\ I A * A ^ U. S. DEPARTMENT OF AGRICUL BUREAU OF ANIMAL INDUSTRY. Bulletin No; 73. D. E. SALMON, D. V. M., Chief of Bureau. THE BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK UNDER LABORATORY CONDITIONS. BY LORE A. ROGERS, Expert in Dairy Bacteriology, Bureau of Animal Industry. SEP 23 1952 L/BRARY WASHINGTON: 60VT. PUBS. f?OOM GOVERNMENT' PRINTING OFFICE. California gional cility 1905 ORGANIZATION OP THE lU'KKAT OP ANIMAL INW STKV. r /;/<;/: D. K. SALMON, ]). V. M. Axxixttnit -Chief: A. I>. MICI.VIN, I*. V. S. r/,;,/('/rr/.Y K. B. JONKS, LL. M., M. D. /><;/'/// Dirixinii: I'll). II. WKHSTKK, M. S.. chic!'; ( 'i. ufKNci: I'.. l,\\i:, I',. S.. assistant chief. Inspect ion Diriximi: A. M. KAURI MITON, I'.. S., I >. V. M., chief. ' Diriximi: RICHARD \\'. UH-KMAN, Ph. (i., V. M. IX, chief. N. M. S. Aiiixt: W. S. I). HAINKS. Animal HiixlKintlmiin: <;KOKUE M. KOM.MKI., I>. S. A. Librarian: BEATRICE C. OBERLY. LABORATORIES. Dii-ixifin: MARION DORSET, M. !., chief. Pathological. IHvision.' .Jonx K. MOHI.ER, A. ."M., V. M. IX, chief. Dirision: BRAYTON H. RANSOM, B. Sc., A. M., acting zoologist. EXPERIMENT STATION". Superintendent: E. C. SCHROEDER, M. I). V. ; expert assistant, \V. K. COTTON. INSPECTORS IN CHARGE. Dr. F. W. Ainsworth, Union Stock Yards, Pitts- burg, Pa. Dr. M. O. Anderson, care Geo. A. Horinel & Co., Austin, Minn. Dr. Don C. Aver, Post-Office Building, South Omaha, Nebr. Dr. G. S. Baker, 6th and Townsciid sts., SHU Fran- cisco. Cal. Dr. L. R. Baker, South St. Joseph, Mo. Dr. A. E. Behnke, room 432. Federal Building, Milwaukee. \Vis. Dr. John A. Bell, Watertown, N. Y. Dr. S. E. Bennett, room 338, Live Stock Exchange Building, Kansas City, Knns. Dr. E. L.Bertram, care J. S. Gilmore, Davenport, Iowa. Dr. Fred Braginton, care Continental Packing Co., Bloomington, 111. Dr. J. J. Brougham, care Missouri Stock Yards, St. Louis, Mo. Dr. G. W. Butler, care Drummond Bros., Eau Claire, Wis. Dr. J. B. Clancy, National Stock Yards, 111. Dr. Lowell Clarke, room 320, Quincy Building, Denver, Colo. Dr. Joel E. Cloud, care the Agar Packing Co., I>os Moines, Iowa. Dr. CharlesCowie, Ogdensburg, N. Y. Dr. David Gumming, 912 Lapeerave., Port Huron. Mirh. Dr. Robert Darling, care Chas. S. Hardy, San Diego, Cal. Dr. E. T. Davison, Rushville, Nebr. Dr. J. F. Deadman, Sault ste. Marie, Mich. Mr. Albert Dean, room 328, Stock Yard Station, Kansas City, Kans. Dr. K. L. De Wolf, care Chas. Wolff Packing Co., Topeka, Kans. | Dr. Geo. Ditewig, care Union Stock Yard*, Cin- cinnati, Ohio. Dr. E. P. Dowd. care White, Pevcy A: Dcxu-r Co., Worcester, Mass. Dr. O. E. Dyson. 316 Exchange Bnildini:, Union Stock Yards, Chicago, 111. Dr. Geo. C. Faville, P. O. box 796. Norfolk. Va. Dr. J. W. Fink, care Swift . box 1145. Salt Lakt- City, Utah. Dr. A. A. Holcombe, Aurora, 111. Dr. U. G. Houck, care Swift & Co., Fort Worth, Tex. Dr. W. E. Howe, care Western Packing Co., l>en ver, Colo. Dr. Julius Huelson, care Jersey City Stock Yard- Co., Jersey City, N. .1. Dr. F. W. HnntiiiKtnn, U. S. customs >fli-e. ;. T. R. R. wharf, Portland, Me. Dr. Robert Jay, care Jacob E. Decker ,v Son, Mason City, Iowa. Dr. G. A. Johnson, Kxchange Building. Sioux City, Iowa. Dr. James Johnston, care U. S. consulate. 26 Chapel St.. Liverpool. England. (Concluded on third page of cover.) U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. Bulletin No. 73. D. E. SALMON, D. V. M., Chief of Bureau. THE BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK UNDER LABORATORY CONDITIONS. BY LORK A. ROGERS, Expert in Dairy Bacteriology , Bureau of Animal Industry. WASHINGTON: G O V K R N M I XT P R I N 1 INC, n F K I C F, . I <)0 S . FKR OF TRANSMITTAL U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY, Wa*hinffton, D. C., May 11, 1905. SIR: I have the honor to transmit herewith a manuscript entitled '* The bacteria of pasteurized and unpasteurized milk under labora- tory conditions," by Lore A. Rogers, expert in dairy bacteriology in this Bureau, and to recommend its publication as Bulletin No. 73 of the Bureau series. Respectfully, D. E. SALMON, Chief of Bureau. HON. JAMES WILSON, Secretary of Agriculture. Dy. 63. 2 CONTENTS. Pago. Introduction "> Pasteurization S Kxperiments with raw and pasteurized milk 11* Influence of lactic bacteria on the development of peptonizing bacteria 2S Conclusions 80 Summary 80 Bibliography 81 8 THE BACTERIA OE PASTEURIZED AM) UNPASTEURIZED MILK UNDER LABORATORY CONDITIONS. INTRODUCTION. The rapidly increasing size of cities, with the resulting removal of a large proportion of the consumers from the source of supplies, has necessitated marked changes in the transportation and storage of vari- ous food products. This has been, with many articles of food, a sim- ple question, but with others of a more perishable nature the problems involved have been more complex and dillicult. This is especially true of milk. In this case we have a food in almost universal use that is subject to rapid changes, affecting not only its appearance and taste, but also its value and even its safety as a food. The-^- changes are brought about by bacteria which are normally present in milk, even when it is collected under the best sanitary conditions, and which lind there favorable conditions for rapid multiplication. Thus we fre- quently rind that, as a result of this growth, in a comparatively few hours milk may become entirely unfit for use. In the smaller cities and towns the producer usually delivers the milk directly to the consumer, and the problem of a good milk supply is simply one of sanitary barns and a proper cooling and handling of the milk. In the larger cities, however, tjie demand is too great for the immediately adjacent country to supply, and the producer is so far removed from the consumer that a middleman is necessarv. A lanjv proportion of the milk used in the larger cities of this counlrv i- shipped by rail, a few companies usually buying the milk from the farmers and distributing it from milk depots in the cities. Some of the milk used in Boston comes 14." miles (')." The spread of the city of New York into the surrounding counlrv. together with the increasing value of land for truck fanning, lia- forced the dairy farms farther and farther awav. until now milk for use in New York City is collected along the banks of the St. Lawrence River. .">.">(> miles away. (') In Chicago the conditions are somewhat dif- ferent, and practically all of the milk used there i> produced within loo miles. "Superior li^un-s icl'rr t<> tin- l>ihliuliy at tin- rml "I ihr Imllclin. 6 BUREAU OF ANIMAL INDUSTRY. Long shipments have necessarily lengthened very materially the time which elapses between the production of the milk and its con- sumption. A considerable part of the milk is held ten or twelve hours on the farm before it is delivered at the milk station, and another dela\ r frequently occurs at the city depot before the milk is distributed. Much milk reaches Boston from 18 to 30 hours old. (') The greater part of the New York milk is from 12 to 36 hours old when it reaches the city. Great care is now used to hold the milk as nearly as possible in its original condition. On the long hauls the milk is cooled at the receiving station and ice is used to hold down the temperature in transit. Notwithstanding all the precautions observed, the milk, as it is delivered to the consumer in the city, is usually in a state far from satisfactoiy. On the average farm the conditions are such that the milk becomes heavily contaminated with various kinds of bacteria. If the milk is not cooled at once, multiplication of these soon begins and continues with more or less rapidity, which depends largely on the temperature. Even in milk held at comparatively low temperatures the increase may be great. A few examples of the bacterial content of city milk will serve to illustrate the extent of this contamination. Sedgvvick and Batehel- der ( 3 ) found in 57 samples of Boston market milk from 30,000 to 4 r 220,000 bacteria per cubic centimeter. Hill and Slack (*) tabulated the results of the examination of 2,394 samples, nearly all taken as the milk arrived in the city, as follows: Per t-ent. Below 100,000 bacteria per cubic centimeter 42 Between 100,000 and 500,000 per cubic centimeter 29. 75 Between 500,000 and 1 ,000,000 per cubic centimeter 9. 75 Between 1,000,000 and 5,000,000 per cubic centimeter 12. 75 Above 5,000,000 per cubic centimeter 5 Uncountable spreaders 0. 75 Park ( 5 ) gives the average of 20 samples taken from cans immediately on arrival in New York in March as over 5,000.000 per cubic centi- meter. The average of 13 samples delivered to tenement houses in midwinter was 1,977,692 per cubic centimeter, while early in Septem- ber it was 15,163,600 per cubic centimeter. In the better districts at the same time the number was considerably less. Bergey ( fi ) found the average of 10 samples collected at railroad stations in Philadelphia in July to be 4,802,355 per cubic centimeter. It is readily seen from these figures that the bacterial contamination of the city milk supply, especially of the large cities, is uniformly great. It is well established that spec! fie diseases are not infrequently spread through the milk supply. Aside from this important phase of the question, which can not be discussed here, it is well known that bac- teria are able to bring about in milk many changes of a highly unde- sirable nature. These vary from decompositions scarcely affecting the BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK. 7 taste to the formation of products of a very toxic nature; the most familiar change is the souring, or curdling, resulting from the acid fermentation of the sugar. While this spoils the milk for drinking purposes, the group of bacteria which brings about this change is not considered deleterious. Indeed, they are introduced in the form of artificially prepared cultures in the manufacture of butter and many kinds of cheese. On the other hand, there is normally present in milk a group of bacteria which acts on the nitrogenous constituents the casein and albumen- and their decomposition products. These bac- teria secrete enzymes, or digestive agents, which act entirely inde- pendently of the cell, and are capable of bringing about digestive action greatly in excess of the needs of the organism. The rennet usually secreted with the proteolytic, or digestive, enzymes curdles the milk, which is slowly digested until in time the curd may be entirely replaced by a clear serum. In some cases the digestion may go on without previous curdling. As milk is ordinarily used, this process is not sufficiently advanced to affect the appearance or taste apprecia- bly, although it would probably be unusual to find milk twenty-four hours old in which some change of this nature had not taken place. The products of this decomposition may be entirely harmless even when taken in considerable quantities, or. on the other hand, they may have a very toxic action. The, cases of ptomaine poisoning from milk occasionally reported arc in all probability due to bacteria of this class. Such cases are so infrequent and usually of such obscure origin that they need not be considered in a discussion of the general milk supply. However, it is generally recognized that children, and especially children under one year old. may be very seriously atl'ected by milk containing many bacteria of this type. The relation between the condition of the milk and the amount of intestinal trouble in young children has been demonstrated by the investigations of I 'ark and Holt (') in New York City. They found that this relation held for very young children only, and did not hold for children over three years old. even when they were fed milk containing large numbers of bacteria. They were unable to rind any relation between specific varieties of bacteria occur- ring in tlie milk and the health of children. Forty per cent of the cultures tested caused death when injected intraperitoneally. but only one of I .'lit caused illness or death when fed to young kittens. It is probable that the intestinal troubles of children are not all caused bv one or two specitic varieties of bacteria, but that they may be caii>ed by any one of a number of widely distributed ^pecie-. which may under certain circumstances produce abnormal conditions in the digestive tract. This may be brought about either through the direct action of the bacteria in the intestines or indirectly by the formation of toxins in the milk itself. 8 BUREAU OF ANIMAL INDUSTRY. The great bacterial contamination of milk is now well recognized, and the public interest in this question is shown in the municipal con- trol which is now very generally exercised over the milk supply. The control usually takes the form of inspection for adulteration and the presence of antiseptics, licensing of dealers, and in some cases the inspection of the dairy farms, with certain requirements regarding their sanitary condition. In a very few cities it is required that the milk shall not be above a certain temperature when delivered, and that the bacteria shall not be above a certain limit. The city regulations of Boston require that milk shall not contain over 500,000 bacteria per cubic centimeter. Another method which has been adopted with good results in some cities is the inspection of the dairies and milk by some organization, usually a medical society. Dairies meeting the requirements of the society are given a certificate and are allowed to advertise their milk as certified by the society. The so-called model, or sanitary, dairies furnishing milk under exceptionally clean conditions are becoming somewhat numerous, but, as they are obliged to sell their product at an advance over the usual price, they have not yet become an impor- tant factor in the general milk supply. All of these methods are designed to prevent the contamination of milk by bacteria or to inhibit their growth while the milk is being transported from the farm to the consumer. Although they have doubtless resulted in a distinct im- provement, it is evident that they have by no means produced a perfect milk supply. An ideal milk would be collected under perfect sanitary conditions from healthy cows and held during transportation at such a temperature that the few bacteria which are found in it, even under the best conditions, would have no opportunity to develop. A milk supply of this nature will come only through a long slow process of education and regulation. In the meantime milk dealers have resorted to various methods to inhibit or destroy the bacteria already present in the milk. The objection to the use of antiseptics is so strong, in this countiy at least, that this method need not be considered. PASTEURIZATION . A method which was at one time looked upon as in some respects a solution of the question of the city milk supply is the application of heat to milk in such a manner that the greater part of the bacteria is destro3'ed without seriously affecting the character of the milk. This process is known as pasteurization, from the fact that it was first, used on an extensive scale in the wine industry of France on the recommen- dation of Pasteur. In the application of this term to milk it is fre- quently confused with sterilization, which involves the complete destruction, not necessarily by heat, of all the bacteria present. As commonly practiced, however, pasteurization did not produce the BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK. 9 results expected and its use gradually declined. Nevertheless, the statistics of 200 cities and towns collected by the Dairy Division ( 8 ) show that considerable milk is still pasteurized, and the practice is probably gaining somewhat in favor. Of the 1<>3 cities answering an inquiry regarding the amount of milk pasteuri/ed. H2, or about 50 per cent, stated that more or less milk was pasteurized. Usually a small amount only was treated, but in some cities it was given as from 10 to 50 per cent of the total supply. Various methods of pasteurization are employed, nearly all of them being efficient if properly used. To be efficient, pasteurization should destroy practical!}' all of the bacteria in the vegetative stage. This may be accomplished by the application of a comparatively high degree of heat for a short time or of a lower temperature for a longer period. In addition to being effi- cient, the method should not give the milk a decided cooked taste. In all probability this fault has been responsible for the decreased use of pasteurization. Milkmen have been careful to adopt a temperature low enough to avoid a cooked taste, and as a result a small part only of the bacteria has been destroyed, and the increased keeping quality of the milk has not been sufficient to repay the extra labor. Further- more, a method to be practical must be economical of heat, labor, and time. A method applicable to a few gallons of milk can not be economically applied to several hundred or a thousand gallons. The simplest apparatus for pasteurizing milk is a vat surrounded by a water jacket, with some arrangement for heating the water. In the machines of this type the milk is heated to the desired temperature and held for some time, usually !") to 80 minutes. The temperature required for efficient pasteurization is dependent on the length of exposure. The thermal death point of the tubercle bacillus, which is the most resistant of the known nonspore-forming pathogenic bacteria, is usually taken as the criterion for the proper pasteurizing temperature. Recent work by Theobald Smith (") and Russell and Hastings ("') has shown that this bacillus may be destroyed by an exposure for !." to _'<> minutes to a temperature of f'.o ( '. (14-O F. ). provided that the milk is thoroughly stirred to prevent the formation of a lihn on the surface. In practice a slightly higher temperature >^ 71 ('. (l.V> !><> F.) is usually adopted. This has been found to be the highest temperature that can be used without causing a distinct cooked taste. Another apparatus which has been generally adopted where milk is pasteurized on a large scale involve* the continuous flow of the milk. In this case the milk Hows over a heated surface. \\ here it is raised to the required temperature, and passes on in a continuous stream over the cooler. In one machine of this tvpe the milk parses into a cylin- der, when' it is thrown by rapidly revolving paddle* in a thin sheet on the inner surface of a >tcain jacket. The milk i* heated almo*t 2!M>o:; No. 7:> (>.">-- 10 BUREAU OF ANIMAL INDUSTRY. instantly to any temperature desired and passes into the cooler after a very short exposure. In an improved form of this machine the out- going hot milk is partially cooled by flowing back over the incoming cold milk. One pasteurizer of this type is so arranged that the milk flows between revolving cylinders containing hot water. In another type the milk flows slowly through a vat holding revolving disks tilled with water which is heated by a jet of steam. On account of the short exposure the temperature necessary to insure efficient pasteurization is considerably higher than that used in the machines giving a longer exposure. Harding and Rogers, ( n ) working with a machine of the continuous rapid-heating type, found that it was very efficient at 80 or 85 C., but inefficient at 70 C. The higher temperatures (80-85C.) are recommended by Bang, ( 12 ) because the tubercle bacillus is certainly destined at these tempera- tures, even with the very short exposure secured with these machines, and the milk, in addition to having an improved keeping quality, may be guaranteed to be free from pathogenic bacteria. More recent work by Russell and Hastings ( 13 ) indicates that a temperature considerably below 80-85 C. will insure the destruction of the tubercle bacillus, even with the short exposure obtained in continuous pasteurization. Numerous objections, aside from the extra expense and labor, have prevented the general adoption of this practice. One of the most valid is that pasteurization ma\ r be used to correct the faults of insani- tary conditions and thus retard the progress of hygienic methods. Milk collected under cleanly conditions and properly transported and delivered does not need pasteurization. Another serious objection in the eyes of the dealer is the changed condition of the fat globules. The cream separates out more slowly, giving the buyer the impression that the milk is deficient in fat. The taste of the milk is also changed more or less, especially if the continuous machines with the higher temperatures are used. The question of the comparative digestibility of raw and cooked milk has been much discussed and investigated, but can not be considered as definitely decided. It is very generally believed that milk which is highly heated is somewhat less digestible than raw milk, and may in some cases cause pathological conditions due to improper nutrition. On the other hand, it his been shown in numerous instances that chil- dren thrive on pasteurized milk. The observations of Variot ( u ) made on 3,000 infants among the poorer classes of Paris fed on milk steril- ized at 108 C. throw some light on this important question. Three or 4 per cent of these children could not use this milk, but the remain- der, including those that had been retarded in their development by gastro-intestinal troubles, did well under this treatment. No scurvy or rachitis was observed in these children. The statement has frequently been made, and is now generally BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK. 11 accepted, that the bacteria developing in pasteurized milk are much more undesirable than those growing in raw milk, and that for this reason pasteurized milk ma}' become actually dangerous as a food, while the taste and appearance remain unchanged. It is a matter of common observation that pasteurized milk frequently decomposes with a rank odor without souring, while unheated milk simply turns sour, curdles, and remains unchanged for some time. The reason for this is very evident. In the unheated milk the lactic bacteria develop enor- mously, forming acid so rapidly that the growth of the peptonizing bacteria is checked or completely prevented. In the pasteurized milk, on the other hand, the lactic bacteria are usually all destroyed, leavi?ig a clear field for the development of the more resistant spore-forming peptonizing bacteria. Conn and Ksten ( 1!i ) state that the development <>f lactic bacteria serves as a protection both to the 1 milk and the person drinking it, since it prevents the growth of other bacteria. Flugge,( lfl ) on whose work most of the objection to pasteurized milk is based, heated milk for a short time at !>o -i)5 C. and isolated the surviving bacteria, which he studied in pure culture. These he found to be mostly of the peptonizing and the anaerobic butyric acid forms. Some of the former produced dangerous toxins. WeberC 7 ) found that most of the bacteria developing in so-called sterili/ed milk were of the hay or potato bacillus type. Flugge's toxin- forming bacteria were found three times in the 15o flasks examined. It is easy to see how the presence of considerable numbers of bac- teria of this class in milk fed to infants might cause serious results either through the product ion of their toxins in the milk or after being carried themselves to the digestive tract. That this does not always hold true for pasteurized milk is well illustrated by sonic recent work by Park and Holt( : ) in New York City. In this work the conditions were controlled as well as it was possible in inVestigat ion^ of t hi- nat tire. About ;">O babies were selected from the tenement houses and divided as accurately as possible into two e<|iial lots. All were fed on milk modified at one of the Straus milk depots and were treated in the same way. except that one half were fed on milk pasteurized at Hi.~ F. for thirty minutes and the other half received the same milk without heat ing. The raw milk averaged in the morning I.-JOIMKIH bacteria per cubic centimeter and the pasteurized about Ij'iin per cubic centimeter, while in the afternoon of the same day they contained respectively about jo. ooo. i io<> and r.o.ooo bacteria per cubic centimeter. In di-cus-ing the results of their investigations the authors say: Within OIK- week 'JO of the -7 infants put on the r;i\v milk suffered from moderate or severe diarrhea, while during the same time only ."> cases of moderate and none >f severe diarrhea occurred in those taking pasteurized milk. Within a month s of the -7 hud to he changed from raw hack to heated milk, hvrause of their continued 12 BUREAU OF ANIMAL INDUSTRY. illness; 7, or 25 per cent, did well all summer on raw milk. On the other hand, of those receiving the pasteurized milk, 75 per cent remained well, or nearly so, all summer, while 25 per cent had one or more attacks of severe diarrhea. There were no deaths in either group of cases. Very similar results were obtained from a second trial made in the same manner during the following summer. Doctors Park and Holt were, however, unable to find that pasteurization of the milk affected the amount of intestinal trouble in older children. These results may be taken as conclusive evidence that, under cer- tain circumstances at least, poor city milk is rendered a safer infant food by pasteurization. It must be remembered, however, that this work was done under carefully controlled conditions and that the milk was given out in small bottles only, so that it had to be used before there was time for any great development of deleterious bacteria. Park and Holt, in the paper cited above, express the belief that intestinal troubles in children fed on poor milk which had been pas- teurized were due to changes in the milk which were not neutralized by heat. Liibbert, ( 18 ) on the other hand, states that the toxic prop- erties of a milk culture of one of Fliigge's peptonizing bacteria were destroyed by heating. The results touching on this question are not conclusive, but it seems probable, when the comparatively stable nature of the known toxins is considered, that the toxicity of badly infected milk would not be decreased by the heat received in ordinary pasteurization. We have seen b} T this brief review 7 of the literature on milk supply that, notwithstanding the increasing stringency of municipal regula- tions and efficiency in their enforcement, the milk is usually badly contaminated, frequently even to the point of becoming positively dangerous when used as a food for infants; that some authorities consider pasteurization an advisable remedy for this condition, and that this method is used in man} T American cities. On the other hand, it appears that some people consider that the bacteria which develop in milk after pasteurization make it more dangerous than poor raw milk. EXPERIMENTS WITH RAW AND PASTEURIZED MILK. It is undoubtedly true that bacteria of a very undesirable nature may develop in the clear field left by the destruction of the lactic bacteria, but how soon they develop after pasteurization, what num- bers they must attain before the milk becomes dangerous, and how their development compares with the growth of similar bacteria in indicated milk, are questions which have not yet been answered. The writer has been unable to find the results of any investigations giving the quantitative bacteriological examination of pasteurized milk beyond the number present immediately after pasteurization. In BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK. 13 undertaking the work, the results of which are given in this paper, it was not intended to attempt to demonstrate that it is advisable to pas- teuri/e or that it is inadvisable, but to determine the rapidity of bac- terial development in heated milk and to compare it, quantitatively and qualitatively, with the Horn of raw milk. It was planned at the begin- ning to make this investigation more comprehensive, but certain cir- cumstances have interfered to prevent its completion at this time. It is believed, however, that the results already obtained are of sufficient value- to warrant their publication in this incomplete condition. The milk used in this work was all obtained from a large dairy with an excellent reputation. When delivered, the milk, which was the mixed milk of several herds, was 24 to 3<> hours old. The first nine samples were held overnight in a refrigerator to allow bacterial development corresponding to that of the oldest city milk. The remaining samples were pasteurized when received that is. when 24 to 80 hours old. Although no comprehensive bacteriological examinations of the milk supply of Washington, I). ('., have been published, the results of the following determinations, made (with (he exception of Nos. 1 and 2) in March and April, indicate that these may be taken as fairly representative of the milk delivered by the large Washington dairies during the warm months: TAHI.K I. lliirti'rin in milk from rurtoux xoiircex, . < '. No. Ohtuim-d in,!,. l.iu-ticm-id. T " lal .'""" inj* "<- teriii. I'lrfiiit. Prrr.i: l'(Ki f> City dairy . r.'tl li;.s.">o. IKKI do . . . IJ('> S'l. (KKI . . In carrying out the work outlined in the introduction it is essential first of all that the pasteurization bo efficient. The method by which this result is obtained is not important. For the sake of convenience a small model made for this work was used, with a temperature of *."> ('.(!*."> F.). This apparatus consisted of a small trough, with an inlet and an out let tube at opposite ends: a series of revolving hollow disks, so arranged that steam could be blown through them, served the double purpose of heating and stirring the milk. The milk flowed in at one end. was heated to the required temperature, and passed out and over the surface of a cooler which brought the temperature down to about -J'i-2.") ( 1 . The temperature adopted (1X.~> F.) is higher than is ordi- narilv used, and milk treated in this wav has a distinct cooked taste; 14 BUREAU OF ANIMAL INDUSTRY. hut, since this work YVJIS arranged only to study the bacteria grow ing in efficiently pasteurized milk, this fact was of no importance in this instance. Three samples were collected in sterile flasks one from the milk before pasteurization and two of the pasteurized milk. One of the latter was placed in a refrigerator kept uniformly at 10 C. (50 F.), and the other was kept (with the exception of a few samples which were held in the laboratory) in an incubator holding uniformly at 20 C. (68 F.). In like manner half the samples of raw milk were held at 20 C. and the other half at 10 C. Gelatin plates were made from each sample at the time of pasteurization, and at intervals of 6, 12, and 2-i hours, up to 96 hours, or until the milk curdled. Two and one- halt' per cent lactose gelatin was used. A series of dilutions in sterile water was made so as to get as nearly as possible 300 or 400 colonies on a plate. When the lactic bacteria became numerous the liquefiers were counted on plates containing several thousand colonies. Although this method is probably inaccurate it enables one to count many of the less numerous species, which would be missed in a dilution suited to the lactic forms. All small spherical solid colonies were counted as lactic-acid bacteria. While this grouping would probably include many inert forms the error would not affect the results seriously. The plates were incubated at 20 C. until the spread of liquefying colonies made it necessary to count. In many cases this became necessary before some of the slower-growing colonies had appeared. No attempt was made to study the various species beyond what was necessary to determine the relationship of the colonies occurring on the different plates. In the tables the bacteria are arranged in four different groups the total bacteria, the acid-forming bacteria, the peptonizing, or liquefying, bacteria, which are subdivided into the slow and rapid liquefiers, and the inert bacteria, or those having no appreciable effect on milk. The action on milk was determined by transferring a num- ber of type colonies from each sample to litmus milk and incubating for several days at 30 C. The acidity of each sample of milk was titrated against foNaOH with phenolphthalein as an indicator when the gelatin plates were made, and the appearance and taste of the milk usually noted when it curdled, or at the end of 96 hours. BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK. 15 TABLK II. Physi&d condition and '.tantr of the milk. Sample No. Treatment. em per- , iture. 11 21 31 12 22 32 13 28 33 14 24 34 15 26 3f> 16 '26 36 17 27 37 18 28 38 19 29 39 110 210 : :ti2 113 213 S13 111 214 311 Raw C. Hour*. Room. 72 Room. 72 10 VJO 10 % Room. 1)6 10 % Room. 48 Room. 24 10 96 10 96 Room. 96 1(1 96 Room. 72 Room. 96 10 96 10 % 20 4S 10 96 20 IS 20 4s 10 72 10 90 20 I.S 10 91 ; 20 4H 20 IS 10 96 10 % 20 is 10 96 20 4S 20 IS 10 96 Id 9*1 20 IS III 96 211 21 20 Is 1(1 '.HI 111 '.'(', 20 Is Id % do Raw do Kuw . do Kuw Pasteurized . do. Kuw Pasteurized do Raw .[ Pasteurized do Raw . . . Pasteurized .do . .. Raw Pasteurized do Raw Pasteurized ....do Raw Pasteurized .. do Raw . . Pasteurized > C 1 . usually curdled in 4S to T'J hours with an acid taste, although at times there were indications of peptoni/ation. In the raw milk at 1" (.'. the curdling was much retarded and the taste was affected by presence of digesting bacteria in one or two cases. The pasteuri/.ed milk held at '20 C., on the other hand, usually had a rennet curd at the end of 4S 16 Bl'KKAU OF ANIMAL INDUSTRY. hours, with a disagreeable taste, although in a few instances the lactic bacteria were evidently not entirely destroyed and formed an acid curd in 48 hours. The pasteurized milk held at 10 C. almost without exception remained perfectly sweet to the taste and unchanged in appearance for 9fi hours or more. TABLE III. Aridity of milk exprewed an per cent of lactic III ill! v ige of lactic HC 12 24 <] after lapse of 48 72 96 hours Per H. Per ct. \ Per ct. l'n-,-1. l',r,-t. I'irct. Per cl. Raw milk kept at 20 C 0.131 0. 1(18 0.254 0.4-19 O.(i% 0.711 .KM .134 .127 Raw milk kept at 10 C 13C 152 1(17 177 '33 388 497 Pasteurized milk kept at 10 C . . 134 129 131 l'>9 129 130 13 4) 1 As would naturally be expected, the increase in acidity of the un heated milk was much more rapid at 20 C. than at 1< (.-. The average of the increase in acidity of the pasteuri/ed milk at 2<> C. does not express the true state of affairs, as the increase was usually very slight, except in a few instances when the rapid multiplication of lactic bacteria caused a sudden and marked increase in the acidity. In the pasteuri/ed milk at 10 ('. there was usually no increase what- ever, and in one lot only was there sufficient acid to afl'ect the taste. The complete results of the bacteriological counts are given in Table V. These 4 are summarized in Table VI. which contains the mean of the total bacteria, and in Table VIII. which contains the mean of the peptoni/ing bacteria. TAKLE V. HaHeria i>er cubic centimeter . SAMPLK No. 11. RAW MII.K KEIT AT ROOM TK-MPHKATTHK. Number of bacterin found after lapse of hours."' 6 hours. 12 hours. I 24 hours. is hours. (Jroup. 72 hour-. % hours. Total bacteria Total lactic 835, 000 12,825,000 75,032,500 1)71, 500. (XXI ">T.O. (125, (*X> 150. 937. (XXI I! 500 25 UX) 1) ' > 5 (XXI 1 932 6(HI .">70 2. 11"> r.'T.ixxi 3*. x (I 30 3.~>~) 2, -Mxi '.'_'i i. u ni Total liqueliers. . . Slow liquetiers . . . Inert . 111 311 58<> 4. MO 1.7^1 191. SKI 37. llu.UXi a The ii>;e of the milk >fiveii in the tables is reckoned from the time of pasteiiri/alion. l> Dilution too low. 18 BUREAU OF ANIMAL INDUSTRY. TABLE V. Bacteria, per cubic centimeter of milk, arranged in groups Continued. SAMPLE No. 12. RAW MILK KEPT AT 10 C. Group. Number of bacteria found after lapse of hours. 6 hours. 12 hours. 24 hours. 48 hours. 72 hours. 96 hours. Total bacteria Total lactic 103,925,000 139,500,000 153,000,000 266, 000,000 618,000,000 152,500,000 354,000,OCC Total liquefiers... 75,000 100,000 500,000 Rapid liquefiers . . Slow liquefiers ... Inert SAMPLE No. 22. PASTEURIZED MILK KEPT AT ROOM TEMPERATURE. Total bacteria Total lactic 70 23 50 1,200 1,475,000 1,175,000 7,500 40, 740, 000 40, 440, 000 150,000 308,000,000 298,750,000 Total liquefiers... Rapid liquefiers .. 4 8 800 800 Slow liquefiers Inert 225,666 150,000 9, 250, 000 SAMPLE No. 32. PASTEURIZED MILK KEPT AT 10 C. Total bacteria Total lactic 70 70 30 60 70 6 60,600 69,800 300 Total liquefiers... Rapid liquefiers .. 4 4 2 10 Inert 500 SAMPLE No. 13. RAW MILK KEPT AT ROOM TEMPERATURE. Total bacteria Total lactic 33, 707, 500 32,945,500 762,000 83, 062, 500 168, 812, 500 80,830,500165,625,000 2,232,000 3,187,500 231, 226, 4, 1, 750, 000' 937, 500 812,500: 812,500 . 947, 946, 260, 000 ! . . 750,000 Total liquefiers ... Rapid liquefiers . 500,000 3, 000 000 j 1 I SAMPLE No. 23. PASTEURIZED MILK KEPT AT ROOM TEMPERATURE. Total bacteria Total lactic 4,040 29,320 (a) (0) . .. Total liquefiers ... 11 50 SAMPLE No. 33. PASTEURIZED MILK KEPT AT 10 C. Total bacteria Total lactic 4,040 6,390 46, 150 843,000 Liquefied. 34,375,000 1,875,000 247. 750, 000 29, 250, 000 12 190 720 4,500 5, 000, 000 3.000,000 2, 625, 000 2, 375, 000 3.000.000 27, 500, 000 215, 600, 000 a Dilution too low. b Flask broken. BACTERIA OF PASTEURIZED AND UNPA8TEURIZED MILK. 19 TABLK V. Hucteria prr cnbic centimeter of milk, arrangeil in tjrtmpn Continued. SAMPI.K No. 14. RAW MII.K KBIT AT 10 O. Group. Number of bacteria found a ft IT lapse of hours. (> hours. 12 hours. 24 hours. 4H hours. ! 72 hours. % hours. Total bacteria .... 11,125,000 10,125,000 49,250.000 3Hx, 500, 000 26tf, 600, OOOktt. 750. 0004,102, 000, 000 Total lactic. Total li(|lieliers... 140,000 Rapid lit)iiefiers.. 55,000 457, (KK) a, 312, 000 5,250,000 750. (KK) 9, 337, 500 IM.OOO.OOU 11,625,000 Slowliquetiers H5 000 . 2. 562. 000 . . . SAMPI.K No. 24. I'ASTKCKI/.KII MII.K KKPT AT R )OM TEMPKKATCKK. Total bacteria 50 Total lactic 50 30 , 500 49, 600, 000 471, 500, 0001 , 643, 000, 000 Total li(|uetiers... 10 Rapid liquctiers .. 10 ... 10 2,750 10 2,750 8,' 360, 666 1,540,000 6, f-20, (XX) 90, (KK), (KK) 37.500.000 . 52. 500, 000 . 61,000,000 Slow liquctiers Inert . .... ! f>, 7f)() 41,240,000 :5N1 500 000 1 SAMPI.K No. 34. I'ASTKCKI/.KD MII.K KKIT AT 10 c. Total bacteria 50 Total lactic 50 160 I4H.OOO 21,600,000 277,250,000 Total liqiictiers... 10 . . 50 30. 9(X) 11,500 19 400 1 1 , 200. 000 46.750.000 50 Slow liqucticrs TKMPKKATI RK. SAMPI.K No. 5. RAW MII.K KKPT AT ROOM Total bacteria .... X. 12:5, 751) l:i. Total lactic 2,991.250 12. Total liquefiers. .. 1:52,500 Rapid lii|iieliers .. 70. (XM) Slow liqiieticrs .. . 62.500 Inert .. 725, (KK 66, 62.", 000 15*. 750, (XX) 100, (KK) 6:5, !7. r ,(KK) I3S, 5, (KKI 4.125.000. 5(KJ, (KK) 2,750,000 5, ,")(K). (KK) l.:V75, (KK) . lS,:i(KI,(XKI 9. 750, (KK) . Total bacteria Total lac(i( SAMPI.K No. 25. I'ASTKIKI/.I:I> MII.K KKPT AT ROOM TKMPKHA ITKK. l,(XXIi ;{,525.(KKI I) 21X1 1 I'HK'I :5.525 (XXI Rapiil li(|iicticrs 21X1 I,(XKI 3. 525. (KKI Slow Hi|iieliers Inert .. ; A.MPI.K No. :;."i. --I'.xsTKi KI/.K.II MII.K KKPT AT lo r Total bacteria .... 'I'otal lactic llll 50 10 2,790 .'I.MIO r,, i:to 7. 7211 Total lii|iiclicrs... o 30 o o HO 2t> 20 _1> in Inert .. 2. 7-.X) 5.Si(l 6, UK) 7.7OO 20 BUREAU OV ANIMAL INDUSTRY. TABLE V. Bacteria per cubic centimeter of milk, arranged in groups Continued. SAMPLE No. 16. RAW MILK KEPT AT 10 C. Group. Number of bacteria found after lapse of hours. 6 hours. 12 hours. 24 hours. 48 hours. 72 hours. 96hoaw. Total bacteria .... Total lactic 5, 326, 000 4,560,000 125,000 75,000 50,000 650,000 21,500,000 18,000,000 1,500,000 600,000 1,000,000 2, 000, 000 30,875,000 29,200,000 1,400,000 1,100,000 300,000 275,000 75,000,000 70,550,000 2, 500, 000 2, 500, OuO 314,000,000 283, 250, 000 24,250,000 21,250,000 3,000,000 6,500,000 Plates liquefied. 480,500,0 878, 00(1, (XX) 99,000,000 65,500,000 33, 600. 000 3,600,000 Total liquefiers... Rapid liquefiers . . Slow liquefiers . . . Inert 1,750,000 SAMPLE No. 26. PASTEURIZED MILK KEPT AT 20 C. Total bacteria Total lactic 20 40 220 1,040,000 960,000 80,000 10,000 70,000 1,581,000,000 1,581,000,000 Total liquefiers... Rapid liquefiers.. 6 30 10 20 90 90 Inert SAMPLE No. 36. PASTEURIZED MILK KEPT AT 10 C. Total bacteria Total lat-tic. . . 20 40 20 30 110 2,430 2,430 67,900 67,880 20 Total liquefiers... 30 10 20 10 SAMPLE No. 17. RAW MILK KEPT AT 20 C. Total bacteria Total lactic Total liquefiers 37, 950, 000 34,726,000 3, 075, 000 136,000,000 110,750,000 24,500,000 317,000,000 314, 750, 000 1,750,000 500,000 1,250,000 500,000 78, 000, 000 ""i, 666, 666 452,500,000 452, 350, 000 25, 000 Rapid liquefiers.. Slow liquefiers 25,000 3,050,000 150,000 1,250,000 23,250,000 750,000 1,000,000 25,000 125, 000 SAMPLE No. 27. PASTEURIZED MILK KEPT AT 20 C. Total bacteria Total lactic 2, 220 2,800 75,500 72, 920 280 15, 650, 000 15, 552, 500 30,000 1,227,500,000 1,218,250,000 760,000 500,000 Total liquefiers... Rapid liquefiers.. 10 10 280 300 30,000 67,500 250,000 7, 500, 000 SAMPLE No. 37. PASTEURIZED MILK KEPT AT 10C. Total bacteria Total lactic 2, 220 2.X60 2,350 3,130 :?6,500i 6,715,000 1 Total liquefiers... Rapid liquefiers .. 10 10 6 io io 126! 76,666 90 30' 36,380 6,645,000 ; BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK. 21 TABLE V. Bacteria per cubic centimeter of milk, arranged in groups Continued. SAMPLE No. 18. RAW iMiLK KKPT AT 10 C. Number of bacteria found after lapse of hours. 6 h< mrs. 12 hours. 24 hours. 58, 500, 000 52,500,000 5,7.50,000 3,500,000 2, 2-50, 000 1,250,000 ED MII.K Ki 116,000 48 hours. 72 hoi re. 96 hours. Total bacteria .... '2, 600, 000 9, 5 Total lactic ' 2, 125,000 8,t> 75,000 '24,250,000 35,W)0 22,700,000 00,000 1,225,000 75,000 775,000 25, 000 -150, 000 50, 000 325, 000 ). '28. I'ASTEI'KIX ,HO 421 1*9,500,000191.500 183,500,1X10 4, 500, 000 000 215,500,1.00 . ... 189,700,000 1,800 000 Total liquefiers... 125,000 8 Rapid liquefiers .. 25,000 5 Slow liquefiers 100, 000 2 Inert f:0 (X 1 1,500, (XX) 1 475. 000 3,000 (XX) 325 000 1,500 000 . IT AT 20 C. 9, loo, ooo SAMPLE N Total bacteria.... 80 Total lactic Total liqiieliers... Rapid li(|iietk'rs .. Slow liqiieliers 10 -90 10 170 120 75, 000 55, 000 20,000 875,000 125 000 450, (XX) . .. Inert SAMIM.K N Total bacteria 80 Total lactic >. US. I'ASTKCKIX.KH Mll.K KEPT AT 10('. (K) 10 10 70 8 500 532, 500 Total liqiieliers. . . Rapid liquetiers .... 10 10 750' 750 Slow liquerlers Inert 7 7611 . 3-', 500 SAMIM.K No. lit. RAW MII.K KEPT 10. 875, 000 10, 5.50, 000 '225, 000 100,000 125 (XH> AT 20 ('. 93,61X1,000 Total bacteria ... 10,287,500 . 2'.l. I'ASTK.ritlUKI" Mll.K Kl 220 111) 111 .,,.,. A .,. .j () o ,, (") Total liqiieliers... 10 Rapid liqiieliers .. 10 Slow liqllcliers 5 10 5 10 111 110 Inert . . SAMIM.K N o. :>. I'ASI K.riti/KD MII.K KKPTAT IOC. Total bacteria 15 Total lactic Ml 10 '.HI 70 1 '.XHI '.'. OOO Total liqiicliers..., 10 Rapid liqiieliers .. II Slow liqiietiers 10 11 10 11 10 II ii 10 . 200 2OO Inert .. i TIKI '.' IMKI 22 BUREAU OF ANIMAL INDUSTRY. TABLE V. Bacteria per cubic centimeter of milk, arranged in groups Continued. SAMPLE No. 110. RAW MILK KEPT AT 10 C. Group. Number of bacteria found after lapse of hours. 6 hours. 12 hours. 24 hours. 48 hours. 72 hours. % hours. Total bacteria Total lu:tic 252, 500 185,000 67,500 40,000 27,500 1,082,500 862, ,">00 210,000 132,500 77,500 10,000 8,200,000 6, tiOO, 000 2, 575, 000 1,225,000 1,350,000 25,000 57,000,000 40, (MO, 000 15, %0, 5, 210, 000 10, 750, 000 1,000,000 211, 250, 000 637, 000, 000 181, 375, 000 516, 550. 000 26,875,000 16,750,000 19,875,000 10,000,000 7,000.000; 0,750,000 3,000,000 3,700,000 661,250,000 i;:;7, 000,000 19, 250, 000 13, 175, (XX) 6,125,000 5,000,000 Total liquefiers... Rapid liquefiers . . Slowliquefiere Inert SAMPLE No. 210. PASTEURIZED MILK KBIT AT 20 C. Total bacteria 40 60 40 150 a7,500 Total lactic Total liqu<'fiers . . . 20 10 150 2, 500 Rapid liquefiers 20 10 150 2 500 Slow liquefiers Inert SAMPLE No. 310. PASTEUKIZED MILK KEPT AT 10 C. Total bacteria Total lactic 40 30 35 40 30 I 200 2,800 Total liquefiers 10 5 30 150j 2 HOO Rapid liqueflers . 5 20 150J 10 10 2 800 Inert SAMPLE No. 111. RAW MILK KBIT AT 20 C. Total bacteria Total lactic 7,050,000 o, 847, 500 180, 000 20,000 160,000 22,500 106, 450, 000 10. : >,300,IXH) 825, 000 175,000 650,000 325, UOO 229, 437, 500 225, 187, 500 3,500,000 .'00, 000 3. 000, 000 750, 000 543, 000, 000 540, 750, 000 2, 000, 000 625, 000 1,375,000 250,000 799, 500, 000 799, 300. 1)00 125,000 TotHl liquefiers... Rapid liquefiers . . Slow liquefiers 125, 000 75,000 SAMPLE No. 211. PASTEURIZED MILK KKPT AT 20 C. Total bacteria 70 835 185 38, 700 I") Total lactic 3(1,900 20 35 90 1 , 400 397 500 Rapid liquefiers 20 10 30 . 22, 500 25 60 1 400 375 000 Inert 800 400 SAMPLE No. 311. PASTEURIZED MILK KKPT AT 10 ('. Total bacteria Total lactic 70 95 1 , 001) 920 1,030 273, 900 25, 920. 000 273,000 25,917,900 20 20 26 10 900 ' 100 20 10 300 20 25 600 2 100 Inert "Dilution too ''Dilution too low. BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK. 23 TABLE V. Bacteria per cubic centimeter of milk, arranged in grouf>* Continued. SAMPLE No. 112. RAW MILK KBIT AT 10 C. Group. Number of bacteria found after lapse of hours. 6 hours. 12 hours. 24 hours. 48 hours. 72 hours. % hours. Total bacteria Total lactic 217,500 170, 000 82,500 12,:iOO 20, 000 15,000 %5,000 900,000 52,500 12,. "SOU 10, 000 12,500 3,012,500 2, 587, 500 325, 000 75, 000 250, 000 100.000 25, 162, 500 23, 162, 500 1,950,000 500, 000 1 , 450, 000 350, (XX) 1.55, 812, 500 221 , 500, 000 ... 144 247,500 Total liquch'ers... Rapid liqiiefiers .. Slow liqiieriers Inert 3,690,000 11,000,000 375 (HIO 3 350 000 7, 875, 000 .... SA.Ml'LK No. 212. I'ASTKfKI/KI) MlLK K KIT AT 20 C. Total Total Total Rapid Slow 1 Inert Total Total Total Rapid Slow 1 Inert bacteria ! 192 lactic 345 495 1.484,800 liqtictiers 5 495 1 184, 8(X) 495 1 48-4 800 iqueliers 5 SAMIM.K N bacteria 192 lactic 1 1 i. 312. I'ASTKI-UI/.KI) MILK KKIT AT 10 C. 132 42 110 110 55 6(KI liqueliers... 5 liqiieflers 15 5 50 50 iqueliers , 5 10 55 550 ] SAMIM.K No. 1 Hi. RAW MILK KKIT AT 20 C. ToUil Total Total Rapid Slow 1 Inert 1 bacteria 1.802,.5(K) 157, 437,. 51X) 629, 000, 000 1. 181. 500.000 lactic 1,776,750 155, 437. 500 628. (XX). (XX) 1. 180. '.V'5. (KXI liqueliers... 20, (XX) '. liqiiefiers.. 750 1 iqueliers 19.250 > 5 750 1 1 50, (XKI 425, (KKI 275, (KKI ."HI. (KKI 75, (KK) 5. 313. l'.\sTKt'i:i/.Ki> MILK KKIT AT 10 ('. i:io :'.>7 no 1,610 : M;.'.I.'.O 11.037. 5(Xl J5o :;. 25u 25O 3.250 liqueliers... Ill llquetiers.. 10 III 20 15 -_'0 10 10 10 10 to 5 10 1 . 5!HI : Nl.7(K 1 1 . 03 1 . 2-VI 24 BUREAU OF ANIMAL INDUSTRY. TABLE V. Bacteria per cubic centimeter of milk, arranged in groups Continued. SAMPLE No. 114. RAW MILK KEPT AT 10 C. ' Oroup. Number of bacteria found after lapse of hours. 6 hours. 12 hours. 24 hours. 48 hours. 72 hours. 96 hours. Total bacteria Total lactic 38,000 33,250 3,250 2,500 750 1,500 260,000 202,500 50,000 45,000 6,000 7,500 3,025,000 2,725,000 225,000 225,000 27,687,500 25,862,500 1,600,000 1,600,000 313,000,000 305,125,000 5,375,000 3,750,000 1,626,000 Total liquefiers . . . Rapid liquefiers.. Slow liquefiers ... Inert 12,500 12,500 75,000 225,000 2,500,000 SAMPLE No. 214. PASTEURIZED MILK KEPT AT 20 C. Total bacteria .... 122 50 520 25,000 1,275,000 Total lactic Total liquefiers 10 10 460 25,000 1,275,000 Rapid liquefiers . . 10 10 460 25,000 1,275,000 Slow liquefiers . . . Inert SAMPLE No. 314. PASTEURIZED MILK KEPT AT 10 C. Total bacteria 122 60 40 380 2,000 54 255 000 Total lactic Total liquefiers... Rapid liquefiers . . Slow liquefiers . . . 10 10 10 10 10 10 15 15 10 10 100 100 4,000 4,000 Inert 370 1,900 54,251 000 TABLE VI. Average number of bacteria per cubic centimeter in all samples under each treatment. Description and treat- ment of sample. Number of bacteria after the lapse of hours. 6 hours. 12 hours. 24 hours. 48 hours. 72 hours, i 96 hours. Raw milk kept at 20 C. . . 13,522,331 74,142,857247,651,250 467,910,714 1,501,335 124, 783, 928 1,026 608, 079, 166 320,337,388 254,678,542 15, 119 568, 718, 500 236, 941 , 250 975, 500, 000 308, 041, 666 562, 650, 000 2,462,492 37,088,456 1 Pasteurized milk kept at 20 C 246 17,640,428 245 426 6,028 31,457,833 38,406,785 308 378 Raw milk kept at 10 C... Pasteurized milk kept at 10 C Probably the most striking thing in these tables, especially to the person not familar with the bacteriology of milk, is the enormous number of bacteria usually found in the raw milk after standing two or three days. This number always reached several hundred million per cubic centimeter, and occasionally went as high as 1,000,000,000. Even higher numbers have been obtained ( l5 ). Accepting Cohn's ( 19 ) calculation of the weight of a single bacillus as 0.000,000,001,571 milligram, the weight of 1,000,000,000 bacteria, a number not infrequently found in a cubic centimeter of milk, would be over 1 milligrams. In other words, over H tenths of 1 per cent of the total weight of the milk has been converted into plant tissue. But even this does not tell the whole truth. In the course of their growth the bacteria have converted a considerable proportion of the BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK. 25 milk solids into by-products of growth. If, as is usually the case, when such large numbers are found, the bacteria are of the lactic- acid class, the decomposition is of a comparatively harmless nature, although in each cubic centimeter of milk 5.7 milligrams of lactose may have been converted into t> milligrams of lactic acid; on the other hand, if many bacteria of the liquefying group are present, each tiny cell, by secreting its digestive enzymes, minute in the individual but potentially great in the aggregate, is able to produce results all out of proportion to its infinitesimal weight. The influence of low temperatures in retarding the growth of bac- teria is well known, but it may be of interest to compare the increase under the conditions of this experiment. In considering the multipli- cation of a single cell in the raw milk, it should be remembered that at the time the count began the bacteria had already passed the period of rapid increase. For this reason the increase of the bacteria in the pasteurized milk, as tabulated in Table VII, can not be compared with the increase in the raw milk under similar conditions. TABLE VII. Increase of n shiyi:' oncti'i ium in out' cubic centimeter of in life. Number of bacteria found after tin- lapse of Description an. "> is.:! :.'. II.'.) -2. . r > KHW milk at 10 ('... 1 1.7 2.2 7.1 II.) 17.1 31.1 Pasteurized milk at 20 (' .. 1 1'Hsteuri/ed milk at 10 (' .. 1 2-1.2 I'.. r> :>o7. 1'.i'.t '.H'.7. iu7 ;;. 9S1.U32 l.'i I. 1 C>. 2 : Id.dal H:i.21,S It will be seen that the progeny of a single cell in the raw milk held at 10' C. had not readied in !M' hours the number attained in the raw milk at % Jo (\ in '24 hours. In the pasteuri/ed milk the conditions are different in that the count beginning when the milk contained a verv few bacteria only extends over the period of rapid increase. The bacterial multiplication was very rapid in the pasteuri/ed milk held at iJO" ('..a single organism reproducing itself three hundred thousand times in 4* hours. The pasteuri/ed milk held at lo ('. presents a striking contrast: the multiplication was very slow Tor the liiM 4 s - hours, and at the end of '.' hours the increase \va^ far below that in the pasteuri/ed milk at the higher temperature. Returning to the actual numbers of bacteria in the milk we lind that at the time of pasteuri/ation the mean of the samples held at :io ( '. was 13.ooo.ooo per cubic centimeter, while the milk held at lo ('. shows a slight Iv higher number. This probably represents poor rather than average city milk, but may be taken a-- a fair -ample of milk needing pasteuri/ation. The reduction of this number to ill."> per cubic centimeter may be considered as etlicient pastouri/ation. probably a 26 BUREAU OF ANIMAL INDUSTRY. higher efficiency than would be obtained under ordinary commercial conditions. The variation from the mean was slight; the minimum number found was 20 per cubic centimeter and the maximum 4,040. The latter was so exceptional that the results from this sample (No. 33) were excluded in making up the means. Milk under ordinary conditions is held in a refrigerator at 10 to 15 C. and is consumed usually within 12 hours or at most 24 hours after delivery. The milk held at 10 C. will therefore represent normal conditions more nearly than that held at 20 C., but it will be of interest to study the bacterial development in the milk held at the higher temperature. In the raw milk over 200,000,000 bacteria per cubic centimeter were found at 12 hours, while the same quantity of pasteurized milk contained about 6,000 onl} 7 ; 12 hours later the latter number had increased to over 1,000,000, and at 48 hours the difference in the total bacteria present in the two cases was small. There is more or less variation from these means in individual cases, but the frequency of their occurrence makes it safe to predict that pasteurized milk held at 20 C. for 24 hours or more will have a high bacterial content. The growth of bacteria in the pasteurized milk at the low tempera- ture is so slow that at 48 hours after pasteurization the number of bacteria was less than that usually found in sanitaiy milk, and even at 96 hours was not much greater than the total number in the milk before it was heated. The results obtained at this temperature were uniform, showing much variation at 72 hours and 96 hours only. At these two periods two or three exceptionally high results have raised the general mean considerably above that of the remaining counts. Milk showing an acidity of about 0.25 per cent has a distinct sour taste, and would not be considered fit for use after it had reached this condition. The average raw milk had reached this stage at 48 hours, while the pasteurized milk was, so far as the total bacteria show, in good condition 24 and even 48 hours later. At 20 the difference was somewhat less although, as will be pointed out later, the nature of the bacteria developing in the pasteurized milk at the higher temperature has some influence on the length of time during which it may be con- sidered fit for use. It is, however, very unsafe to judge the quality of the milk by the total number of bacteria present without regard to their nature. A few hundred thousand bacteria of certain species may be more harm- ful than a hundred million of another class. For this reason it will be more profitable to consider the bacteria grouped under the head of peptonizing bacteria, as this group includes the bacteria generally acknowledged to be the cause of the most undesirable changes in the milk. It is difficult to secure accurate counts of liquefying bacteria, especially when they are growing with lactic-acid bacteria, but the BACTERIA OF PASTEURIZED AND UNPASTEURIZED MILK. 27 results given in Table VIII may be taken as a fair representation of the growth of this group under these conditions. TABLE VIII. Average number of peptonizing bacteria per cubic centimeter of mUk. Description and treatment of sample. Haw milk at 20 C hours. (> hours. 12 hours. 24 hours. 48 hours. 72 hours. 96 hours. 621 82 571 _M i 1 - 7 4,905,3X5 11 505,333 11 1,814,583 188 1,518, 6(i6 9 2,927,857 259,8311 5, 272, 500 15 700,000 2, 411.Ha 11,708,750 3, 143 1,375,000 31,225,000 12,781,250 56, 219 Pasteurized milk at '20 C. ... Raw milk at 10 C 32,918, 4,251, 750 219 Pasteurized milk at 10 C In making up the mean for the raw milk at 20 C C. those determina- tions in which some unusual circumstance made the count evidently inaccurate are not included. In this milk the mean shows a rapid increase of liquefying bacteria for the first , ooo.ooo in !MJ hours, although the lactic bacteria were present in large numbers from the beginning. An examination of Table 111 shows that the acidity of this milk at '.> hours was o.2:> percent only, which is a little below the mean for 4* hours. Kxclud- ing this exceptionally high figure the mean number of liquetiers pres- ent at !M> hours would be lo.X'.tl .. indicating a slight falling oil after 72 hours. The counts of the liquefying bacteria in (lit 1 pasteurixed milk at 1<> C\ indicate that there was scarcely any growth for IS hours, and even after IMi hours the number found was no greater than that of the unheatcd milk 72 hours earlier. In other words, the pasteurixed milk was in better condition, so far as bacteriological examination shows. 28 BUREAU OK ANIMAL INDUSTRY. when 48 hours old than tho unhcatod milk at the time of pasteuriza- tion. In many cases there were very few liqueriers even at 9H hours. The variation in species occurring in these samples of milk was not great. Three or four species of liquetiers were almost uniformly present and all multiplied more or less, although, after the influence of the lactic-acid bacteria began to be felt, this number was usually reduced to one or two varieties. The most common and persistent of these was the hay bacillus. Two or three species of liquefiers usually survived pasteurization. In the hearted milk held at 20 C., the great increase was confined, almost without exception, to the hay bacillus. The rapid growth of the colonies of this organism usually prevented the determination of the slower growing, less abundant species. The hay bacillus developed much more slowly in the pasteurized milk at 10 C. For this reason it was possible to ascertain the presence of a few other species, none of which could be called predominant. The bacteria included under the heading "inert" were mostly of a group forming white colonies spreading slightly on the surface of the gelatin. In the pasteurized milk, however, and especially the part held at 10 C. , there frequently occurred a considerable increase of a variety forming small round colonies resembling the colonies of the lactic group, but without appreciable efi'ect on milk. The lactic-acid bacteria which survived pasteurization were found, in nearly every case, to be identical with the predominating lactic species in the cor- responding unheated milk. The qualitative difference in the flora of the pasteurized and unpas- teurized milk consisted in the absence, with a few exceptions, of the lactic group from the latter, the small reduction in the number of varieties of peptonizing bacteria, the rapid multiplication of the hay bacillus in the heated milk at 20 C., and the presence in some cases o"f large numbers of inert bacteria in the pasteurized milk held at 20 G C. INFLUENCE OF LACTIC BACTERIA ON THE DEVELOPMENT OF PEPTONIZ- ING BACTERIA. Taking up the question of the protective action of the lactic group, we find that when both the lactic and the peptonizing bacteria are growing together in milk, as in the unheated milk in these experiments, there was a rapid increase in the latter for a short time, followed by a period of slow decrease, or at least a complete prevention of multipli- cation. This was not confined to a single species, but included all liquefiers found in these milks. Reference to Table IX shows that this inhibition was coincident with the development of from 0.25 per cent to 0.3 per cent acidity: BACTKRIA OF PASTEURIZED AND UNPASTEURIZED MILK. 29 TABLK IX. Relation of acidity of milk to development of peptonizivg hacteria. Percentage of acidity and number of bacteria after the lapse of hours. (hours. 12 hours. 24 hours. 48 hours. 72 hours. 9fi hours. [Acidity .131 621,577 .1:58 73, 650 . 1GS 4,905,333 . 153 306, 400 . 254 1,814,583 .168 1.5-12, 400 .449 2,927,000 .183 5, 827, 000 .6% 700,000 . 245 9, 200, 500 .711 1,375,000 .417 12,781,250 20 [Peptonizing bacteria fAcidity . 551 10,891,666 10 [Peptonizing bacteria In the raw milk at 20 C., the growth of the liquetiers was checked between , which, as has already been explained, showed an exceptionally slow increase in acidity, there was little increase after 4s hours, when the acidity had reached 0.245 per cent. The samples (Nos. 21. 22, 2b', and 27) in which there was a sudden multiplication of lactic-acid bacteria, with a correspondingly increased acidity in the pasteurized milk, were accompanied by a sharp decrease in the liquefying bacteria. Bouska, (") who has done some extensive work along this line, states that, while the major part of the inhibitory action exercised by the lactic bacteria on />. subtil-!* is due to lactic acid, it is also evident in media containing no sugar. Thus it seems evident that the protective influence of the lactic group of bacteria becomes effective only when the acidity has reached a point that renders the milk undesirable for food. The tables given in the paper by Conn and Ksten. ('") previously quoted, show a more or less rapid increase of liquetiers for a time, depending on the tem- perature at which the milk was held. Although the acidity is not given, the number of lactic bacteria present makes it probable that the liquetiers increased until the milk was at least slightly sour. This applies, however, to the number of liquetiers present, and does not take into account the effect of the acid reaction on the proteolytic en/vmes secreted by this group of bacteria. The pastetiri/ed milk held at 20 C. frequently curdled is hours after pasteuri/.ation. with a very disagreeable taste, although the mean number of peptoni/ing bacteria found at this time was only ^.5iH>.iMKi per cubic centimeter, while the raw milk containing several times as many bacteria of this group, in addition to the lactic hacteria. usually remained unchanged except for a slight sour taste. It must be remembered, however, that the aciditv was sufficient to obscure any change except a decided one. In one instance (sample No. !'). in which the raw milk contained hours to about 5,000.000 per cubic centimeter and then decreased slowly. In the heated milk the peptonizing bacteria increased rapidly after 12 hours, and the milk was usually curdled in 4S hours, with a disagree- able taste and odor. Occasionally lactic bacteria survived pasteuriza- tion and multiplied rapidly after 24 hours, completely inhibiting the peptonizing bacteria. J//7/- held at 10 C. (50- P.). In unheated milk the growth of the bacteria and the consequent curdling of the milk was much retarded. The average milk did not contain sufficient acid to affect the taste until it was over 4.S hours old. The proportion of peptonizing to lactic bacteria was greater than at the higher temperature, and the taste of the milk occasionally showed the influence of the former. In the pasteurized milk the bacteria increased very slowly, and in nearly every case the milk was unchanged in taste and appearance. % hours after pasteurization. In only 2 of 14 cases was there a marked increase of peptonizing bacteria. The predominating bacteria were species having little or no effect on milk. The lactic bacteria inhibited the development of the peptonizing bacteria only when they had developed sufficient acid to render the milk unfit for use. It seems probable that the acid had a distinct inhibitory action on the proteolytic enzymes of the peptonizing bacteria. ( 1 ) WlIITAKEU, . M. Tht' milk supply of Boston and other New Knjrland cities. I . S. Department of Agriculture, Bureau -L's. Boston, .Ian. 14. isirj. (4) HIM., II. \V., and SI.VCK, K. II. Bacterial count* of Boston's milk supply. Am. Jrn. Pub. llyir. and .Irn. Mass. Aasoc. Boards of Health, v. 14, No. 4, pp. L'M-LMS. Bston, Nov., 1 ( .'04. 32 BUREAU OF ANIMAL INDUSTRY. (5) PARK, W. H. The great bacterial contamination of the milk of cities. Can it be lessened by the action of health authorities? Jrn. of Hyg., v. 1, No. 3, pp. 391-406. Cambridge, July, 1901. (6) BERGKY, D. H. Sanitary supervision of the collection and marketing of milk. Univ. of Penn- sylvania Med. Bui., v. 17, No. 5-6, pp. 187-192. Phila., July-Aug., 1904. (7) PARK, W. H., and HOLT, L. E. Report upon the results with different kinds of pure and impure milk in infant feeding in tenement houses and institutions in New York City. A chemical and bacteriological study. Med. News, v. 83, No. 23, pp. 1066-1078. N. V., Pec. 5, 1903. (8) ALVORD, HENRY E., and PEARSON, R. A. The milk supply of two hundred cities and towns. II. S. Department of Agriculture, Bureau of Animal Industry, Bui. 46. Wash., 1903. (9) SMITH, THEOBALD. The thermal death point of tubercle bacilli in milk and some other fluids. Jrn. of Exper. Med., v. 4, No. 2, pp. 217-233. N. Y., March, 1899. (10) RUSSELL, H. L., and HASTINGS, E. G. The thermal death point of tubercle bacilli under commercial conditions. Wis. Agr. Exp. Sta., 17th Ann. Rept. for year ending June 30, 1900, pp. 147-170. Madison, 1900. (11) HARDING, H. A., and ROGERS, L. A. The efficiency of a continuous pasteurizer at different temperatures. N. Y. Agr. Exp. Sta., Bui. 172, pp. 507-530. Geneva, Dec., 1899. (12) BANG. Some experiments on the temperature necessary for killing tubercle bacilli in milk. Trans, of the Brit, Cong, on Tuberculosis for the Prevention of Consumption, Lond., July 22-26, 1901, v. 3, pp. 392-398. Lond., 1902. (13) RUSSELL, H. L., and HASTINGS, E. G. Effect of short periods of exposure to heat on tubercle bacilli in milk. Wis. Agr. Exp. Sta., 21st Ann. Rept. for year ending June 30, 1904, pp. 178-192. Madison, 1904. (14) VARIOT, G. Valeur nutritive du lait de vache sterilis a 108 pour 1'allaitement artificiel. Compt, Rend. Acad. des Sci., t. 139, No. 23, pp. 1002-1003. Paris, Dec. 5, 1904. (15) CONN, H. W., and ESTEX, W. M. The effect of different temperatures in determining the species of bacteria which grow in milk. Storrs Agr. Exp. Sta., 16th Ann. Rept. for year end- ing June 30, 1904. pp. 27-88. Middletown, Conn., 1904. (16) FLUGGE, C. Die Aufgaben und Leistungen der Milch-Sterilisirung gegeniiber den Darm- Krankheiten der Sauglinge. Ztschr. f. Hyg. u. Infekt., Bd. 17, Hft. 2, pp. 272-342. Leipz., 1894. (17) WEBER, A. Die Bakterien der sogenariten sterilisierten Milch des Handels, ihre bio- logischen Eigenschaften und ihre Beziehungen zu den Magendarmkrank- heiten der Siinglinge, etc. Arbeiten aus dem kaiserlichen Gesundheitsamte, Bd. 17, Hft. 1, p. 108-155. Berlin, 1900. (18) LtJBBERT, A. Ueber die Natur der Giftwirkung peptonisi render Bakterien der Milch. Ztschr. f. Hyg. u. Infekt., Bd. 22, Hft, 1, pp. 1-11. Leipz., June 25, 1896. (19) COHN, FERDINAND. tlber Bakterien, die kleinsten lebenden Wesen. 47 p. 17 cm. New York, Henry Holt & Co., 1889. (20) BOI-HKA. F. W. Etudes sur Pantagonisme entre les baeteriesdn groupe des ferments lactiques et celles du groupe dvi Bacillus subtilis. Rev. gen. du lait. v. 3, No. 1, pp. 1-11. Oct. 15, 1903. O Dr. K. ('. Joss, rare Carstelis Paeking Co.. Taeo- , ma, Wash. Dr. J. S. Kelly, euro Blomcr i Miehael CM., Quiney, 111. Dr. V. D. Ketehum, South St. I'aul, Minn. Dr. A. Long, care Sperry & Barnes, New Hnvcn. Conn. Dr.C. Loveberry, room 102, Custom-House (new), Dr. H. D. Mnync. M alone. N. V. Dr. Louis Mets,ker, room 22, N. T. Armijo Build- ing. Albuquerque. N. Mex. Dr. .1. Miller, care John Morrell & Co., Ottumwa, Dr. C.L. Morin, St. Albans, Vt. Dr. \v. .1. Murphy, Care Springfield Provision Co., Brightwood, Mass. Dr. \V. N. Neil. care John Cvidiihy Co., Wichita, Kane. Dr. V. A. Norgaard, Honolulu, Hawaii. Dr. F. M. IVrry, Fort Fairlield, Me. Dr. <;. W. Po'pe, Animal Quarantine Station, Athcnia. N*. J. Dr. II. T. Potter. Calais, Me. Dr. .1. (). F. 1'riee, eare Brittain & Co., Marshall- town, Iowa. leeond page m Dr. R. A. Ramsay, Fargo, N.Dak. Dr. A. G. G. Richardson, 707 Empire Building, Knoxville. Tenn. Dr. A. E. Rishel, eare Cudahy Packing Co., Los Angeles, Cal. Dr. W. H. Rose, IK Broadway. New York, N. Y. Dr. F. L. Russell. Orono. Me. Dr. .1. F. Ryder, HI Milk st., Boston, Ma-s. Dr. K. ['. SehatTter, care Cleveland Provision Co., Cleveland. Ohio. Dr. C. A. Schaufler, 134 South Second st., Phila- delphia, Pa. Dr. Tnos. W. Scott, care The Rath Paeking Co., Waterloo, Iowa. Dr.T. A. Shipley, care T. M. Sinclair & Co. (Ltd.), Cedar Rapids, Iowa. Dr. N. C. Sorensen, care Kingan & Co., Indian- apolis, I nd. Mr. Win. II. Wade, Animal Quarantine Station, Halethorp, Md. Dr. H. N. Waller, 109 West 12d st., New York, N. Y. Dr. G. W. Ward, Newport, Vt. Dr. B. P. Wende, Live Stock Exchange Building, Kast Buffalo, N. Y. Dr. W. H. Wray, 34 Strcatham Hill, London. S.W., England. University of California SOUTHERN REGIONAL LIBRARY FACILITY 405 Hilgard Avenue, Los Angeles, CA 90024-1388 Return this material to the library from which it was borrowed. JAN University < Southern Library