desirable since the milk must afterward be handled again. DAIRY METHODS 137 A common method of cooling in large dairies is by causing it to run over a metallic surface that is artificially cooled. This cools and aerates the milk at the same time.* Aeration.-The practice of aerating at the time of cooling the milk has become very general in the better dairies, and it is undoubtedly of considerable value. It is accomplished by al- lowing the milk to flow in a broad stream and a thin layer over artificially cooled metallic surfaces. So far as concerns its effect upon the bacteria of milk it is of no value at all, for it does not decrease the numbers but, on the other hand, tends slightly to increase them. It is easy to see that if the milk is for some time exposed to a large surface of air, it will be constantly absorbing bacteria. The number thus obtained, however, is not great, and more probably come directly from contact with the aerator itself. Both together do not furnish enough to over- come the advantage to the milk from aeration. It is, however, claimed by Marshall1 that aeration increases the subsequent rapidity of bacteria growth, since it removes the carbon dioxide that is normally present in the milk which has a slight checking action on bacteria growth. This has, however, been questioned by others. The purpose of the aeration is both to cool the milk by contact with cold surfaces and to remove some of the so- called "animal odors" that are commonly present. It should be noticed that most of the "animal odors" are purely filth odors, due mostly to manure. Milk that is drawn under perfectly clean conditions, without chance for contamination or entrance of manure, does not need aeration for the purpose of removing odors. Such milk is best bottled at once without aeration and cooled in the bottles. The need of aeration is directly propor- tional to the amount of filth that enters the milk after it is drawn. If aeration is adopted it should be carried on in a place where the air is as pure as possible. To aerate in a room where * Marshall. Ann. Rep. Mich. Bd. of Agri., p. 259, 1902. Barthel. Rev. Gen. d'Lait, III., p. 294, 1904. 138 PRACTICAL DAIRY BACTERIOLOGY the dust of the barn has a chance to enter is bad. Aeration improves the flavor of milk that is produced under any but the very best of conditions, but it does not decrease the number of bacteria and is of no value to milk that is to be used in butter making or cheese making, except for its rapid cooling of the milk. Filtering Milk.-The practice of filtering milk to remove the dirt has been adopted in some places, especially in Europe. The filter is commonly made of layers of sand of different degrees of fineness, and in the best methods the milk is forced through the filter from below upwards. These filters are not very widely in use. So far as bacteria are concerned they do not accomplish anything, for they are never fine enough to remove them. They do remove some of the dirt. They are difficult to clean thor- oughly and are not, in general, to be recommended.1 Centrifugal Force as a Method of Cleaning Milk.-In some dairies the use of centrifugal machines has been adopted for cleaning milk. In doing this the rapidity of the rotation of the machine is not as great as when it is desired to separate the milk from the cream. Careful testing shows that this treatment certainly does remove a large part of the dirt from the milk. One who examines the mass of sediment that collects in the drum of such machines cannot doubt that a large amount of dirt is removed. Further, an examination of the milk itself and an estimation of the milk which it holds shows that there is a noticeable reduction. Curiously enough, however, such a proc- ess does not reduce the number of bacteria. It certainly would be expected that, if it removes dirt, it would at the same time reduce the bacteria. On the contrary, the number of bacteria usually appears to be increased by passing through the centrif- ugal machine, and is practically never reduced. The reasons 1 Weigmann. Milchztg., p. 289, 1901. Weil. Milchztg.. p. 739, 1901. Vieth and Martiny. Zeit. f. Fleisch u. Milch Hyg., XI., p. 326, 1901. DAIRY METHODS 139 for this are probably three. I. The centrifugal machine itself is somewhat complicated and is extremely difficult to get thor- oughly clean. After using, it is, of course, washed, but it is difficult or impossible to wash the bacteria all away; and when the next lot of milk is passed through it, there may be some contamination of milk by bacteria from the machine. Probably, however, this is a small item. 2. In the method of determining bacteria, as shown on page 273, the number is determined from the numbers that grow in gelatin, and it is assumed that there was in the original milk one bacterium for each colony. It not infrequently happens that bacteria are just about to divide, and if such a half-divided bacterium is in the milk that has been tested, the two halves being so closely together will make only one colony, and will be counted as one, whereas they will be broken apart by the centrifugalization and appear as two col- onies. 3. It frequently happens that several bacteria may cling together in the original milk, when the whole bunch would produce a single colony and be counted as one. When, however, milk is passed through the centrifugal machine the agitation it receives is apt to break the bacteria apart and thus increase the number of isolated individuals. The result would be a larger number of colonies in the gelatin plates, and these when counted would give a greater number of bacteria in the milk passed through the machine. Hence, although centrifugalization can- not really increase the number of bacteria, nor does it improve the keeping quality of the milk, it does increase the number counted on a gelatin plate. In spite of this fact, it is beyond doubt a very useful means of cleaning milk, since milk that has been thus centrifugalized is deprived of most of its filth.1 1 Dunbar and Kisler. Milchztg., p. 753, 1899. Rolet. Rev. Gen. d'Lait, I., p. 185, 1901. Eckles and Barnes. Bui. 59, Iowa Exper. Sta., 1901. Severin. Cent. f. Bact., II., xiv., p. 605, 1905. 140 PRACTICAL DAIRY BACTERIOLOGY INFECTION AT THE CREAMERY AND FACTORY The primary infection of milk is at the barn and dairy, but a secondary infection may occur elsewhere, either in creameries or factories or milk distributing stations, and it is necessary to guard milk in these places. For example, many creameries that handle milk cans are careless about their washing, and even if they thoroughly wash them, will only adopt the system of a steam jet for a few seconds for the purpose of sterilizing. Many factories do even worse than this; they allow dirty, un- washed cans to lie around the factory for some time or permit the farmers to carry them away unwashed. Any factory that depends upon the farmer to wash the cans will suffer for it. The factory cans circulate from one farm to another, and if a farm once becomes contaminated with bacteria, such trouble is sure to be distributed through the factory to the whole com- munity. The remedy is to cut it off at the creamery, and this can be done only by thorough cleaning and sterilizing of the cans at this central station. In many separating stations and cheese factories a very un- fortunate custom has arisen that distributes all kinds of bacteria and makes it impossible for the dairyman to furnish a good product. The farmer brings his milk in cans and carries back to his farm whatever skim milk chances to be running through the separator at the time. By the time he has reached home, especially in summer, the skim milk is apt to be sour. The farmer may now attempt to wash the cans after emptying them, but this is really impossible. The cans cannot be properly cleaned on the farm, and fresh milk is put into them at the next milking. Under such conditions it is impossible to produce good milk. The conditions are still worse in some cheese factories, where the farmer takes back whey to his home. The whey from the cheese vats is stored in tanks or barrels from which the farmer fills his milk cans. The whey tanks are not washed, DAIRY METHODS 141 and this whey is frequently in an advanced state of fermenta- tion. It is sure to pollute the milk cans beyond the possibility of cleansing by any ordinary washing. Under such conditions trouble is sure to arise, and by this means not infrequently such infections as slimy milk, or other mischievous fermentations, are widely spread. The remedy is either to furnish separate receptacles for returning the by-products to the farm, or to pas- teurize them before giving them back to the farmer. The latter is by far the better and is already adopted by law in some countries. In all cases the whey should be returned to the farm as quickly as possible. The creamery should be the pattern for the farmer, but, alas, it is sometimes only a pattern of careless- ness and filth, and the individual dairies will always be worse than the creamery. In the disposal of wastes, too, many factories or creameries are greatly at fault. The practice of allowing any kind of waste to accumulate close to the factory is undoubtedly a fruitful cause of trouble. The draining systems should have plenty of running water to carry away all wastes and to empty them at a con- siderable distance from the creamery. Dirty cans, or cans filled with refuse from the milk, should not be allowed to stand around the rooms where fresh milk is placed, or upon platforms in con- tiguity with cans containing fresh milk. The water which is used in factories for cooling milk should be as fresh and clean as possible, and any kind of dirt in it is greatly to be deprecated. It must always be recognized that although water is used to cool the can only, and is not meant to touch the milk, it is quite impossible to keep cans of milk in water and handle them as rapidly and as roughly as they have to be handled without al- lowing the water to splash upon the covers, and hence to get into the milk itself. The milk supply company in the city is frequently at fault for troubles which it attributes to the farmer. It is not un- common to find the cans in which milk has been shipped to the 142 PRACTICAL DAIRY BACTERIOLOGY city simply emptied of milk and shipped back again without washing. By the time they reach the farm they are in a very bad condition, smelling strongly, filled with dried milk and bad condition of filth. Such dirty cans a farmer cannot possibly clean and even if they are sent to a creamery where there is plenty of live steam the thorough cleaning is extremely difficult and almost impossible in the few moments that can be given to each can. The next milk that goes to the city in these cans is badly contaminated even at the start and cannot reach the city in good condition. The city distributing company blames the producer when the real trouble is the failure to wash the cans clean at the time they are emptied. All cans should be washed in the receiving station as soon as they are emptied and shipped back in a clean condition. That this rule is not followed any one may prove to himself by simply looking at and smelling of the empty cans he may find at a railroad station. DISINFECTION OF THE BARN OR DAIRY It sometimes happens that a disinfection of the barn or dairy is a matter of necessity. This may be due either to the appear- ance in the milk of some unusual or troublesome fermentation, like slimy milk, which the dairyman finds difficult to eradicate by ordinary means, or it may be necessary to rid the premises of the germs of some contagious disease. The thoroughness of the disinfection needed will be dependent upon the circum- stances of the case, and it should be done with especial care where it is necessary to get rid of some contagious disease. Disinfection of dairy premises is not an easy matter, and to do it thoroughly is extremely difficult. If a barn is to be dis- infected, one must first remove all filth by scraping, and thoroughly clean the floors, walls and ceilings by the use of hot water, scrubbing completely from the floors and cracks all filth that may be accumulated. After thorough washing some dis- DAIRY METHODS 143 infectant must be applied. In choosing the disinfectant for the purpose one must be guided by conditions. One of the best is corrosive sublimate, % oz. to two gallons of water; but this is intensely poisonous, and is unsafe to use where cattle are to be subsequently stalled, unless absolutely thorough washing with water follows the use of the sublimate. Corrosive sublimate also corrodes metals badly and cannot be used upon metallic surfaces. Chloride of lime, 6 oz. to a gallon of water, is an extremely efficient disinfectant, its disinfecting power being due to the chlorine which it contains. It is less harmful than corrosive sublimate, but its odors make it sometimes more ob- jectionable. No other disinfectants are as satisfactory as these two. A partial disinfection, which is sometimes quite sufficient, may be obtained by applying a thorough coat of whitewash, which may be put upon the walls, ceilings and floors with a spray pump. This also makes the room lighter and brighter, and is eminently useful. Indeed, all barns should have a coat of whitewash applied, at least twice a year, if they are to be kept in proper condition. Disinfection of the dairy proper follows the same general methods described above, excepting that the premises are kept cleaner, so that the original cleaning and scraping are commonly not so necessary. The use of the corrosive sublimate is espe- cially undesirable here, and the use of chloride of lime must be followed by thorough airing to remove the odor before the dairy is used again. If the nature of the room is such that it can be tightly closed, it may be disinfected by formalin. The application of the formalin is rarely advisable, excepting in the hands of someone who thoroughly understands it, and the de- tails of the method need not be given here. The treatment of dairy utensils by boiling water and steam is usually sufficient, and if thorough, quite sufficient for practical disinfection. CHAPTER VII TREATMENT OF MILK FOR MARKET It is impossible to make any statement as to the number of bacteria liable to be found in milk. There may be as few as 3,000 or as many as 200,000,000 per c.c. Even in fresh milk the number varies widely, as we have seen, and curiously enough wide variations may occur on the same farm, under apparently similar conditions. Sometimes a dairy that is pro- ducing milk regularly with few bacteria will, in one or two days, find the number enormously increased, without being able to find any explanation for the fact. The number found at any later hour in any sample is still more variable and will depend upon three factors: 1. The cleanliness of the conditions under which the milk was produced. 2. The age of the milk. 3. The temperature at which it has been kept. Of these three factors the latter is most important in determining the numbers. There has grown up in recent years a strong demand that the number of bacteria in milk offered in market shall be kept low. While the presence of large numbers is liable to a very errone- ous interpretation, no doubt it is desirable to keep the bacteria down to as small a number as possible. The question how this can be done is forcing itself more and more on the attention of the milk producer. We have noticed in the last chapter the various devices which are being adopted in dairies for reducing the original bacterial contamination. We have next to notice methods adopted for placing milk containing as few bacteria as possible in the hands of the consumer. 144 TREATMENT OF MILK FOR MARKET 145 TRANSPORTATION The problems of transportation resolve themselves into two: i. Temperature. 2. Protection from contamination. The various devices by which these ends are reached cannot be con- sidered at length here. In this country milk handlers have learned to use ice freely, and by means of it milk may be trans- ported for long distances upon the railroad and still be fairly cool and sweet when it arrives at its destination. The ice car has made this practicable. To get the best results the milk should be cooled to as low a temperature as possible before be- ing placed in the car, and then kept cold during transportation by the use of plenty of ice. It is hardly necessary to say that the cooler the temperature maintained the more satisfactory the result. In transportation the milk is commonly sent in cans. For practical reasons of economy this has seemed to be necessary for large supplies of milk, although it would be a far better plan to transport it in glass jars, if this were feasible. The cans used in transportation should have a tightly fitting cover, which overhangs the top of the can and thus prevents the entrance of dirt. But the cans ordinarily used are themselves the source of much trouble and the origin of much of the bacterial contamination of the milk. A glance at the battered, rusty and dirty milk cans that one finds at a railroad station will convince him that not a little of the bacterial content of city milk comes from transportation. Of course, the expectation is that these cans will be washed be- fore milk is placed in them again, but battered, rusty cans are practically incapable of being washed clean by any- method adopted in an ordinary dairy. Cleaner cans, more frequent re- placing of old with new ones, would do much toward improving the condition in which the milk reaches the city. In the handling of the milk after it reaches the city there is another chance for contamination. The story is simply that 146 PRACTICAL DAIRY BACTERIOLOGY of the dairy repeated, and the same precautions are necessary. Cleanliness and sterilization must be the watchwords. The use of glass jars is a very great improvement over the older FIG. 55-RESULTS OF THE ROU.GH USAGE ON THE RAILROAD It is almost impossible to clean the can on the left method of delivering from cans, chiefly because they are sure to be cleaner. They are smooth so that they are easily washed and transparent so that dirt is quite sure to show. Then, too, after they are once filled and covered with their caps they are quite well protected from external contamination on their jour- ney from the factory to the consumer. All of this contributes to the purity of the milk. Of course, all glass bottles must be thoroughly washed and sterilized. TREATMENT OF MILK FOR MARKET 147 INFECTION IN THE HOME The consumer of the milk needs a caution concerning his method of keeping it as well as does the farmer for producing it. It makes no difference how fine a quality of milk may be furnished, it will not remain in good condition unless properly treated. In many cases faults and complaints that arise from the consumer are due to his own carelessness and not to the character of the milk which he receives. He places milk in a pitcher that has not been carefully washed and that still con- tains some bacteria from the last sour milk which it held. The milk is placed in a warm room and allowed to stand for some hours before being placed in an ice chest. If placed in an ice box, this is presumably filled with ice, but the ice frequently runs low, and the temperature rises. Under such conditions, especially in summer weather, trouble is sure to rise, and the milkman will receive the complaint that the milk will not keep when the trouble is due entirely to the treatment from the cus- tomer. For his own advantage the customer should be careful that the milk vessel in which the milk is placed has been thor- oughly cleaned, that it is kept cold, that it is not allowed to be- come warm under any condition, and that it is not placed in a position where dirt or bad odors can reach the milk. If these suggestions are followed, the complaints of bad milk will cer- tainly be lessened. THE USE OF PRESERVATIVES The use of preservatives in milk is at the present time to be unhesitatingly condemned. Whether in time perfectly harmless preservatives may be discovered cannot be predicted. But in this country the use of preservatives is, at present, illegal and cannot be recommended under any circumstances. It is, there- fore, unwise here to give any directions as to the use of such materials. 148 PRACTICAL DAIRY BACTERIOLOGY The preservatives that have been most commonly used for the purpose may, however, be briefly mentioned. The most com- mon- in use in this country is formalin. This is a clear liquid, which is a very efficient germicide, and is the basis of most, if not all, of the liquid preservatives on the market under different names. It is very efficient as a preservative of milk, even as small a quantity as one part in 40,000 (one teaspoonful to 15 gallons of milk) noticeably enhancing its keeping property.1 Whether this small amount injures the digestibility of he milk is not yet settled,2 although in general formalin makes proteids difficult to digest, even one part to 2,000 being said to do so. But at all events formalin is an adulterant and, as such, for- bidden by law in most places in the country. It is easily detected in milk by chemical means.3 Borax, boracic acid and salycilic acid have also been used, but they are all even more objectionable than formalin. Most of the preservatives of the market put up in powdered form contain one or more of these substances. The use of hydrogen peroxide (H2O2) has been very strong- ly urged in recent years as a preservative in milk.4 This material, when added in small quantity, about one gram per liter (1 oz. to 9 gals.) has the advantage of rapidly disappearing from the milk, especially if subsequently heated.5 After being in the milk for a little, one atom of oxygen that it contains is loosened and passes off, leaving the substance as plain water H2O. Meantime it has destroyed most of the bacteria. It seems to be an efficient and perfectly harmless preservative. It is, 1 Lowenstein. Zeit. f. Hyg., xlviii., p. 239, 1904. Schaps. Zeit. f. Hyg., 1., p. 247, 1905. 2 Chester and Brown. Bui. 71, Del. Exper. Sta., 1905. Trillat. Rev. Gen. d'Lait, III., p. 380, 1904. 3 Eury. Milchw. Zent., iii., p. 173, 1907. * Budde. Nord. Mejeri. Tidn., 1903. Barthel. Nord. Mejeri. Tidn., 1893; also Rev. Gen. d'Lait, II., p. 289, 1903. 0 Rosam-Pilsen. Cent. f. Bact., II., viii., p. 769, 1902. Lukin. Cent. f. Bact., II., xv., p. 20, 1905. TREATMENT OF MILK FOR MARKET 149 however, expensive, and it has not yet been brought into practi- cal use anywhere. It has hardly been tried in this country. The use of oxygen for disinfection has also been strongly recommended. This gas, when passed in quantity into milk under proper conditions, serves as a partial disinfectant. Oxygen gas is, of course, perfectly harmless, and, moreover, it rapidly passes off from the milk, so that it can hardly be called an adulterant. Its use would, therefore, seem to offer little ob- jection, if it can be found to be both practical and cheap. No practical method of using it to purify a milk supply has as yet been offered to the public, and none is in use at present, but it offers promise for the future. STERILIZATION A few years ago it was thought that the solution of the milk problem lay in destroying bacteria by heat; and there developed rapidly a system of milk sterilization. This was first recommended by physicians as possibly making the milk more digestible by cooking, and subsequently was strongly advocated by bacteriologists for destroying bacteria and rendering milk perfectly safe. By sterilization is meant the treatment of milk to a tem- perature sufficient to destroy all bacteria. The word, however, has been used in two different ways, which has given rise to a slight confusion. In order to absolutely sterilize milk it is necessary to use a temperature higher than that of boiling water, for milk is practically sure to contain spores of bacteria that cannot be killed by simple boiling. In order to destroy them a temperature of 2200 to 2300 is necessary, and to obtain this the milk must be heated in sealed chambers under steam pressure. Only milk so heated can really be called sterilized, and this milk is rarely found outside of a bacteriologist's labo- ratory. In common use, however, the word sterilizing refers to milk that has been simply boiled. The boiling of the milk 150 PRACTICAL DAIRY BACTERIOLOGY destroys most of the bacteria that fail to produce spores. The acid bacteria are thus killed since they are not spore producers. Such boiled milk will keep for a much longer time than ordinary milk. Inasmuch, however, as it contains bacterial spores these will in time develop and cause its destruction. The milk is, therefore, not really sterilized. It should be called boiled milk, but in popular language the word sterilization is frequently applied to simple boiling. At one time there was a vigorous attempt made to place upon the market absolutely sterilized milk. This product was sealed in bottles and subjected to heat of steam under pressure, and since it was sterilized it was supposed to keep indefinitely. Milk sterilization has undergone a variety of changes in public opinion, but at the present time it has become almost wholly abandoned as a method of treating milk on anything like a large scale. The sterilization of milk produces a number of chemical changes, which seem to interfere with its digestibility, so that the weak stomachs of infants and invalids cannot so easily digest and assimulate it. While many experiments have shown that sterilized milk is capable of perfect digestion by strong, vigorous persons, the evidence is fairly abundant that for weak stomachs it is not as satisfactory a food as raw milk.1 Sterilized milk, moreover, has the taste of boiled milk, which is unpleasant to many people. It has also been found that in spite of the high heat and the greatest care even such highly heated milk is not always sterilized and, hence, will not keep.2 Tests of the milk from many companies showed none in which the milk could be relied upon as always sterile, and if it could not be depended upon, the value of the process was practically nothing.3 1 Doane and Price. Bui. 77, Md. Agri. Exper. Sta., 1901. Dukes. Lancet, p. 1857, 1901. Variot. Rev. Gen. d'Lait, iv., p. 238, 1905. 2 Fliigge. Zeit. f. Hyg., xvii., p. 272, 1894. 8 Weber. Hyg. Rund., p. 688, 1901. Sidler. Arch. f. Hyg., xlvii., p. 327, 1903. TREATMENT OF MILK FOR MARKET 151 The process of boiling milk in the home for keeping it or using it as an ordinary article of diet is fairly common. It is more common in Europe than in America, and in the larger cities in Europe most of the milk which is directly con- sumed is first boiled. With those in our largest cities who do not have ice chests the custom of boiling the milk is also common, this being done for the purpose of keeping it longer. Such sterilization or boiling certainly has some ad- vantages. Sterilized milk will never distribute any of the specific diseases above referred to, nor will it be the cause of diarrheal troubles due to the excessive growth of bacteria in milk. The inconvenience of boiling the milk, the development of the "boiled taste" that it produces, together with the fact that such milk is somewhat less digestible than the raw milk have prevented a wider adoption of this method. It is prac- tically certain that the solution of the milk problem of our cities is not along the line of sterilizing or boiling. PASTEURIZATION OF MILK This procedure has recently become very widely known. The name was derived from Pasteur, who devised the method for treating wines and thus protecting them from various unde- sirable fermentations. The essence of the process is the heating of a liquid to a moderate temperature, sufficient to destroy many but not all of the bacteria, and insufficient to produce any very great chemical changes. The^heating should be followed by rapid cooling. Pasteurization destroys a large part of the bacteria in milk, and this reduction in number greatly increases its keeping property.1 The temperature at which pasteurization is performed varies considerably in different methods. In some cases a temperature as low as 1400, maintained for an hour or more, is adopted; in other cases the temperature of 1600 is used for a short time, pos- 1 Bitter. Zeit. f. Hyg., viii., p. 240, 1890. 152 PRACTICAL DAIRY BACTERIOLOGY sibly io minutes. Sometimes a temperature slightly above the lat- ter is adopted, but maintained for a shorter time, not more than a minute. Many machines and devices invented as pasteurizers produce quite different temperatures, and the effect, as would be expected, is very different with different methods. This must be borne in mind in trying to explain the effect of pasteurization on milk, and in determining its value.1 The chief effects of pasteurizing are: I. The maintenance of pasteurizing temperatures destroys a large part of the lactic bacteria. Inasmuch as these produce no spores they are quite easily killed, and as a consequence pasteurized milk will not ordinarily sour. When subsequently it shows signs of spoiling, as it will always do in time, the changes which appear are quite different from those in normal, unpasteurized milk.2 2. The temperatures adopted are generally sufficient to kill the germs of specific diseases, excepting tuberculosis. Typhoid fever, scarlet fever and diphtheria germs are all destroyed by com- paratively low temperatures, and pasteurization is quite sufficient to kill them. Five minutes exposure to 1550 will kill typhoid germs, and probably also those of scarlet fever and diphtheria. Whether pasteurization will destroy the tuberculosis bacillus will depend entirely upon its thoroughness. These organisms can be destroyed by a temperature of 1600 maintained for 10 minutes.3 At lower temperatures a longer application of heat is necessary. At a temperature of 1400 the bacillus will be destroyed,4 if the temperature is maintained for a couple of hours under conditions which prevent the formation of a scum on the surface of the "milk."B Hence, the efficiency of pas- 1 Freeman. Arch. f. Pediatrics, 1896. Rogers. Bui. 73, Bu. An. Ind., 1905. 2 Severin and Budinoff. Cent. f. Bact., II., xiv., p. 463, 1902. 3 Bang. Rev. Gen. d'Lait, I., p. 425, 1902. Barthel and Stenstrom. Cent. f. Bact., I., xxx., p. 429, 1901. * Smith. Jour. Exper. Med., p. 217, 1899. B Hesse. Zeit. f. Hyg., xxxiv., p. 346. 1900. Russell and Hastings. Rev. Gen. d'T.ait, III., p. 34, 1903. Russell and Hastings. Ann. Rep. Wis. Exper. Sta., 1904. TREATMENT OF MILK FOR MARKET 153 teurization in destroying the tuberculosis bacillus will depend upon the temperature and the length of the process. It is quite certain, however, that pasteurization as it is now frequently adopted in our cities, which means only a momentary heating of the milk at a temperature of about 1600, is not sufficient to destroy the tuberculosis bacillus. The effect of pasteurization upon the bacteria that are prob- ably associated with diarrheal diseases is as yet unknown, in- asmuch as we do not know what they are. If, as it is believed, these troubles are due to the very great growth of some com- mon bacteria, the use of pasteurization will be efficient in re- moving part of the danger of such troubles by destroying most of the germs. Practical experience has also shown that pas- teurization is a useful procedure in the attempt to control the diarrheal troubles during hot weather. It is a matter of great importance to know whether pasteur- ization of milk has any effect upon its digestibility or ease of assimilation. The heat applied to it is not sufficient to produce the chemical changes which occur in milk when it is boiled or sterilized, or the taste which develops in boiled milk, nor do the albuminoids of the milk coagulate as they do when it is boiled. Laboratory experiments have seemed to show that the digestibil- ity of the milk is not impaired. There has been a general feel- ing in the past that while sterilization injures the digestibility of the milk, pasteurization does not do so. Recently, however, it has been claimed that pasteurization does impair the value of the milk, though laboratory experiments do not show it. It is insisted that its constant use has, in some cases, seemed to result in a certain type of disease, called rickets, that has been attributed to improper assimilation. The instances of this char- acter are few and, as yet, somewhat uncertain. They have, however, produced in recent years something of a prejudice against the use of pasteurized milk. They are too rare as yet to constitute any valid argument against pasteurization.1 1 Doane and Price. Bui. 77, Md. Exper. Sta., 1901. 154 PRACTICAL DAIRY BACTERIOLOGY The efficiency of pasteurization differs much with the method adopted. When well done it may reduce the bacteria very greatly, for example, from 3,000,000 per c.c. to 10,000 per c.c., or even less. If less efficient the number at the close of the process would be perhaps some hundreds of thousands. The efficiency depends upon several factors. Dirty milk jars, or jars with dirty rubber rings, produce trouble, and other careless methods of handling decrease the value of pasteurization.1 The most considerable factor affecting it, however, is the temper- ature, together with the length of exposure of the milk to the temperature. In general it is best to use lower temperatures for a longer time than higher ones for a shorter'period, and it is doubtless best to pasteurize at a temperature no higher than 140°, if the process is maintained for a long enough period, e.g., two hours; for this destroys all disease germs and produces the least change in the milk.2 It is also important to do the heating in such a way as to avoid the formation of a scum on the surface of the milk, for such a scum makes' the process less efficient in destroying disease bacteria. This can be done by some device to produce a constant agitation. Pasteurization is sometimes adopted in a household on a small scale and sometimes in a central station for large quantities of milk. When adopted in the household it can be done by a very simple, easily devised apparatus. Figure 56 shows an apparatus which makes it possible to pasteurize the milk without the necessity of using a thermometer. The milk is placed in bottles, which are placed in a receptacle that has been partly filled with boiling water. The whole apparatus is then set aside to cool. The milk lowers the temperature of the water, and the hot water raises the temperature of the milk until they each reach the same temperature, after which the whole apparatus cools down. By proper adjustment of the size of the bottles and the 1 Swellengrebel. Cent. f. Bact., xii., p. 440, 1904. 2 Jensen and Plattner. Rev. Gen. d'Lait, iv., p. 419, 1905. TREATMENT OF MILK FOR MARKET 155 amount of the water it is possible, after a little testing, to ar- range the apparatus so that the milk will acquire just the temperature of pasteurization before it begins to cool. The use of such an apparatus is extremely simple, and after it is once adjusted can be successfully manipulated, even by those who do not understand the use of the thermometer. Home pasteur- ization has come to be a very common practice. The danger to children from tu- berculosis and other troub- lesome diseases in the milk has not yet been guarded against by any public means. Hence, there is a growing tendency on the part of people of intelligence who are bringing up children on milk to pasteurize it. This plan is to be recommended in cases where the child must depend upon milk the source of which cannot be strictly relied upon. Per- haps such pasteurized milk is not as valuable a food as unpasteurized milk from absolutely reliable sources, but with the conditions in our cities, it is almost impossible to find milk that can be strictly relied upon to contain ijo dangerous disease germs. Under these cir- cumstances pasteurizing is the only safe method by which a young child can safely be fed upon milk. FIG. 56-SIMPLE IMPROVISED METHODS OF PASTEURIZING WITHOUT THE USE OF A THERMOMETER (RUSSELL) 156 PRACTICAL DAIRY BACTERIOLOGY PASTEURIZING ON A LARGE SCALE In the last few years the plan of pasteurizing the milk on a large scale has come to be frequently adopted. This is done sometimes in creameries in connection with butter-making, and more frequently in some of our large cities for treatment of the general milk supply. In some countries the pasteurization of cream for butter-making is practically universal, but this is not true in America. In the pasteurization of the public milk supply the purpose has not been primarily to protect the public but to keep the milk from souring. Milk distributers have found it difficult to furnish milk that will keep without preservatives, but have learned that the application of heat enables them to do so. For this reason pasteurization has become widely adopted by large milk companies. The pasteurization of milk on a large scale involves the use of special machinery, which it is not the purpose of this book to describe in detail. Two different principles are involved in the different pasteurizing machines.1 One class is called discon- tinuous pasteurizers. (Fig. 57.) In these the milk is placed in a large receptacle and then heated, usually by steam running through coils of pipes of special devices. During the heating it is constantly agitated and is retained at the desired temperature for any length of time that may be wished: after which it is cooled by the running of cold water through the same pipes, or by some other device. It is then removed as pasteurized, and more milk takes its place. The second class is called continuous pasteurizers. Here the milk flows into a machine in a constant stream, and after passing through a series of conduits in which it is heated to the desired temperature, it is passed through others where it is once more cooled, and finally emerges from the machine in a continuous stream of cool, pasteurized milk. The efficiency of continuous pasteurizers depends naturally upon 1 Weigmann. Milchztg., p. 417, 1901. TREATMENT OF MILK FOR MARKET 157 the length of time that the milk remains in the machine. If the milk runs too rapidly, it will not be sufficiently heated; if it runs too slowly, it may be overheated; and the results, there- fore, will vary widely.1 Either the continuous or the discontinuous method may be efficient with proper care. The first is sometimes called in- stantaneous pasteurization since the actual heating of the milk FIG. 57-PASTEURIZER OF THE DISCONTINUOUS TYPE The milk is placed in the inner compartment. (D. H. Burrell & Co.) is very brief, and the second is called prolonged pasteurization. It is obvious that the prolonged or discontinuous method will commonly be the more efficient. In the use of the continuous method it is a great temptation to hasten the process, thus re- ducing the time by running the current of milk too rapidly to allow a sufficient heat, and thus reducing the efficiency of the 1 Russell. Ann. Rep. Wis. Exper. Sta., 1905. 158 PRACTICAL DAIRY BACTERIOLOGY process. Indeed, the milk is commonly heated no longer than one minute. While either method may thoroughly pasteurize the milk, there is a larger chance of error in the continuous pasteurizers, and practical study has shown that, as they are commonly used, the pasteurization by the continuous pasteurizer is less thorough than by the use of the discontinuous machines. As frequently used in the large milk supply companies, the pas- teurization is of little value in improving the healthfulness of the milk. The milk is brought to the required temperature for an instant only, just sufficient to reduce the number of acid bacteria and thus increase the keeping quality of the milk, but not long enough to have much influence on the disease germs. There is, to-day, a move being made to bring this under proper inspection. If pasteurization can be properly controlled and placed under inspection so that its efficiency can be assured, it will be a very simple means of removing from the milk the danger of disease distribution. In pasteurization the cooling of the milk after heating is of as much importance in preserving it as the original heating. The process does not kill all bacteria, and those remaining will subsequently grow rapidly unless the milk is cooled at once. From this it also follows that the milk should bl used as quickly as possible, for while it will keep longer than unpasteurized milk the bacteria are growing in it all the while, and, since the acid bacteria are lacking, the putrefactive germs will have a greater chance to grow than usual. Old pasteurized milk is, therefore, worse than sour milk. It should be recognized also that only fresh milk should be pasteurized. After it has begun to sour the heat may curdle it, and it is never wise to use this treatment upon milk that contains more than 2 per cent. acid. Pasteurization is sometimes applied to cream to enhance its keeping and enable it to find a market. The cream keeps well, but it loses some of its consistency. Such cream appears thinner than before treatment, and it will not whip as will ordinary TREATMENT OE MILK FOR MARKET 159 cream. Its consistency may be restored by adding a little of a material called "viscogen." 1 This is made by adding a strong solution of cane sugar to freshly slacked lime and allowing the mixture to stand until the upper part of the mixture is clear. This clear liquid is poured off and added to the cream in propor- tions of i part to 100 or 150 parts of cream. This restores the consistency to the cream, but since it is an addition of a foreign substance its use is illegal. The effect of pasteurization upon the fat of milk proves a detriment in another way, since it interferes with the ready separation of the fat from milk. When the fat of normal milk is examined under the microscope the globules are found to be bunched together in little groups; but the fat globules of pasteurized milk are nearly separate from each other. This interferes with the cream rising as usual and there is no sharp line separating the cream and the milk after standing. This does not interfere with the use of the milk, but, if it is desired to separate the cream from the milk it cannot be done so easily as with raw milk. In the feeding of infants it is some- times desired to separate the top third for separate use, this third containing the most fat. Pasteurized milk cannot be readily treated in this way, so that the absence of a "cream line" is a real disadvantage. TYPES OF MARKET MILK The various suggestions for improving the quality of milk above given will, when followed out, produce a type of milk far different from that which has been common in our markets in past years. But it is impossible to carry out these various direc- tions without expense. It is impossible to produce a high grade of milk for a low price. To obtain a type of milk that is being demanded by health officers will involve an expenditure which will evidently raise the price of milk. As soon as the public is 1 Babcock and Russell. Bui. 44 Wis. Exper. Sta., 1896. 160 PRACTICAL DAIRY BACTERIOLOGY ready to pay the cost for the precautions suggested, the dairy- man will be willing to adopt them, but he cannot do so and furnish milk at the small price that has been paid for milk ob- tained under poorer, more careless conditions. As a result of this we find in our markets, to-day, several different types of milk. Sanitary Dairies.-Near our large cities there have been ap- pearing in the last decade sanitary dairies, in which all possible precautions are taken to produce milk of the highest grade. In these dairies the precautions given above are carefully followed out, and the greatest care is taken to produce a product above suspicion. The milk from these dairies frequently sells at twice the price of ordinary market milk, and even higher, 20 cents per quart being sometimes paid. Such milk is certainly more reliable and less likely to produce trouble than ordinary milk. Although there is already a large demand for such milk, these sanitary dairies do not meet the milk problem, for only a very small portion of the milk supply will ever be thus pro- duced. The higher prices naturally prevent the people at large from purchasing it, and it is only the small number of persons, who have been willing to pay any price necessary to obtain a good product, who will receive the benefit. At the present time, therefore, sanitary dairies, while they have their place, do not reach the root of the milk problem. That the precautions adopted in such places are efficient in reducing the number of bacteria is shown by the following figures obtained from the averages of a large number of samples of milk from four different sanitary dairies: NUMBER OF BACTERIA IN MILK FROM SANITARY DAIRIES Dairy No. I in winter 930 per c.c. Dairy No. 1 in summer 1.050 " " Dairy No. 2 1.400 Dairy No. 3 2,000 Dairy No. 4 280 TREATMENT OF MILK FOR MARKET 161 In Dairy No. 4 a separate milking room was used (Fig. 47). These numbers are very small, and the value of care in the dairy can be seen when the figures given above are compared with a similar series of averages from some common dairies. The best result in this second series was 60,000 per c.c., and the worst was 19,000,000 per c.c., in the milk, at the time it was delivered at the milk company's collecting station. Certified Milk.-A somewhat different method of reaching the same end is by the production of certified milk. This is milk from dairies that have been inspected and passed upon by a certifying board as producing milk of satisfactory character. The Certifying Board is not always constituted in the same way, but it is usually in some way connected with health officials. The methods that are taken by these Boards to assure themselves that the milk is satisfactory are not always the same. They usually, however, involve an inspection of the dairies and a frequent chemical and bacteriological analysis of the milk. Dairies whose methods and whose milk meet a certain standard thus set by the Board receive a certificate for a certain length of time, which will be renewed later, if there are no reasons for the contrary. Such certified milk has certain advantages and certain disadvantages. The purchaser knows that he is obtaining milk that has been produced under at least fair con- ditions. He must pay a little more for it, although the price is always less than that of the milk of sanitary dairies, because less care and expense are involved in its production. But, on the other hand, no sample of milk, even if it is certified, can be guaranteed as free from disease germs or other contamina- tion. The Certifying Board can certify only as to the general conditions of the dairy that produced the milk, never as to any particular sample of milk. It sometimes happens that certified milk is of no better character than the ordinary market milk, and sometimes it is not even so good. It is, however, on the average safer, and is becoming quite widely used. 162 PRACTICAL DAIRY BACTERIOLOGY Not a few farmers in the vicinity of our larger cities have recognized the possibility of furnishing certified milk and ob- taining a slightly higher price for their product. It is not diffi- cult for any farm to produce such changes in its dairy methods as will enable it to obtain the certificate of such a Board. The necessary precautions are not great or expensive, and inasmuch as a somewhat higher price may be obtained the plan is a very promising one and is being more or less adopted by up-to-date dairymen. Whether this plan of certifying milk in our larger cities is to develop into a more general method of procedure or whether some more radically different method of furnishing the city with milk is to be adopted cannot yet be stated. The Production of Certified Milk.-While ordinary dairies are unable with the present price of milk to adopt all of the precau- tions suggested and advised above, there is no farm where certain changes cannot be made with so little expense as hardly to raise the price of production, but at the same time sufficient to produce a marked improvement in the milk. The production of certified milk does not necessarily involve great outlay, but may be done with fair success upon any good farm. It may be useful, therefore, to enumerate the simpler precautions which are most useful in improving the grade of the milk, the adop- tion of which will usually enable a dairyman to furnish certified milk. The following precautions are arranged according to their importance: I. Veterinary inspection of the herd and testing all animals with tuberculin, followed by the rejection of all reacting animals. 2. Washing and sterilizing milk vessels and all dairy utensils. This should be done more thoroughly than is common on the ordinary farm. 3. The cleaning of the cows, clipping hair on the udders and hindquarters, the moistening of the udder and underside of the flanks before milking. TREATMENT OF MILK FOR MARKET 163 4. The use of a covered milk pail. The best are made of aluminum, since they are spun without seams. Milk should never be allowed to sour in them, for this will so infect them with bacteria that it will be very difficult to clean them. These pails should be used for no other purpose except to hold milk. 5. Covering the cans tightly when filled with milk. 6. Cleaning the cow stalls and barns. Anything short of clean stalls is repulsive. 7. Immediate cooling to as low a temperature as possible short of actual freezing. 8. Feeding after milking. The other precautions which have been suggested above are of less striking effect. They may, perhaps, be placed in the fol- lowing order: Discarding fore-milk-clean hands and clothes for the milker -clean bedding for the cows-removal of milk from stall im- mediately after milking-ceiling of stables kept free from dust, preferably plastered. There is no reason why any farm cannot adopt the precautions of better kept cows, cleaner stables, covered milk pails and more careful washing of the vessels, and every farm which will pay greater attention to these will obtain at once an improved grade of product. Indeed, many a dairyman by the simple adoption of these precautions, No. 1 to No. 8, will readily obtain the certificate of the Certifying Board. Condensed Milk.-For many years condensed milk has been widely sold on the market. In condensing, a considerable por- tion of the water is removed by evaporation, and then fbr the purpose of preserving it a considerable percentage of cane sugar is often added. There is thus obtained a condensed product with a large amount of sugar, and one which will, when placed in sealed cans, keep for a long time. The large amount of sugar present very materially injures the value of the milk as a food, rendering it less digestible. For this reason, while condensed 164 PRACTICAL DAIRY BACTERIOLOGY milk is useful for some things, it can never be substituted for milk in an ordinary diet. It has undoubtedly a place in our food products, but it does not, and it cannot, take the place of milk for most purposes. Unsweetened condensed milk is also upon the market. This material is sterilized by heat after being condensed to the desired volume. It is then sealed and it keeps simply because it is steril- ized. It has the advantages and disadvantages of sterilized milk, and while an article of value for some purposes it adds little to the solution of the milk problem of cities.1 The preservation of sweetened condensed milk is due to its great sugar content, which prevents the growth of bacteria. But sugar will not prevent the growth of yeasts. We have already seen (page 9) that yeasts can grow in sugar solutions. While bacteria do not usually spoil condensed milk it is sometimes found, to the misfortune of the manufacturer, that yeasts may ferment it. Immense losses have occurred in the condensed milk industry in the last few years by the growth of fermenting yeasts. Bacteria can usually be found in condensed milk also, but they do not develop. Hence, if yeasts do not produce trouble, the material keeps well for a long time. Evaporated Cream.-This is a milk product which has been on the market for a few years, although not very widely used. It consists of milk with a varying amount of cream, evaporated to less than half its bulk and thoroughly sterilized by heat. It is then sealed in cans and will keep indefinitely. It has some of the disadvantages of sterilized milk, having much the same taste. Little is known of its digestibility, although laboratory tests2 have shown that it is equally digestible with raw milk. Like condensed milk it has its place among food products, but it will not replace raw milk as an article of food. 1 Kasdorf. Rev. Gen. d'Lait, I., p. 73, 1901. 2 Mojonnier. Med. News, 1905. TREATMENT OF MILK FOR MARKET 165 Concentrated Milk.-A new method of treating milk has been recently devised which offers some exceptional advantages. The process is covered by patents, but it consists in the withdrawal of a considerable portion of the water from the skim milk, and the subsequent remixing of the product with the cream in proper proportion. In its preparation the use of heat by special devices is adopted, and the skim milk is treated for about two hours to a temperature of 1400. This reduces it to a thick mass which resembles cream. The cream, which has meantime been pas- teurized, is now mixed with it in proper proportions, and the material is then known as concentrated milk. When the proper amount of water is subsequently mixed with this concentrated milk, 3 parts of water to I part of milk, it is restored to its original condition so closely as to be almost indistinguishable from fresh milk. It tastes the same, it curdles with rennet, cream rises upon it in much the same way as on fresh milk, and so far as experimental tests have determined, its digestibility is not in any degree impaired. The advantages of concentrated milk are several. I. Its treatment at a temperature of 1400 for two hours absolutely destroys all disease germs, including the tuberculosis bacillfls, so that there is no possibility of the product distributing in- fectious diseases. 2. The treatment reduces the number of bacteria so greatly that the chance of the milk being the cause of diarrheal troubles is practically removed. 3. The condensa- tion of the milk increases the proportion of milk sugar. This produces a substance in which bacteria do not grow readily, although they grow rapidly enough if it is diluted with its normal amount of water. As a result concentrated milk may be kept, if cooled, for a very much longer period than any type of fresh milk. If kept at a temperature of below 500, it will not only keep sweet for six to eight days, but at the end of this time the number of bacteria will be surprisingly few, for example: in a series of tests, after six days the milk contained less than 166 PRACTICAL DAIRY BACTERIOLOGY 500,000 bacteria per c.c. After some days the bacteria will begin to develop and eventually become abundant, but during the first few days after its preparation it is possible to transport such material for long distances, and yet place it on sale in a city in a better condition, at least so far as bacteria are concerned, than most of the milk which now reaches the larger markets. 4. The expense of the transportation of such milk is very much reduced because of the decrease in bulk. 5. The concentrated milk is itself pasteurized, and because of its method of prepara- tion is thoroughly pasteurized. The condensation of the milk is not possible by this method except by long-continued heating at, at least, a temperature of 1400, and this is sure to complete the pasteurization. This low temperature is not sufficient to give any cooked taste or apparently to produce any chemical changes except the evaporation of the water, in this respect being much better than pasteurization at a higher temperature; but it is sufficient, as shown by careful tests, to destroy the pathogenic bacteria of milk. For these reasons concentrated milk promises great aid in solving the milk problem of cities, since it offers at a low price milk that cannot be distinguished from ordinary milk, and yet can be guaranteed as free from pathogenic bacteria; and it will keep well. Attempts are being made at present to put this product upon the market in this country. Milk Powder.-Many attempts to reduce milk to a powder have been made with more or less success.1 The devices for this are several and need not concern us. In all cases where milk is reduced to a powder it is capable of being kept for a long time. Where the milk contains the cream, however, the reduction to the dry powder does not prevent its eventually be- coming rancid, and powdered whole milk will not keep indef- initely. Powdered skim milk will keep without much trouble. 1 Jensen. Rev. Gen. d'Lait, IV., 1905. Jluyge. Rev. Gen. d'Lait, HL, n. 320, 1904. TREATMENT OE MILK FOR MARKET 167 These products have come to be well known in the last few years, and there are several of them, made by different processes, now on the market and widely used. None of them, however, really takes the place of fresh milk, because in no case has it been found possible to redissolve the powder again and get a milk that behaves like fresh milk. They are useful, especially in cooking, and in a variety of other ways, but it is doubtful if dried milk will ever replace the fresh milk of our cities. Frozen Milk.-A method of meeting the milk problem by freezing the milk into cakes and pressing it in forms of bricks has been adopted in some localities in Europe.1 The difficulty of its success has been in part that the freezing changes the character of the milk slightly, so that it is not always possible to melt the bricks of ice and obtain a good quality of milk. At all events this process has not been widely adopted and does not, at the present time, seem to promise a solution of the milk problem. 1 Hittcher. Milchztg., p. 292, 1902. Bischoff. Arch, f." Hyg., XLVII., p. 68. CHAPTER VHI PUBLIC PROBLEMS A number of very vital questions have arisen in the last ten years concerning the relation of milk to the health of our com- munities, and the problem of the milk supply is becoming a very pressing one. Although this problem has not yet been solved, a number of factors have been fairly well settled. In the first place it may be stated beyond peradventure that cheap milk is always poor milk, and an improvement in the quality of the milk must mean an advance in price. We have just seen in the last chapter what a great amount of care is necessary to guard the milk from various sources of undesirable contamination and to preserve it after it has been produced. It is impossible to produce good milk at a low price, and the only hope for a better quality of fresh milk is that the public should be ready to pay a price sufficient to reimburse the producer for the extra care necessary to produce it. THE CITY MILK SUPPLY The condition in which milk reaches our larger cities varies greatly under different conditions. The bulk of it is brought in upon ice cars and consequently kept fairly cool; but the temperature varies greatly, with the different seasons of the year, with the amount of ice used and the care that can be given. It is the ice car that has made it possible to concentrate in our large cities the immense milk supply that is demanded. Since New York demands a quantity of milk so great as to require it to reach 400 miles for part of this supply, it is evident that only with the use of the ice car can this demand be met. It is also 168 PUBLIC PROBLEMS 169 evident that such milk cannot be furnished in the city in a very fresh condition. In fact, in some of our cities a large part of the milk is 48 hours old when distributed. Some of it may not be more than 24 hours old, but only a very small portion of it, from dairies close at hand, can be regarded as really fresh. It is not a matter of very great importance whether the milk is fresh or not, if it is only possible to keep it for the length of time necessary under conditions in which the bacteria will not develop too abundantly. If, therefore, it is kept sufficiently cool, milk from a distance may be, at the end of 48 hours, in a much better condition than milk from nearer dairies that has been brought into our cities, fresh, but that has not been properly cooled. The distance of transportation, therefore, is not neces- sarily a factor to render milk less wholesome, provided it is properly handled. There has been a very great improvement in the conditions regulating the milk supply in the last 15 years. The milk is produced on farms with more care; farms in general are more cleanly, some of them very much more cleanly than formerly; and more attention is paid to the quality of the milk. This improvement in the dairy has resulted in an improvement in the milk, and at the present time our larger cities are supplied with a better grade of milk than before. Curiously enough, these statements cannot be made in regard to the milk of the smaller communities, for these are sometimes at a disadvantage in comparison with the larger ones. In some states the larger cities have very stringent rules regulating the milk that can be sold, rules which exclude the milk from many of the poorer classes of dairies. But these same dairies, whose milk cannot be sold in a city, may send their product to the smaller towns and dispose of it without difficulty. This condi- tion is doubtless only temporary, for laws will probably be adopted by which the smaller communities will be as well pro- tected as the larger. 170 PRACTICAL DAIRY BACTERIOLOGY Need of Public Inspection.-The recognition of the close asso- ciation between the purity of the milk supply and public health has given rise to a large amount of work on the part of health boards and others directed toward the control of the milk supply. There is no food more easily adulterated than milk. Water can be added to it, and a variety of disinfectants and preserva- tives can be put in it without j:he consumer having any suspicion of it. For this reason the regulation of the milk supply has demanded a special set of rules quite different from those ap- plicable to other foods. The individual is quite incapable of protecting himself, and he must, therefore, depend upon the expert knowledge of public officials, whom he must appoint for the purpose. The difficulty of the problem has rendered the methods of inspecting and controlling the public milk supply still more or less uncertain and tentative. Some of the rules and regulations that have been adopted and are now in force are undoubtedly of high importance, and some probably are mistakes which will be corrected as more information and better ideals prevail. The ends to be reached by a public control of the milk supply are three: I. Chemical purity. 2. Freedom from specific disease germs. 3. Clean milk. We are not concerned here with chemical problems, and the matter of chemical purity is mentioned here only because the same set of officials are likely to be called upon to consider chemical and bacteriological problems. The methods of testing milk may be found in books designed for that purpose. At this point it is only necessary to state that the chemical purity of milk is fairly well insured by public milk inspection. The dif- ferent states have different laws as to the standard which a pure milk should reach, and they are in general so efficiently en- forced that the public milk supply has a very fair grade of chemical purity. CHEMICAL PURITY PUBLIC PROBLEMS 171 The specific disease germs liable to be distributed by milk we have already noticed. There seems to be only one method by which milk can be guaranteed as free from disease germs. This method is by treating the milk to a heat sufficient for pasteuriza- tion. Inspection of the dairies will undoubtedly reduce the danger of disease germs getting into milk; but recognizing the wide occurrence of bovine tuberculosis, the difficulty of detecting it and the fact that the cows may have this disease in the udder unsuspected; recognizing, too, that cases of walking typhoid fever are possible, unsuspected by the patient or his friends, it will be seen that no kind of dairy inspection, no matter how rigid, can free the milk from the possibility of disease germs. No subsequent care or cooling of the milk can destroy them, if they have found entrance into the milk. Under these cir- cumstances the sole practical method of assurance that the milk shall contain no specific disease germs is by adopting pasteur- ization or sterilization. All other methods of controlling the milk supply must be looked upon as a means of improving its gen- eral cleanliness and wholesomeness, and reducing the chances of the presence of the disease germs; but none of them can guarantee that the milk will be free from pathogenic bacteria. The danger of infecting the milk by germs of contagious dis- eases is so insidious that even in the best kept dairies and with the best intentions it will sometimes happen. Some of the san- itary dairies, kept under the highest condition of cleanliness, have been suspected of having distributed disease. Therefore, it follows that no methods are known by which a supply of fresh untreated milk can be positively guaranteed as free from disease germs. It must be recognized, however, that dairy inspection and the methods adopted for the improvement of the quality of the milk in general do greatly improve the milk by decreasing the chances of the distribution of the disease germs, but they do not entirely remove them. FREEDOM FROM SPECIFIC DISEASE GERMS 172 PRACTICAL DAIRY BACTERIOLOGY So far as concerns the likelihood of milk being the cause of the diarrheal troubles, the matter stands differently. These ap- pear to be associated with the development of bacteria in the milk, and any of the dairy methods already pointed out, which reduce the multiplication of bacteria, and which bring about a condition where the milk on sale in our markets shall contain a comparatively small number, will probably tend to reduce these dangers. CLEAN MILK The desirability of having clean milk needs no emphasis, but the methods by which this can be accomplished, and the methods by which it can be determined whether milk is clean, are not so simple. How is it possible to study miscellaneous samples of market milk and determine whether each was produced under conditions of proper cleanliness and with proper care? This question in one form or another has been asked over and over again; but up to the present time it has received no satisfactory answer. The method of determining this by actually estimating the amount of dirt in the milk, as indicated on page 297, is quite impractical. The accuracy of the dirt determination is so uncertain and the results in the hands of the different people show so much variance, that it has hardly been seriously sug- gested that the determination of the dirt in milk shall form a part of the municipal inspection. While such determination is of value, it is as yet impractical for the purpose of controlling or testing the public milk supply. A method that has been gradually coming into adoption for this purpose is the determining of the number of bacteria in the milk. It is fairly easy to determine, with moderate accuracy, the number of bacteria, and if we assume that the value of the milk is in proportion to the smallness of the number of bacteria, it is fairly easy to reach conclusions as to its wholesomeness. Recognizing, as we have done, that bacteria-free milk is impos- PUBLIC PROBLEMS 173 sible to obtain except by sterilizing, it follows that all market milk will contain some bacteria. Hence it is necessary, if this is to be our means of determining quality, to set a standard as to the number of bacteria that may be legitimately found in proper market milk. How large a number is consistent with the proper condition of the milk when it is placed upon the market ? PUBLIC CONTROL OF THE MILK SUPPLY The growing recognition of the dangers in the milk supply and the inability of the individual to handle the problem for himself has led to a demand that the public health officials shall in some way guarantee the purity and healthfulness of the whole supply. The necessity for such a control is apparent, but it is not so evident how it can be brought about. Three quite different methods of reaching the end have been advocated: 1. The use of standards. 2. Dairy inspection. 3. Compulsory pasteurization. THE USE OF STANDARDS Milk standards may be arbitrarily fixed for chemical and bacteriological purity, and by constant analysis the dairies or dealers that fail to reach the standard may be determined. For chemical purity this works fairly well. For bacterial purity its value is not so clear, for even after a standard has been set, the enforcing of it is difficult and of doubtful wisdom. Numerical Bacterial Standard.-The actual numbers of bac- teria found are very great in the milk of large cities. In Chicago, a long series of tests showed it to range from 100,00.0 to 1,000,000 per c.c.; in New York, from 250,000 to 5,000,000: in Baltimore, from 1,500,000 to 4,500,000. The milk delivered to factories ranges from 5,000.000 in winter to 30,000,000 in summer. In small towns and cities the average is considerably lower than in the larger cities because of nearness to the source PRACTICAL DAIRY BACTERIOLOGY 174 of supply, 10,000 to 50,000 being frequently found; but even in these places many samples of milk show much larger numbers.1 With such wide variations it clearly becomes difficult to de- termine upon any standard of numbers. At present, only in a few places has the attempt been made to establish such a stand- ard. Boston has tried to enforce a standard of 500,000 per c.c., while it has been doubted whether New York could reach a standard of 1,000,000. Smaller standards than either are aimed at by some smaller cities, and for special grades of milk 30,000, or even 10,000, are sometimes demanded. Of course, it is not to be assumed that the milk that comes to New York is twice as dirty as that which reaches Boston, and hence it is evident at the outset that the number of bacteria alone is not a satis- factory means of determining the cleanliness of the milk. The method of determining the number of bacteria in milk is fairly simple. Special collecting cases are provided, con- taining vials capable of being closed and cooled with ice. (Fig. 58.) These are filled with samples of milk to be tested, from the cars, delivering wagons or elsewhere. In collecting the samples it is necessary to stir thoroughly the contents of the can or jar which is to be sampled, and, of course, to de- termine and record with accuracy its source. Care in collecting the sample is of the utmost importance, for the milk at the bot- tom and the top of a can may be quite different. It is also customary to test the temperature of the milk at the same time and make a careful record. The samples thus collected are to be packed in ice and carried immediately to the laboratory. In the laboratory they should be immediately tested by ordinary methods, agar culture media being used in most cases at a dilu- tion of 1,000 (see page 273), the plate being incubated at a tern- 1 Jordan. Analyses of Chicago Market Milk. Chicago, 1904. Park and Bebb. N. Y. University Bui. I., No. 2, 1901. Harrison. Trans, of Canadian Inst., vii., p. 467, 1902-3. Bergy. Univ, of Pa. Med. Bui., 1904. PUBLIC PROBLEMS 175 perature of 98°. With this method the plates may be counted in 24 hours, and the number of bacteria in the original sample of milk determined. The method of testing by the microscope, given on page 285, is adopted in some places with success, and gives a very much FIG. 58-COLLECTING CASE FOR MILK SAMPLES The samples are removed from the cans with sterilized pipettes, and placed in tubes. These are placed in weighted racks (.A) and put into the center compartment of the carrying case. more rapid method of determining whether the sample of milk has such a small number of bacteria in it that it falls below a standard of 500,000 per c.c., or whether the number is so large as to run far beyond this. By the culture method at least 24 hours must elapse; by the microscopic method the results are fairly quick. In using the microscopic method it is desirable, if the microscope shows that the number of bacteria is probably in the vicinity of the 500,000 limit, to make agar plates at once in order that the test of the microscopic method may be con- 176 PRACTICAL DAIRY BACTERIOLOGY firmed by the test of the plating. If, however, the number of bacteria in a single microscopic field is not more than 20. to 30, the plating is unnecessary for determining that the number of bacteria is within a 500,000 per c.c. standard. Significance of the Milk Standard.-When we consider the real significance of determining the number, we are forced to conclude that it is less than could be desired, and indeed it be- comes questionable whether as now done its real value is worth its cost. The number of bacteria in any sample of milk depends upon three factors and not upon one. It is true that the original filth or cleanliness of the dairy, together with the care in pro- ducing the milk, is a factor contributing thereto, but the age of the milk and the temperature at which it is kept are, also, important. Indeed, as we have seen, the age and temperature are of more significance in determining the number of bacteria in milk than is the question of the original cleanly or filthy conditions. It is true that, even if fresh samples of milk are examined, the number of bacteria in the different samples will vary. Milk produced under good conditions may have as few as from 5,000 to 10,000 per c.c., while that produced under poor conditions may have from 100,000 to 200,000, or in some cases, perhaps, half a million per c.c. In other words, the cleanliness of the original conditions of milk production will determine variations in the number of bacteria in the milk when strictly fresh, ranging from a few thousand to a few hundred thousand. But, on the other hand, age and temperature run these few thousands up into millions, and it is not uncommon to find the number of bacteria rising as high as from 10.000,000 to 20,- 000,000 per c.c. Hence, the high numbers present in our city milk are dependent upon the age of the milk and the temper- ature at which it has been kept rather than upon the cleanliness of the conditions in which the milk is produced. Roughly speaking we may say that variations in number below 200,000 or 300,000 are due primarily to differences in the cleanliness PUBLIC PROBLEMS 177 and care of the milk production; whereas variations in numbers from half a million upwards to several millions must be attrib- uted to differences in the age and temperature of the milk. Furthermore, where we have very high numbers the reason lies practically always in a too warm temperature rather than any other factor. But these high numbers of bacteria usually consist of the harmless lactic acid type, and the fact that the numbers have run up to 20,000,000 may not detract in the slightest degree from the wholesomencss of the milk. High numbers of bacteria, therefore, do not necessarily mean un- wholesome milk; while, on the other hand, milk may have a small number of bacteria and yet contain enough distinct specific disease germs to be decidedly dangerous. One other feature connected with the numerical standard is to be noticed. The method used for determining the numbers by no means gives them all. As stated on page 175, the number is determined by diluting 1,000 times, plating upon agar and incubating at 98°. This really gives only a small part of the bacteria present. If the plates arc made on gelatin and in- cubated at 700, much higher numbers will be found. For ex- ample, a sample of milk diluted 1.000 times, plated on agar and incubated at 370, gave 3.000,000 bacteria. The same milk diluted 10,000 times, plated at the same time on gelatin and incubated at 200, gave 30,000,000. Usually there will not be found quite so large a difference, but there is always a smaller number found by the ordinarily adopted method than by using gelatin and incubating at lower temperatures. Hence, a numerical de- termination never gives the actual number of bacteria in the milk, but only of the numbers that grow upon agar and at temperatures of 98°. Objections to a Numerical Standard.-For these reasons it becomes a serious question as to whether a milk standard is of much value. There are several objections which arise against its adoption. 178 PRACTICAL DAIRY BACTERIOLOGY I. The methods of analysis can never be of value in con- demning any particular sample of milk, but only in giving con- demnation of the dairy that produces the milk or the dealer who distributes it. It usually requires 24 hours before the number of bacteria can be determined, and before this the milk has, of course, been consumed. Even if the number of bacteria can be obtained two or three hours after the sample is received, it is quite too late to prevent the distribution and consumption of any particular sample of milk. All that can be done then is to condemn all of the milk from a certain source on the ground that one or two of the samples of milk have shown a number of bacteria above a certain standard. This is, of course, an unsatisfactory method of dealing with the question, but it is really the only possible one. 2. The use of a numerical standard is likely to be extremely unfair, condemning milk from some sources that should not be condemned and passing milk from other sources that should not be passed. A numerical standard, as we have just seen, is simply a somewhat indefinite gauge of filth, temperature and age combined. One dairy may have conditions of the most ex- treme slovenliness, and yet the proprietor is shrewd enough to cool the milk to a low temperature by the use of ice and thus check the development of bacteria. Under such conditions, though the milk is filthy and the conditions that surround his dairy are most unwholesome, the milk would readily pass the inspection, because the simple use of ice has prevented the bacteria from developing. The milk is likely to contain much that renders it unwholesome, but it will pass inspection. On the other hand, a dairy at a little greater distance from the city might be kept under conditions of scrupulous care in the milk production, and yet because of the use of less ice and thus less cooling, the milk before reaching the city would contain large numbers of lactic bacteria. Such milk would be condemned, and yet it would have nothing unwholesome in it. For it must PUBLIC PROBLEMS 179 be remembered that it does not render milk unwholesome simply to have a considerable number of bacteria in it, provided these are of the ordinary lactic acid type. The result is that the milk of the well kept dairy may be condemned, while the milk of the slovenly kept dairy may pass inspection, simply because in one case a cooler temperature has been used, in spite of the fact that the cooler temperature has not rendered the milk more wholesome. This evidently makes a numerical standard unfair to the dairyman. 3. A numerical standard can under no conditions insure the wholesomeness of milk. The wholesomeness of milk is de- pendent upon the question as to whether it contains the types of bacteria that are liable to produce trouble and not at all upon the simple question of the number of bacteria present. A numerical standard primarily aims at the question of temper- ature and not at the question of cleanliness. Under these con- ditions it is evident that no sample of milk that falls under a numerical standard can be guaranteed as wholesome and none that pass beyond it can be condemned as unwholesome. 4. The enforcing of a numerical standard is very difficult. Where it has been attempted the success of enforcing it has been questionable. It is doubtful whether at the present time it is possible by any legal measure to enforce a rule which would require a numerical bacteria standard of milk. The only way that it has been enforced is by certain communities having the power to determine what milk may and may not be sold on the market. Where this is done the milk inspector can refuse to allow the sale of milk that comes from dairies producing milk with high numbers of bacteria. This has been done in some places with some success. Value of a Numerical Standard.-In spite of these conditions a numerical standard has certainly proved to be of value. Its value, however, is indirect rather than direct. Where the numerical standard has been made fairly small, it has been 180 PRACTICAL DAIRY BACTERIOLOGY practically impossible for the dairy to meet that standard under the old conditions. When a standard is adopted the dairyman is usually supplied with directions for improving the methods and producing a better milk. The application of these methods becomes more and more rigid as the dairyman finds that his milk is likely to be condemned or rejected, because it fails to reach the bacteria standard. There is no question that with the improving of the dairy conditions and care in the production of the milk there results an improvement in the grade and a greater wholesom^iess of the milk; hence it is that the adop- tion of numerical standards has had a tendency to raise the quality of the milk furnished in the city. As fast as the dairy- man can be taught to use better methods in dairies, so fast will the danger associated with this product be reduced and tend to disappear. Care means safety, and the extent of the care is the extent of the safety. Inasmuch as numerical standards do bring about greater carefulness on the part of the dairyman they have been and are of emphatic value. Practically it has been found that in cities where a numerical standard has been set and where an attempt has been made to enforce such a numerical standard as thoroughly as possible, there begins al- most at once an improvement in the quality of the milk. The number of bacteria becomes smaller, and the death rate of chil- dren falls in proportion. Hence, a numerical standard is of value in raising the type of milk and even reduces the danger of its being the cause of either specific or general diseases. Qualitative Analysis of Milk Bacteria.-It is evident that a very much better knowledge of market milk could be obtained if it were possible by some simple means to determine not only the number of bacteria but their kinds. Such a complete analysis is not possible by any means yet known. The detection of dis- ease germs is, as yet, practically out of the question. But a bacteriological analysis may be made that gives much more in- formation than the simple determination of numbers, since the PUBLIC PROBLEMS 181 determination of the relative numbers of the different types of bacteria enables us to draw some useful conclusions as to the age and purity of the milk. Such an analysis is also very useful when the attempt is being made to locate the source of special troubles that a cheese factory may experience. The method of making such a qualitative analysis is given on page 304. To make it of any value it is necessary to know how to interpret the results.1 To do this intelligently requires considerable experience with the work, but a few general rules can be given. The first thing to do is to estimate in percentages each of the types found. Then, in determining the meaning of the different percentages, the following facts should be considered: If the number of bacteria is very high, 5,000,000 to 20,000,000, the percentage of Bad. ladis acidi should range from 20 per cent, to 50 per cent. If the percentage is lower than this, it indicates that this large number of bacteria contains many suspicious organisms, and that the milk should be condemned. If the per- centage is as high as 80 to 90 per cent., the milk may be re- garded as unsuspicious, even though the number of bacteria mount up into many millions. If the original sample has only a few hundred thousands, little inference can be drawn as to the healthfulness of the milk from the percentage of types of bacteria. If, however, the number of liquefiers is high, it in- dicates excessive filth contamination, while, if these are few and the acid organisms numerous, it indicates a high quality of milk. Large numbers of liquefiers are always an indication of undesirable filth contamination. These specifications are not very definite, but they cannot be made more definite from the fact that at present a more accurate qualitative analysis appears to be impractical. 1 Conn and Esten. Ann. Rep. Storrs Exper. Sta., 1902-3. 182 PRACTICAL DAIRY BACTERIOLOGY SIGNIFICANCE OF DIRECT MICROSCOPICAL STUDY Other facts can be determined by microscopic study which give information of value concerning the milk besides the de- termination of the numbers of bacteria. It will commonly be found that the specimen prepared as described in experiment No. 17 will show a number of rather large, irregular cells (Fig. 59). These rep- resent cells from the cow and are commonly spoken of as leucocytes. If, how- ever, the udder should have any affection by which pus is being discharged, the pus will also appear in the milk and appear identical with the leucocytes. Does the presence of leucocytes in milk, therefore, indicate pus formation in the ud- der? That it does not nec- essarily do so is indicated by the fact that practically all milk shows some leucocytes. They are identical with blood cells, and some of them appear at all times to pass through the milk gland into the milk where they cannot be distinguished from pus cells. Their presence does not, therefore, mean the formation of pus. But if they are especially abundant, it is believed to indicate pus formation and to make the milk suspicious. Different observers have attempted to give some estimate as to the number of these cells that may be found in pure milk so as to make a standard by which it may be judged. But the number given as a standard has varied with the method adopted FIG. 59-SEDIMENT FROM CENTRIFUGAL- IZED MILK AS IT APPEARS UNDER THE MICROSCOPE The large cells are leucocytes; the smaller ones bacteria PUBLIC PROBLEMS 183 for detecting them. Stokes, using one method, concluded that milk should be condemned if it contained more than 12 leuco- cytes per field of the microscope when studied with a one-twelfth inch immersion lens.1 Stewart's method modified by Slack (see page 285) gives slightly different results, and when this method is used it has been assumed that milk should be condemned if it contains 25 or more cells to the field, which is equivalent to about 100,000 per c.c.3 Doane's method of determining the number of leucocytes is more accurate and uniform. As de- scribed in experiment 18, it gives much higher numbers.3 Doane regards 500,000 leucocytes per c.c. as sufficient to render the milk suspicious and 1,000,000 per c.c. as indicating inflamma- tion of the udder. None of the methods yet devised can be regarded as very accurate or satisfactory. Considerable variations are found in successive determinations of the number of leucocytes in the same specimen of milk, and there is certainly no knowledge at hand to-day which enables us to say how large a number is sufficient to suggest that the milk really contains pus. All that can be said is that if the number per field of the microscope is much beyond 25, or the number over 500,000 per c.c., the milk may be regarded with suspicion. When mixed market milk shows a large number of leucocytes, it probably indicates that some of the cows supplying the milk are suffering from in- flammatory troubles of the udder, and if the milk of a single cow should show large numbers, it probably indicates inflam- mation. But our information is, to-day, admittedly too small to make the determination of much value, and as a means of municipal examination of milk the determination of the number of leucocytes is not very satisfactory? 1 Stokes. Ann. Rep. of St. Bd. of H., Maryland, 1897, 1898 & 1901. 2 Stewart. Am. Medicine, ix., p. 486. Slack. Jour, of Infec. Dis., p. 214, 1906. 3 Doane. Bui. 102, Maryland Exper. Sta. Savage. Jour. Hyg., p. 123, 1906. 4 Rullmann and Trommsdorff. Arch. f. Hyg., LIX., p. 224, 1906. 184 PRACTICAL DAIRY BACTERIOLOGY The value of a microscopic study may be somewhat increased by a careful examination of the types of bacteria found, since the presence of inflammation is accompanied by Streptococci in the milk. But, as we have already seen, Streptococci are practically always present in milk, it being almost impossible to draw milk from the most healthy cow without these bacteria getting into the milk. The presence of Streptococci in milk can, therefore, not be considered as meaning much of anything. The ordinary Streptococci of milk, however, are commonly not found as long chains but as isolated spheres, or in pairs. The pus forming cocci frequently grow in long chains. If, there- fore, in a microscopic study there appear many long chains of Streptococci, this again may render the milk suspicious.1 In short, if in making a microscopic examination, as described in experiment No. 17, the number of bacteria per field appears to be more than from 40 to 50, or if the number of leucocytes is more than 50 per field, or if there appear many long chains of Streptococci, the milk must be regarded as suspicious, requiring further study, or, perhaps, an investigation of the dairy where it was produced. DAIRt INSPECTION During the last few years there has been an increasing recognition of the value of public dairy inspection. This plan though first suggested some 12 years ago in this country was first adopted by some of the large dairy companies in Europe.2 In more recent years its value has been recognized, and the practice has been slowly but surely extending until at the present time it has become quite common in certain sections of the country. Five years ago a dairy inspection was scarcely known in the United States, but some of the larger dairy com- panies began first to organize a system of inspection of the 1 Bruning. Exper. Sta. Rec., xvii., p. 496, 1905. Kaiser. Arch. f. Hyg., Ivi., p. 51, 1906. 2 Conn. Ann. Rep. of Ct. Bd. of Agri., 1895. PUBLIC PROBLEMS 185 dairies that furnished their milk, and later some of our cities have adopted a similar plan. At the present time the plan of inspection of dairies by public officials is becoming quite com- mon and is rapidly increasing.1 A dairy inspection simply means that the dairies furnishing milk are visited periodically by some persons who are interested in the supply from the consumers' end and are not especially interested in the dairies themselves. They are familiar enough with the proper conditions in a dairy to make such a visit in- telligently and to determine whether the dairy is carried on under proper conditions. These inspectors are supposed to make their appearance without any special warning, so that they will find the dairy in its normal, ordinary conditions. They are supposed to examine into all its details of milk production and' distribution, to make recommendations to the dairymen as to improvements, and to make report to some headquarters as to whether conditions in each dairy are suitable for warranting a continuation of the dairy as a source of milk supply. In such a dairy inspection the irispector must naturally take into consideration all the factors which contribute to the whole- someness and cleanliness of the milk. He should consider all the phases of the dairy which we have outlined in the last two chapters. A summary of the points which should be examined by him may, however, be useful. His attention should be given primarily to the following factors: i. General cleanliness in the barn and dairy. 2. The condition of the cozes. By this is meant (a) whether they have been tested with tuberculin and demonstrated to be free from tuberculosis; (b) whether there are any signs of dis- 1 Bergy. Penn. Dept, of Agri., 1900. Pearson. 17th Ann. Rep. Bu. An. Ind., 1900. Goler. Charities, Aug. 5, 1905. Goler. Scheme for the Sanitary Control of the Municipal Milk Supply. Rochester, 1905. Goler. Am. Jour, of Nursing, 1903. Goler. Am. Medicine, 1903. 186 PRACTICAL DAIRY BACTERIOLOGY ease in the cows, especially of udder disease; (c) whether they are kept in a condition of cleanliness or are allowed to become caked with manure; (d) whether they are given exercise; (e) the kind of food which is used; (f) the time of feeding relative to milking. 3. The source of the dairy water. The source of the water which is the cow's drink is a matter of far less importance than the water used in the dairy for washing the milk cans and other utensils. This source of supply should be beyond suspicion. In suspicious cases, samples of water should be collected and sent to a proper laboratory for analysis, to determine whether the water is fit to be used in the dairy. 4. The condition of the barn; whether it has plenty of light and air, whether the manure is removed frequently, and whether dirt and dust are allowed unduly in and around the cows' stalls. 5. The method of disposing of the manure; whether it is allowed to accumulate in the barn, under the barn or nearby, or whether it is frequently removed to a considerable distance. 6. The method of milking; whether the milkers are careless or slovenly in their work, thus encouraging the entrance of great quantities of filth into the milk or udder of the cow, or whether they are intelligent and cleanly in their actions so as to produce milk under the best conditions. 7. The condition of the milker. This should refer to (a) the matter of his health, inasmuch as all suspicious cases of in- fectious diseases should be rigorously excluded from the dairy; (b) cleanliness of the milker before going to the milking. That the milker should make a careful toilet and wear clean clothes has already been emphasized and needs to be carefully watched by the inspector. 8. The method of washing and sterilising the milk jars, cans and other utensils. 9. The treatment of the milk after milking. This should include the question of aeration, attention being especially given PUBLIC PROBLEMS 187 to devices used for cooling the milk to as low a point as possible. io. Bottling of the milk, if this is done in the dairy, or the method of placing the milk in the cans if the milk is to be car- ried in this manner. ii. The method of transporting the milk. This should in- clude the temperature of the transporting receptacles, the clean- liness of these receptacles, and the method of handling them, and whether they are washed before they are returned to the farmer. Recognizing that there are many things to be taken into con- sideration, it becomes evident that it is impossible to formulate any standard of cleanliness, which the dairy must attain in order to pass inspection. At best much must depend upon the per- sonality of the inspector, and it will be to a large extent the matter of his judgment whether any dairy is in proper condi- tion, where an improvement can best be made, and when a dairy must be excluded from furnishing the public milk supply. Difficulties of Dairy Inspection.-While a dairy inspection has its emphatic advantage, and while it is unquestionably growing in popularity, certain somewhat serious objections have been raised against it. i. It is difficult, at least at present, to get trained men who can properly serve as inspectors. Since there are so many mat- ters upon which they must pass judgment, and since the per- sonal attitude of the inspector is of so much importance, it is manifestly desirable that none except those who thoroughly understand dairies should be employed as inspectors. It is hardly possible to give to an untrained individual instruction which will enable him to carry on a satisfactory inspection. Trained men, however, are hard to find, and while there is no difficulty in getting persons who will serve as inspectors for the sake of obtaining a salary, it is not so easy to obtain men who are satisfactory. If inspectors are not properly equipped 188 PRACTICAL DAIRY BACTERIOLOGY for carrying on this work, they are pretty apt to produce more trouble than good and will certainly produce constant friction. 2. The value of dairy inspection is slight unless such inspec- tion is frequent. There is little advantage in an inspector visit- ing a dairy and, if he finds it in poor condition, making recom- mendations and a report, and then going away and not coming back for several months. The farmer is very apt to pay no attention to the directions given him. To be of any efficiency, therefore, the inspection should be frequent. Where there are large numbers of dairies contributing to a milk supply, it is necessary that there be a large number of inspectors, if the dif- ferent dairies are to be visited with any degree of frequency. To do this will require the expenditure of an almost prohibitive sum of money. An inspector, to be of any value, must have a good salary, for a poorly paid man is worse than useless. Con- sidering the thousands of dairies furnishing milk to large cities, all of which must be inspected, it becomes a question whether a public inspection does not involve more expense than can be properly met. This, at all events, is one of the obstacles to an introduction of dairy inspection. Whether the money thus spent would not be one of the very best investments is another ques- tion. At all events, in the early stages of this attempt at intro- ducing milk inspectors, these difficulties are prominent. 3. The enforcement of the rulings of the dairy inspectors has practical difficulty. It is one thing to have an inspector tell the dairyman to clean up his barn and make various improve- ments; it is quite another thing to get him to do it. Dairy in- spection was first adopted by milk companies, and they have had very little difficulty in enforcing their regulations by a very simple means. These companies, in many cases at least, pay for their milk a trifle higher price than the ordinary price, and in such cases they can very easily refuse to receive milk from any dairy that does not reach the standard which they them- selves set. Such a method of enforcement is very efficient. In PUBLIC PROBLEMS 189 some of our cities a somewhat similar method can be and has been adopted. Cities have usually the power of making regu- lations concerning the food products that are sold in the market, and thus of regulating the sale of milk. The cities can put into the hands of a health board, or any other com- mission, the right to determine what milk shall or shall not be sold in a city. This commission can then take the ground that milk from dairies which their inspectors condemn cannot be sold in the city. This may effectually prevent the sale of such milk in the city, and, if properly carried out, will bring a strong influence to bear upon the dairyman. Another method of enforcing the instructions of the inspector might be by public statute. This is the most difficult and unpromising of all. The difficulty of enforcing laws is well known, and it is doubtful whether the punishing of a dairy because it has not reached a certain flexible standard of cleanliness would be possible. There is one method by which an enforcement of dairy in- spection might be reached, and that is by statutes which put into the hands of proper health officers everywhere the right to give licenses to the milk distributors in the communities or to withdraw them. If every milk distributor, in small towns as well as in large, requires a license to sell milk, then the officials would have the power of enforcing upon the dairyman the demand that the dairy and the milk should be kept in proper condition, and that the suggestions of the dairy inspectors should be followed. Such licenses are required in some places, especially in larger cities, but there is no such general rule at the present time. No other general method seems to be possible by which instructions of dairy inspectors can be enforced. Inspector and Farmer.-The relation of the inspector and the farmer should be one of the most friendly nature, and it should be the purpose of each to help the other. It is no advantage to the farmer to deceive the inspector, nor is it any advantage to the inspector to be so rigid and severe in his condemnation as 190 PRACTICAL DAIRY BACTERIOLOGY to make it impossible for the farmer profitably to carry on his business. The purpose of the inspection is not to drive the dairyman out of business, but to bring about an improvement of the conditions under which the milk is produced. Any farmer would prefer to furnish good milk rather than poor milk. It is eminently desirable that the farmer and the milk inspector should, therefore, work together, and their attitude toward one another should be not that of hostility but of helpfulness. Of course, the extent to which this attitude will be developed will depend largely upon the individuals. Some inspectors will never take a friendly attitude to the farmer, and some farmers will always look upon the inspector as his enemy; but the friendly relation should be the aim of both the dairyman and the in- spector. Withal it must be recognized by dairy inspectors and all public officials that the application of proper dairy methods in •the dairy depends wholly upon the readiness of the public to pay the price. No amount of law will force the dairyman to adopt methods that he cannot afford at the price paid for his milk. To obtain a quality of milk that is beyond suspicion re- quires the raising of the price paid to the producer above that which has been common in the last few years. It will always be possible, doubtless, to purchase good milk and poor milk. One cannot purchase silk for six cents a yard, but he can purchase a poor grade of cotton cloth. So we could not expect to buy first class milk which is beyond suspicion for a sum of money that is insufficient to reimburse the dairyman who em- ploys satisfactory methods. The whole problem is one of price. The attitude of the public toward the producer should be not one of hostility but one of mutual assistance. The public ought to agree to pay willingly for whatever safeguards it demands for its milk, and as soon as this is done so soon will the dairy- man be ready to adopt these safeguards. Rules and regulations will even then be necessary, but only to guide, not to force the producer. PUBLIC PROBLEMS 191 COMPULSORY PASTEURIZATION It has been evident from the facts already considered that it is extremely difficult, probably impossible, to put safeguards around the milk sufficient to guarantee positively its wholesome- ness. But this can be done by the simple plan of compulsory pasteurization. It is impossible to guarantee that the milk that comes to our cities contains no harmful bacteria, but it is per- fectly possible to destroy them by heat after the milk reaches the city, and thus guarantee the milk that is sold to the con- sumer. Should the officials who have power in the matter in- sist upon pasteurization of all milk offered for public consump- tion? Over this question there is a growing agitation.1 Compulsory pasteurization has been adopted in some of the countries in northern Europe for all the milk products that reach the creameries, cheese factories, etc., but the suggestion of adopting it for the ordinary milk supply is new. In its favor is urged the fact that only thus can the public be positively pro- tected against tuberculosis and other diseases distributed by milk. No dairy inspection, however much money is put into it, can give such a guarantee. Dairymen cannot all be made careful and cleanly, and even if they were, there would still be the danger from tuberculosis and walking typhoid. Pasteur- ization alone can insure freedom from disease germs. On the other hand, it is urged that although pasteurization is desirable and useful, it should be left to the individual to determine whether he wishes to adopt it. To be of value it must be thor- ough. We have noticed how inefficient the process may be when improperly carried out, and hence to be of real value it must be under public control, and under the inspection of the proper authorities. This would involve compulsory pasteuriza- tion, which would make it ordinarily impossible for the in- dividual to obtain unpasteurized milk, if he wished to do so. 1 Brown. Arch, of Pediatrics, p. 251, 1903. 192 PRACTICAL DAIRY BACTERIOLOGY It is claimed that pasteurization slightly reduces the digestibility of milk and hence should not be compulsory. It is also urged that pasteurization will set a premium upon carelessness and render nugatory all attempts to improve the conditions in the dairies. If the dairyman knows that the bac- teria which get into his milk will be killed by heat before the milk reaches the consumer, he will inevitably take less pains to keep them out, and consequently the dairies will fall back again into the condition of slovenliness from which they have been slowly emerging. Such a result is certainly not to be desired, and this is a valid argument against compulsory pas- teurization, although it may be a question whether it is not bet- ter to have dirty milk rendered safe by pasteurization than clean milk that may contain tuberculosis and occasionally typhoid fever bacilli. For these reasons the question of the wisdom of compulsory pasteurization is still unanswered. As yet, it is not anywhere adopted as a public measure, although not a few milk companies have adopted it as a means of protecting their milk from souring, frequently not informing their patrons of the fact. Compulsory public pasteurization is being strongly urged in some places, and it may possibly be the coming method of procedure. CHAPTER IX BACTERIA AND BUTTER-MAKING It is evident that cream and butter will contain bacteria. They are present in the milk, they will be sure to be in cream when separated from the milk, and in the period that precedes butter-making they develop in great numbers. They are always present in cream whether sweet or sour, always present in but- ter whether fresh or stale. In butter-making it seems to be, at least according to present demands, absolutely necessary that the dairyman should have at his command large numbers of certain kinds of bacteria. The processes in butter-making are planned to insure the presence of the proper kinds and to stim- ulate their development rather than to check their growth. In- deed, in some cases, butter-makers have found it difficult to make satisfactory butter, because of too rigid attempts to ex- clude bacteria from their milk, and hence from their cream. For the butter-maker, therefore, bacteria are friends rather than foes. CREAM RIPENING The relation of bacteria to butter-making is associated with the phenomenon of cream ripening. The origin of the ripening of the cream is due to the early conditions of dairying when the butter was made wholly in small farms, and it was rarely possible to churn oftener than once in two or three days. It was necessary on such farms to allow the cream to accumulate until a sufficient quantity was collected to make good churning. During the period in which the cream was collecting it was sure to undergo the changes which have subsequently been spoken 193 194 PRACTICAL DAIRY BACTERIOLOGY of as ripening. When later the making of butter was concen- trated into creameries, time for accumulating the cream was unnecessary, but the creameries soon found it necessary to keep their cream for a time before churning it in order that it should assume a condition similar to that of the soured cream on the isolated farm. From this came the process of ripening cream, which has been well-nigh universal. The purposes of ripening the cream are several. I. It is thought that the given quantity of cream will yield a larger amount of butter after ripening than when churned sweet. 2. It is certain, also, that cream will churn more readily when ripened than when sweet, although this does not apply to cream obtained by the separator, but rather to cream obtained by the gravity method. 3. Butter from well-ripened cream is thought to keep better. 4. But the primary reason for the ripening of the cream is in the development of the flavor to the butter. Sweet cream may be churned and butter obtained from it, but it will not have the ordinary butter flavor, showing rather the milder characteristic taste of sweet cream. This is the objec- tion to the butter extractors-machines that separate the cream from the milk and churn it into butter in one process-since the fact that the cream was not ripened prevents the butter from acquiring the desired flavor. Doubtless the changes that occur in the cream during the period develop in it the flavors char- acteristic of butter. Sweet cream butter, however, is in considerable demand in some places. In Europe there is a general tendency to prefer a milder flavor in butter than is preferred in America. An American traveling in Europe is apt to find the butter tasteless, while a European is apt to complain of American butter as too strong. In many parts of Europe a very slight ripening of the cream, or no ripening at all, is adopted, and the butter thus obtained has very little flavor. Frequently the butter is not salted and is thus more delicate than the highly flavored butter BACTERIA AND BUTTER-MAKING 195 of America. Some people are very fond of such mild butter. A demand for sweet cream butter has developed in America; but at the present time this demand is small and forms only a very small part of our butter product. There has been a change in public taste concerning butter during the last 25 years. At present the demand is for a milder flavored butter than a gen- eration or so ago, and a strong, high flavored butter will not find, to-day, as ready a sale as it formerly would. Butter that would formerly have sold readily as first grade would now be regarded as too strong and ranked as second or third quality. No doubt there has been, even in the United States, a general tendency for a milder type of butter. Whether this tendency will develop further until our markets demand a large amount of sweet cream butter cannot, of course, be predicted. The Cause of Cream Ripening.-The ripening of cream is a phenomenon of bacteria growth. The bacteria which are in the cream find it an excellent medium for food, and if kept at a fairly warm temperature during the ripening period, their devel- opment is quite rapid. For the 12 to 24 hours of ripening the bacteria multiply, and by the time the cream is ripened and ready to be churned they are present in prodigious numbers. Many analyses of ripened cream have been made, disclosing the fact that whereas in the sweet cream bacteria may be 2,- 000,000 to 3,000,000 per c.c., in the same cream when ready to churn there may be about 500,000,000 per c.c. The numbers at the time of ripening, however, vary widely, being sometimes as low as 200,000,000, or even lower, and sometimes as high as 2,000,000.000 per c.c. There does not seem to be any especially close connection between the completeness of,the ripening and the number of bacteria. In some thoroughly ripened cream samples there may be two or three times as many bacteria as in others, and yet no difference in the completeness of the ripening can be seen. The growth of bacteria in the cream produces critical 196 PRACTICAL DAIRY BACTERIOLOGY changes which considerably modify its nature.1 The lactic acid bacteria always develop lactic acid, and the cream becomes sour; but there are other changes also. We do not yet know what all these changes are nor to what extent they contribute to the ripening phenomenon. That the other changes have something to do with the production of the flavor in butter is evident from the fact that a butter flavor cannot be produced in the cream by adding lactic acid to it, and if the ripening were wholly the result of the souring, the addition of lactic acid should produce the same result as normal ripening. Growth of Bacteria During the Ripening.-The study of the growth of bacteria during the ripening of cream is quite sug- gestive and instructive. At the outset the cream is quite cer- tain to contain numerous kinds of bacteria, differing much, of course, in different samples. In the composite cream of a cream- ery the varieties will be very numerous, while in cream of a private dairy they may be less numerous. During the ripening the cream is commonly kept between 6o° and 700, at which temperature many bacteria develop rapidly, but not all kinds with equal vigor. During the first few hours of the ripening there is found to be a general increase in the number of bacteria of nearly all kinds that were present in the original cream, so that after six or eight hours there will almost universally be found higher numbers of all species of bacteria than were found at first. During this time, however, the lactic acid bac- teria, especially of the Bact. lactis acidi type, have been in- creasing more rapidly than the others.2 In the very fresh cream these bacteria may have been, and commonly are, com- 1 Conn. Ann. Rep. Storrs Exper. Sta., 1890, pp. 91, 93 and 94. Conn. Cent. f. Bact., II., ii., p. 409, 1896. Ill, p. 177, 1897. Conn and Esten. Cent. f. Bact., II., vii., p. 743, 1901. Conn and Esten. Ann. Rep. Storrs Exper. Sta., 1900, 1901. Storch. Cent. f. Agr. Chem., 1891. Haywood and McDonnell, Bui. 44 Penn. State Col. Exper. Sta., 1899. 3 Conn. Bacteria in Milk and Its Products. P. Blakistons Son, 1903. Conn and Esten. Ann. Rep. Storrs Exper. Sta., 1901. BACTERIA AND BUTTER-MAKING 197 paratively few in numbers, forming sometimes not more than i to 2 per cent.; but their percentage rises rapidly until they come to form an appreciable portion of the whole. After sev- eral hours, the time varying with different specimens, the acid bacteria constitute a large percentage of the whole. From this time, after they form perhaps 50 per cent, of all of the bacteria present, the other species begin to be injuriously affected by the growth of the lactic bacteria, probably by the acid produced. The acid forming germs still continue to increase in numbers, while the others cease to grow so rapidly. After a time they begin to diminish and may finally largely or wholly disappear. The result of this is that during the last stages of the ripening there may be present in the cream nothing but acid bacteria, which sour the cream and produce the final changes in the ripening. It will thus be seen that the ripening of the cream may be divided into two stages: In the first the growth of the miscel- laneous species of bacteria continues, and all types may become more or less abundant. In the second the acid-forming germs take possession of the cream and gradually force the others into the background and, finally, may crowd them out entirely. Both of these stages doubtless contribute to the final product. Without the proper lactic organisms it is impossible to get the proper flavored butter. But butter made from pasteurized cream and ripened by pure cultures of lactic acid bacteria does not develop so much flavor as that in which the original bac- teria are allowed to grow with the acid germs. Hence, it is probable that the development of the miscellaneous bacteria in the first phase of the ripening has not a little to do with the final butter flavors. Concerning the matter, however, nothing is positively known, the problems being too complex to have ad- mitted of any solution at the present time. 198 PRACTICAL DAIRY BACTERIOLOGY THE NEED OF CONTROLLING THE RIPENING The butter-maker needs bacteria, but he wants the right kind. The character of the butter obtained from the cream is closely dependent upon the kind of bacteria which develop in it. Many experiments have been performed of making butter from cream ripened with different species of bacteria, which have shown that very different results come from the use of different bacteria.1 Some species produce unpleasant taints, bad tastes, bitter tastes and other undesirable results; many species have been found to be quite negative in their action on the cream and resulting butter. Other species, again, develop pleasant tastes and odors, and are advantageous and useful in the cream ripening. It chances, fortunately, that the latter is true of most of the lactic acid bacteria. Since in the ordinary bacterial growth in cream the lactic bacteria in the end finally get the upper hand and grow at the expense of all of the others, it commonly happens that the ripening produces a good flavor, and a satisfactory butter is obtained. If this were not true, butter-makers would never have adopted this process of cream ripening, for, of course, ripening has been adopted because it has been found to be commonly suc- cessful in giving a desirable product. Unfortunately, how- ever, the favorable species of lactic bacteria do not always get the upper hand in the cream ripening. It sometimes happens that there are present in cream large numbers of vigorous bacteria just as capable of rapid growth as are the desirable lactic acid germs. In these cases the unusual bacteria may develop abundantly and produce a variety of uncommon changes in the cream, with the result of giving an undesirable flavor to the butter. Such a phenomenon explains the occasional ap- pearance of bad tasting butter. 1 Conn. Ann. Rep. Storrs Exper. Sta., 1894. Eckles. Cent. f. Bact., II., iv., p. 730, 1898. BACTERIA AND BUTTER-MAKING 199 When butter was made upon the small farm the occasional appearance of an improper ripening was a matter of no very great importance. The butter is rarely so bad that it cannot be eaten, and the farmer who made his own butter did not particularly trouble himself if, for a few days, or even for a few weeks, his butter had a stronger taste than usual. When, however, butter came to be made in creameries, the necessity for producing a uniform product was greater. When a cream- ery is producing hundreds of pounds of butter per day, and when the market price will depend upon the character of the butter, it is a matter of more importance to obtain a desirable flavor. A good flavored butter will yield a high price, and poorly flavored butter will inevitably yield a lower price. Under these circumstances, the question of financial failure or success may hinge upon flavor, and this in itself depends upon the ripening of the cream. Hence, in recent years all cream- eries have come to recognize the vital importance of a proper control over the ripening. Inasmuch as this ripening is a bac- terial problem it follows that the success of a creamery is, to a large extent, dependent upon the control of its bacteria. Flavor and Aroma.-The general character of the butter is determined by both flavor and aroma. The term flavor refers to the taste of the butter, whereas the term aroma refers rather to the smell. These two factors do not appear to be the same, and in some cases, at least, they have a different origin. Some bacteria give rise to a desirable flavor in butter but do not give rise to an aroma; others give rise to an aroma without much flavor; and different species still produce neither acid, flavor nor aroma.1 It is not understood from what materials the flavors and aromas are produced.2 That they are not caused by the acid in the cream alone has already been mentioned. They are not due primarily to the fat, as is shown by the fact that they will appear in ripened skim milk.3 That they are 1 Conn. Ann. Rep. Storrs Exper. Sta. 1894. - Weigmann. Milchztg., p. 193, 1897. • Mickels. 22d Ann. Rep. Wis. Exper. Sta., 1905. 200 PRACTICAL DAIRY BACTERIOLOGY caused by certain bacterial decomposition products is evident; but whether they are the result of the action of bacteria upon the casein or upon the albumen, or the milk sugar, or the fat, is not yet settled. ' The question is a complex one, but the probability is that flavors and aromas are not due, in ordinary butter, to any one kind of bacterium or to any one kind of de- composition, but that quite a variety of processes may contribute thereto. It is certain, however, that the production of both flavor and aroma is more or less independent of the production of acid. By this it is not meant that the bacteria producing acid may not also produce flavors and aromas, but that the two phenomena of acid production and flavor production are not identical. What evidence we have to-day indicates that the production of flavor and aroma is not primarily associated with the common lactic acid bacteria, and must be attributed in a measure to certain other types of organisms.1 CONTROL OF CREAM RIPENING If the quality of butter is so closely dependent upon the nature of the cream ripening, it is extremely desirable to control this phenomenon. In the earlier periods of farm butter-making no attempt was made to do this, and the result was a great ir- regularity in the butter. As the industry has become more con- centrated greater stress has been laid upon the cream ripening, until at the present time the butter-makers have this process largely under control. Twenty years ago little attention was given to cream ripening; to-day it receives more care perhaps than any other phase of butter-making. The methods of regu- lating it are several. 1. Regulation of Temperature.-The temperature which is recommended for cream ripening has varied all the way from 1 Grimm. Cent. f. Bact., II., viii., p. 584, 1902. Severin. Cent. f. Bact., II., xi., p. 202, 1903. Van der Leek. Cent. f. Bact., II., xvii., p. 467, 1906. BACTERIA AND BUTTER-MAKING 201 500 to 750. In earlier years, higher temperatures have been adopted. There are certain important reasons for the higher temperature. In a well-ripened cream there must be an abun- dance of lactic acid bacteria, and these do not grow well below 6o°. Certain other species of bacteria, which produce a bitter taste, are more likely to develop at low temperatures than at high. If, therefore, cream is ripened at too low temperatures, the acid organisms do not grow so well, and there is a greater chance for the bitter bacteria to develop sufficiently to produce an undesirable flavor. On the other hand, at too high temper- atures other types of bacteria, especially gas-producing forms, and others equally undesirable, are particularly favored. A temperature from 65° to 700 appears, on the whole, most likely to avoid both of the difficulties, and under ordinary circum- stances the best results can be obtained by a temperature from 65° to 70 0. In warmer weather it may be desirable to use a lower and in cold weather a somewhat higher temperature. 2. Duration of Ripening.-The duration of the ripening varies very much with the conditions. Sometimes cream when brought to a creamery is already nearly sour and has, therefore, become ripened even before the butter-maker receives it. In other cases, especially in winter, it will be not only very sweet, but will contain small numbers of bacteria, requiring a much longer ripening. Moreover, milk produced under good dairy conditions, clean and fairly free from bacteria, will ordinarily require longer ripening than milk that has .been produced under less favorable conditions, and that consequently already contains bacteria in great numbers. The length of time will vary also with the temperature, being, of course, longer at lower temper- atures. To determine when the cream is sufficiently ripened the butter-maker has two methods. One is the general appear- ance to his eye and taste, the other is by determining the acidity. The production of the acid may not be the whole of cream ripening, inasmuch as it has little to do with the flavor and 202 PRACTICAL DAIRY BACTERIOLOGY FIG. 6o.-THE USE OF BURETTES FOR DETERMINING THE AMOUNT OF ACIDITY aroma; but, as a rule, flavors develop parallel with the acidity, and if the acidity becomes too high the flavors become too strong. It is a fairly easy matter to determine the acidity of BACTERIA AND BUTTER-MAKING 203 cream, and as a practical procedure it has become common to determine the degree of ripening from its amount of acidity. The general practice of butter-makers is to ripen the cream until the acidity reaches about .5 to .65 per cent, of lactic acid. This is determined by the well-known methods given in other places and need not be repeated here (Fig. 60). If the cream is very rich, containing a higher per cent, of fat than usual, the ripening should not be continued to quite such a high per cent, of acidity. The reason for this is that the acidity is de- pendent upon the fermentation of milk sugar, and if the per cent, of fat is very high, there is a smaller amount of milk sugar than usual, and the acidity will not rise so fast; hence, the ripening becomes completed with a lower acidity than usual. In using the acid test as a method of determining the ripening of cream it must be remembered that this test only gives the grade of acidity, and while this is a gauge it is not the whole ripening. 3. Use of Starters.-The greatest change that has taken place in butter-making in recent years is the adoption of more and more careful methods of regulating cream ripening by the use of starters. Twenty years ago they were only occasionally used; to-day their use is well-nigh universal. A starter simply consists of a lot of cream or milk, which has become already soured, and is added to the mass of cream in the cream vat for the purpose of controlling the ripening process. It was first used by butter-makers in cold weather only. In the early days of creameries it was found that during the cold weather the cream frequently would not ripen quickly, and though left for the ordinary time in the cream vat, it was not ready to churn when desired. This was easily obviated if the proper amount of sour cream was added to it. This was called "starting" the ripening, and hence the name starter. Although used originally only to start the process, it has been found that starters have other advantages. By their use butter-making 204 PRACTICAL DAIRY BACTERIOLOGY can be much more accurately controlled. 1. The flavor and aroma of the butter resulting from the ripening may be to a large extent modified by the use of starters and rendered more uniform. 2. A greater uniformity in the product has been found possible by using a properly prepared starter. The mat- ter of uniformity, especially in the handling of butter, is of much practical significance. It is of importance to the manu- facturer that his cream should reach its proper stage for churn- ing at practically the same time every day, otherwise the whole routine of the creamery is disturbed. By the use of starters of the proper type and used in the proper way, the ripening can be so controlled that the churning can take place at prac- tically the same time every day. 3. The use of starters has frequently been found valuable in avoiding undesirable taints and tastes in the butter. These unpleasant flavors, which even in the best creameries occur occasionally, can frequently be checked or remedied by the use of a considerable quantity of a proper starter. THE MAKING OF STARTERS Home Starters.-Under this head is included a type of starter that can be manufactured in the ordinary dairy; for it consists of nothing more than a good type of sour milk or cream. In order to obtain this, it is only necessary to select several quarts of milk, preferably from a cow that gives a good quality. If a creamery wishes to make such a starter, it will select the milk from one of the better kept dairies among its patrons. This milk is then placed in a clean, sterilized pail, or can, covered to keep out the dust, and placed at a temperature of from 65° to 700. After 24 to 48 hours the milk should show signs of souring, and after it has become decidedly sour, but not yet curdled, it is to be used as a starter. In the preparation of such a starter, evidently all that is needed is a fair quality of milk, clean pails and a proper temperature. It must be noted, how- BACTERIA AND BUTTER-MAKING 205 ever, that it requires some skill on the part of the butter-maker to know whether the starter that he thus obtains is of the best character, and whether it should be used or thrown away, and another obtained. Starters made in this way are not sure to be uniform, inasmuch as the different samples of milk may contain different types of bacteria, and experience is needed on the part of the butter-maker to know whether the starter thus obtained is satisfactory. Starters from Commercial Cultures.-In the last few years there has been placed upon the market quite a variety of com- mercial cultures for the purpose of controlling butter-making. These are prepared by bacteriologists and consist of many types of bacteria that have been found by experiment to produce a satisfactory ripening. The bacteria have usually been isolated from ripening cream, and have been chosen from a large num- ber of tested species which promise to produce a desired type of ripening and, consequently, the best grade of butter. These commercial cultures are commonly called pure cultures, which means that they consist of one species of bacteria only. We have already noted that the flavors, aromas and acidity are probably not produced by the same organisms. Probably no bacterium combines in itself the requisite powers of producing all three factors. Pure cultures, therefore, might not be ex- pected to give all the qualities desired in a first class butter. To meet this difficulty some of these commercial cultures consist of a mixture of bacteria of two or more kinds, in order to produce a starter which will furnish at the same time flavor, acidity and aroma. These bacteria cultures, whether pure or mixed, are developed in a bacteriologist's laboratory until the organisms are in great abundance, and are prepared for market in various ways. In some cases they are mixed with some inert powder and sold in a dry form. In other cases they are sent in the form of a bouillon culture much like those described on page 295. Others again are cultivated in milk and sent out as milk cultures. 206 PRACTICAL DAIRY BACTERIOLOGY The question of the purity of the commercial cultures is of the greatest importance. Inasmuch as they consist of living organisms it is absolutely necessary that the cultures should contain the organisms desired and no others. It would be of no great value to add to a lot of cream a mass of bacteria, un- less it contained the desired organisms and no others. During the first years that such cultures were put on the market they were very apt to be impure, and even at the present time they are not absolutely reliable. It is an extremely difficult matter to produce bacteria in quantity without their becoming con- taminated with undesirable species; and in the commercial cultures it occasionally happens that unexpected and undesired species of bacteria or molds find their way into the cultures, and injure or ruin them. It also sometimes happens that the culture, though pure, may lose its vigor, for bacteria retain their greatest vigor only under the most favorable conditions. As better methods of cultivating bacteria are learned, the diffi- culties of impure cultures are disappearing, and the cultures are becoming more and more reliable each year. At the present time the commercial cultures can be fairly well depended upon. But even to-day the cultures put out by one of the most reliable firms occasionally contain species that are not wanted. Absolute purity and uniformity of the cultures have not yet been reached. In preparing a starter from a commercial culture the dairy- man must remember that he is dealing with living organisms and must handle it accordingly. It must not be heated, for this would kill the living organisms. It must be used, if possible;. hile fresh, for the bacteria in the culture begin some days after to lose their vigor, and eventually die. An old commercial cul- ture, therefore, is useless. The culture must be treated in such a way as to give the bacteria the greatest chance for developing, since the growth of these organisms produces a starter. The cultures are usually sealed when they leave the bacteriologist's laboratory. Care should be taken, if a commercial article be BACTERIA AND BUTTER-MAKING 207 used, to see that the seal of the package is unbroken. If the seal is broken, there has been an opportunity for contaminating bacteria to enter. The culture, if not used immediately upon receipt, should be kept in a cool, but not cold place; for example, in a refrigerator, not on the ice but near it. At this temperature they will keep best. Lastly, in most cases, the z.hole cultiire should be used at once, and the dairyman should make no at- tempt to use half a culture and reserve the other half for a later date, especially if it be a liquid culture. It is almost im- possible for him to remove half of such a culture from its package without so contaminating the other half that, if pre- served for use at a later date, it is no longer reliable. Preparation of a Commercial Culture.-To prepare a starter some plan must first be adopted to increase the number of bac- teria, for the package bought in the market does not contain enough of these organisms to furnish a starter for any con- siderable quantity of cream. If only a small amount of starter is needed, for example, a quart for a few gallons of cream, the procedure is as follows: A quart of skim-milk is placed in a glass jar and sterilized either by boiling, or better by pasteurizing, by submitting it to a heat of 1800 for half an hour, stirring frequently to insure uniform heating. The milk is then cooled by placing it in cold water, and when it has reached a temperature of 8o° the com- mercial culture, from a freshly opened package, is stirred in thoroughly; the whole is covered to keep off the dust, and placed at a temperature of about 65°. When the milk has become quite sour, but before it has curdled, it is ready to use as a starter. It is much better to use the starter before curdling takes place, for two reasons: 1. While the milk is still liquid it is easy to distribute a starter uniformly through the cream that is to be ripened, so that with a little stirring the whole mass of cream is inoculated with the bacteria. After the milk has curdled it is much more difficult to distribute it uniformly 208 PRACTICAL DAIRY BACTERIOLOGY through the cream. 2. The lactic acid bacteria which are espe- cially desired in the cream appear to be most vigorous just be- fore the souring. Before the milk has soured or after it has curdled, they do not seem to have so much power as they do just before curdling. Care should be taken never to allow the material to cool to a temperature much below 65°. If it does, undesirable bacteria are liable to grow, and the growth of the desired organisms is checked. If a larger amount of starter is needed, for a creamery where there are hundreds of gallons to be ripened, the preparation of a starter must be continued farther. The starter is prepared in exactly the same way up to the last step. Meantime several gallons of cream or skim-milk have been pasteurized, the amount depending upon the size of the churning. This should be cooled to 8o° at about the time when the starter prepared according to the last paragraph has become soured. This starter is then poured into the new lot of pasteurized milk or cream, the mixture is covered to keep out the dust, placed at a temper- ature of about 65°, and when it has become well soured, it is a starter of sufficient quantity to be used in a vat of hundreds of gallons of cream. This process is spoken of as a "building up process." Evidently the whole process is only a means of in- creasing the number of bacteria by growing the culture in a considerable amount of pasteurized milk. The starter thus prepared is added to the cream in varying proportions, the larger the amount the quicker the ripening, a quick ripening being, however, generally undesirable.1 Some- times as much as 1 part of the starter to 10 parts of cream is used; in other cases a smaller amount is used and sometimes more. After the cream in the vat, into which the starter has been inoculated, has been ripened for the proper length of time and is ready to churn, a certain quantity of it is removed, placed in a clean can, and set aside to serve as a starter for the next 1 Dean. Ont. Agr. Col. Exper. Sta., Farm. Rep., 1901. BACTERIA AND BUTTER-MAKING 209 day's churning. In this way some starter is reserved each day to be used in the cream collected for the day, and thus the or- iginal starter is carried on from churning to churning. After some days, however, it is necessary to resort once more to a new pure culture built up in the same way. The reason for this is that the miscellaneous bacteria in the cream may develop in larger numbers each day, until eventually the cream contains too many of the undesirable organisms. How long a starter may be carried from day to day before a fresh one must be substituted cannot be definitely stated. It is customary to con- tinue the same starter for a week, sometimes for two weeks, but rarely for a longer period than this. THE USE OF A STARTER In Ordinary Cream.-In this case, the starter prepared as above described is simply added to the cream of a day's collec- tion, in proportions which experience has found to be most use- ful, without previous treatment of the cream. In using the starter thus it is evidently desirable that the cream should be as fresh as possible, because the fresher the cream the smaller the number of bacteria in it, and the greater the chance that the starter will overcome them and produce its desired effect. The use of starters in this way is open to a theoretical ob- jection. The cream that has been brought to the creamery al- ready contains bacteria in large numbers and, ordinarily, in considerable variety. These bacteria would themselves produce the ripening of cream, even without any starter, although it would be a slower process than when the starter was added. The effect of the starter added to the cream already filled with bacteria in great numbers will evidently not always be uniform. It may be that the bacteria already present will produce their own effect in ripening quite independent of the starter. It may be, on the other hand, that the starter added in considerable quantity will quite overcome the effect of the smaller number 210 PRACTICAL DAIRY BACTERIOLOGY of bacteria originally in the cream. If the starter added in this way is to have any effect, it must be added in sufficient quantity to overcome the deleterious action of any bacteria that may chance to be present. In practice, however, it is found that the use of starters in this way is of value and, in most cases, there is a noticeable improvement in butter made from cream thus ripened. The results, however, are not absolutely uniform, and even with the use of a large amount of starter, it will some- times happen that the bacteria present in the cream will have more influence than those of the starter, and the butter will suffer. In this country, however, this method of using starters is becoming very widely adopted, and most of the creameries that make use of starters at all use them by the method just described. The use of starters in unpasteurized cream was first begun in this country and has been adopted more widely here than elsewhere. In Pasteurized Cream.-Clearly, if it is possible by a simple method to destroy the bacteria already present in cream before the addition of a starter, we might expect the starter itself to have a more uniform and more pronounced effect. This can easily be accomplished by pasteurization, which, as we have already noticed, destroys a large proportion of the bacteria in milk. This method of using starters was first devised and adopted in Denmark before the method mentioned in the last paragraph was used in the United States.1 It is the only logical method of their use. The pasteurizing of cream is simple and not very expensive, and inasmuch as it produces a medium largely free from bacteria, it is to be recommended. The use of starters in pasteurized cream has become practically universal in Denmark and some of the other countries of Northern Europe.2 There are two reasons for this: I. A higher and more 1 Storch. Milchztg., p. 304, 1890. 2 Steiner. Milchztg., p. 401, 1901. Marcas. Rev. Gen. d'Lait, I., p. 413, 1902. Dean. Ann. Rep. Ind. State Dairy Ass'n, 1903. BACTERIA AND BUTTER-MAKING 211 uniform grade of butter can be obtained in this way. 2. The prevalence of tuberculosis has brought about the enactment of a law requiring all milk that goes through the creamery to be pasteurized. For this reason Denmark butter is always made from pasteurized cream, and this makes it necessary to use an artificial starter, since pasteurized cream will not ripen of itself. The pasteurization destroys practically all of the acid bacteria, and, as we have learned, when the acid bacteria are absent the putrefying bacteria are quite sure to develop. Hence, pasteur- ized milk requires an acid starter to insure a proper ripening. Pasteurization is also said to make churning easier. Although the method just described is the only logical method for the use of starters, it has not yet been adopted to a very great extent in the United States. A small but growing number of creameries use it, but for practical reasons it has not become popular. The pasteurization of the cream requires the purchase of new apparatus, and more work in the handling of the cream, so that it involves some additional expense. Moreover, the but- ter-makers have found by experience that the use of starters In cream without pasteurization in the majority of cases produces results that are quite satisfactory. Hence, whereas the use of starters, either natural or commercial starters, has become well- nigh universal in the better creameries, the pasteurization of cream to prepare it for their use has not made very much head- way in this country. In comparing the value of butter made from pasteurized cream with that made from unpasteurized cream, the former is found to be more uniform; but the butter made in this way is not, at least to the American taste, superior to butter made without pasteurization, due probably to the prevention of the growth of miscellaneous bacteria that occurs before the lactic bacteria develop. Pasteurized cream butter is somewhat milder in flavor than that made from ordinary cream, and the Amer- ican market demands a flavor in its butter somewhat stronger 212 PRACTICAL DAIRY BACTERIOLOGY than that which is popular in Europe. Hence, to the American taste, up to the present time, the butter from pasteurized cream is not superior to that made from unpasteurized cream. In pasteurizing cream for butter-making the methods already described are used. Sometimes the whole milk is run through a pasteurizer, and the cream subsequently separated from it. Sometimes the milk is first run through the separator, and the skim-milk and the cream pasteurized separately afterward. The effect of high temperature is not so marked upon cream as it is upon milk. A temperature of 1800 is quite sure to produce the cooked taste in milk; but this temperature, or even a higher one, can be used upon cream without danger of developing the cooked taste in the butter, for these cooked flavors do not seem readily to affect the fat. A temperature even as high as 185° or 1900 might be used without special detriment.1 It has been thought that the use of pasteurization is deleterious to the grain of the butter, but this can be remedied by cooling the cream to a temperature of 48° before churning. One advantage that comes from the pasteurization of cream in butter-making is that butter made from such cream keeps better than butter made from unpasteurized cream.2 The reason for this is not difficult to understand. Pasteurized cream, sub- sequently inoculated with a pure culture, will contain practically nothing but lactic bacteria, and the lactic bacteria, as we have already noticed, prevent the growth of putrefactive and other organisms. The butter made from such cream will, therefore, show very few of the types of bacteria that are liable to give rise to undesirable fermentation, and for this reason the keeping property of the butter is enhanced. It is even found that the keeping of the butter is increased by the use of pasteurized water for washing the butter after the churning. 1 Dean and Harrison. Bui. 117, Ontario Agr. Col., 1902. 2 Dean and Harrison. Ont. Agri. Col. Bui. 117, 1902. Farrington and Godfrey. Ann. Rep. Wis. Exper. Sta., 1903. BACTERIA AND BUTTER-MAKING 213 A second advantage of pasteurizing cream is that it destroys the disease germs. Butter would seem to offer an opportunity for the distribution of the germs of contagious diseases.1 The ripening and churning do not destroy the pathogenic bacteria, and we naturally expect to find them in butter. Market butter frequently does contain the tubercle bacillus, as has been dem- onstrated by many examinations of market butter in Europe, and in America as well. The pasteurization of the cream is the only means that can be adopted for thoroughly protecting but- ter from this source of danger. It should be stated, however, that although theoretically butter may be a means of distributing any of the contagious diseases which are liable to be in milk, no data are at hand which indicate that this actually occurs. While we have many instances of epidemics due to milk, none have yet been reported that are attributed to butter. Whether this means that butter is not a source of danger, because the ripening process has injured or destroyed the disease germs, or whether it is simply lack of evidence, it is too early to state. It is signif- icant, however, to remember that the development of lactic bac- teria in large numbers has a tendency to destroy other bacteria that may be present. Inasmuch as the ripening of cream re- sults in the enormous development of lactic bacteria, it is pos- sible that this process actually removes from the butter some of the danger of its distributing ordinary diseases. The con- clusion, however, does not apply to the tubercle bacillus. GENERAL VALUE OF STARTERS The almost universal adoption of starters in butter-making is in itself enough to indicate that the butter-makers have found them of value. Briefly summarized, the effect of the wide use of starters has been to raise the general quality of the butter. While perhaps the high grades of butter are not any better to- day than they were before the days of starters, there is a much X Reitz. Cent. f. Bact., II., xvi., p. 719, 1906. 214 PRACTICAL DAIRY BACTERIOLOGY smaller quantity of low grade butter produced. The general result has been a raising of the average by a reduction in the amount of poor butter rather than by an improvement in the best grades of butter. In some creamery exhibits it has been found that the starter butter scores on the average two points higher than butter made without a starter. Very little choice is found between the commercial starters and the home starters, either of them producing a good butter and an improvement over butter ripened without a starter. BUTTERMILK In somewhat recent times buttermilk has come to be recom- mended somewhat widely as an article of diet for weak children or invalids. Buttermilk has always been used as a drink, but not until recently has it been regarded as of special dietetic value. The recommendation of buttermilk for children and invalids is a very striking comment upon the impression that bacteria are necessarily harmful. Buttermilk contains bac- teria in quantities, practically as many as are in cream when ripened. The number will be from 200,000,000 to 500,000,000 per c.c., and sometimes very much higher than this. That such material, containing bacteria in these great numbers, can be recommended for children is demonstration beyond question that instead of being harmful the bacteria may be useful to human beings. The presence of acid bacteria in the human intestines seems necessary to control the normal process of digestion. If these acid germs are not present, the contents of the intestines are much more liable to undergo putrefaction, thus producing trouble. For this reason the taking of large numbers of acid organisms into the stomach seems to be very useful.1 Butter- milk is usually almost a pure culture of lactic acid bacteria, and as such it can be used to very great advantage in cases where acid organisms are needed in the intestines. The same will be 1 Bienstock. Ann. d. 1'Inst. Past., xiv., p. 750, 1900. BACTERIA AND BUTTER-MAKING 215 true of sour milk, which is also frequently recommended for this purpose. I'he relation of bacteria to butter after it is made is of less practical importance than their relation to the other milk products. Butter when first made contains large numbers of bacteria, for a considerable proportion of those in the ripened cream will get tangled with the fat. In butter, however, the bacteria do not find a medium very favorable to growth, and instead of increasing in this medium they soon begin to decrease in numbers. Their development in the first few hours is not always the same. Sometimes they will increase for a few hours after the butter is made; and sometimes they will begin to de- crease in numbers at the very start. The difference is apparently due in part, if not wholly, to the amount of washing given the but- ter. If the butter is washed until all of the albuminous material is washed out, less food is furnished to the bacteria, the environ- ment is unfavorable to them, and they begin to decline in num- bers at once. If less thoroughly washed, the conditions are more favorable, and they may increase for a day or two. But in either case they soon begin to decline in numbers. In a single experiment, for example, the following numbers were found: BACTERIA IN BUTTER Butter, 2 hours old . . 50,000,000 bacteria. Butter, 1 day old . . . 26,000,000 bacteria. Butter, 2 days old . . 2,000,000 bacteria. Butter, 30 days old . . 300,000 bacteria. In this reduction of bacteria the lactic germs are the first to disappear, and the spore-bearing bacteria are those likely to remain in the end. In old butter the number of bacteria is al- ways small, but they never disappear, and spore-bearing organ- isms are sure to be found. The common dairy organism, Oidium lactis, is almost certain to be present in considerable numbers 216 PRACTICAL DAIRY BACTERIOLOGY in old butter, and one of the easiest methods by which a culture of this organism can be obtained is to make a common gelatin plate from a sample of old butter. From the facts just stated it is evident that bacteria will have less effect upon the butter after it is made than upon milk or cheese. RANCIDITY OF BUTTER It is well known that butter will not keep indefinitely. In a few days the fresh aroma of a finely ripened butter disappears, due probably to the dissipation of the volatile gases; but from this time on for many weeks the butter may remain in prac- tically the same condition. If it is kept cool, it may be months before there is any further deterioration. But later there is an inevitable appearance of rancidity, and the butter slowly be- comes stronger and finally unfit for use. The cause of the devel- opment of this rancidity has been difficult to determine, ap- parently because a variety of factors contribute to it.1 It is, in part, probably due to chemical fermentation, produced by enzymes from the milk, and in part to the growth of bacteria. The rancidity is much more likely to occur if the butter is ex- posed to the light, and develops more readily at warm than cold temperatures. At temperatures below freezing rancidity does not occur. If butter is, therefore, kept cool and in large masses, it may be held for a long time without the appearance of any very noticeable strong flavor. In the end, however, the rancidity is practically sure to appear. To what extent bacteria are con- cerned in this change we do not know yet, although most in- vestigators have concluded that they are prominently concerned in the phenomenon. There are some species of bacteria known, the butyric acid organisms, which have the power of producing butyric acid. Oidium lactis, B. butyricus, B. fluorescens, Butter- sdure bacillus of Grassberger and other species have been found 1 M'Kay and Larson. Bui. 71, Iowa Exper. Sta., 1903. Rogers. Bui. 57, Bu. An. Ind., 1904. BACTERIA AND BUTTER-MAKING 217 associated with rancidity. It would be natural to assume, there- fore, that they have some important relation thereto. But the change goes on after the bacteria cease to grow and may occur independently of bacteria. Rancidity may be certainly looked upon as a fermentation change, and the only method the dairy- man has of controlling it is by cool temperatures, by packing the butter in large masses, and by keeping it from the light. It may be delayed by pasteurizing the cream and using pasteurized water for washing, a fact that shows its close relation to bac- teria. Fortunately, it is a matter of no very great importance, because butter can be kept without difficulty for some months, and before it has spoiled it is almost always possible to market it.1 Quite a variety of defects occur in butter, some of which are associated with bacteria, while others apparently have no con- nection with micro-organisms. The former are in most cases due to the growth of undesirable bacteria in the cream, producing there certain by-products, which affect the cream and butter. In some cases, apparently, the troubles are due to bacteria grow- ing in the butter itself. The following list includes the most important of these troubles, which have been demonstrated to be associated with bacterial action. Red Spots.-The development of red spots in butter has been studied in a few cases, and has been found to be due to a growth of micro-organisms." In one case, a yeast, known as a pink yeast, has been found to develop somewhat abundantly in butter, producing a red color, and in other cases investigated, the cause was due to bacteria.3 The trouble is a rare one. BUTTER DEFECTS 1 Reinmann. Cent. f. Bact., II., vi., p. 131, 1900. Shattenfroh and Grassberger. Arch. f. Hyg., xxvii., p. 54; xlii., p. 219; lx., p. 40. Eichholz. Hyg. Rund., xin., p. 130, 1903. Jensen. Jahr. d. Land. d. Schweiz, 1901. 2 Weigmann. Milch. Zent., I., p. 4, 1905, 3 Poda. Milch. Zent., II., p. 97, 1906. 218 PRACTICAL DAIRY BACTERIOLOGY Turnipy-tasting Butter.-It has long been a belief that cows fed upon turnips will produce turnipy-tasting milk and butter. This is true to a certain extent, beyond doubt; but in some of these cases the turnipy taste has been found due not directly to the food eaten by the cattle, but to a certain kind of bacteria growing in the milk. Two or three bacteriologists have in- dependently investigated infections of turnipy-tasting butter, and have discovered bacteria, which when inoculated into milk produce this undesirable taste. Hence, a turnipy-taste is, in some cases, of bacterial origin.1 Cowy Butter.-The peculiar odor which is common around the barn, called "cowy odor," is well known. This odor is some- times noticeable in milk, and it has been assumed that this is because the milk absorbs the odors from the barn or the filth that gets into the milk, which is doubtless usually correct. The same odor is sometimes detected in butter, and the interpretation has been that the odors from the barn have been transferred to the butter in this way. But it has been found that certain bacteria are capable of producing exactly this cowy odor, and there seems no doubt that in some cases this peculiar defect in butter is due not to the barn odors primarily, but to bacteria which have been transferred from the barn through the ripening cream to the butter itself.2 Putrid Butter.-This is a difficulty that is rarely seen, and has only been reported in one or two cases. Jensen found that the defect was due to a certain kind of bacteria growing in the cream and contributing to the ripening. The bacterium in ques- tion is a close ally to certain species found in the intestines (B. f>ittrificus), and the putrid taste is really one of decay. This organism in question is readily killed by heat, and the pasteur- 1 Gruber. Cent. f. Bact.. II., ix., p. 684, 1902. Jensen. Milchztg., 1892. 2 Pammel. Bui. 21, Iowa Exper. Sta. BACTERIA AND BUTTER-MAKING 219 ization of the cream is a perfect remedy for the trouble. Eckles found a similar case which was due to liquefying bacteria.1 Lardy or Tallowy Butter.-If butter is kept in the sunlight for a moderate length of time, it loses its color and flavor and becomes of a lardy and tallowy consistency. In addition to this, however, Storch has found that certain bacteria are capable of producing a similar disagreeable odor in butter.2 Oily Butter.-An organism capable of producing an oily effect on the butter, which is very common in Denmark, and produces considerable loss, has been discovered and studied by Jensen. It is an acid organism which curdles milk readily, but when growing in cream develops peculiar characteristics which give a strong, unpleasant taste. The result upon butter is quite disastrous. Bitter Butter.-Butter with a bitter taste is a rare phenom- enon, but a few cases have been discovered. Here, again, the trouble is due to the growth of bacteria in the cream. The trouble is, undoubtedly, closely associated with that of bitter milk described on a previous page. Moldy Butter.-When butter is kept under conditions favor- ing their growth, molds are not uncommon upon its surface.3 These will grow especially on the outside of butter that is packed in tubs, particularly if the conditions are moist. The molds that grow are nothing more than common species, which are liable to appear on any moist organic surface, if the temper- ature favors their development. There is little difficulty in preventing their growth by having the tubs washed in brine. A better method still is to soak the tubs in melted paraffin, the tubs being dipped in a bath of paraffin melted at 2500. This not only prevents molding but reduces loss in the butter from evapora- 'Eckles. Bui. 59, Iowa Exper. Sta., 1901. Jensen. Cent. f. Bact., ii., p. 409, 1891. 2 Fischer. Hyg. Rund., v., p. 513, 1895. Storch. 18. Rep. Danish Agri. Exper. Sta., 1890. 2 Rogers. Bui. 89, Bu. An. Ind., 1906. 220 PRACTICAL DAIRY BACTERIOLOGY tion, this reduction in loss more than compensating for the cost of the paraffin. If the butter is packed in parchment or waxed paper, soaking this also in strong brine will prevent the develop- ment of the molds. The molds do not particularly injure the butter, but detract much from its appearance. Fishy Butter.-Butter that has been exported to Europe from Australia has frequently been reported as having a fishy taste, and a similar fishy taste has sometimes been found in American butter. In all of these instances the trouble is due to the growth of organisms. It is stated that Oidium lactis will sometimes produce this fishy taste in butter, but a similar fishy taste has been described by Rogers due to yeasts. BACTERIA IN OLEOMARGARINE PRODUCTS The oleomargarine manufacturers have found that the flavors which they wish to imitate can be produced only by the proper action of bacteria. Hence, for many years, they have adopted methods for increasing the number of bacteria, so as to obtain for their product as close an approximation to the typical butter flavor as possible. The methods by which they do this are closely related to those of cream ripening. They first place in a vat a certain quantity of milk, cream or skim-milk; usually whole milk is used for the purpose, but fre- quently skim-milk and sometimes, especially for special grades, cream has been used. In this mass of milk is placed a consider- able quantity of a starter, prepared essentially in the same way as before described. The whole is raised to a temperature which stimulates the growth of bacteria, and is allowed to ripen in the normal fashion. In the meantime the manufacturer prepares various fats, which he is to use to make up the bulk of his product. These fats are not always the same, dififerent manu- facturers using dififerent materials. In this country the founda- tion of oleomargarine is chiefly cotton-seed oil, lard and stearin, BACTERIA AND BUTTER-MAKING 221 mixed in proportions to give a constituency to the final product, which is adapted according to the climate where it is to be used. If, for example, the material is to go to a warm climate a larger amount of stearin is included, while if it is to go to a colder climate, more lard. These oils are melted and mixed, and then, after the milk just mentioned above has become fully ripened, it is mixed in the oils in definite proportions, one to five being not uncommon. After thorough mixing the product is drawn off under cold brine, where it immediately hardens into a product resembling butter in most respects, except the color. The mate- rial was formerly artificially colored to resemble butter more closely. As a result of the present law the coloring matter is usually left out, and the oleo products are white. The oleo- margarine itself consists of oils which have very little flavor, but the mixture is flavored by the products of bacteria growth in milk. The flavoring is, therefore, essentially identical with the flavor of butter. As long as the manufacturers of this product were able to color their product so as to imitate butter in appearance, the product which they manufactured was very easily sold as but- ter. It is sometimes of such high grade that it is almost impos- sible to distinguish it from butter by taste or smell, and only by chemical means can it be detected. The product is perfectly healthful, and in some respects even more wholesome than but- ter. The manufacturers of this product are usually more care- ful in their processes than the ordinary farmer. The oleo con- tains less filth, and there is less chance of its containing disease bacteria than there is in ordinary butter. In short, so far as cleanliness and wholesomeness are concerned, a good oleo product is superior to much of the butter on the market. Now that a law practically prevents the coloring of oleomargarine it is no longer possible for it to be sold on the market as butter. It appears, therefore, as an oleo product, and in this form nothing can be said against it as a wholesome substitute for butter. 222 PRACTICAL DAIRY BACTERIOLOGY One reason that the oleomargarine products have acquired such a large market is because the'manufacturers so quickly learned that the butter flavor which they wished could be ob- tained by the aid of bacteria growing in milk. They seized upon this idea earlier than butter-makers did, and utilized this flavor-producing power of bacteria some years before the but- ter-makers had awakened to the necessity of controlling their cream ripening. CHAPTER X BACTERIA IN CHEESE Although cheese is, from a chemical standpoint, the most nutritious of all of our foods, people do not usually eat it be- cause they realize its nutritious value. It is eaten chiefly for its flavor, since it adds a relish to many another food that may be flavorless. In this respect its value is very great, for flavors have a very important part in the economy of digestion. If we look upon cheese, therefore, both from its nutritive and its flavor value, we shall see that it is one of the most valuable of foods. In making cheese the milk is usually first curdled with rennet, after which the curdled casein is separated from most of the water which drains from the curd as zu hey. A large part of the milk sugar is lost since it is dissolved in the whey, while most of the fat remains entangled with the casein. About two-thirds of the food material of the milk is thus retained in the cheese. The value of the cheese is largely dependent upon its ripening, because it is this process which brings it into a proper condition for eating. The fresh cheese contains all the original casein of the milk, but it is rather hard, difficult of digestion and flavor- less. The completed cheese is softer, easier to digest, and it contains the desired flavor. RIPENING OF CHEESES The ripening of cheeses is a very complex phenomenon, and one as yet only partly understood. This is due partly to the intricacy of the subject, which has presented many difficult 223 224 PRACTICAL DAIRY BACTERIOLOGY problems, and partly also to the fact that there are very many different kinds of cheeses, and the ripening of the different types is not by any means due to the same agency. There are no fewer than 250 varieties of cheese in France alone, and. although other countries have hardly as many, the varieties elsewhere are also numerous. These varieties may be fairly well arranged into two groups: (1) the hard cheeses, and (2) the soft cheeses, although there are some intermediate forms. The ripening of the hard cheeses is very different from that of the soft, and the ripening of the different types of the soft cheeses varies greatly one from the other. Each kind of cheese must, therefore, be studied as a special problem, and while the ripening of some kinds of cheeses has been thoroughly studied and is quite well understood, that of others remains wholly for the future to explain. At the present time, there- fore, we can give only some general statements regarding cheese ripening and its relation to micro-organisms. To understand the phenomenon in question we notice, first, that the changes occurring in the ripening can be divided into three classes: (1) physical changes affecting the general ap- pearance of the curd; (2) chemical changes affecting its com- position; (3) chemical changes which develop the cheese flavors. Chemical and Physical Changes.-These may be considered together, because they are doubtless closely connected. A very noticeable physical change takes place in the texture of the curd. When first made this is hard, rather dry, and somewhat tough and elastic; but as the ripening occurs its texture changes, its elasticity disappears, and it turns eventually into a softer constituency, which differs very much in the different types of cheeses. The softness of the resulting cheeses is dependent largely upon the amount of moisture it contains and the temper- ature. The larger the amount of moisture left in the cheeses, other things being equal, the softer it will become; and the higher the temperature at which the cheese is kept, the softer BACTERIA IN CHEESE 225 it tends to become. Dry cheeses are always hard, and even the soft cheeses may be hard after they become dry. The chemical changes which occur are very profound. The first to occur is, in most cases, the development of lactic acid, and in nearly all, if not all, types of cheeses this souring is necessary for the subsequent steps. After the acidity develops the changes go on very much more slowly; but they usually consist of a gradual reduction in the amount of acidity until finally the cheese may become slightly alkaline. At the same time the casein is undergoing a series of changes by which it is gradually reduced from the condition of a raw proteid (casein) to the condition of peptone or proteoses, which are similar products. Proteids are, as a rule, insoluble in water, but after they have been converted into peptones or proteoses, the prod- ucts are soluble. This change is essentially similar to that occurring in the digesting of the proteids in the stomachs of animals, and the changes in the ripening of cheese are very similar to those of digestion. For this reason a ripened cheese is to be looked upon as an already digested food. With several of the soft cheeses, like the Brie and Camembert, the digestion is very complete, and the ripened cheese is an almost wholly predigested product. Such cheeses are very easily handled by the digestive organs and form a very easy assimilable food. The extent to which this breaking down of the casein into soluble products occurs varies very much in the different types of cheeses. In the hard cheeses it is less complete than in the softer ones; and in the latter class the change may be so great as to reduce the casein largely into soluble products. The change also varies with the age of the cheese, for it is a phenomenon that progresses week by week as the cheese ripens and does not become complete until the whole process is finished. This general process is commonly called digestion.1 1 Van Slyke and Hart. Ann. Rep. N. Y. Exper. Sta., 1903. Van Slyke and Hart. Bui. 261, N. Y. Exper. Sta., 1905. Harding. Bui 237, N. Y. Exper. Sta., 1905. 226 PRACTICAL DAIRY BACTERIOLOGY Flavors.-The production of flavors is of no less importance than the chemical digestion of the cheese. At the present time, however, there is a very profound ignorance concerning the real source and cause of cheese flavors. They are without doubt the products of decomposition. They appear in the cheese only toward the end of the ripening process, and are regarded generally as due to the end products of decomposition. The proteids and proteoses, that result from the digestion of the caseins, do not themselves have any of these peculiar cheese flavors; but toward the end of the ripening some of the material seems to be still further broken down into the simpler end products which show the flavors characteristic of cheese. The nature and cause of these flavoring substances we do not def- initely know, but further reference to the matter will be made later. BACTERIA IN CHEESE RIPENING In studying the relationship of micro-organisms to cheese ripening, we can best understand the subject if we consider separately the chief types of cheese. There is no question but that the ripening processes of hard and of soft cheeses, although phenomena somewhat closely related, are very different from each other, and are produced by very different agents. We shall understand the subject better if they are considered separately. Sour Milk Cheese, Dutch Cheese, etc.-There is one type of milk product, commonly called cheese, that is not in any proper sense a true cheese. Under this head are included cheeses that consist of nothing except the curd of sour milk. In making them the milk is allowed to sour under the action of the lactic acid bacteria until it is well curdled.1 Sometimes, however, a little rennet is added to the milk in order that the curdling may occur more quickly than it would under the action of the lactic bacteria alone. After being thus curdled, the curd is separated 1 Hall, Van Slyke and Hart. Bui. 245, N. Y. Exper. Sta., 1904. BACTERIA IN CHEESE 227 from the whey by draining and finally by squeezing, by various processes, until it is reduced into a fairly hard pulp. It may then be run through mills which grind it up into a very fine, smooth mass. This product may be molded into cakes or short cylinders and wrapped in tinfoil or paper, to be sold at once and eaten fresh. This type of cheese has long been known in our households as Dutch cheese, cottage cheese, Smearcase, etc. Such cheeses seem to be easily digested. They are the result of the action of bacteria upon the milk with no other agency seemingly concerned, and are primarily due to the growth of the lactic acid species. In recent years, our dairymen have devel- oped a large market for two very similar cheeses, which they, however, sell under the name of Neufchatel, and Cream cheese. These two cheeses, so abundant in our markets, are scarcely any different from the old cottage cheese of our homes, except that rennet is used to hasten the curdling, and the curd is after- wards ground between rollers in a mill, which renders it smooth. American Neufchatel cheese is, therefore, nothing more than a sour curd, not ripened, and having no real cheese flavors; a very different product from a European Neufchatel cheese. This product should not really be classed under the head of cheeses, but as a sour milk curd. Sometimes such sour milk curds are ripened into a real cheese {Harz cheese), but this is not done in America.1 The ripening that occurs is similar to that of some of the soft cheeses described below. THE RIPENING OF HARD CHEESES Hard cheeses are much more used in the United States than any of the soft varieties. The most common kinds are the American or Cheddar cheese, the Edam cheese, the Pineapple' cheese, and the Swiss cheese. In all of these cheeses the es- sential feature of the manufacture is that after the milk has 1 Eckles and Rahn. Cent. f. Bact., II., xiv., p. 676, 1905. Eckles and Rahn. Cent. f. Bad., II., xv., p. 726, 1906. 228 PRACTICAL DAIRY BACTERIOLOGY been curdled by the use of rennet, the whey is extracted as completely as possible, first by cutting the curd and draining, and then by pressing under a high pressure until all of the whey which can be squeezed out is removed. This makes a hard, tough mass, which is put aside for ripening. The details of manufacture differ, of course, in different types of cheeses. In the Swiss cheese the milk is sweet when it is curdled, while in the American or Cheddar cheese the milk is allowed to be- come sour. Each of the other types of hard cheeses has its own special method of manufacture, with slight differences in the product. The ripening of these cheeses involves both digestive and chemical changes, and the production of flavors. To bring about these changes there are, at least, two different agents, either or both of which may play a part in the process. The cheeses contain both enzymes and bacteria. Enzymes.-In all cheeses there is present a certain quantity of enzymes, capable of digesting casein, derived from, at least, two sources: I. The rennet used in cheese curdling is derived from the stomachs of sucking mammals, and because of its method of preparation rennet will always contain considerable quantities of pepsin, the juice that carries on the digestive func- tions in the mammal's stomach. It is impracticable to prepare rennet from a stomach without its containing some pepsin. 2. It has also been shown by Babcock and Russell that milk itself,1 when secreted, contains a ferment, which they have called galactase, and which has also the power of digesting casein. This is either secreted by the cow or by bacteria growing in the milk ducts of the udder. Both pepsin and galactase are capable of converting casein into peptones or proteoses, and during the ripening period they are both acting slowly but constantly. By some it is believed that they are, to a large extent, responsible 1 Babcock and Russell. Ann. Rep. Wis. Exper. Sta., 1900. BACTERIA IN CHEESE 229 for the changes of casein, which constitute the ripening of the cheese. Bacteria.-The cheeses also contain bacteria in great numbers, and usually in some considerable variety. Bacteria are present, of course, in the original milk, and when the milk is placed in the vat for curdling, their number will be considerable, differing very slightly in the different kinds of cheeses. When the cheese is made of sour milk (Cheddar Cheeses), their number will be very high, for the reasons already indicated, whereas, if cheese is made of sweet milk (Swiss Cheese), the number will naturally be much less. Bacteria, too, are added to the milk with the rennet which always contains vast numbers, some of which are useful, some detrimental and some indifferent.1 It has even been suggested that the useful bacteria (acid bacteria) be artificially added to rennet to hold the others in check. The bacteria which are in the cheese at the beginning grow rapidly. For some days after the cheese is made they increase rapidly in numbers, reaching a maximum usually in about five days, when they may be as many as 650,000,000 per gram.2 After this they decrease. The decrease in cold-cured cheeses is less rapid than in those cured at a higher temperature. The bac- teria found are chiefly lactic acid bacteria, two common types being found, the others that may be present at the outset soon disappearing.3 During the ripening they lose their acid- producing power to a considerable extent, this fact perhaps favoring the development of taints. Chemical Changes.-There are thus, at least, two different factors that may contribute toward the chemical changes of ripening cheeses-the growth of bacteria and the action of the chemical ferments. Which of these two factors is the prom- inent one in cheese ripening is a much discussed question. 1 Thoni. Land. Jahr. d. Schweiz, 1906. 2 Harrison and Connell. Rev. Gen. d'Lait, HI., 1903. Harrison. Rev. Gen. d'Lait, V., p. 409, 1906. 3 Wenzirl. Cent. f. Bact., II., vi., p. 785, 1900. 230 PRACTICAL DAIRY BACTERIOLOGY When cheese ripening was first studied by Duclaux, it was be- lieved by him that the ripening was primarily the action of bacteria. We have already seen that certain kinds of bacteria have the power of changing casein to peptone-the liquefying type (see page 40)-and Duclaux, finding such organisms more or less abundant in cheeses, reached the conclusion that the change from casein to peptone in the cheese ripening was due to the growth of these peptonizing bacteria. This he argued strongly and held for many years, supporting his position by the claim that when treated by heat or antiseptics, so as to destroy bacteria, the bacteria would not ripen. Later the fact was more and more clearly seen that the liquefying bacteria are not common in cheeses, especially in the better grades. 1 f present at the beginning they rapidly decrease in numbers and come to be very few, if not absent entirely, a fact which forced the conclusion that they cannot contribute materially to the ripening of cheeses. More recently, Gorini has advanced the view that certain "acid liquefiers"-i.e., peptonizing bac- teria that at the same time produce acid-are intimately con- nected with the ripening.1 But there does not yet appear to be much evidence for this. These facts led to a suggestion by Freudenreich that the ripening is due really to the lactic acid bacteria.2 Lactic acid bacteria, as we have already seen, do not liquefy gelatin and do not ordinarily have any power of changing casein to peptone. As they grow in the milk, they produce lactic acid which curdles the milk, after which they apparently cease to act upon it at all. Hence, it would not seem that they could digest cheese. Freu- denreich,3 however, showed that if the acid which they produce 1 Gorini. Cent. f. Bact., II., viii., p. 137, 1902. Gorini. Rev. Gen. d'Lait, iii., pp. 289, 405, 1904. 2 Freudenreich. Tirage special d. 1'Ann. agricole de la Suisse, 1905. 3 Freudenreich. Rev. Gen. d'Lait, I., p. 112, 1901. Freudenreich. Land. Jahr. d. Schweiz, 1897, 1899. Freudenreich and Thoni. Cent. f. Bact., IL, xiv., p. 34, 1905. BACTERIA IN CHEESE 231 is neutralized by the presence of some alkaline, like carbonate of soda, the bacteria continue to grow, and eventually produce the peptonization of the casein. Moreover, he was able to show that the grade of the cheeses is very closely dependent upon the growth of lactic bacteria. He found that certain types of lactic bacteria, inoculated into the cheese, resulted in a fine grade of cheese, while others resulted in a poorer product. He found, also, that cheeses from which lactic acid bacteria were excluded by aseptic milking would not ripen normally, while they would do so if the acid germs were present.1 All of these facts to- gether led him to the conclusion that it is this peptonizing power of the lactic acid bacteria, under certain conditions, which is responsible for the chemical changes that take place in the ripening cheese.2 This conclusion, however, has not been very generally accepted; for while others have confirmed his conclusion that the lactic acid bacteria under these conditions do produce a certain amount of peptonization of the casein, the action is extremely slow and not very complete, and it has not seemed to most chemists that the phenomenon in question is sufficiently explained by this slow action of the lactic acid bac- teria. The bacterial explanation of the ripening has been thrown into uncertainty by Babcock and Russell, who showed that cheeses, if kept in a vapor of chloroform, which will prevent bacteria growth entirely without checking the action of the enzymes, will undergo the chemical changes of ripening, al- though they, will not acquire the desired flavor. As a result, a third theory has been advanced that the chem- ical changes of digestion are due to the action of the enzymes? These enzymes are always present, some in the original milk and some added with the rennet, and it is only natural to sup- pose that during the long period of ripening they will carry on 1 Freudenreich. Land. Jahr. d. Schweiz, 1902. 2 Jensen. Milch. Zent., II., p. 393, 1906. 3 Babcock, Russell and Vivian. 17. Ann. Rep. Wis. Exper. Sta., 1900. Jensen. Landw. Jahr. d. Schweiz, 1900. 232 PRACTICAL DAIRY BACTERIOLOGY the peptonizing of the casein, which is their distinctive char- acteristic.1 The conclusion regarded as most probable to-day, therefore, is that the chemical changes, whereby the caseins are broken down to peptones and proteoses, are due to enzyme action rather than to bacteria, and the enzymes that produce the action are partly present in the original milk (galactase), and partly added to it with the rennet, which is used to curdle the milk (pepsin).2 Flavors.-The source of the cheese flavor is even more un- certain than that of the chemical action. The peptones and proteoses, which are obtained by the enzyme digestion of the caseins, do not possess any noticeable flavor, and certainly do not show any trace of cheese flavor. The digestion of the casein, which may have been produced by the enzymes present, will not result in the flavoring of the cheeses. Moreover, the cheeses ripened in chloroform vapor, which allows the enzymes to act, but prevents bacteria from growing, though they ripen, do not develop flavors.3 These flavors must be due to some other cause than enzyme action. That they are the end product of chemical decomposition seems to be extremely probable. In many cases, they are associated with ammonia; and ammonia, as is well known, is one of the final products of proteid destruc- tion. It is generally believed that these end products are the result of the action of bacteria or some other micro-organisms. The only known agency that commonly produces the complete destruction of proteids is bacteria, and while the matter has never been put to any satisfactory test, the most probable ex- planation seems to be that these cheese flavors are the result of the final bacterial decomposition. Against this view, however, has been urged the fact that in 1 Van Slyke, Harding and Hart. Bui. 203, N. Y. Exper. Sta., 1901. 2 Marcas and Huyge. Rev. Gen. d'Lait, vi., p. 27, 1906. 3 Van Slyke and Hart. Bui. 231, N. Y. Exper. Sta., 1903. Harding. Bui. 237, N. Y. Exper. Sta., 1903. Van Slyke, Harding and Hart. 20. Ann. Rep. Wis Exper. Sta., 1902. BACTERIA IN CHEESE 233 the well-ripened cheeses hardly any bacteria, except lactic acid organisms, are present, and this class of bacteria does not, so far as is known, have any power of producing cheese flavors. Some bacteria, if they grow in proper abundance in milk, will in time develop well known cheese flavors; but these organisms have not been found in old, strongly flavored cheeses. Whether they have anything to do with the production of cheese flavors is therefore uncertain. It has been suggested by Rogers 1 that the flavor of cheeses is due to the bacteria which grow in them during the first few days. Liquefying bacteria are found during this early period, and before the miscellaneous bacteria disap- pear, as they do later, some of these liquefiers may secrete from their bodies substances, possibly enzymes, that continue their action in the cheese slowly, but for a long time. Although the bacteria that produce them soon die, the chemical ferments which they have produced continue their activity until they finally produce the new products that give the flavor. On the other hand, Harrison and Connell2 think the lactic bacteria most closely associated with flavor. All of this, however, is at present largely theoretical. We know that the digestion of the casein, whether produced by pepsin or by galactose, does not produce the cheese flavors. We know that certain kinds of bacteria are capable of producing these flavors, and while the matter has not yet been settled, the probability seems to be that the flavoring of the cheese must be attributed to bacterial action. Other Functions of Bacteria in Cheese-ripening.-Whether or not bacteria are the source of the flavor of hard cheeses, there is no doubt that they do play a very important part in cheese- making in several distinct directions. As has already been indicated, the first step in the ripening of the cheese is the souring of the curd, and this is produced by lactic acid bacteria. It seems to be quite necessary that this should take place in 1 Rogers. Bui. 62, Bu. An. Ind., 1905. 2 Harrison and Connell. Cent. f. Bact., II., xi., p. 637, 1904. 234 PRACTICAL DAIRY BACTERIOLOGY order that the proper chemical changes be obtained. In the manufacture of the Cheddar cheese the development of a proper acidity in the curd is necessary to produce the proper texture in the cheese, and, also, to bring about the proper ripening. If the milk sugar is washed out of the cheese so that lactic acid cannot be formed the ripening does not progress normally; and very badly ripened, badly flavored cheeses are the result.1 This is due to the fact that in such cheese the acid bacteria do not grow, while the liquefiers develop abundantly. A study of the chemistry of the ripening of cheeses has shown that the con- version of the casein into the desired digested products occurs only when lactic acid is present to combine with them in forming certain new products. Thus the development of lactic acid in the cheese is a necessary first step in the ripening, and inasmuch as this lactic acid is dependent upon lactic bacteria, the ripening is closely associated with these organisms. Weigmann believes further that the lactic bacteria prepare the way for other species that grow later and furnish the flavors; but this is not yet proved. In the ripening of Swiss cheeses the relation of bacteria is somewhat different. These cheeses, as already noticed, are made from sweet milk rather than sour. The Swiss cheeses when properly ripened contain large holes, a distinctive char- acteristic of this peculiar type. These holes seem to be due to the carbon dioxide gas produced by certain types of bacteria, but it is not the common lactic acid bacteria that produce them, for these do not give rise to gas. Nor does it seem to be the common gas-producing lactic bacteria referred to on page 34; for these, as noticed later, are apt to ruin cheeses. The normal holes of the Swiss cheeses are due to a different species of bacterium, which has recently been described.2 But neverthe- less the Swiss school of dairymen has learned that the ripening 1 Babcock and Russell. Cent. f. Bact., II., ix., p. 757, 1902. 2 Freudenreich and Jensen. Cent. f. Bact., II., xvii., p. 529, 1906. BACTERIA IN CHEESE 235 can be very much better controlled if the curd contains a culture of proper lactic acid bacteria, although it is not yet known exactly what action they have in the ripening. As a result of these facts, cheese makers have in recent years learned that the use of lactic acid starters in their milk is decidedly advantageous. This has been independently worked out and adopted in Europe and in America, and in the last few years the practice of using pure lactic starters in milk which is to be made into cheese has been rapidly growing. It has been found that this practice enables the cheese maker to con- trol much more accurately his ripening cheese and to reduce the number of failures. The reason why the inoculation of a lactic starter tends to reduce the failures in cheese-making can be easily understood from the facts already considered. The lactic acid bacteria have the power of checking the growth of other germs, and even of destroying them entirely. When, therefore, the milk has a large quantity of lactic bacteria devel- oping rapidly in the curd, the other bacteria, which might, under different circumstances, produce unpleasant flavors and tastes, thus injuring the cheese, are prevented from increasing. The lactic bacteria protect milk from putrefactive and other changes which would result from the growth of miscellaneous bacteria; but in the handling of milk this is a matter of little importance, for the milk is used so quickly. In the handling of cheeses, however, this protecting action of the lactic bacteria becomes very much more important and is indeed the secret of good cheese. The cheese curd lies for weeks, or even months, in a moist condition, and there is opportunity during all this time for the growth of bacteria. If a proper lactic organism is present at the outset, the cheese will be protected from the various putrefactive types that would otherwise be likely to injure it. Their absence in sufficient quantity is responsible for many of the defects to be noticed later.1 1 Peter. Cent. f. Bact., xiv., p. 321, 1905. 236 PRACTICAL DAIRY BACTERIOLOGY For these reasons, then, the cheese industry is learning the prime importance of a strong lactic fermentation in milk to be made into cheese, and in order to bring this about it is rapidly adopting the method of using starters. The starters used in cheeses are essentially identical with those used in but- ter-making, and they are used in much the same way. Home starters are frequently made and inoculated into the milk, and the use of commercial starters is also rapidly growing. It has been suggested that acid bacteria be inoculated into the rennet to prevent the growth there of undesirable bacteria. It is in- teresting to find that types of lactic bacteria which are useful in butter-making are not always satisfactory in cheese-making. In some experiments, for example, it has been found that bac-* teria that give a fine flavor and aroma to butter, produce a bitter taste and a ruined product when used in cheese-making. This simply indicates that the types of lactic bacteria are different, at least so far as concerns their final action on the casein. The use of starters in the cheese industry seems to be firmly estab- lished at the present time, and is practically sure to extend, for it is one of the methods of safe-guarding cheese against un- desirable fermentations; and it is almost sure to become recog- nized as even more necessary than in butter-making. BACTERIA IN EDAM CHEESE It is an interesting fact to learn that, some years before the relation of bacteria to cheese-making was studied, the Holland cheese makers had practically learned the value of bacteria cultures in making Edam cheeses.1 For many years they have used a starter known as slimy whey, which was added to the milk that was to be made into cheese. This material contains a large quantity of a species of a coccus, Strep. Hollandicus, that makes the milk slimy, and when used in cheese-making regulates the ripening so that the cheeses mature more rapidly and more 1 Bockhaut and Ott de Vries. Rev. Gen. d'Lait, v., p. 1, 190S. BACTERIA IN CHEESE 237 uniformly than when made without such a starter. Not all Edam cheeses are made by the use of such a slimy whey, but its use has become quite prevalent. The cheeses made from it are no better than the best cheese made without it, and indeed some think they are not quite so good; but they are more uni- form, there is less chance for failure, and they ripen in four weeks instead of six. It has been recently shown that the use of a pure culture lactic starter in Edam cheese works just as well as the slimy whey. PASTEURIZATION IN CHEESE-MAKING In the manufacture of hard cheeses the use of pasteurization as a method of preparing the milk for the reception of a starter has not been successful. Pasteurization has an effect upon milk, such that it does not curdle by rennet in exactly the same way that it would otherwise do. This matter is one of comparatively little importance, for the curdling property can be restored by the addition of a calcium chloride to the milk. But experience has shown that, for some reason as yet unknown, cheeses made from pasteurized milk will not ripen normally. Whether this is due to the fact that pasteurization destroys certain of the enzymes necessary for ripening, or to the fact that it kills some of the necessary micro-organisms, or to both facts combined, is not yet ascertained. Pasteurization, however, has hitherto been found impracticable for the hard cheese industry. What may develop in this line in the future cannot be stated.1 Temperature of Ripening.-The ripening of hard cheeses is a slow process, requiring weeks and sometimes months before completion. The higher the temperature, the more rapid the ripening, and the less the uniformity2 of the cheeses. The 1 Milchztg, p. 582, 1904. Henzold. Milchztg., p. 262, 1901. Tiemann. Milchztg., p. 386, 1901; p. 212, 1902. Klein and Kirsten. Milchztg., p. 6, 1901. 2 Farrington and others. Ann. Rep. Wis. Exper. Sta., 1903. Babcock, Russell, Vivian and Baer. Ann. Rep. Wis. Exper. Sta., 1901. See also Bui. 49, Bu. An. Ind., 1903, and 20. Ann. Rep. Bu. An. Ind., 1903. 238 PRACTICAL DAIRY BACTERIOLOGY common practice of leaving the cheese in a fairly cool room to ripen at normal temperatures has produced a fairly rapid ripening, giving a more or less strong cheese of the type that has been common on the market. In the last few years, how- ever, experiments have been undertaken to determine the effect of ripening at lower temperatures. The results have been highly satisfactory, for it is found that if the cheeses are placed in ripening rooms at temperatures below 56°, though ripening goes on much more slowly, it is more uniform, more satisfac- tory, and there is a noticeable improvement in the product. The use of low temperatures for ripening is quite new, but has ex- tended rapidly, and at the present time a large amount of cheese is thus ripened. DEFECTS IN HARD CHEESES Most of the defects which injure a cheese, sometimes com- pletely destroying it, are due to the growth within it of undesir- able organisms. There is quite a variety of these, some of which are rare, some of which are fairly common, and some of which cause immense losses to cheese makers the world over. The most important we will briefly consider. Gassy or Swelled Cheese.-The phenomenon spoken of as gassy or swelled cheese is perhaps the most common of cheese faults. It is due to the development inside of the curd of a considerable quantity of gas, which fills it full of holes and causes it to swell and lose its shape. Sometimes the holes are extremely numerous and small, and sometimes they are fewer but of larger size; and, in any case, they are un- desirable. The abundance of the gas produced varies greatly. Even good cheeses are apt to show some gas holes, but the amount is so small it does no especial injury to the cheese. Sometimes the gas is so abundant as to cause the cheese to burst, in which case it is, of course, completely ruined. Between these extremes are all kinds of intermediate grades. In some BACTP2RIA IN CHEESE 239 cases, the development of the gas is accompanied by an unusual fermentation and an unpleasant taste and smell. The appear- ance of much gas always injures and sometimes totally ruins the cheese. The cause of the trouble is the development of gas-producing bacteria. Several different species are known, which have this power of developing gas in great quantities in FIG. 6l-PIECES OF CURDLED MILK One curdled by the common Bad. lactis acidi, showing no gas; the other by Bad. aerogenes, filled with gas bubbles that would spoil a cheese the ripening cheese. Most of them, perhaps all, belong to the type which have been referred to in Chapter II as Bad. aero genes type. (Fig. 61.) As pointed out in that chapter, the different varieties of this type vary much in the amount of gas they produce; sometimes the quantity is very slight, some- times it is extraordinarily large, and it is easy to understand how different strains and different quantities of bacteria will produce grades of gasiness in cheeses. It is to be remembered that the gas which produces the swelled cheese is a different thing from the gas which produces the normal holes in the Swiss 240 PRACTICAL DAIRY BACTERIOLOGY cheese. Swiss cheeses also suffer from gas production, but the phenomenon is totally different from the one which produces the ordinary holes so familiar in that type, and is caused by different species of bacteria. Remedies for gassy cheese are chiefly in the way of pre- vention. not of cure. Indeed, it may be stated concerning all the defects of cheeses that, after they have once made their appearance, there is practically no remedy. They are slow in developing, and usually are not seen until the cheese is so far ripened that the injury is beyond repair. The cheese makers' defence is in guarding against them; and for this purpose various methods are within his reach. One of the most satisfactory is the use of a large, vigorous starter of common lactic acid bacteria. The protecting power of this type of bacteria will prevent the growth of many others, and if the non-gas-producing acid organisms are present in the milk in sufficient quantity, the gassy organisms will be held in check. A vigorous culture of slimy whey is said to be efficient in preventing swelling of Edam cheeses, and it will do the same in other types of cheese.1 A second factor in preventing gas formation is the use of low temperatures. As pointed out on a previous page, the gas-producing organisms develop readily at high temperatures, 8o° to ioo°. At lower temperatures, on the other hand, they do not grow very readily, while the Bad. lactis acidi type does grow vigorously. If, therefore, the curd is kept at low temperatures, the develop- ment of the gassy curd and the gassy cheese is much lessened.2 Cheeses that will become gassy at 750 will not do so at 60°. It is possible by a cool temperature so to check the develop- ment of gas that a fair cheese may be produced from milk, which contains enough of gas bacteria to ruin it. A third partial preventive is the use of more salt in the cheese, for this 1 Bockhaut and Vries. Cent. f. Bact., II., xii., p. 89, 1904. 2 Spallanzani. Rev. Gen. d'Lait. IV., p. 477, 1905. BACTERIA IN CHEESE 241 in a measure prevents the development of the gas organisms. A fourth preventive is the use of the ferment or curd test of the samples of milk, as shown on page 258. By the use of this ferment test the cheese maker can discover whether the milk from any of his patrons contains too large a number of gas-producing organisms. If so, he can discard the suspicious milk, and thus avoid danger of excessive gas production in his general product.1 Bitter Cheese.-Large losses have been produced in cheeses by the development of a bitter taste, a trouble probably always due to the growth of micro-organisms. In one extended infection of this sort, the bitterness was traced by Harrison to yeasts (Torula amari) found in the vats, in the cans, and other utensils which held the milk to be made into cheese.2 (Fig. 26.) A thorough cleaning and sterilization may be efficient in removing the difficulty. In other cases, a similar trouble has been due to the action of bacteria from sources not yet understood, developing vigorously and producing strong, bitter-tasting products.3 One of the common sources of such trouble is the liquefying bacteria. These, as we have seen, are always likely to be in the milk, but their growth in the cheese is held in check by the lactic acid bacteria. Sometimes the acid organisms are not vigorous enough to restrain them, and they develop too abundantly. As we have earlier noticed, they may produce bitter products, and these affect the cheese. Large losses are sometimes due to such a cause, the whole output of a factory being affected in this way for a long time. Remedies against these troubles can be found only on the same 1 Freudenreich. Land. Jahr. d. Schweiz, 1890. Weigmann. Land. Woch. f. Schlesw.-Holst., 1890. Thoni. Land. Jahr. d. Schweiz, 1906. Hastings. 22d Ann. Rep. Wis. Exper. Sta., 1905. Russell and Hastings. Bui. 128, Wis. Exper. Sta., 1905. Weigmann. Milch. Zent.. IL, p. 441, 1906. 2 Harrison. Rev. Gen. d'Lait, L, 1902. 3 Freudenreich. Land. Jahr. d. Schweiz, 1894. Weigmann. Milchw. Zent., IT., p. 441, 1906. 242 PRACTICAL DAIRY BACTERIOLOGY lines already pointed out, cleaning of the milk vessels, more care in the production of the milk, and the use of larger quantities or more vigorous starters of lactic acid organisms, to check the growth of other bacteria. Putrid Cheese.-Sometimes milk becomes acted upon by putrefying bacteria; soft spots appear upon the surface, which may become larger and eat their way into the cheese, and produce a more or less slimy appearance upon the cheese, even to the center. The extent of the trouble will depend upon the abundance of the putrefaction. The trouble is undoubtedly due to the growth of putrefying bacteria, but not much is known about the matter at present. Fruity or Sweet Cheese.-This is a phenomenon which occurs sometimes over widely extended districts, and detracts from the character of the cheese without always ruining it. It is characterized by a peculiar sweet taste, which, although not unpleasant, spoils the flavor of the cheese and thus injures the sale of the product. This trouble has been studied by Harding and found to be due to a yeast, which gets into the milk.1 Rusty Spot of Cheese.-This is a defect occurring in the cheese during the ripening, characterized by rusty, red spots on the outside and, indeed, not infrequently throughout the whole cheese. The cheese loses its value, and may in the end become quite ruined, if the trouble develops sufficiently. The cause is a bacterium, B. rudcnsis, which has been studied by Harding and Connell.2 Tn all these cases the only remedy is the preventive one of a greater guard placed over the milk supply, a more careful cleaning and steaming of the milk vessels, until the organisms producing the troubles are eliminated. 1 Harding, Rogers and Smith. Bui. 183, N. Y. Exper. Sta., 1900. 2 Harding, Rogers and Smith. Bui. 183, N. Y. Exper. Sta., 1900. Harding and Smith. Bui. 225, N. Y. Exper. Sta., 1903. BACTERIA IN CHEESE 243 The difficulty in dealing with all cheese defects is that one cannot be sure, until weeks after he has applied his remedy, whether it is sufficient to eradicate the trouble. The defects usually appear weeks after the cheese is made; if, there- fore, the trouble should appear, and a dairyman should insti- tute a most careful renovation and cleaning of his vats and cans, it will be weeks before he can be sure that he has reached and removed the difficulty. This makes the handling of the cheese problem an extremely difficult one, and it emphasizes strongly the lesson that only by constant watching can one be successful in producing uniformly a good product. More than any other dairy product is cheese dependent upon the presence and growth of the proper micro-organisms. SOFT CHEESES The essential difference between a hard cheese and a soft cheese is the quantity of water left in the cheese after the making, the soft cheeses having a very much higher percentage of moisture. To bring about this condition the method of manufacture is designed to retain the whey in the curd. After the milk is curdled the curd is sometimes dipped out directly into forms provided with holes in their sides, through which the whey drains naturally without the application of any pressure. (Fig. 62.) In other cases, the curd is cut in the vat, but the curd and whey together are dipped into forms for draining. In all cases, the whey simply drains naturally without pres- sure. As a result, there is produced a cheese which is softer in texture and which contains a very much higher quantity of water than is allowed to remain in the hard cheeses. This large amount of water produces a ripening of a totally different character from that which occurs in the hard cheeses. The process of ripening is different, the agents that bring it about are different, and the final result is very dif- 244 EXACTICAL DAIRY BACTERIOLOGY ferent from that in the hard cheese; moreover, the ripening is liable to greater variations in the soft cheeses than in the hard cheeses and is more difficult to control. The difficulty of controlling the ripening can be readily understood when one appreciates the effect of the large amount of moisture present. Bacteria, molds and yeasts all find a suitable medium for growth in the wet curd of the soft green cheese, and unless FIG. 62-FORMS USED IN MAKING AND DRAINING CAMEMBERT CHEESES the progress of the ripening is exactly right, the cheese maker may expect the development of kinds of micro-organisms that are unfavorable to his product and that will spoil his cheeses. The experience of soft cheese makers bears out this theoretical conclusion, for the variety of soft cheeses is very great, and a large number of cheeses are ruined by improper ripening, due to the growth of undesirable organisms. Soft cheeses are much less uniform in character than hard cheeses. They differ very greatly in texture and in flavor, and are subject to a large number of defects that injure or ruin them. They are, in BACTERIA in cheese 245 short, more difficult to make with success than the hard cheeses, largely, if not wholly, because the water they contain offers such a favorable medium for the growth of bacteria and other micro-organisms. Partly for this reason, also, they are the higher priced. Two or three types of soft cheeses have been carefully studied, and their ripening is fairly well understood. We may, therefore, give in some little detail the facts known concern- ing the ripening of three of these types of soft cheeses, the three selected being those that are most popular in our markets, and those which bring the highest prices: the Camembert, the Roquefort and the Limburger. These types have been sub- jected to a more thorough investigation, and more is known concerning their method of ripening than of any other kinds of cheese. Roquefort cheese is not strictly a soft cheese, since the cuid is pressed, but it is softer than the typical hard cheeses, and moreover its ripening is more like that of the soft cheeses than the hard. Hence, we class it here. Of these three types, one is ripened by molds primarily, one by molds and bacteria together, and the third by bacteria without molds. THE CAMEMBERT TYPE This includes the Camembert and the Brie cheeses, which are essentially alike, differing only in size and shape, and some- times in their amount of fat, but made and ripened in the same way.1 The French Coulommier cheese also belongs to the group, and the ripened Neufchatel. The latter is not found in this country. The ripening process is essentially the same in all cases; all have a moldy rind, and the resulting cheese is very much alike. These cheeses were first manu- factured in France, where they have been made for a century; but from that country they have been distributing themselves ' Epstein. Arch. f. Hyg., XL., p. 1, 1902; XLV., p. 354, 1902. Thom. 22d Ann, Bep, Bu, An. Ind., 1905. 246 PRACTICAL DAIRY BACTERIOLOGY over the neighboring countries in Europe. An attempt has also been made to manufacture them in America, but hitherto with only partial success.1 While it has been possible to manu- facture cheeses with the same qualities as Camembert, it has hitherto been impossible to do so with sufficient uniformity to make the industry a perfect success. The reason for the lack of uniformity is quite easy to understand when one has a knowledge of the real nature of the ripening. The ripening of the Camembert cheese, the best illustration of this type, has been so thoroughly studied that it is fairly well understood. The first phe- nomenon that occurs is the souring of the curd, which is brought about by a Bad. ladis acidi type of organism. These grow in the milk previous to the addition of the rennet, although the milk is not allowed to become very sour be- fore curdling. But they continue to grow during the curdling and for a day or two after the cheese is made. If by any chance the lactic bacteria fail to develop vigorously, a ruined cheese is sure to result. The second step in the ripening is the appearance on the surface of the cheese of a species of mold, which has been named Penicillium camemberti.2 (Fig. 63.) This mold appears in from two to four days and is, at first, of a pure white color; later, when it begins to produce spores, it becomes a steel gray, but never a deep blue like the common mold. (Fig. 64.) It is a species of mold that apparently does not occur in FIG. 63-PENICILLIUM CA- MEMBERTI Showing method of form- ing spores (Thom.) 1 Conn, Thom and others. Ann. Rep. Storrs Exper. Sta., 1905. Thom. Ann. Rep. Storrs Exper. Sta., 1905. 8 Thom. Ann. Rep. Storrs Exper. Sta., 1905. BACTERIA IN CHEESE 247 America, but it is very common in Europe in the sections where Camembert cheese is made. Its absence from America is the chief reason why America has been unable to make Camembert cheese; and where successful cheeses have been produced, it has been by importing and inoculating this species of mold FIG. 64-FOUR COLONIES OF A COMMON MOLD ON SOFT CHEESE Penicillium camemberti into the American cheese factories. This white mold grows on the surface of the cheese, but does not penetrate below the surface. After about two weeks it reaches its limit of growth, forms spores and dries down to a somewhat thin crust. With the white mold there commonly develops, also, a considerable quantity of Oidium lactis, see page 55, and without much doubt this organism contributes in a measure to the resulting 248 PRACTICAL DAIRY BACTERIOLOGY product.1 The growth of these two organisms together neu- tralizes the acid of the curd at the surface of the cheese and renders it slightly alkaline. In the meantime the mold has secreted a chemical ferment, an enzyme, which has the power of digesting the curd. As fast as the acidity of the curd is reduced and the enzyme secreted, the latter acts upon the curd, changing it from a hard con- sistency to a soft texture. This softening begins over the whole of the cheese, immediately underneath the mold on the surface, and extends its way toward the center. At first, the cheese is a hard, solid curd from surface to center, but as this enzyme acts beneath the mold there is seen a thin layer of softened material. This layer grows deeper and deeper as it encroaches upon the curd. The enzyme produces a profound change in the casein, converting it first into peptones and similar bodies; later, these break down into still simpler bodies, or "end products," among which ammonia may always be detected. These latter end products give the flavor, and appear to be produced by bacteria rather than by the action of the enzymes secreted by the mold. During the ripening the cheese will be found to have a core of a sour, acid curd in the center, sur- rounded by a layer of soft, digested material. (Fig. 65.) The cheese ripens thus from the surface inward and is not com- pletely ripened until the soft layer reaches the center. One of the difficulties in making a satisfactory cheese is to have the ripening reach the center so as to ripen the cheese com- pletely before it becomes too soft at the outer edge-over- ripe. The degree of softness varies greatly. Sometimes the cheese becomes so soft that it will run out like cream when the cheese is cut, a condition that is prized by some connoisseurs. More commonly it is of a waxy or soft, buttery texture, not soft enough to "run." The waxy brands are less highly flavored and in general more popular. 1 Maze. Rev. Gen. d'Lait, iv., p. 472, 1905. BACTERIA IN CHEESE 249 The flavors are not due to the enzyme digestion but to the end products of decomposition. In the case of this cheese, as in the hard cheeses, no positive knowledge is at hand as to the exact source of the flavor. That it is not due to the mold alone is certain from the fact that the softened cheese may be nearly tasteless, if a pure culture of mold has completed the ripening. The peculiar Camembert flavor is beyond doubt associated with some of the micro-organisms growing on or in the cheese. Bac- FIG. 65-CAMEMBERT CHEESE, CUT OPEN TO SHOW THE SOFTENING OF THE CURD teria commonly grow upon the surface of the cheeses, producing, late in the ripening, a red-brown color characteristic of many good cheeses; but some cheeses with an ideal flavor do not show this red color, and hence it does not appear essential. These reddish-brown areas appear chiefly where the layer of mold has been rubbed or torn away. The Oidium lactis is certainly con- cerned in the flavoring of the cheeses, at least in some cases, for if the Oidium is too abundant the flavor is ruined, while, if it grows moderately, the flavor is ideal. Apparently the flavor 250 PRACTICAL DAIRY BACTERIOLOGY is affected also by the type of lactic starter; for some acid organisms, when used for starters in the cheese, produce dif- ferent flavors in the final product from those produced by others. All that can be safely stated at the present time is, that while the digestion is due to the mold, the flavors are the result of the action of other organisms, probably bacteria aided by Oidium lactis, and that various types of undesired flavors are due to the over-growth of Oidium or some other mischievous bacteria. The manufacture of these cheeses in Europe has been carried on only by empirical methods; for the micro-organisms present in European dairies appear to be the ones that are needed to produce the desired ripening of the Camembert cheese. All that the cheese makers need is to make their cheeses according to a regular rule and expose them to the organisms that are natural to their dairies. Sometimes, even in Europe, these methods do not produce a sufficient inoculation of the white mold, and the cheese suffers in consequence, and when the same methods were tried in America the proper mold never appeared. In earlier years, when our cheese makers tried to adopt the same method of cheese-making, they produced a different type of cheese. The molds which grew upon the surfaces of such cheeses in our dairies were not the white mold, but either a blue mold or the Oidium lactis, and these produced very different results from the white species, spoiling the cheeses. Hence it was found necessary to prevent their growth. But in doing this, and not knowing that he needed to substitute the white mold, the cheese maker produced a type of cheeses different from the real Camembert, with bacteria and yeasts on its surface instead of molds. To be sure he called them Camembert, but they were really nearer a Limburger cheese, and no one who knew the imported product would fail to see a great difference between them. The real Camembert was mild and delicate; the American type strong and tasted of incipient putrefaction. BACTERIA IN CHEESE 251 In the last few years, aided by the work of the agricultural department, an attempt has been made to introduce into this country the micro-organisms necessary for the production of the cheeses, and to inoculate them artificially into the dairies, with the hope of making on this side of the Atlantic a real Camem- bert cheese. The result of these experiments has shown that it is perfectly possible to make such cheeses, and many have been made that are equal in character to the imported ones. It has proved, however, to be extremely difficult to handle the organisms in a manner that is uniform and certain to produce desired results. At the present time, this industry has not been placed upon a firm footing. The cheeses are made in consider- able quantities in this country at the present time, but while some of them are equal to the imported, the quality of the gen- eral product is still below that of the imported cheese. We may confidently expect, however, that success will be achieved in this line in the not distant future. In the manufacture of the Camembert type of cheese in America the difficulties experienced are due to the undue growth of organisms that are deleterious and that produce un- pleasant results. Sometimes the Oidium lactis grows too lux- uriantly upon the cheese; when it does so it prevents the mold from getting the proper growth, and the resulting cheese has a very strong, unpleasant taste. Sometimes the class of liquefy- ing bacteria get into the milk, and the lactic acid bacteria do not grow sufficiently to prevent their developing. During the period of ripening the moist curd furnishes them every op- portunity to grow, and they develop bitter tastes, as we have learned they will do, if they grow too luxuriantly. In some cases, the mold develops too luxuriantly, the cheese digesting too rapidly under the rind and becoming much too soft here before the ripening reaches the center. A variety of other troubles appears in the manufacture of these cheeses, all of which are due to the improper control of the various organisms 252 PRACTICAL DAIRY BACTERIOLOGY concerned in the ripening. It will be recognized that in the manufacture of a product which requires the proper succession of three or four distinct species of micro-organisms, the problem of their control becomes extremely difficult, and it is not sur- prising that the artificial use of these cultures has not always FIG. 66-WORTHLESS CAMEMBERT CHEESE Showing porous texture produced by gas-forming bacteria been successful. When we remember, too, that the curd is so full of moisture that it offers the very best medium for bacteria growth we can readily understand the difficulties that the maker of soft cheeses may meet. (Fig. 66.) These various troubles must be met by different methods, but there are two general plans of action that help to meet them all. The first is that of pasteurizing the milk. While, as al- ready pointed out, pasteurizing the milk does not seem to be practical with the hard cheeses, since the cheeses do not ripen. BACTERIA IN CHEESE 253 it is perfectly feasible with the soft cheeses. By thorough pas- teurizing most of the mischievous bacteria in the milk can be killed, and then, if proper lactic bacteria and mold cultures are subsequently inoculated into the milk, the ripening will take place normally and more uniformly. This procedure has been adopted in some of the better and more scientific factories in Europe, and is also being taken up in this country in the manu- facture of these cheeses. The manipulation of the pasteurized milk needs to be slightly different, but offers no great difficulty. The second general safeguard for the- cheese is the use of a large amount of a vigorous lactic acid starter to hold in check the growth of the organisms that would otherwise be likely to produce trouble. By these two methods it will probably be pos- sible to control the ripening and to avoid the irregularities that have too frequently arisen. This type includes the Roquefort, the Stilton and the Gorgon- zola cheeses. Their method of manufacture is somewhat dif- ferent, but, in all cases, the essential character of the cheese is produced by the growth of the same species of mold within the cheese. They do not have a moldy rind and show no ex- ternal evidence of the ripening agent. Anyone familiar with these cheeses will remember that when ripened they are filled with green masses and streaks, and microscopic study shows that these are the spores of a species of mold that has been named Penicillium roqueforti. (Fig. 67.) It is much like the Camembert mold, only that the latter is white, while the Roque- fort mold, when it produces spores, is a blue green. In the ripening of the Roquefort, Stilton, etc., the molds do not grow on the surface, as in the case of Camembert cheese, but they grow within the cheese; and to bring this about the texture of the curd is made somewhat porous, so that it contains abundant air spores within, into whicji the mold can grow. THE ROQUEFORT TYPE 254 PRACTICAL DAIRY BACTERIOLOGY In the manufacture of the typical Roquefort cheese, the fol- lowing general procedure is adopted. The cheese maker begins by cultivating the necessary mold upon bread; and after the moldy bread has produced a great quantity of spores, the mass is dried and ground into powder. All true Roquefort is made from the milk of the sheep, although cow's milk is sometimes used, in part, in making an inferior grade.1 Goat's milk is not used for these cheeses, contrary to popular impression. After curdling the milk with rennet in the usual way, and draining the curd, it is placed in a form in a thin layer, and over the top of the layer is strewn a quantity of powdered, moldy bread with its thousands of spores. Over this is placed another layer of curd, and more mold spores; and then a third layer of curd over all. The mold is thus planted within the cheese. The whole is then pressed by moderate pressure in a form. After a few days the cheese becomes hard enough to be removed from the form, and is next placed upon a machine which punches it full of holes by means of small needles. The purpose of this is to allow air to enter into the center of the cheese, thus furnishing the molds in the center with the air they need for growth. The cheese is then put into the ripening room, where the molds develop, growing partly upon the surface, but primarily within. The surface of the cheese is sometimes covered with a growth of bacteria, forming a slimy layer, and does not show any trace FIG. 67-PENICILLIUM ROQUE- FORTI Showing method of forming spores (Thom.) 1 Milchztg., p. 152, 1903. BACTERIA IN CHEESE 255 of the mold; but within the cheese the mold grows into the cracks and air spaces, and into the holes punched by the needles, in time permeating the whole. As the molds grow, they develop a peculiarly peppery, piquant taste, which is character- istic of the Roquefort cheese. Just before the cheese is fully ripe they taste bitter, but this taste disappears as the final flavor develops. A good Roquefort cheese is only possible when there is a luxuriant growth of these molds within the cheese, no surface growth being allowed. It is interesting to note that although the Stilton and Gorgonzola cheeses are ripened by the same mold, the method of manufacture does not commonly in- volve the artificial inoculation with molds, reliance being placed upon the natural inoculation with mold spores from the air or the dairy utensils. The molds grow, however, and produce a similar flavor to that of Roquefort. The makers of these cheeses sometimes punch them full of holes to stimulate mold growth. In the Gorgonzola cheeses, especially, this punching is not done till the cheeses are a month old, or more, and fre- quently masses of bacteria are carried by the punching needles into the holes from the surface. These may grow within the cheeses, producing discolored areas which detract from the quality of the cheese. Most of the Roquefort cheeses are made in South France, or, at least, ripened there. The reason for this is that their proper ripening requires a low temperature and high moisture conditions, and in South France there are large, damp, lime- stone caves, partly natural and partly artificial, where the ripening can go on to perfection. These caves have a tempera- ture of about 6o° and not 400, as sometimes stated. Three companies have obtained possession of these caves, and they have in their hands practically all of the Roquefort business of the world. Roquefort cheeses are sometimes made in other places, but they are practically all sent to these companies for ripening. 256 PRACTICAL DAIRY BACTERIOLOGY The manufacture of the Roquefort cheese in the United States is yet to come. There seems to be no reason why a cheese can- not be made in this country, which is ripened by the Roquefort mold and which will have the Roquefort flavor as a result; but it is not likely that a real Roquefort can ever be made in Amer- ica, because, as already stated, the typical Roquefort is made of sheep's milk. It is doubtful if Americans will ever be con- tented to raise sheep and milk them; and unless the sheep's milk is used the true Roquefort cannot be made. Stilton cheese and Gorgonzola cheese are, however, made from cow's milk and ripened by the same mold that is found in Roquefort. These cheeses can certainly be made in this country. Stilton has al- ready been made in Canada, and there is no reason why the manufacture cannot be undertaken and developed in the United States. It will require some experimenting and doubtless some new methods and special conditions for ripening, but there will practically be less difficulty in making this type of cheese than in making the Camembert type. This type of cheese, originally produced in Germany, is now made in great quantities in this country. It is a soft cheese that is ripened wholly by the agency of bacteria, without any mold growth. No extended study of the ripening of the Lim- burger cheese has yet been made, but its essential nature is as follows:1 After being drained into a form that is firm enough to handle, the cheeses are placed in a ripening cellar. Every few days they are removed from the shelves and rubbed over with some liquid, water being commonly used, although vinegar is some- times put into the water. The surface is thus kept constantly moist. Because of this constant moisture on the surface of the THE LIMBURGER TYPE 1 Freudenreich. Rev. Gen. d'Lait, i., p. 477, 1902. Roi. Milchztg., p. 163, 1903. BACTERIA IN CHEESE 257 cheese, molds cannot grow upon it, for they need a damp, not a wet surface; but a quantity of bacteria grow instead. These ripen the cheese, doubtless by the secretion of chemical fer- ments, although the process has not as yet been fully studied. The resulting cheese contains very high flavors, closely re- sembling those of decay, and the cheeses rapidly putrefy when they become old. If they are marketed at the right stage, the flavors are not strong enough to be disagreeable, and many persons are very fond of them. The Limburger type of cheese includes Bachstein and some others. GUARDING THE MILK SUPPLY Cheese seems more likely to be injured by unfavorable bac- teria than other milk products. The reason is that it remains for such a long time, during the ripening, in a condition more or less favorable for bacteria growth. Any mischievous organ- isms that get into the milk have, therefore, a great chance for producing trouble before the cheese is ready for market. This is even more true of the making of soft than of hard cheeses. No cheese maker can produce good cheese from milk filled with mischievous bacteria. The cheese maker must, therefore, be on constant guard to protect his milk and all other materials from undesirable bacterial contamination. Several practical suggestions for testing and guarding milk are, therefore, espe- cially appropriate at the close of this discussion on cheese. Since a cheese factory is likely to receive milk from many different farms it will happen that the milk from the different sources will not be alike. It may be that while most of the milk is good, that from a single farm contains a quantity of mis- chievous organisms. If this milk is mixed with the rest, it may spoil the whole; but if the infected dairy can be detected, and its milk excluded from the rest until improved, the whole output will benefit. To enable the cheese maker to do this there have been devised the Fermentation and the Curd Tests. 258 PRACTICAL DAIRY BACTERIOLOGY The Fermentation Test.-This consists simply in taking small samples of milk from the different dairies, putting them in separate vials or bottles, placing them in a warm temperature, about 900, for a few hours, and carefully examining the results. If the milk curdles in the normal fashion, with a clean smooth curd, the sample may be regarded as satisfactory. If large numbers of gas bubbles appear in any sample of milk, or if ex- ceptionally unpleasant odors arise, it may be assumed that the milk in question contains something that is liable to produce trouble, and it will be wise, therefore, to refrain from mixing the milk from the individual patron with the general day's col- lection. Under these circumstances, a visit to the creamery may soon show the source of trouble and suggest a remedy.' The Curd Test.-The curd test is similar in its purpose, and is designed primarily to prevent gassy cheese.2 It consists in placing in clean vessels-fruit cans will serve perfectly well for the purpose-about a quart of the milk of each patron. This is heated to 900, and about ten drops of rennet added to it. after which it is allowed to stand until it curdles. The curd is cut, and the whey poured off occasionally, the whole curd being allowed finally to mat together. For this purpose it should be kept at a temperature of about 98°. After it has become well matted, the curd is cut open and examined. In the case of good milk, the curd will be fairly smooth, showing only occasional mechanical holes. If, however, the curd proves to be full of pores, indicating the presence of gas within the curd, it is prac- tically certain that the milk contains some of the gas-producing organisms, which are liable to give rise to trouble. Under these circumstances, the milk of the patron in question should be looked upon with suspicion. These tests are used quite commonly in some cheese-making countries, but have not, as yet, a very wide application. There 1 Dugelli. Cent. f. Bact., II., xvii., p. 37, 1907 * Babcock, Russell and Decker. 12th Ann. Rep. Wis. Exper. Sta., p. 148, 1895. BACTERIA IN CHEESE 259 are some instances where they may be very useful. In the or- dinary working of a cheese factory it is hardly possible to use the tests for the milk of any particular day, for it is impractical to wait long enough to test the different samples of milk before they are mixed together in a general curdling vat. But the tests may be very useful in detecting whether the milk from any one farm is constantly "off," so as to be a danger to the general supply. For tracing the source of trouble the tests may thus be of great value. The cheese maker should constantly bear in mind the danger of contamination of his milk. Sometimes "off" flavors in cheeses have been traced to bacteria in the dairy water supply. Old barrels of souring whey are a source of trouble. The practice of soaking rennet in sour whey to extract the rennet material is sometimes common, but it is a bad practice, for such a mixture furnishes a most excellent medium for bacteria growth, and troublesome yeasts and bacteria have been traced to such a source. Dirty cans sent back to the farm without sterilization, slovenly methods in the barn, and, in short, all of the sources of bacterial contamination previously mentioned are very likely to trouble the cheese maker as much, if not more, than any others connected with the dairy industry. PART II LABORATORY DIRECTIONS FOR EXPERI- MENTS IN DAIRY BACTERIOLOGY. LABORATORY WORK IN DAIRY BACTE- RIOLOGY It is expected that the experiments in this section shall ac- company the study of the text. Recognizing, however, that different classes of students will have different times and con- veniences for laboratory work, the two parts of the subject have been separated. It must be emphasized, however, that the prac- tical work is fully as important as the theoretical discussion, and no one can really understand the latter without the former. The course of experiments outlined will probably be too long for short courses. For this reason the important experiments that should be performed without fail are indicated by *. These are fundamental. The others may be taken as time per- mits. It will frequently be a good plan to divide the experi- ments not thus marked among different sections of the class, having each section report the purpose, method and result of such experiments to the whole class. The directions for these experiments are given in sufficient detail, so that anyone with a fair knowledge of laboratory meth- ods can follow them without other instruction. The private student of dairying may, therefore, carry out this laboratory course by himself, although, of course, it will take more time in this case than if adequate personal instruction were ob- tained. Special emphasis should be placed upon the necessity of care- fully labeling every culture made and recording in a note book each experiment, its purpose and results. 263 264 PRACTICAL DAIRY BACTERIOLOGY It may not always be possible for the student to follow the order of experiments given. There is no necessity for following the order given except that numbers i to 13 should precede all others. LIST OF APPARATUS NEEDED Steam sterilizer. Autoclav. Hot air sterilizer. A common gas oven used for cooking will do. Stew pan for cooking media. Flasks-1 liter and 1% liter. Test tubes-Board of Health pat- tern. Petri dishes-4 inches in diameter. Pipettes-1 c.c., 2 c.c. A few larger ones are also convenient. Each of the smaller pipettes should have a glass tube to contain it, sealed at one end. Wire baskets to hold test tubes. Test tube racks. Burette holder and 4 burettes. Evaporating dishes. Measuring cylinders-1 liter and 100 c.c. Counting plate or counting card. Platinum wire to be fused into glass rods. Common tumblers are frequently more convenient than test tube racks for holding test tubes. Culture oven, with constant temperature of 98°. This is ex- pensive and may be omitted. FIG. 68-TEST TUBES-BOARD OF HEALTH PATTERN 265 LABORATORY WGkk 1W DAIRY BACTERIOLOGY Bunsen tumr/s. Forceps-Common and Cornet forceps. Microscope, including a Vu-inch immersion lens and plenty of slides and cover glasses. A counting plate or counting cards. Peptone. Salt. Beef extract. Gelatin-gold label. Agar Agar. Litmus, dry in cubes. Absorbent cotton. Common cotton, good quality. MATERIALS Phenolthalein. Alc^....i. Corrosive sublimate. NaOH. HC1. Immersion oil. Methylen blue. Normal NaOH Normal HC1 These solutions will best be bought from dealers. They may be made by methods described in No. 2 and 3, but the beginner will hardly be able to make the Normal HC1 successfully. Dextrose, lactose and saccharose. PRACTICAL WORKi *No. 1. Washing Glassware. All glassware used in bacte- riological work must be thoroughly washed. No special direc- tions need be given save that hot water and soap are necessary. New glassware should be treated first with i% HC1. Used glassware that contains the remains of gelatin or other media must first be boiled in water, preferably containing a little sal 1 The following laboratory manuals may be found useful as books of ref- erence: Moore. Laboratory Directions for Beginners in Bacteriology. Ginn & Co., 1900. Frost. A Laboratory Guide in Elementary Bacteriology. The Macmillan Co., 1903. Gorham. A Laboratory Course in Bacteriology. W. B. Saunders, 1901. Van Slyke. Methods of Testing Milk and Its Products. Orange Judd Pub. Co.. 19°6. 266 PRACTICAL DAIRY BACTERIOLOGY soda or powdered soap. After boiling, wash well in hot water and then rinse thoroughly in clear cold water; drain and allow to dry. The student may begin by thus washing 50 test tubes (Board of Health pattern, Fig. 68), 2 liter flasks, 1 dozen Petri dishes and several I c.c. pipettes. After drying, plug the test tubes and FIG. 69-PIP- ETTE AND GLASS TUBE TO HOLD SAME WHEN STERILIZED. FIG. 70-HOT AIR STERILIZER flasks with cotton, place the pipettes in pieces of glass tubing closed at one end and plugged with cotton at the other (Fig. 69). Place all the articles in the sterilized oven (Fig. 70), and heat to 155° C. (3io0 F.) for one hour. This sterilizes them and 267 LABORATORY WORK IN DAIRY BACTERIOLOGY they are ready for use. All glassware subsequently used should be treated in the same way. *No. 2. Normal NaOH Solution. To make this with absolute accuracy special chemical methods are necessary. A solution which is nearly accurate, sufficiently so for all purposes of this work, can be made by dissolving 40 grams of pure NaOH (in sticks) in one liter of water. This should be kept in a tightly closed bottle. It does not maintain its strength long and should frequently be made fresh. To make 1/10 normal NaOH, one part of normal is mixed with nine parts of distilled water. *No. 3. Normal HCL This is more difficult to make, and it will usually be best to buy it, already made, of dealers in chemical supplies. It can be made as follows: Add 50 c.c. of chemically pure HC1 to 450 c.c. of distilled water. If it is of the right strength, one c.c. of it will exactly neutralize one c.c. of normal NaOH. Place one c.c. in an evaporating dish, dilute with 10 times its bulk in water, add a few drops of phenolthalein, and then exactly one c.c. of normal NaOH. If the mixture turns red, it means that there is not enough acid in the solution, and a little more should be added. If no red appears, add a drop or two more of 1/10 normal NaOH, and see if the red color appears. If it does, the HC1 solution may be regarded as normal. If, however, it requires several drops to produce the red color, the HC1 solution is too strong and must be weakened by adding water. By adding a little acid or water as may be indicated, and testing repeatedly, the HC1 solution may be brought to a strength where it will exactly neutralize the same amount of NaOH. When this point is reached, it is to be labeled Normal HC1. *No. 4. Preparation of Bouillon. Measure out the following: Water I liter Liebigs extract of beef 3 grams Common salt 5 grams Peptone 10 grams 268 PRACTICAL DAIRY BACTERIOLOGY Place in some dish in which the mixture may be boiled, and carefully weigh the dish and its contents. Dissolve the mixture at a temperature of about 1500 F. (6o° C.), after which weigh again and restore the evaporated water by bringing the weight to its original mark. Bacteria culture media need to have a proper grade of acidity. If the amount of acid is either above or below the desired point the bacteria either do not grow at all or not very readily. The method of bringing the bouillon to the desired point is some- what complicated and must be described in detail. a. Measure out five c.c. of the bouillon, place it in an evap- orating dish with ten times its bulk of water, and bring nearly to a boil. While still hot add to it a few drops of phenolthalein.1 This is a solution which is colorless so long as it is acid, but turns red as soon as it becomes alkaline. The bouillon is always acid and consequently the phenolthalein will make no change in its appearance. b. Place the evaporating dish under a burette containing 1/10 normal NaOH. Take the reading of this solution carefully in the burette. Allow the solution to drop from the burette into the evaporating dish, drop by drop, stirring carefully. (See Fig. 60.) As long as the material remains acid no color will appear. Allow the solution to drop into the dish until a faint red color makes its appearance and does not disappear upon stir- ring. When this point has been reached the contents of the evaporating dish are at a neutral point. c. Taking the reading for the burette, determine how many cubic centimeters of the solution have been used to neutralize the 5 c.c. of bouillon. Divide by 5 to determine the amount necessary to neutralize 1 c.c. and multiply by 995 to determine the amount necessary to neutralize the rest of the bouillon. d. Add to the entire quantity of bouillon enough NaOH to neutralize it, but in doing so use normal NaOH instead of 1/10 18 per cent, dry phenolthalein in 50 per cent, alcohol. 269 LABORATORY WORK IN DAIRY BACTERIOLOGY normal. It will, of course, require only i/io as many cubic centimeters, as calculation has shown above would be required of i/io normal. This will bring the culture media to the neutral point. e. This medium is now not acid enough for the best bacteria growth. The best reaction is one in which there is 15 cubic centimeters of normal HC1 to each liter of the media (1.5%). Con- sequently to this bouillon there should be added 14.9 c.c. of normal HC1 which will give the desired acidity, 1.5%. An example will show this more clearly. Suppose that it required 1.5 cubic centimeters of the 1/10 normal NaOH to neutralize 5 c.c. of the bouillon. Dividing this by five would give .3 c.c. required to neutralize 1 c.c. Multiplying by 995 would give 298 c.c. required for neutralizing a liter. Dividing by ten to reduce this to normal NaOH will give 29.8 c.c. Therefore, 29.8 c.c. normal NaOH is added to the bouillon, giving the neutralizing point. After this, 14.9 c.c. of normal HC1 added to the material will give an acidity, 1.5%. f. After obtaining the desired acidity, bring the bouillon to a brisk boil for 15 minutes, after which enough water must be added to restore to the original weight. Heat once more to boiling and filter through filter paper into a sterile flask. Place about 10 c.c. in a few test tubes. Replace the cotton plug in both the flask and test tubes; place the test tubes and the flask in a steam sterilizer and steam for 20 minutes (Fig. 71). Put aside at a temperature of the room for 24 hours, then steam FIG 7I_STEAM STERilizer PRACTICAL DAIRY BACTERIOLOGY 270 again for 20 minutes. Set aside another 24 hours and steam again. The bouillon is now sterile and can be kept indefinitely. FIG. 72-FILTERING CULTURE MEDIA THROUGH ABSORBENT COTTON "1 he upper funnel containing cheese cloth, the lower absorbent cotton If the bouillon or any part of it is to be made into agar or gelatin as shown below, the neutralization and sterilization may be omitted. *No. 5. Agar Culture Medium. a. To 500 c.c. bouillon prepared as in (3) add 1.5% of agar LABORATORY WORK IN DAIRY BACTERIOLOGY 271 (7.5 gm.). Allow the agar to soak in the cool bouillon for % hour. Place in a stew pan and record the exact weight of FIG. 73-FILLING TEST TUBES WITH A MEASURED AMOUNT OF A CULTURE MEDIUM 272 PRACTICAL DAIRY BACTERIOLOGY dish and contents. Boil until all the agar is dissolved. Weigh again and restore to original weight by adding water. b. Determine acidity. Neutralize and adjust reaction to 1.5% HC1, as described in 4, c. c. After obtaining the proper acidity bring the mass to a brisk boil, and while it is boiling add to it the white of an egg, previously mixed with a little water. Boil hard for 20 minutes. d. Weigh, and add water to bring it back to its original weight. Bring to a boil. e. While boiling, as in d, place in a large funnel a consider- able quantity of absorbent cotton. Over the top of the funnel, or in a second funnel over the first, place some cheese cloth (Fig. 72). Pour the hot agar mass through the cheese cloth into the absorbent cotton. It will run through the cotton and may be caught below in a flask. It should be transparent, clear, and of a light yellowish-brown color. f. From the flask fill a number of sterilized test tubes, putting about 10 c.c. into each. (Fig. 73.) Replace the cotton plug. In filling these tubes it is best to use a funnel, and care should be taken not to allow the agar to touch the tube where the cotton is afterwards to be inserted, as it will stick to the cotton. g. Place the tubes, together with what remains in the flask, in a steam sterilizer, figure 71, and allow the steam to act on them for 20 minutes. Then remove and set aside in the temper- ature of an ordinary room. h. 24 hours later put the material again in a steam sterilizer, and sterilize for % hour. i. 24 hours later sterilize again in steam for % hour. The material is now finished and may be preserved for future use. j. Agar slants. After the last steaming, while the agar in the tubes is still hot and liquid, lay 20 of them down on a table with their mouths slightly raised so that the agar will form a slanting surface in the tubes. (Fig. 74.) Allow7 them to harden in this position. LABORATORY WORK IN DAIRY BACTERIOLOGY 273 *No. 6. Determination of the Number of Bacteria in Milk. It is always first necessary to dilute the milk with sterilized water, since ordinary milk contains so many bacteria that, un- less diluted, the numbers will be too large to be estimated prop- erly. Milk contains such a widely varied number, however, that it is difficult to determine beforehand how much to dilute the material; and one must be guided, when possible, by knowledge as to the age of the milk. Where a miscellaneous lot of milk FIG. 74-HARDENING AGAR SLANTS is to be studied for this purpose, probably the best method is to dilute it 1,000 times. In order to dilute milk 1,000 or any other definite number of times, it is necessary to have flasks and vials measured to hold certain definite amounts. It is most convenient to have some holding 4 c.c., 9 c.c., 19 c.c. and 99 c.c. (Fig. 75.) From them the desired dilution can readily be obtained. a. Fill a number of these vials to the mark with water, plug with cotton and sterilize. This can best be done in an autoclav. (Fig. 76.) In using this autoclav place a small amount of water within it and then the vials to be sterilized. Replace the cover, fastening it down tightly with the screws designed for the purpose, and light the gas. Leave the cock open until steam begins to come out, and then close. Allow the steam pressure to rise to 15 lbs. and maintain this pressure for one hour. Allow the autoclav to cool before opening. The vials may then be 274 PRACTICAL DAIRY BACTERIOLOGY removed ready for use and, since they are sterile they will keep indefinitely. They will be called water blanks. (Note.-The autoclav may be used to sterilize other liquid media. If it is used for gelatin care must be taken not to allow the pressure FIG. 75-VIALS FOR MILK DILUTION to rise above 5 lbs., or the gelatin will refuse to harden sub- sequently. The successful use of the autoclav for sterilizing gelatin requires considerable experience.) b. Shake the milk to be tested, very vigorously, in order to insure uniformity, and, with a sterile pipette remove one c.c. to one of the vials holding 9 c.c. of water. Shake, and with a fresh pipette transfer one c.c. to a second water blank of nine c.c. Shake again, and with a fresh pipette transfer one c.c. LABORATORY WORK IN DAIRY BACTERIOLOGY 275 to a third similar water blank. This last vial will contain i/ioo of a c.c. of milk and one c.c. of it will contain 1/1,000 of a c.c. It will readily be seen that any modification of the dilution may easily be made. If two water blanks only are used, the dilution will be 100. If four are used, it will be 10,000. If very high dilutions are desirable, for example, 1,000,000, six vials can be used, or it is better to proceed somewhat differ- ently. Place 99 c.c. of water in each of three flasks or bottles, and ster- ilize. To one add 1 c.c. of the milk to be tested, and, after a thorough shaking, transfer 1 c.c. into a second flask con- taining 99 c.c. Transfer 1 c.c. of this to a third flask and 1 c.c. of this last will contain 1/1,000,000 of a c.c. of milk. By very simple calculation any desired dilution may be obtained. c. Melt four test tubes of the agar medium in hot water and cool to a temperature of about 500. Into each tube place, with a sterilized pipette, 1 c.c. of the diluted milk and then mix thoroughly by gentle shaking. After the mixing pour the con- tents of the tube into a sterilized petri dish (Fig. 77) and cover immediately. As soon as the agar hardens place the petri dish, now called an agar plate, in an incubating oven at a temperature of 370. It is always well to make at least four such plates in order that an average may be taken for the final result, since no one plate can be relied upon for very great accuracy. After 24 hours at 370 examine the plates; they will be covered with FIG. 76-AUTOCLAV, FOR STERILIZING UNDER STEAM PRESSURE 276 PRACTICAL DAIRY BACTERIOLOGY small dots, each of which represents what is called a colony of bacteria. They are supposed in all cases to be a mass of bac- teria that have developed from a single individual in the orig- inal milk which has been fixed in its position by the hardened agar. If every bacterium in the original milk has thus grown and produced a colony, it is only necessary to count the colonies FIG. 77-PETRI DISH in each agar plate to find the number of bacteria in 1/1,000 of the original c.c. of milk product, which was the amount taken. Count the numbers on each plate; add them together and divide by 4 to determine the average number. Multiply by 1,000 to give the number of bacteria per c.c. of the milk. For convenience in counting, the bottom of the plate may be divided into small areas by marking with a wax pencil. If the number is very great use a counting plate or counting cards. These are divided into areas of 1 sq. centimeters. Place the plate over the card and count the number of colonies on each of several of the 1 cm. areas and obtain the average. Determine by the card the number of such areas in the whole plate, and by multiplying calculate the total number of colonies on the plate. The box shown in figure 78 will be found very convenient in counting the colonies. It consists of a shielded lens, and a slate LABORATORY WORK IN DAIRY BACTERIOLOGY 277 base with a divided circle. It is made by the International Scientific Instrument Co. It will be evident that the number thus obtained will always be an under estimate of the number of bacteria, because some bacteria will not grow in the agar culture medium. There are also other facts which decrease the number of colonies, so that the numbers thus obtained will never be equal to the total number of bacteria. *No. 7. Gelatin Culture Media. a. Measure out and place in a stew pan a definite quantity of the bouillon prepared as above de- scribed and add to it 12% of a first grade gelatin, "Gold Labe! Gelatin'' being preferred. Allow the gelatin to soak in the bouillon till soft and al- most melted. Weigh the whole material, with its containing dish, and record weight. Boil briskly for five minutes, then add water to restore original weight. b. Determine acidity and bring reaction to 1.5% HC1, as described in 2, b to e. c. Boil 15 minutes. Add the white of an egg and boil briskly for 15 minutes. Replace the water of evaporation. d. Heat once more to boiling and filter through absorbent cotton, as illustrated in Fig. 72. e. Fill 25 test tubes with about 10 c.c. each; sterilize the tubes and the rest of the gelatin in a flask, by steaming in FIG. 78-COUNTING-BOX (International Instrument Co.) 278 PRACTICAL DAIRY BACTERIOLOGY steam sterilizer 20 minutes. Allow to stand 24 hours in room temperature, and steam again for 20 minutes. 24 hours later steam a third time. It is now sterile and may be reserved for future use. *No. 8. Litmus Gelatin. For the study of milk bacteria it is very desirable in most cases to detect the production of acid. For this purpose a litmus culture medium is used, the prepara- tion of which is as follows: a. To 500 c.c. bouillon add 15% gelatin and 1% lactose (milk sugar). The rest of the process up to the point of filling the test tubes is exactly as above described in 5, a to e. In filling test tubes, exactly 8 c.c. of the medium is to be placed in each of the test tubes, and the test tubes are to be sterilized on three successive days. b. Prepare a litmus solution by weighing out 50 grams of solid litmus (the litmus cubes preferred). Soak for 24 hours in 5 times its weight of water; filter through filter paper, and a deep blue solution will be obtained. This is too alkaline, and it is necessary to bring its reaction to the same point as the reaction of the gelatin-namely, 1.5%, as follows: c. Measure out 5 c.c. of the litmus solution in an evaporating dish; dilute with 10 times its bulk of water, and heat. Add to it from the burette 1/10 normal HC1, drop by drop, until the blue turns from a deep blue to a faint red. This gives the neutral point of litmus. d. By the reading on the burette before and after neutraliza- tion determine the number of c.c. required to neutralize 5 c.c. of the litmus. If we divide by 5 to give the amount required to neutralize one c.c. and multiply by 495, we shall have the number of cubic centimeters necessary to neutralize the whole litmus solution. If this amount of acid is added it will bring the litmus solution to the litmus neutral point, which is different from the phenolthalein neutral point used in 4, b. The litmus neutral point is 2.5% more acid than the phenolthalein neutral LABORATORY WORK IN DAIRY BACTERIOLOGY 279 point, and it is desired to make the reaction 1.5%. To do this several methods are possible, the simplest of which is as fol- lows: To the litmus solution add the amount of HC1 necessary to bring it to the phenolthalein neutral point, and then add 10 c.c. per liter of normal NaOH to bring it back to 1.5%. An actual example will make this clear. Suppose we start with 500 c.c. and find that it requires 1.5 c.c., 1/10 normal HC1 to neutralize 5 c.c. 1.5 4- 5 = .3 x 495 = 148.5 c.c. 1/10 normal required for 495 c.c. of solution. 148.5 -4- 10 = 14.8 c.c. normal HC1. Add 14.8 c.c. to the lit- mus solution. .495 x 10 - 4.9. Hence, by adding 4.9 c.c. normal NaOH, the litmus solution will be brought to the desired reaction, 1.5% acid. Since this solution and the gelatin made in a are of the same degree of acidity, they may be mixed in any proportion without changing the reaction. e. Sterilize the litmus solution in the same way as the gelatin. The method of combining the gelatin and litmus will be men- tioned below. No. 9. Litmus Agar. This is prepared by adding to ordinary agar (5) milk sugar and enough litmus solution to give it a blue color. It has some advantages over plain agar, since it differentiates acid colonies. It is not so useful as litmus gelatin. *No. 10. Gelatin Plates. Gelatin culture media offer a num- ber of advantages over agar, since they enable us to tell more satisfactorily the kinds of bacteria which may be present in the milk. It is more difficult to use, however, and results more frequently in failure, for reasons to be noted presently. In making gelatin plates from milk, it is desirable to dilute the milk to such an extent that the number of bacteria in each plate shall be not more than 500 or 600, preferably not more than 200. If the numbers are higher than these, there is likely to be trouble and inaccurate determinations. For this reason, 280 PRACTICAL DAIRY BACTERIOLOGY to make a successful plate with gelatin, it is necessary to know something of the age of the milk, in order that the proper dilu- tion may be obtained. If milk is not more than 24 hours old, and has been kept fairly cool, a dilution of 1,000 will ordinarily be satisfactory; sometimes a higher dilution is necessary. This difficulty of determining the amount of dilution is the difficult point of the gelatin method. For this reason, when gelatin is used for estimating bacteria in milk, it is desirable to make several plates of different dilutions in order that the proper one may be reached in one at least. Procure some milk from 12 to 24 hours old. Dilute it as above described so that one vial has a dilution of 1,000, another of 10,000, and another of 100,000. Add 1 c.c. of each dilution to separate tubes of the melted gelatin, thoroughly mix and pour into the Petri dishes, replacing the cover. It is best to make at least two such Petri dishes from each solution. Place at first in a cool place to harden. The plates must be kept in a temperature of not much over 700 or the gelatin will melt. For this reason it is difficult to use gelatin in summer. The bacteria at 700 do not grow as rapidly as they do at 370, and the colonies do not begin to appear, usually, for 2 days. The ap- pearance of the colonies in the gelatin plates is much different from that in agar plates, as will be noticed later. *No. 11. Litmus Gelatin Plates. This requires only a slight modification of the process described in the last paragraph. Dilute the milk as above. To several tubes of gelatin prepared in 8, a, add 2 c.c. of the litmus solution prepared in 8, b to c. Melt and mix thoroughly. Place in each tube 1 c.c. of the diluted milk, then pour into the Petri dishes, after hardening, keep at about 700. After the colonies have grown for two or three days, those which produce acid will be found to be red, or surrounded by a little red halo where the blue litmus has been turned to red. It is, therefore, easy by examination to determine the number of acid producing bacteria in such milk. LABORATORY WORK IN DAIRY BACTERIOLOGY 281 From the fact above stated it will be evident that for a gen- eral determination of the number of bacteria in samples of milk whose origin and age are unknown, it is easier to use agar with a dilution of 1,000 than gelatin. If we have an approximate idea of the number of bacteria, so that the proper dilution may be determined, the use of litmus gelatin will enable us not only to determine in numbers, but will give the means of fairly well separating the different kinds of bacteria. Gelatin, therefore, will give most of the information that agar gives, and consider- ably more under conditions where gelatin can satisfactorily be used. The numbers obtained from plain gelatin plates are usually higher than those obtained from litmus gelatin, and the numbers from either plain gelatin or litmus gelatin are much higher than the numbers from agar. Determine the numbers of colonies in both the plain gelatin plates and the litmus gelatin plates. Isolate several colonies as in the following: *No. 12. Isolation of Colonies. The bacteria which develop on plates prepared as above described, produce clusters, as al- ready mentioned, called colonics. In many cases it is pos- sible to tell the type of bacteria from the shape and appear- ance of the colony. A study of the plates, especially the litmus gelatin plates, will, therefore, frequently enable us to determine, not only the numbers, but something concerning the kinds of bacteria. The bacteria are now to be isolated from the plates. Sterilize a platinum needle (Fig. 79) in a flame, then dip the tip of it into one of the colonies. When it is re- moved, a number of the bacteria will adhere to it, and these can then be transferred to one of the slant agar tubes, by re- moving the cotton plug and drawing the tip of the wire gently over the surface of the agar. Sterilize the needle again before laying it down. Label the tube by some number, recording in a note book the appearance of the colony, and its source. *No. 13. Purification of Cultures. Presumably the culture 282 PRACTICAL DAIRY BACTERIOLOGY made in No. 12 will be a pure culture. By this term is meant that it will contain only one kind of bacteria. If the colony on the plate comes from a single bacterium, as has been assumed, the colony of bacteria will contain a single kind of organism. It frequently happens, however, that a single colony may come from two bacteria, side by side, in which case it may not be a pure culture. In order to be sure of having a pure culture, it is always necessary to purify it, as follows: a. Fill some test tubes with about 10 c.c. of water, and sterilize one hour in an autoclav. These are water blanks. b. After the cultures of No. 12 have grown from one to two days, remove with a platinum needle a small amount of the bacterial growth and transfer it to a water blank. Shake thoroughly so as to distribute the bacteria uniformly. Transfer with a platinum loop, two loopfuls of this to a second water blank, for the purpose of more highly diluting the bacteria. Again after a thorough shaking, trans- fer two loopfuls of this second tube to a tube of melted gelatin. Shake gently, to distribute the drop of water uniformly through the gelatin, and pour out into a Petri dish. In about two days the plate will be dotted with colonies. Pick out one with a platinum needle, inoculate upon an agar slant, and it will almost invariably be pure. This purification of cultures must be done in all subsequent work. If it is not done, errors are sure to result from the mixture of different kinds of bacteria together, and all the work will be rendered worthless. *No. 14. Preservation of Cultures. After having once been planted on the surface of agar slant, agar culture may be kept alive, as long as the agar remains moist; and it may be studied fig. 79 platinum NEEDLES LABORATORY WORK IN DAIRY BACTERIOLOGY 283 at any time. But when the agar becomes dry, the bacteria usually die. If, however, the tube be sealed with parafin or sealing wax, it can be kept for months, in many cases for years, without dying. After purifying the cultures, carefully label, and reserve them for future use. *No. 15. Microscopic Study. Prepare one or both of the fol- lowing staining solutions: Methylene Blue Saturated alcoholic solution of Methylene Blue 15 c.c. Potassium Hydrate (1: 10,000 )x 50 c.c. Fuchsin Solution Saturated alcoholic solution of Fuchsin ... 5 c.c. 5% solution of carbolic acid 45 c.c. Place an ordinary cover-glass in a special pair of forceps (Fig. 80) and in the center place a very small drop of sterilized FIG. 80-FORCEPS FOR HOLDING COVER-GLASSES WHILE STAINING or boiled water. With a platinum needle remove a very small quantity of the bacteria growth from the surface of an agar tube of No. 14 and place in the water drop. Stir this drop with the platinum wire, to distribute the bacteria, and spread as widely as possible over the cover-glass. Allow to stand in the air until dry, and then pass the cover-glass through a gas flame quickly three times. The purpose of this is to fasten or fix the bacteria firmly to the glass so that they will not wash away. It is necessary to avoid using too much heat, for over-heated 1 To make this solution add 1 c.c. of a 10 per cent. KOH solution to 99 c.c. of water, and then add 5 c.c. of this to 45 c.c. of water. 284 PRACTICAL DAIRY BACTERIOLOGY bacteria will not stain. Place on the cover-glass enough of one of the staining solutions to cover it completely, and allow the specimen to stand undisturbed for several minutes. The length of time varies with the conditions: usually 2 to 5 minutes is long enough. Wash off the stain with water. The preparation is then ready to study with a microscope. If only a few mo- ments' study is desired, the cover-glass can now be placed on a glass slide, in a drop of water, with the bacteria side down, and studied directly with the microscope. If, however, it is desired to preserve the specimen for future study, dry the cover- glass, then place a drop of Canada balsam on a glass slide, and upon this put the cover-glass, bacteria side down. Study the preparations thus made with a microscope. For this purpose it is necessary to have high magnifying powers. Because of the minuteness of the bacteria, practically nothing can be made out concerning their shapes, with a magnifying power less than about 1,000 diameters, and to obtain this it is common to use a 1/12-inch immersion lens. Place the prepared specimen on the microscope stage, and put a drop of immersion oil on top of the cover-glass. Lower the 1/12-inch lens into the immersion oil, and then carefully focus upon the bacteria. If the microscope has an Abbe condenser or a diaphragm below, it is best to have this widely open, for the colored bacteria are more easily seen with an open diaphragm. The bacteria are very small, and even with this magnifying power it may sometimes be difficult to make out their shape; but ordinarily their general appearance can easily be made out. Examine in this way all the cultures isolated in No. 14, and sketch. *No. 16. Comparison of Yeast and Bacteria. Rub up in a watch glass a bit of a cake of yeast with a little water. Place a drop of the liquid on a slide and examine directly with the microscope, using a 1/6-inch objective. Dry a little of the material on a slide and stain exactly as in staining bacteria (No. 15). Study with the immersion lens, comparing the yeast LABORATORY WORK IN DAIRY BACTERIOLOGY 285 cells as to size and shape with bacteria already studied. Hunt for cells showing budding. If these are not found they may be obtained in quantity by planting a little yeast in a weak solution of molasses in water and allowing to grow for a few hours in FIG. 8l-CENTRIFUGE FOR MILK TESTING (International Instrument Co.) a warm place. Make a sketch of the yeast cells with buds, showing their relative size to that of bacteria. No. 17. Direct Microscopic Study of Milk. For this a centrifuge is needed, having tubes with straight sides, and closed at the bottom with a rubber cork. (Fig. 81.) Place 2 c.c. of the samples of milk to be tested, after thorough shaking, in the tubes. It is necessary to fill enough tubes with milk from dif- 286 PRACTICAL DAIRY BACTERIOLOGY fercnt samples, to balance the centrifugal machine on both sides. Rotate in the machine at a rate of 2,000 to 3,000 per minute, for 5 minutes. Remove the tubes from the machine and they will be found to have a layer of cream at one end, and a small slimy deposit at the other end next the cork. Holding the tube with the cream end down, remove the cream with a platinum loop, and pour out the milk gently. A slimy sediment will be left attached to the cork. Carefuly remove the cork with its adhering sediment, and smear over the surface of a glass slide, with a drop of sterile water, to cover an area of exactly four square centimeters. Special slides marked with wax pencils into such areas are needed. This distribution of the sediment must be carefully done in order that it shall be uniform. After the thorough distribution, dry in gentle heat, or without heating, and stain by flooding the surface with dilute of methylene blue for a short time, and then wash the stain away. After again drying, the material may be mounted under cover-glass, or it may be examined immediately with an immersion lens without a cover-glass. There will be found on the slide a considerable number of stained bacteria, usually showing a variety of forms, and also a varying number of large cells, most of which have deeply stained nuclei, but unstained bodies. These latter are leucocytes, and represent cells of the character of the white blood cells from the cow, which have found their way into the milk. In some cases, leucocytes represent pus cells and are in- dicative of inflammatory changes in the animal's udder. Where the numbers are small, however, they give no such indication, for the normal milk of healthy udders will usually show con- siderable numbers of these leucocytes. To interpret the meaning of what i§ found under the microscope in these prepa- rations, see page h84. No. 18. Doane's Method of Determining Leucocytes. Place 10 c.c. of milk in the tubes of a Centrifugal and rotate at about 2,000 per minute for four minutes. Carefully remove the cream LABORATORY WORK IN DAIRY BACTERIOLOGY 287 from the surface with a bit of cotton on the end of a rod, being careful not to leave any of the cream in the tube. By means of a small siphon remove the* milk, keeping the tip of the tube just below the surface so as to avoid disturbing the sediment. Siphon away the milk to within about one-eighth of an inch of the sediment. Add two drops of a saturated alcoholic solu- tion of methylene blue to the tube, and, after thoroughly mixing, place the tube in boiling water for two minutes to aid the stain- ing. Add enough water to bring the total bulk either to one or two c.c., according to the amount of sediment. This will give a blue mass of stained sediments. To count their number an ordinary blood counter is used. This will have a counting chamber marked off into squares and holding exactly one-tenth of a cubic millimeter. Fill this counter with the stained sedi- ment, cover with a cover-glass, allow to stand for about one minute for the leucocytes to settle, and then, placing under a microscope, count the number of leucocytes found in a single ruled square and calculate the numbers in the whole chamber. Remembering then that the chamber contains one-tenth of a cubic millimeter of the stained sediment, it is easy to calculate the number of leucocytes in the original milk. This should be calculated upon the basis of a certain number per c.c., and good milk should not have more than 500,000 per c.c. No. 19. Staining of Tubercle Bacilli. The staining of tubercle bacilli is rarely needed in dairying, but it is sometimes useful. The method is as follows. Make Carbol Fuchsin as follows: Basic fuchsin i part. Absolute alcohol io parts. Carbolic acid (1-20 of water) . . . 100 parts. Prepare, also, a 20% solution of hydrochloric acid in water. Spread the specimen to be stained (sputum or milk sediment from a centrifuge in the last experiment) upon a glass slide, and dry slowly in moderate heat. After drying, flood the slide 288 PRACTICAL DAIRY BACTERIOLOGY with the above stain, and allow it to stain for five minutes, warming slightly. Wash off the superfluous stain, and place in the acid solution, until all tracds of red have been removed, which will require, usually, from about three to five minutes. Wash off the acid, dry and flood the slide for about half a minute with methylene blue solution. Wash again, dry and examine with a compound microscope, without a cover-glass. The tubercle bacilli will be found, by this method, to stain red. whereas other bacteria will be stained blue. It requires a little experience to be successful with this method of staining, but it is fairly easy after a little practice. *No. 20. Qualitative Bacteriological Analysis. This is very difficult and can be successfully undertaken only after consider- able experience. A qualitative analysis of milk must begin with an estimation of the number of bacteria to be expected, in order that a proper dilution may be selected to give 200 to 1,000 col- onies per plate. If the age and temperature of the milk are known, an approximate guess may be made. If the milk is less than 12 hours old and is kept moderately cool, a dilution of 100 and 1,000 may be chosen. If from 12 to 24 hours old, 1,000 to 10,000 dilution will be better. If the milk is kept as warm as 200, higher dilutions are needed, sometimes as high as 1,000,000 in warm milk 24 to 36 hours old. If it is kept cool, 500, but if 48 hours old, it will be likely to require 10,000 to 30,000. These numbers are, unfortunately, not very definite. Secure some milk not more than 6 hours old. Dilute 1,000 times and make several litmus gelatin plates exactly as in ex- periment 11. It is always best to use two or three different dilutions in order, if possible, to insure proper results in one set, at least. At the end of two days examine the plates to see whether rap- idly liquefying colonies are liable to spoil the plates. If lique- fiers are abundant the plates must be studied at once; but if not, it is best to leave them for from 4 to 6 days before study. 289 LABORATORY WORK IN DAIRY BACTERIOLOGY When the plates are ready, count as follows: a. The number of liquefying colonies. These may roughly be separated into those that liquefy rapidly and those that liquefy slowly, giving two easily distinguishable types. b. Count the number of Bact. lactis acidi (see page 30). c. Count the number of B. aerogenes (including B. coli). These can be recognized by their brilliant acid, large surface colonies and frequent gas bubbles. d. Count the number of colonies that neither produce acid, nor liquefy gelatin, and group them together as neutral species. This last group will contain many different kinds of bacteria, and may, with experience, be differentiated into several other types; but it is hopeless for the beginner to do much more than is indicated above in qualitative analysis. *No. 21. Separation of the Common Species of Bacteria from Milk. Allow some milk to stand at about 700. As soon as it begins to sour, but before it curdles, dilute 1 c.c. 500,000 times and make litmus gelatin plates. Allow to grow four days at 70 0 unless liquefiers make it impossible. a. Determine the total number of bacteria in 1 c.c. of milk. b. Determine the number of acid bacteria per c.c. c. Determine the number of liquefiers per c.c. d. Determine the percentages in each case. e. Isolate a colony of Bact. lactis acidi. This may be recog- nized as follows: It is an intensely acid colony, rather opaque, always below and never on the surface. It is small, only just visible to the naked eye and when examined under a low power microscope it frequently, though not always, shows a slight roughness, looking like minute spines around its edge. (Fig. 16.) With a platinum needle lift out dne of these colonies and inoc- ulate into a gelatin tube by stabbing the needle directly into the gelatin as shown in figure 82. After growth purify as described in experiment 13, label and set aside for future use. f. Look over the plates made in this and the last experiment 290 PRACTICAL DAIRY BACTERIOLOGY and see if any large (usually about the size of a pinhead) colo- nies appear on the surface, white in color and intensely acid. If so isolate one, preferably one that shows a gas bubble be- FIG. 82-MAKPNG GELATIN STABS neath or beside it. Inoculate upon an agar slant, labeling it Bad. aerogenes Whether it is really that species will be determined later. If no gas bubbles appear, isolate several of LABORATORY WORK IN DAIRY BACTERIOLOGY 291 the large, acid, surface colonies and some of them will probably prove to be the species desired. In the same way isolate and inoculate on agar slants one rapid and one slow liquefier, and several of the neutral type of colo- nies. After about two days' growth purify the cultures and reserve for future study. *No. 22. Milk Agar. Add 1.5% agar to some skim milk. Af- ter half an hour boil (better to heat in an autoclav) until the milk curdles into a custard. Replace water of evaporation-ad- just reaction to 1.5% acid-filter through absorbent cotton- place in tubes and sterilize in the usual way. After the third sterilization slant the tubes and allow to harden. This milk agar is not so transparent as ordinary agar, but the lactic bac- teria grow upon it more readily. Inoculate the purified cultures obtained in No. 21 upon the milk agar slant, and reserve for future use. No. 23. Oidium Lactis.. Procure a little soft cheese from market, preferably of the Camembert or Brie type.1 Pour out into a Petri dish a tube of plain gelatin, and into another a tube of litmus gelatin. Allow to harden. With a platinum needle scrape off a little of the growth on the rind of the cheese and touch it upon the surface of the gelatin at several spots. It is well to try several parts of the cheese rind in this way. Cover, and after two days' growth it will usually be possible to find spreading colonies of Oidium on the plates. They are thin, and spread rapidly over the surface; they may be recognized under the microscope because at first they are seen to be composed of threads like a mold, which soon break up into short sections. The colonies usually become about % inch in diameter. (See Fig. 29.) *No. 24. Cheese Mold. On the same plates, after 2 or 3 days, 1 If such soft cheese is not to he found conveniently, Oidium ladis can usu- ally be found in samples of old tub butter. 292 PRACTICAL DAIRY BACTERIOLOGY will appear a white mold. This grows more slowly than the Oidium, and its threads do not break up into spores. Spores form on the surface upon special branches. (See Fig. 63.) Both the Oidium and mold may be isolated for future study if time permits. No. 25. Abnormal Fermentations. If any unusual types of fermentation can be found, like slimy milk, bitter milk, tainted milk, etc., they may be studied to advantage. Gelatin plates should be made from such milk and the different kinds of colo- nies isolated. After purifying, these should be inoculated into sterile milk to see if any of them produce the trouble seen in the original milk. If such is found it may sometimes be traced to its source by hunting for it in various places around the dairy ; c. g., in the milk cans-cows' teats-dairy water-hay dust, etc. No definite directions for this can be given, for each problem will need different treatment. *No. 26. Potato Tubes. Carefully wash and peal a large po- tato. With a special cutter (Fig. 83), or with a broken test tube with sharp edges, cut out some potato plugs. Cut each obliquely in two parts and soak in running water 12 hours. In the bottom of some large test tubes place a little cotton and enough water to cover it. Place a single potato slant in each tube (Fig. 83) and sterilize by one-half hour steaming on three successive days. *No. 27. Fermentation Tubes. To 200 c.c. of bouillon pre- pared as in (4) add 2 grams of lactose (milk sugar). To a sec- ond 200 c.c. add the same amount of dextrose (grape sugar), and to a third lot add saccharose (cane sugar). Dissolve by heating -filter through filter paper and pour into fermentation tubes (Fig. 20), completely filling the closed arm and about one- half the bulb. Sterilize as usual. If gas collects in the arm, remove by tilting the tube. *No. 28. Milk Tubes. Procure some fresh skimmed milk. Test the reaction with litmus paper and if acid add sufficient NaOH to bring to neutral point of litmus paper. Place in tubes, about LABORATORY WORK IN DAIRY BACTERIOLOGY 293 10 c.c in each. Sterilize by steaming one-half hour for three successive days. No. 29. Testing Characters of Bacteria. Inoculate the sev- eral bacteria previously isolated and purified, into various media. In doing this it is best to make first a fresh agar slant from each FIG. 83-CUTTER FOR MAKING POTATO PLUGS, METHOD OF CUTTING THEM AND PLACING THEM IN TUBES bacterium to be tested, so that the inoculation may be made from a culture not over 24 hours old. Inoculate with a small amount of the growth from the agar surface, the following: a. An agar slant, b. A gelatin stab. c. A tube of plain bouillon, d. A dextrose fermentation tube. e. A lactose fer- 294 PRACTICAL DAIRY BACTERIOLOGY mentation tube. f. Two milk tubes, one to be kept at 700 and the other at 98°. g. Two potato tubes, one at 700 and one at 9<°'°. Allow the cultures to grow several days, examining each FIG. 84-. YPES OF GELATIN STAB GROWTHS a. Filiform, b. Beaded, c. Echinate, d. Villous, e. Arborescent FIG. 85-TYPES OF GELATIN LIQUEFACTION a. Crateriform. b. Napiform, c. Infundibuliform. d. Saccate, e. Stratiform day. For each species of bacterium make a careful record, noting especially the following points: Agar slant. Note the type of surface growth as compared with figure 19. Morphology. Stain and study with the microscope, noting shape-formation of chains-spores-determine motility. (See No. 31.) Gelatin stab. Note liquefaction-needle growth-surface LABORATORY WORK IN DAIRY BACTERIOLOGY 295 growth-color. Compare the growth with the different types shown in figures 84 and 85, and determine with which each agrees. Note the amount of elevation of the surface growth and compare with figure 86. Bouillon. Note turbidity-scum-sediment. Fermentation tubes. See below (No. 30). Milk tubes. Note at 700 and 98° the development of acid as shown to litmus paper-curdling-separation of whey-appear- ance of gas bubbles-subsequent softening of the curd, called digestion. Potato tubes. At 700 and 98°. Note color of growth- abundance of growth-texture of growth-discoloration of potato. By such characters as above determined, different kinds of bacteria are distinguished and described. For a complete descrip- tion more characters than those mentioned are necessary. The characters of the cultures which have been named, Bad. ladis acidi and Bad. acrogencs, should be compared with those given on page 30 to see if they agree with the characters there given. *No. 30. Fermentation Tubes. Inoculate a series of fermen- tation tubes (dextrose, lactose and saccharose) with different bacteria. Among those chosen should be one culture of Bad. ladis acidi, and one of Bad. acrogencs. In two days, note whether gas has been produced in the closed arms, and, by means of litmus paper, whether the bouillon has become acid. If gas shows in the fermentation tubes, determine the gas ratio as follows: Without disturbing the gas, fill up the bulb to the top with a 2% NaOH. (By a mark, note the level of the gas in the glass arm.) Place the thumb over the opening in such a way that there will be no air bubble between the thumb and the surface of the liquid. Now, invert the tube, allowing the gas to flow out into the bulb, and. by turning back and forth, mix the gas with the NaOH solution in the bulb, keeping the thumb very 296 PRACTICAL DAIRY BACTERIOLOGY tightly closed over the opening. After thoroughly mixing, turn the tube once more so that all of the gas will be in the closed arm. Remove the thumb; it will usually be found that the level of the gas in the closed arm rises. If so, it will be due to the fact that COa has been dissolved by the NaOH solution. By determining the amount of the gas before and after the test, the proportion of CO2 to the gases not thus dissolved may be ob- tained. This is called the gas ratio. Some gas-producing species of bacteria produce large quantities of carbon dioxide, and small quantities of other gases, while other species produce no carbon dioxide. From one or two of the test tubes inoculated above, that show an acidity, determine the amount of acid produced. For this purpose remove from the tube either 5 or 10 c.c. of the liquid, dilute it with 10 times its bulk of water, heat and titrate, as described in experiment No. 4, b. No. 31. To Determine Motility. Inoculate a little of the bac- teria growth of No. 21 into a test tube of bouillon. Allow it to FIG. 86-TYPES OF SURFACE ELEVATION 1. Flat. 2. Raised. 3. Convex. 4. Pulvinate. 5. Capitate. 6. Umbilicite. 7. Umbonate grow for about 24 hours. By this time the liquid will become somewhat cloudy. Transfer a loopful of this material to a glass slide and place upon it a cover-glass. Place the whole under microscope, preferably using a 1/6-inch objective, and narrow the diaphragm below the stage until most of the light has been cut off. Now focus very carefully upon the bacteria under the cover-glass, and, if they are motile, it will be seen that they are moving around through the microscope field with more or less rapidity. If they are stationary, it may be assumed that they have no flagilla. This test requires great care, for LABORATORY WORK IN DAIRY BACTERIOLOGY 297 with some slowly moving forms it is not always easy to detect the motion with certainty; but it can easily be done with a little study. No. 32. Germicidal Action of Milk. Procure some milk fresh from the cow; dilute i c.c. of this milk 100 times and make 6 agar (or gelatin) plates. Cool the rest of the milk at once to 700. At the end of 2 hours make 6 more plates in exactly the same way as above. In 2 hours more make 6 more plates. After proper time for growth, count the colonies and determine whether there has been a decrease in numbers of bacteria in the first few hours after milking. No. 33. Aseptic Milk. Sterilize a liter flask plugged with cotton. Wash the hands thoroughly in hot water and soap, and put on a clean milking suit. Remove a clean cow from the barn into the open air, brush the dirt from the flanks and udder with a clean brush and then with a damp cloth. Wash the udder and especially the teats with a io% borax solution. After drawing about a dozen jets on to the ground, remove the plug from the flask and, placing its mouth very close to the teat, milk it about half full, and replace the cotton plug. Carry at once to the laboratory and make a litmus gelatin plate immediately, diluting io times. After 2 or 3 days' growth, count the colonies. Are there any acid colonies of the Bad. Ladis acidi present? If time permits, isolate the colonies; purify and determine the general class of bacteria. Allow the rest of the milk to stand at about 700 to see if it will sour normally. No. 34. Effect of Temperature on Species of Bacteria in Milk. Procure some fresh milk and make several litmus gelatin plates, diluting 100 times. Divide the milk in three lots, placing one at about 500, one at 700, and one at 98°. At the end of 24 hours make plates from each, diluting that kept at 500 2,000 times, that at 700 10,000 and 100.000 times, and that kept at 98° 500.000 times. After another 24 hours, make some plates from 298 PRACTICAL DAIRY BACTERIOLOGY the sample at 700, diluted 10.000 times. Allow all plates to grow at room temperature until the colonies are well developed. Determine the total number of bacteria in each sample, and the number and percentage of each kind of bacteria that can be clearly distinguished by its colony. Compare the numbers and percentages of the different species at the different temperatures. *No. 35. Bacteria of the Air. Place one dozen tubes of steril- ized milk-with cotton plugs removed-in various places around the barn, dairy, house and laboratory. Leave undisturbed for 6 hours. Then replace the plugs, carry all to laboratory, and place at about 200. Watch for several days and notice whether all appear to undergo the same kind of fermentation. Do any of them sour normally? Do any remain unchanged? No. 36. Types of Bacteria from the Air. After the tubes in No. 35 show signs of fermentation make a gelatin plate from each, using a loopful of the milk in a water blank and inocu- lating gelatin tubes with a small loopful of the water blank dilution. After proper growth examine the colonies in the dif- ferent plates and compare with each other. What does the ex- periment teach? If time permits, the colonies may be isolated, puriaed and further studied, as in No. 28. *No. 37. Dust Plates. Pour 12 tubes of melted gelatin into Petri dishes; cover and allow to harden. a. During the milking of a cow hold one of the above gelatin plates under the udder of the cow, close to the milk pail; remove the cover so as to allow any dust particles to fall on the gelatin, for half a minute. Replace the cover and carry to the laboratory. b. Place 3 of the flasks at different places in a cow barn several hours after feeding. Remove the covers for 3 minutes. Replace covers and carry to laboratory. c. Repeat b ten minutes after feeding the cows with hay. d. Repeat c after feeding with other foods. e. Expose one plate the same length of time out of doors. LABORATORY WORK IN DAIRY BACTERIOLOGY 299 After the above plates have developed count the colonies on all the plates and compare. No. 38. Bacteria in Hay. Soak a little hay in warm (not hot) water for an hour. Shake well and inoculate three litmus gelatin tubes, the first with one, the second with two and the third with three loops full of the hay infusion. Pour into Petri dishes, incubate and study as usual. Compare the numbers of liquefiers and non-liquefiers. Are there any acid forming colonies ? No. 39. Bacteria in Grain. Repeat the above experiment, using some grain feed instead of hay. *No. 40. Value of Cleaning Milk Bottles. Procure 4 glass bottles in which milk has been standing for a day; ordinary unwashed milk bottles are best. a. Wash one with cold water. b. Wash one with hot water and soap. c. Wash the third and sterilize in steam. d. Leave fourth unwashed. Fill all four with fresh milk. Place all side by side at a room temperature and notice the time of souring in each case. *No. 41. Bacteria in the Milk Pail. Place 100 c.c. of sterile water in a milk pail that has received a simple washing. With a little absorbent cotton rub this water around thoroughly; allow to stand a few moments, and, after another washing around the pail, remove 1 c.c. of this water and plate in agar. Count the colonies. a. Repeat, using (1) a dirty pail; (2) a pail simply steamed for a few seconds in a steam jet, and (3) a thoroughly sterilized pail. No. 42. Bacteria in Manure. With a sterilized platinum loop transfer a small bit of either fresh or dried manure from a cow to a sterile water flask. Mix thoroughly. Transfer two loopfuls of the mixture to a tube of melted agar. Pour into Petri dishes and after growth count the number of colonies. To determine 300 PRACTICAL DAIRY BACTERIOLOGY the actual number in manure it is necessary to weigh the original bit of manure and make subsequent calculations. No. 43. Isolation of B. Coli. Repeat No. 42, using litmus agar. If any strong acid surface colonies appear, choose one that shows a gas bubble if possible; isolate, inoculate on an agar slant, and purify as usual. From the purified culture inoculate a glucose fermentation tube. If gas appears, examine the cul- ture and determine whether the bacterium is a short motile rod. If so, the species is probably B. coli; if it is not motile, it is probably B. aerogenes. Compare with page 34. No. 44. Bacteria on Hair. Melt a tube of agar and one of gelatin, and pour into Petri dishes. After they have hardened place upon the surface of each one or two hairs from the flank of a cow. Two or three days later examine, and note the numerous bacterial colonies growing along the course of the hairs. *No. 45. Fore Milk. Draw the first 3 sets of milk from one teat of a cow into a sterile test tube. Now milk the cow about one-half dry and draw 6 more jets in a second sterile tube. Remove to laboratory and plate at once in plain gelatin, diluting 10 times. Compare the numbers and kinds of colonies in the two sets of plates. No. 46. Bacteria on the Hands. Wash the hands thoroughly in 200 c.c of sterile water. Place 1 c.c. of this water in a tube of melted agar. Count the colonies and calculate the number of bacteria removed from the hands by the washing. No. 47. Estimation of Dirt in Milk.1 Procure some milk from a clean and carefully kept dairy and also some from a dairy where the milking is done in a careless, slovenly fashion. De- termine the amount of dirt in the milk in each case, as follows: a. First method. Place a quart or a liter of each sample of milk in a tall glass cylinder, and allow it to stand for several 1 This experiment is impossible without a chemist's balance and apparatus for drying, such as used in quantitative chemistry and analysis. LABORATORY WORK IN DAIRY BACTERIOLOGY 301 hours. A deposit of dirt will collect at the bottom, which may easily be seen, by looking through the glass. With a siphon carefully remove the milk to within % inch of the bottom, but do not disturb the sediment. Fill up with clean water and allow to settle again Once more, after settling, siphon off the liquid, and fill the glass with water. Repeat this operation several times, until the water becomes fairly clear. Then, after allow- ing the material to settle again, siphon off a considerable portion of the liquid, and after thoroughly mixing the sediment with the remainder of the water in the cylinder, pour it through care- fully weighed filter paper; wash the filtrate by means of water through the filter paper. Remove the filter paper and dry, until it comes to a constant weight. Weigh. The difference in weight between this and the original weight of the filter paper will give the weighed amount of dirt collected. b. Second method. Place a definite quantity of milk, usually io c.c., in one of the glass tubes of a centrifugal machine. Set the machine in motion and rotate rapidly for ten minutes. By this time the sediment will mostly be thrown to the smaller end of the tube. The cream and milk are then to be carefully re- moved from the sediment with a pipette, and the tube filled up again with clean water and centrifugalized again. This is to be repeated until the water becomes clear, after which the sedi- ment in the bottom of the tubes is to be carefully washed out with clean water upon a filter, and the rest of the procedure is as above. c. Third method. Procure some bolting cloth of the finest mesh. The meshes should be fine enough to retain all the visible particles, but to allow fat particles to pass through. Place in a ribbed funnel, moisten with water, and pour through it one quart (or liter) of milk. Wash the material left on the cloth with water and then, with a mixture of ether and alcohol, equal parts. Wash the dirt on to a carefully weighed filter paper. Dry thoroughly and weigh as above. PRACTICAL DAIRY BACTERIOLOGY 302 *No. 48. The Covered Milk Pail. Milk two cows, one into an ordinary milk pail with a flaring, open mouth and the other into a covered milk pail. Determine the amount of dirt in the milk in each case. Immediately after the milking, in each case, make a quantitative count of the number of bacteria, comparing the results together, and determine the value of a covered milk pail. The next day repeat the experiment, reversing the two cows. *No. 49. Advantage of Immediate Cooling. Immediately after milking, cool, by aerating, part of the milk, and place in steril- ized jar. Fill another similar jar with milk not cooled. Fill a third jar with milk cooled by immersing at once in iced water. Place all three jars side by side in an ordinary room and com- pare the time of souring and curdling in the three specimens. Cool night's milk immediately, as cool as possible. The next morning fill two jars, one with the night's milk that has been cooled and the other with the milk warm from the cow. Place both at a temperature of an ordinary room and compare the time of souring and curdling in the two cases. No. 50. The Effect of Careful Milking. Compare the effect of milking, for six successive days, with and without precau- tions. The first three days use open milk pails and make no special attempt at cleanliness. The second three days use closed milk pails, groom the cows, wash the udders and perform the milking out-of-doors instead of in the closed barn. In each case make a quantitative analysis of the number of bacteria in the milk and compare the results. *No. 51. Sterilization by Boiling. Make a set of bacterial plates from a sample of milk in gelatin. Then boil the milk for five minutes in a flask closed with cotton and make another set of plates, diluting by io. Allow the boiled milk to stand for 24 hours and make another set of bacterial plates, diluting by 100. Allow the rest of the boiled milk to stand in the laboratory until it shows signs of fermentation. Determine whether it has LABORATORY WORK IN DAIRY BACTERIOLOGY 303 soured, and describe any other notable change. After the plates have had an opportunity to grow, count the number of bac- teria. No. 52. Sterilization by Autoclav. Sterilize milk in a plugged milk flask for 15 minutes in the autoclav at a pressure of 15 pounds. Allow to stand for several days and then make gelatin plates from it and determine whether the milk was completely sterilized or not. *No. 53. Pasteurizing Milk. Obtain some milk that is from 12 to 20 hours old. Make a set of plates on agar, diluting by 1,000. Divide the milk into two lots. Heat one lot to a temper- ature of 1400 for one-half hour, stirring frequently. Heat the other lot to 1600 for 10 minutes. Allow both to cool and make agar plates from each, diluting by 100. After plates have grown, calculate the number of bacteria before and after pasteurizing at the different temperatures. a. Repeat the last experiment, making plates, however, in litmus gelatin and calculating the number and percentage of acid colonies in the milk before and after pasteurizing. No. 54. Pasteurizing Machines. If a pasteurizing machine is available, test its efficiency by several experiments. In each case a thoroughly mixed sample of milk should be first care- fully tested by gelatin plates, and corresponding sets of plates should be made from the milk after pasteurization. Use the machine under varying conditions of temperature and time of pasteurizing, and determine the efficiency under the different conditions. No. 55. Condensed Milk. Open a can of condensed milk and remove with a sterile platinum loop, or a sterile knife, a quantity of the condensed milk and dilute with sterile water. Make a series of bacterial plates with a loopful of the diluted milk and determine whether the condensed milk was sterile. If colonies develop in the plates, examine with the microscope and deter- mine whether they are yeasts or bacteria. 304 PRACTICAL DAIRY BACTERIOLOGY "No. 56. Quantitative Analysis of Miscellaneous Milk Sam- ples. Obtain milk samples from several milkmen, learning, so far as possible, the age of each sample of milk. These should be collected in sterile bottles and kept on ice until they can be experimented with in the laboratory. Dilute each sample of milk 1,000 times and make agar plates from the final dilution; incubate at 98°. After 24 hours count the number of bacteria in each plate and compare the samples of milk. No. 57. Qualitative Analysis of Miscellaneous Milk Samples. With the specimens above collected it will be useful to attempt a qualitative analysis, though this will be more difficult. The dilution of the milk must be varied according to its age and temperature. If it is fairly fresh-only a few hours old-a dilu- tion of a thousand times is satisfactory. If it is 12 hours old, a dilution should be, at least, 10,000; and if 24 hours old, it should be as high as 100,000, or higher. The amount of dilu- tion necessary may be determined by a direct microscopic study. Make and stain a slide from each sample of milk, as directed in No. 17. Count the number of bacteria per field. If the average number per field is less than 10, dilute the milk 100 times. If it is as high as 100, dilute 1,000 times; and if still higher, dilute in the same proportion. After diluting the milk make six litmus gelatin plates from each sample. It is best to have three of these plates diluted twice as highly as the other three. After cooling, place at a temperature of 700. After two days, examine and determine whether there are any rapidly liquefying colonies that are likely to destroy the plates; if so, make a study of the plates at once. If possible, however, keep the plates for four days before studying them. Study of plates.-After the plates have grown (2 to 4 days) study as follows: a. Determine total number of bacteria per c.c. of milk. Com- pare with the total number found on the agar plates. LABORATORY WORK IN DAIRY BACTERIOLOGY 305 b. Determine the number of liquefying colonies. c. Determine the number of acid bacteria per c.c. Are they of the Bad. ladis acidi type? d. Calculate the percentage of acid bacteria-liquefiers and miscellaneous bacteria in the milk. By reference to page 181 draw a conclusion as to which samples were probably badly contaminated. *No. 58. Bacteria in Fresh Cream. Dilute one c.c. of freshly separated cream with sterile water. If this is separated by a separator, the dilution should be about 1,000. If the cream has been separated by the gravity method, the dilution should be higher, since the cream is older, and should be as high as 100,000. After diluting, make a series of four plates in litmus gelatin, incubate at 700 and study as described in No. 56. *No. 59. Bacteria in Ripened Cream. Repeat experiment No. 57, using, however, some ripened cream that is just ready for churning. In this case the dilution must be much higher, and probably never less than 1,000,000. After incubating determine the variety and numbers of bacteria present. No. 60. Analysis of Cream at Successive Intervals. Obtain some sweet separator cream, and set it to ripen in the ordinary way. Make a series of litmus gelatin plates from the cream, when first separated, and others at 6 hours later, 12 hours later and at the time of churning. The dilution of cream in the suc- cessive experiments should be increased from about 1,000 to 1,000,000. Incubate at 700, and carefully determine the number of bacteria, their types and percentages. Note particularly the increase in the percentage of lactic acid bacteria at the successive periods of the ripening. No. 61. Analysis of Butter Milk. Make an analysis of the bacteria in butter milk in litmus gelatin. The dilution in this case should not be less than 1,00,000. No. 62. Analysis of Butter. Weigh out upon accurate chem- ical scales 5 grams of freshly made butter. Place this in a sterile 306 PRACTICAL DAIRY BACTERIOLOGY mortar, with 9.5 c.c. of sterile water. Rub the water and the butter together thoroughly, so as to distribute the bacteria as uniformly as possible through the water. This mixing should be continued for some time, for, at best, many of the bacteria will remain clinging to the fat. Dilute this mixture 10,000 times and make a series of agar or litmus gelatin plates. Incubate and count as usual. After 24 hours make a second series of plates from the same sample of butter, and repeat again in two days and one week. Obtain, if possible, a sample of butter several months old, and make similar analysis. In all $ases determine the numbers, and, if possible, the proportion of acid bacteria and the liquefiers. No. 63. Home Starters. Obtain some clean milk from a thor- oughly healthy cow; place in sterile vessels, and cover to keep out the dust. Set aside at about 65° until the milk is soured but not quite curdled. Examine carefully by taste, by smell and by general appearance, to determine whether the curd seems to be of a type favorable for butter-making. It should be smooth in appearance, and have a clean, sharp taste and pleasant odor. Use this, if convenient, as a starter for ripening cream. *No. 64. Making a Starter from a Commercial Culture. Sterilize a quart of milk by boiling half an hour, or half an hour in an autoclav at 10 pounds pressure. After cooling to about 8o°, pour into it the contents of a package of commercial butter starter, stir thoroughly, cover and allow to stand at 65° to 70 0 for 24 hours. The milk should by this time be sour and nearly ready to curdle, and may be used at once as a starter. I f a larger amount is needed, pasteurize several gallons of cream by heating to 1550 for one half hour. Cool to 8o°, and pour into it the quart of starter prepared from the commercial cul- ture. Allow to stand at 65°, after which it is ready for use. No. 65. Bacteriological Analysis of a Commercial Culture. Make a bacteriological analysis of some commercial culture to determine whether it is pure or not. For this purpose put a LABORATORY WORK IN DAIRY BACTERIOLOGY 307 small quantity of the culture from the package into a water blank, and, by thorough agitation, distribute evenly through the water. From this inoculate litmus gelatin tubes, some with one, some with two and some with three loopfuls of the diluted cul- ture. Pour into Petri dishes, incubate at 700, and after three days examine to see whether any bacteria can be found in the plate except lactic germs. No. 66. Effect Upon Butter of Pasteurizing Cream. Divide a lot of cream which is to be made into butter into two parts, pasteurize one at a temperature of 1550 for 15 minutes, leaving the other without pasteurization. Add to each the same amount of starter. Ripen in the usual way, churn and make into butter, and compare the products, to see if any difference can be noted between the butter from pasteurized and non-pasteurized cream. No. 67. The Effect of Different Bacteria on Butter. The fol- lowing experiment can be performed only where conveniences are at hand for churning small quantities of cream. Pasteurize a considerable quantity of cream, as above described, and divide into several lots, placing each in a separate vessel. Inoculate each lot with a starter made from different species of bacteria, which have been isolated during the laboratory experiments. Allow the cream to ripen in the normal way for the usual length of time, and churn into butter. Compare the texture, taste and aroma of the butter obtained in the different experiments. No. 68. The Effect of Light Upon Butter. Place two lots of butter, one in a bright light and one in the dark, and after sev- eral days compare them as to appearance, smell and taste. No. 69. Ripening of Cream at Different Temperatures. Di- vide some cream into three lots. Place the usual amount of starter in the cream, and ripen one lot at 500, one at 65° and one at 85°. After proper ripeness has been reached (determined by acid test), churn and compare the butter which is obtained, unsalted, to detect any difference in flavor and aroma. *No. 70. Analysis of Bacteria in Cheese. Determine the num- 308 PRACTICAL DAIRY BACTERIOLOGY ber of bacteria in cheese exactly as above described for butter (see experiment 62), making inoculation into litmus gelatin plates, and determine the varieties of bacteria present. Make a series of plates in this way from a freshly made cheese and another series from some cheese ripened and ready for market. Compare the numbers and kinds of bacteria. No. 71. Determination of Bacteria in Rennet. Dilute a small quantity of the rennet in a water blank and inoculate litmus gelatin plates. Incubate as usual, and examine at the end of two days. This experiment may be performed by the use of agar incubated at 98°. No. 72. The Effect of Gassy Organism on Cheese. Inoculate a small quantity of milk with a starter made from a gassy organism (see page 287). After a few hours add rennet to the milk till curdled, and proceed in the usual way to make the curd into two cheeses. Leave one cheese for a few days in a moder- ately warm room, and notice the swelling due to the formation of the gas bubbles. Place the second cheese in a cool room with a temperature below 6o°, if possible. Compare the formation of the gas bubbles in the two cheeses. No. 73. Isolation of Molds from the Cheese. Obtain from the market a Camembert or Brie cheese. Pour into a Petri dish some litmus gelatin and allow to harden. With a platinum needle scrape off a bit from the surface of the cheese where it shows signs of mold, and inoculate it upon the gelatin by touch- ing the surface with the needle in several places. Cover the dish as usual, and place at 700 for incubation. At the end of two or three days colonies of molds will be found growing on the surface of the plate. Proceed as above described to obtain the mold of the ripened Roquefort cheese. The method of this procedure is the same, except that for inoculating the plate, the platinum needle should be dipped into some of the blue spots, which show in the middle of the Roquefort cheese. LABORATORY WORK IN DAIRY BACTERIOLOGY 309 If time should permit, the molds may be further studied. Allow them to grow for several days, when they will show a change in color, due to the formation of spores. The spores may then be taken up on the tip of a platinum needle and planted on other gelatin or agar plates, or upon agar slants, for preserva- tion. The different species of molds are distinguished from each other by their methods of forming spores and, also, by their color. This subject hardly belongs to this book, but any dairy student who is working upon the problems of soft cheese- making should study the molds sufficiently to become familiar with the white mold of the Camembert, the mold of the Roque- fort, and the common blue molds, which are found almost every- where, but are mischievous organisms in the dairy. No. 74. Bacteria in Soft Cheeses. From either or both of the cheeses in the last two experiments make litmus gelatin plates. After proper growth, study and determine whether there are any bacteria present except lactic acid germs. In most good cheeses of this type the ripened cheese should contain only lactic acid bacteria. ♦No. 75. Effect of Different Species of Bacteria on Milk. In- oculate a series of sterile milk tubes with a large number of different kinds of bacteria. It is well cor this purpose to use all of the species isolated during these experiments, and as many other kinds of bacteria as may be available. Set all of the tubes aside at 700, and at intervals of 24 hours examine each to determine the effect upon the milk of the different species of bacteria. The acidity should be tested by removing a loopful and placing on litmus paper, and special attention should be given to curdling, separation of whey, appearance of gas bubbles, the digestion of the curd and the appearance of odors as a result of the action of the different bacteria. The larger the variety of the different cultures used, the better. INDEX PAGE Acid bacteria, protective action of 84 Acids, normal 267 Acid liquefiers 42 Acidity, adjustment of .... 269 Aeration 137 Aerobic bacteria 17 Agar, culture medium .... 270 plates 275 slants 273,281,282 Air, as a source of bacteria . . 65 bacteria in 295 Alkalies, normal 267 Anaerobic bacteria 17 Analysis, qualitative . . . 288, 304 quantitative 304 Apparatus, list of 264 Aseptic milk 61, 297 Autoclav, use of 273 Bacillus, meaning of term ... 13 cyanogenes 53 coli 37, 57, 70 isolation of 300 significance in milk .... 37 erythrogenes 53 lactis fluorescens ...... 57 foetidus 54 amari 48 saponacei 55 lacto rubifaciens 53 B. lig. amari ........ 48 prodigiosus 53, 57 pyocyaneus 57 sapolacticum 55 subtilis 57 Bacteria, abnormal milk . . . 21, 44 air affecting 17 cold affecting 16 diseased 19 determination of 273 food relations of 18 form of 13 gas-producing .... 36, 70, 74 in grain 299 grouping of 12, 14 PAGE Bacteria, on hair 300 on hands 300 in hay 299 lactic acid 25, 84 in cream 233, 235 types of 38 liquefying 39 in manure 299 microscopic study of .... 283 in milk 173,85,86,105 in milk ducts 60 milker, source of 68 multiplication of 5 neutral 42 in normal milk .... 21, 22, 74 significance in milk .... 176 peptonizing 39 phases of growth 81 in rennet 308 rennet-forming 39 size of 2 temperature affecting . . 15, 75, 77, 297 water affecting 18 Bacterium, meaning of term . . 13 aerogenes . 34, 46, 57, 60, 80, 239, 278 growth at different temper- atures 39 in dairying 35 source 67, 70 capsulatus 57 lactis acidi . . 17,26,32,46, , 60, 80, 74, 181 characters of 30 growth at different temper- atures 38 in butter-making 196 in cheese-making 240 isolation of 29, 289 source 67, 69 viscosus 46 Barn, disinfection of 142 Bouillon, preparation of... . 267 Brie cheese 245 Budding 8 310 INDEX 311 PAGE Butter, aroma of 199 bacteria in 215, 305 bitter 219 cowy 218 effect of bacteria on .... 307 fishy 220 flavor of ' . 199 lardy 219 light effect on 307 moldy 219 oily 219 Pasteurized cream for . 210, 307 putrid 218 red 217 starters 203 tallowy 219 turnipy tasting 218 starters 32,203 Buttermilk 86, 305 use of 214 Butyric acid fermentation ... 43 Camembert cheese .... 225, 245 in America 250 ripening of 246 Carbol fuchsin 287 Centrifugal force for cleaning milk 138 Centrifuge 285 Certified milk 161 Characters of bacteria, testing . 293 Cheddar cheese 228 bacteria in 229, 233 Cheese, bacteria in ... . 233, 308 bitter 48, 241 Brie 245 Camembert 225, 245 Cheddar 228 bacteria in 233, 308 cold ripening 238 Coulommier 245 Cream 227 digestion of in ripening . . . 225 Dutch 226 Edam 236, 240 enzymes in 228 flavor of 232 fruity 242 gassy 238 bacteria in 308 Gorgonzola 253, 255 Harz 227 Limburger 256 Neufchatel 227, 245 Pasteurization 237, 252 poisoning 109 PAGE Cheese, putrid 242 ripening of 223 chemical 229 temperature ■ . 237 Roquefort 253 rusty spot 242 soft 243 bacteria in 309 sour milk 226 starters 32, 235, 236 Stilton 253, 255 sweet 242 swelled 238 Swiss 228, 234 temperature affecting ripening 80 Cholera 104 Coccus, meaning of 3 Colonies 3, 276 Commercial cultures . . . 205,306 bacteria in 306 Concentrated milk 165 Condensed milk . . . 50, 163, 303 Cotton, absorbent 277 Coulommier cheese 245 Counting-box 277 Cow, care of . . . 122 source of bacteria 62 health of 113 Cream, bacteria in .... 195, 305 cheese 277 evaporated 164 Pasteurized 158, 210 Ripening 193, 195 control of ... . 198, 200,201 temperature of 200 preservation of 282 purification of 281 Cultures 2 Curd, digestion of 41 test 258 Dairy, disinfection of 142 inspection of 184, 187 location of 134 personnel 1 sanitary 160 Dairymen, trained 133 Decay 9 Diarrheal diseases 106 Diphtheria 103 Disease bacteria 19 detection of in milk . . 105, 171 Doane's method of determining leucocytes 286 Dust plates 295 Dutch cheese 226 312 INDEX PAGE Edam cheese 236, 240 Enzymes 19 in cheese 228 Feeding before milking .... 121 Fermentation, abnormal .... 292 alcoholic 9,49 butyric acid 43 test 258 tubes 35, 292,295 Flagella 4 Flavor, butter 199 cheese 232 Food relations of bacteria and yeasts 18 Foot and mouth disease .... 98 Fore milk 61, 131,300 Formalin in milk 148 Frozen milk 167 Fruity cheese 242 Fuchsin solution 283 Galactose 228 Garget 44 Gas-producing bacteria . 36, 70, 74 Gas ratio 295 Gassy cheese 238 bacteria in 308 Gelatin culture media 277 plates 279 Germicidal action of milk . 81, 297 Goiddu 52 Gorgonzola cheese .... 253,255 Grain, bacteria in 299 Hair, bacteria on 300 Hands, bacteria on 300 Harz cheese 227 Hay, bacteria in 299 Hydrogen peroxide 148 Hydrophobia 98 Ice cream poisoning ... 78, 109 Infection at creamery 140 at home 147 of milk 107 Isolation of colonies 281 Kefir 51 Kummys 50 Lactic acid, left-handed . . . 35, 39 right-handed 35, 39 PAGE Lactic acid bacteria .... 25, 84 (See Bacterium lactis acidi) in cream 233, 235 types of 38 Leben 52 Leucocytes 182, 286 Doane's method of determin- ing 286 Light, effect on butter .... 307 Limburger cheese 256 Litmus agar 279 gelatin 278 plates 280 solution 278 neutralizing 278 Liquefying bacteria 39 Lock-jaw 98 Lophotrichic 5 Manure, bacteria in 299 removal of 115 Masun 52 Methylene blue 283 Micrococcus, casei amari ... 48 lactis albus 57 lactis citreus 57 lactis varians 57 meaning of term 13 Microscopic study of bacteria . 283 of milk 182, 285 Milk, agar 291 aseptic 61,297 bacteria in 299 bacteriological analysis of . . 174 bitter 48 blue 52 centrifugal force for cleaning 138 certified 161 chemical purity of 170 city supply 168 cleanliness of 172 colored 54 condensed 50, 163, 303 concentrated 165 control of supply 173 cooling 135 advantages of 302 diluting for testing 274 dirt in 71 estimation of 300 detection of diseased bacteria in 105, 171 filtering 138 fore milk 61, 131, 300 formalin in 148 frozen 167 INDEX 313 PAGE Milk, germicidal action of. 81,297 inspection of 107 microscopic study of . . 182, 285 Pasteurization 153 powdered 166 pus in 58, 182, 286 qualitative analysis of ... 180 quantitative analysis of . . .273 red 53 slimy 44 soapy 55 souring 22 sour milk cheese 226 species of bacteria affecting 309 sterility when secreted ... 58 thunderstorms and souring . 23 tuberculosis, use of .... 92 turnipy tasting 54 typhoid distribution 98 Milk ducts, bacteria in ... . 60 Milker, a source of bacteria . . 68 Milking machines .... 129, 130 room 120 Milk bottles, cleaning of . . . 299 Milk pail, bacteria in 299 covered 126, 302 Milk vessels, care of . . . 123, 145 a source of bacteria .... 67 Mold in Camembert cheese . . 246 cheese 291 Roquefort cheese 254 Molds, isolation of 308 Monotrichic 5 Motility 4 Mycelium 10 Neufchatel cheese .... 227, 245 Neutral bacteria 42 Normal milk bacteria . . 21, 22, 74 Numerical standard, objections of 177 value of 179 Oidium lactis . 55, 57, 247, 249, 251 isolation of 291 Oleomargarine 220 Oxygen as a preservative ... 149 Parasites 19 Pasteurization .... 84, 151, 303 compulsory 191 home 154 in cheese-making .... 237, 252 of cream in butter-making 210,307 on a large scale 156 Pasteurized cream .... 158,210 PAGE Pasteurized milk, digestibility of 153 Pasteurizing machines . . 156, 303 Penicillium Camemberti .... 246 Roqueforti 254 Pepsin 20 Peptonizing bacteria 39 Peritrichic 5 Phenolthalein ...... 267,268 Plants or animals 14 Potato tubes 292 Powdered milk 166 Preservatives, use of 147 Protective action of acid bacte- ria 84 Pseudomonas . . . 14 Putrefaction .... 4 ... . 9 Qualitative bacteriological anal- ysis 288, 304 quantitative 304 Rancidity 216 Rennet, bacteria in 308 forming bacteria ...... 39 Right-handed lactic acid . . 35, 39 Ripened cream, bacteria in 195, 305 Ripening of cheese 223 chemical 229 temperature 237 Roquefort cheese 253 Rusty spot of cheese 242 Sanitary dairies 160 Saprophytes 19 Sarcina 13 Scarlet fever 104 Slimy milk 44 Soapy milk 55 Soft cheeses 243 bacteria in 309 Souring milk 22 Sour milk 86 Sour milk cheese 226 Species of bacteria, effect on milk 309 Spirillum 14 Splenic fever 98 Spores, production of 6 Stables, location of 114 Standards 173, 179 Staphylococcus aureus .... 60 albus 60 Starters, home 204, €06 in butter-making 203 in cheese-making .... 235, 236 making of 204, 207 314 INDEX PAGE Starters, use of 203, 209 value of 213 Sterility of milk when secreted 58 Sterilization 149 Streptococcus 107, 184 Sterilization, by autoclav . . . 303 by boiling 302 Sterilizing media 270, 277 Streptococcus lactarius . 26, 27, 57 meaning of term 13 pyogenes 60 pyogenes albus 37 pyogenes aureus 57 pyogenes citreus 57 Stilton cheese 253, 255 Stripping 131 Sweet cheese 242 Swelled cheese 238 Swiss cheese , . 228, 234 Tallowy butter 219 Temperature affecting, bacteria 15 cheese ripening 80 cream ripening 307 numbers of bacteria .... 75 species of bacteria ... 77, 297 Thermophiles 16 Thunderstorms and souring of milk 23 Titrating 265 PAGE Torula amari 49 Toxic poisoning 107, 108 Transportation 145 Tuberculosis, creameries source of 93 bacilli, staining of 287 distributed by milk .... 89, 94 extent of 88 Tuberculous milk, use of ... 92 Turnipy tasting butter .... 218 milk 54 Typhoid distributed by milk . . 98 Udder, bacteria in 59 brushing of 126 moistening 125 Viscogen 159 Washing glassware 265 Water blanks 273, 282 Water as affecting bacteria . . 18 Yeasts 8, 12 food relations of 18 in condensed milk 50 in bitter milk and cheese 49, 241 study of 284 multiplication of 8 Yoghourt 52 STANDARD BOOKS PUBLISHED BY ORANGE JUDD COMPANY NEW YORK CHICAGO Ashland Building People's Gas Building 315-321 Fourth Avenue 150 Michigan Avenue Any of these books will be sent by mail, postpaid, to any part of the world, on receipt of catalog price. We are always happy to correspond with our patrons, and cordially invite them to address us on any matter pertaining to rural books. Send for our large illustrated catalog, free on appli- cation. First Principles of Soil Fertility By Alfred Vivian. There is no subject of more vital importance to the farmer than that of the best method of maintaining the fertility of the soil. The very evident decrease in the fertility of those soils which have been under cultivation for a number of years, combined with the increased competition and the advanced price of labor, have convinced the intelligent farmer that the agriculture of the future must be based upon more rational practices than those which have been followed in the past. We have felt for some time that there was a place for a brief, and at the same time comprehensive, treatise on this important subject of Soil Fertility. 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It is printed on fine paper and illustrated with many full-page photographs that were taken with the especial view of their relation to the text. 336 pages. 6^ x 9 inches. Bound in cloth, with gold stamp- ing. It is unquestionably the handsomest agricultural refer- ence book that has ever been issued. Price, postpaid, . $2.00 Clean Milk By S. D. Belcher, M.D. In this book the author sets forth practical methods for the exclusion of bacteria from milk, and how to prevent contamination of milk from the stable to the consumer. Illustrated. 5x7 inches. 146 pages. Cloth $1 00 (5) Farm Grasses of the United States of America By William Jasper Spillman. 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A piactical guide to the cultb vation and propagation of Fruits, written from the standpoint of the practical fruit grower who is striving to make his business profitable by growing the best fruit possible and at the least cost. It is up-to-date in every particular, and covers the entire practice of fruit culture, harvesting, storing, mar- keting, forcing, best varieties, etc., etc. It deals with principles first and with the practice afterwards, as the foundation, prin- ciples of plant growth and nourishment must always remain the same, while practice will vary according to the fruit grower's immediate conditions and environments. Illustrated. 265 pages. 5x7 inches. Cloth $1.00 Plums and Plum Culture By F. A. Waugh. A complete manual for fruit growers, nurserymen, farmers and gardeners, on all known varieties of plums and their successful management. This book marks an epoch in the horticultural literature of America. 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A complete treatise on water supply, canal construction, reservoirs and ponds, pipes for irrigation purposes, flumes and their structure, methods of applying water, irrigation of field crops, the garden, the orchard and vineyard, windmills and pumps, appliances and contrivances. New edition, revised, enlarged and rewritten. Profusely illustrated. Over 500 pages. 5x7 inches. Cloth $2.00 Forest Planting By H. Nicholas Jarchow, LL. D. A treatise on the care of woodlands and the restoration of the denuded timberlands on plains and mountains. The author has fully described those European methods which have proved to be most useful in maintaining the superb forests of the old world. This expe- rience has been adapted to the different climates and trees of America, full instructions being given for forest planting of our various kinds of soil and subsoil, whether on mountain or valley. Illustrated. 250 pages. 5x7 inches. Cloth. $1.50 (14) The New Egg Farm By H. H. Stoddard. A practical, reliable manual on producing eggs and poultry for market as a profitable business enterprise, either by itself or connected with other branches of agriculture. It tells all about how to feed and manage, how to breed and select, incubators and brooders, its labor- saving devices, etc., etc. Illustrated. 331 pages. 5x7 inches. Cloth $1.00 Poultry Feeding and Fattening Compiled by G. B. Fiske. A handbook for poultry keep- ers on the standard and improved methods of feeding and marketing all kinds of poultry. The subject of feeding and fattening poultry is prepared largely from the side of the best practice and experience here and abroad, although the underlying science of feeding is explained as fully as needful. The subject covers all branches, including chickens, broilers, capons, turkeys and waterfowl; how to feed under various conditions and for different purposes. The whole subject of capons and caponizing is treated in detail. A great mass of practical information and experience not readily obtainable elsewhere is given with full and explicit directions for fatten- ing and preparing for market. This book will meet the needs of amateurs as well as commercial poultry raisers. Profusely illustrated. 160 pages. 5x7% inches. Cloth. . . . $0.50 Poultry Architecture Compiled by G. B. Fiske. A treatise on poultry buildings of all grades, styles and classes, and their proper location, coops, additions and special construction; all practical in de- sign, and reasonable in cost. Over 100 illustrations. 125 pages. 5x7 inches. Cloth. . . ■ $0.50 Poultry Appliances and Handicraft Compiled by G. B. Fiske. Illustrated description of a great variety and styles of the best homemade nests, roosts, windows, ventilators, incubators and brooders, feeding and watering appliances, etc., etc. Over 100 illustrations. Over 125 pages. 5x7 inches. Cloth $0.50 Turkeys and How to Grow Them Edited by Herbert Myrick. A treatise on the natural history and origin of the name of turkeys; the various breeds, the best methods to insure success in the business of turkey growing. With essays from practical turkey growers in different parts of the United States and Canada. Copiously illustrated. 154 pages. 5 x 7 inches. Cloth $1.00 (18) Farmer's Cyclopedia of Agriculture & & A Compendium of Agricultural Science and Prac- tice on Farm, Orchard and Garden Crops, and the Feeding and Diseases of Farm Animals Ey EARLEY VERNON WILCOX, Ph. D. and CLARENCE BEAMAN SMITH, M. S. Associate Editors in the Office of Experiment Stations, United States Department of Agriculture This is a new, practical, and complete presentation of the whole subject of ag- riculture in its broadest sense. It is de- signed for the use of agriculturists who desire up-to-date, reliable information on all matters pertaining to crops and stock, but more particularly for the actual farmer. The volume contains Detailed directions for the culture of every important field, orchard, and garden crop grown in America, together with descriptions of their chief insect pests and fungous diseases, and remedies for their control. It contains an ac- count of modern methods in feeding and handling all farm s^ock, including poultry. The diseases which ^affect different farm animals and poultry are described, and the most recent remedies sug- gested for controlling them. Every bit of this vast mass of new and useful information is authoritative, practical and easily found, and no effort has been spared to include all desirable details. There are between 6,000 and 7,000 topics covered in these references, and it contains 700 royal 8vo pages and nearly 500 superb half-tone and other original illustrations, making the most perfect Cyclopedia of Agricul- ture ever attempted. Handsomely bound in cloth, $3.50; half morocco (•Very sumptuous), $4.50, postpaid flDAlinr limn PAAIDAUV 315-321 Fourth Avenue, New York.N Y. UKANut JUUL* UUM1ANI] People's Gas Building, Chicago, IK. (19)