THE UNIVERSITY OF ILLINOIS LIBRARY <5_o-p . >O AGRICULTURAL LIBRARY BULLETIN No 199 GERM CONTENT OF MILK I. AS INFLUENCED BY THE FACTORS AT THE BARN BY M. J. PEUCHA AND H. M. WEETEK URBANA, ILLINOIS, MAY, 1917 CONTENTS OP BULLETIN No. 199 PAGE 1. FOREWORD 23 2. INTRODUCTION 25 3. THE THREE BARNS IN WHICH THE STUDY WAS MADE 26 4. METHODS OF STUDY 30 5. EESULTS OF THE STUDY 32 Germ Content of the Individual Samples of Milk 40 Average Germ Content of the Milk of the Different Animals 41 Average Germ Content of All the Milk at Different Milkings 43 Comparison of the Results for 1914 and for 1915 46 Number of Bacteria Added to the Milk by All the Barn Factors in Each of the Three Barns 43 6. SUMMARY 51 7. CONCLUSIONS . . .51 FOREWORD The public desire and should be able to obtain a supply of satis- factorily clean and wholesome milk. The representatives of the con- suming public in this matter, the health officials, have frequently adopted the germ content of milk as an index of cleanliness and whole- someness. In this way, the legal limit of germ life permissible in the milk supply as shown by the plate count has been placed in Champaign- Urbana at 100,000 per cubic centimeter and in St. Louis at 5,000,000 per cubic centimeter. No matter what is our personal judgment regarding the wisdom of such bacterial standards, the legally constituted authorities having thus established these limits of bacterial content, the burden rests upon the producer and the retailer to observe them. When adjusting their business methods to such variable limits as those mentioned above, it is important that the dairymen have fairly accurate knowledge of the relative importance of the various dairy operations upon the germ content of the milk. In the general directions which the health officials have formulated for the guidance of the dairymen, great stress has been laid upon the construction and condition of the cow stable. Accordingly, technical studies of the influence of dairy factors naturally included a measure- ment of the influence of barn conditions. The results obtained at the New York Agricultural Experiment Station from such technical studies of the influence of barn conditions were so out of keeping with the ideas of the health officials that it seemed best to redetermine inde- pendently this relationship at this experiment station. The surprisingly accordant results which have been obtained at these two experiment stations should not be understood as counte- nancing dirty methods or dirty milk. They merely point out that earlier impressions, formed in the absence of exact data, did not give a correct value to the importance of barn conditions in connection with germs in milk. Neither should these results be taken as a criticism of health officials. Such' officers are charged with the protection of the public health. Where the facts are available, they utilize them. Where exact information is lacking, they must proceed in accordance with their best judgment even tho they recognize the fallibility of such judgment. The slight effect of barn conditions upon the number of germs in milk was clearly brought out by the extended studies at the New 23 York Agricultural Experiment Station. The studies here reported were made in a different part of the country, in three quite dissimilar barns, by a different laboratory force, using a different method of attacking the problem. The results of this latter study are quite in accord with those obtained in New York. The earlier misconceptions of health officials regarding the im- portance of barn conditions resulted in placing unjust economic bur- dens upon the producer. Now that more accurate data upon this question is available it is to be hoped that these burdens will be more fairly distributed. H. A. HARDING Chief in Dairy Husbandry 24 GERM CONTENT OF MILK I. AS INFLUENCED BY THE FACTORS AT THE BARN By M. J. PEUCHA, ASSISTANT CHIEF IN DAIRY BACTERIOLOGY, and H. M. WEETER, ASSISTANT IN DAIRY HUSBANDRY The studies on which this bulletin is based are a part of an investi- gation begun by H. A. Harding in 1906 at the New York (Geneva) Agricultural Experiment Station. In an introduction to Bulletin 365 of that institution Dr. Harding makes the following statement as to the purpose of the investigation: "When health officials, failing to find other means of characterizing sanitary milk, undertook to specify the conditions under which it should be produced, they were confronted by an almost total lack of detailed information upon this subject. This lack arose from the fact that available studies upon milk sanitation were in the nature of general surveys of the situation. While these general surveys were a necessary preliminary, they gave little informa- tion as to either the absolute or the relative importance of any given dairy operation The importance of the interests involved demands that the needed information shall be furnished as promptly as possible." Investigations toward this end have been carried on at the New York Agricultural Experiment Station since 1906. 1 The aim in these investigations has been to separate the various sources of contamina- tion to which milk is subject and to measure the influence of each on the germ content of milk. The results from these studies point to the conclusion that barn conditions and barn operations have only a small influence upon the germ content of milk. If the above conclusion is true, it will radically change our con- ception concerning the relative importance of the different sources of milk contamination. Since it is an axiom in scientific work that no important results are accepted until they have been verified independ- ^Harding, H. A., Wilson, J. K., and Smith, G. A. Milking Machine: Effect of Methods of Handling on the Germ Content of Milk. N. Y. (Geneva) Agr. Exp. Sta. Bui. 317. 1909. Harding, H. A., and Wilson, J. K. The Modern Milk Pail. N. Y. (Geneva) Agr. Exp. Sta. Bui. 326, pp. 248-281. 1910. Harding, H. A., Ruehle, G. L., Wilson, J. K., and Smith, G. A. The Effect of Certain Dairy Operations upon the Germ Content of Milk. N. Y. (Geneva) Agr. Exp. Sta. Bui. 365, pp. 198-233. 1913. Harding, H. A., and Wilson, J. K. A Study of the Udder Flora of Cows. N. Y. (Geneva) Agr. Exp. Sta. Tech. Bui. 27. 1913. Buehle, G. L. A., and Kulp, W. L. Germ Content of Stable Air and Its Effect upon the Germ Content of Milk. N. Y. (Geneva) Agr. Exp. Sta. Bui. 409, pp. 418-474. 1915. 25 26 BULLETIN No. 199 ently by other workers, it was decided to repeat the study made at the New York Station upon the influence of barn conditions and oper- ations on the germ content of milk. Accordingly, the subject has been restudied at the Illinois Station by a new force of workers, in a new laboratory, and in three barns of distinctly different types. The method of attack in the present study differed from the method used at the New York Station in that all the barn factors were grouped together and their collective influence on the germ content of the milk was determined. In these, as in the previous studies, the utensils were considered as a separate source of contamination and were therefore thoroly steamed before each milking. 1 THE THREE BARNS IN WHICH THE STUDY WAS MADE AND THE DAIRY METHODS EMPLOYED IN EACH In order that the results of this study may be thoroly understood and their significance fully appreciated, it is necessary to give a some- what full description of the conditions and the dairy operations in each of the three barns in which the experiment was conducted. Barn I is a two-story frame building 75 feet long and 45 feet wide. There are approximately 900 cubic feet of space and 9 square feet of window glass per cow. Two rows of iron stalls facing the central aisle and running lengthwise accommodate forty cows. The length of the floor from the manger to the gutter is 5 feet 5 inches in one row of stalls and 5 feet in the other row. The iron stalls used in this barn are known as ' ' Drown ' ' stalls, and are so constructed that it is possible, to a certain extent, to adjust the space inside the stall to the size of the animal. The ceiling and the walls are constructed of matched lumber, are painted, and are without any large crevices. The floor, the gutters, and the mangers are of cement. The feeding materials are stored in a separate part of the building and are brought into the barn thru an end door. During the investigation, the milkings began at five in the morn- ing and at four in the afternoon and lasted an hour and a half. After the morning milking, the cows were turned out into a yard, the manure was taken out, the floors were flushed with water, and the stalls were bedded with sawdust. When feasible, the manure was placed directly on the wagon and taken away. At other times it was dumped about thirty-five feet from the barn in a yard to which the cows did not have access. A tight board fence, six feet high, separated this manure pile from the barn. After the barn had been cleaned, the cows were brought back, fed hay, and cleaned. The amount of time spent on the cleaning of the ir The influence of dairy utensils upon the germ content of milk has been studied separately and the results will be reported later. 1917} GERM CONTENT OF MILK AS INFLUENCED BY BARN FACTORS 27 cows was approximately five minutes to each animal. Occasionally the cows were used for demonstration purposes before classes, and for such occasions their udders and flanks were clipped. This clipping, however, was not practiced regularly for the purpose of reducing the number of bacteria in the milk. Likewise, during the experiment, no systematic attempt was made to clean the cows before each milking. If any of them became dirty prior to the milking, the milker wiped the loose dirt from their flanks and udders with a handful of the saw- dust bedding. In 1914 the udders of the cows were wiped with a damp cloth previous to the milkings, but in 1915 this practice was discon- tinued intentionally. During the milking the cows were fed silage and grain. The hay was brought into the barn usually before the milking was finished, and was distributed into the mangers. This operation frequently caused a considerable amount of dust in the air. The milkers wore milking suits which were changed twice each week. Barn II is a two-story, circular building 70 feet in diameter. There are about 800 cubic feet of space and 9 square feet of window glass per animal. The platform upon which the cows are stanchioned is circular, running around a central ring 45 feet in diameter. Around the outer edge of this platform is the gutter, and between the gutter and the outside brick wall runs a passageway about six feet in width. Especial effort was made in constructing this barn to so equip it that the cows would be prevented from lying down in their own feces. FIG. 1. THE INTERIOR OF DAIRY BARN I 28 BULLETIN No. 199 [May, This was accomplished by varying the width of the platform upon which the cows are stanchioned and by installing adjustable stanchions. By these two means the space for each cow can be adjusted as desired. The brick wall and the wooden ceiling are free from any large crevices, but are rough and not painted. The platform upon which the cows are stanchioned is paved half way around with cork bricks and the other half with creosote blocks. In the center of the barn is a silo 16 feet in diameter and the chutes for the grain and the hay which are stored on the second floor. With few exceptions the daily operations in this barn were about the same as in Barn I. The floor was cleaned regularly, but as a rule was not flushed with water. In cleaning the cows, only about one minute of labor a day was allowed for each animal, while in Barn I a period of five minutes was devoted to that purpose. This reduction in labor in keeping the cows clean was brought about by carefully adjusting to their size the spaces in which the cows were stanchioned. During milking and feeding and in unfavorable weather the cows were stanchioned in the barn ; at other times they were turned out into an acre dry-lot adjacent to the barn. Barn III is a two-story, round, basement barn 50 feet in diameter. Only ten cows occupied it during this experiment, each animal having approximately 1,500 cubic feet of space and 15 square feet of window glass. In the center of the barn are the silo and the grain and the hay chutes. Around these is a circular passageway 10 feet in width. On the outer edge of this passageway are the mangers and the stan- chions, both constructed of wood. There is only a dirt floor and there are no gutters. The brick side-walls and the wooden ceiling are tight but are not painted. During the experiment the cobwebs and the dust were abundant, not having been cleaned from the ceiling for four years previous. The cows were stanchioned only during the milkings. Between milkings they were allowed to roam about in the barn and in the quarter-acre dry-lot adjacent to the barn. A large door leading from the barn into the dry-lot was always open. The floor in the barn was covered with straw once a day, but the manure was allowed to accumu- late on the floor and was removed from the barn only twice a year. The cows were not kept as clean as in Barns I and II, but no manure was allowed to accumulate and to cake on their flanks and udders. These three barns in a general way represent three classes of dairy barns, Barn I being in excellent condition, Barn II being good, and Barn III poor. The difference between Barn I and Barn II as to cleanliness, however, was not very great. On the other hand, Barn III would be classed as a dirty barn, and it is doubtful whether the milk from it would be admitted to the market of some cities. A photograph of each of the three barns is shown in Figs. 1, 2, and 3. 1917] GERM CONTENT OF MILK AS INFLUENCED BY BARN FACTORS 29 FIG. 2. THE INTERIOR OF DAIRY BARN II FIG. 3. THE INTERIOR OF DAIRY BARN III 30 BULLETIN No. 199 METHODS OF STUDY Sterilization. The media used in determining the germ content of the samples of milk were sterilized in test tubes in the autoclave for fifteen minutes at 120 C. In order that all the test tubes might receive the same amount of heat, they were held during the process of steriliza- tion in test-tube racks. All the glassware and other laboratory apparatus withstanding dry heat were sterilized by subjecting them to 160 C. in a dry sterilizer for two hours or more. The pails used for milking in Barn I were steamed for three minutes over a jet. In Barns II and III the pails were steamed in a sterilizer, which was a box constructed of galvanized iron. After such steaming, the pails were always examined for the presence of living bacteria which might have survived the steaming. This examination consisted of rinsing out each pail with 500 cc. of sterile water just previous to milking and then determining the number of bacteria in the rinse water. The examination indicated that all the pails steamed in the steril- izer and 112 of the 130 pails steamed over the jet were free from bacteria. The remaining 18 pails steamed over the jet were not entirely sterile, but the number of bacteria found in them was extremely small and did not affect measurably the results of this study. Taking of Samples. All the samples were taken from the milk of the individual cows when the milker .brought it in pails from the barn into the adjacent milk room. The milk was thus exposed to all the sources of contamination in the barn. After a thoro stirring with a sterile iron spoon fifteen inches long, the desired amount of milk was transferred by means of the spoon into a large test tube. The milk samples were immediately cooled to about 54 C., and were plated, as a rule, within one hour. Dilutions and Plating. Wide-mouthed, glass-stoppered bottles of 250-cc. capacity were used as dilution bottles. This type of bottle was used at the suggestion of Professor W. A. Stocking, Jr., of Cornell University, and was found to be an improvement on the ordinary dilu- tion bottle with a cotton plug. The bottles were sterilized in the dry oven, and just before plating, the required amount of sterile water was introduced into each by ^ means of a graduated pipette. Two dilutions, 1 to 10 and 1 to 100, were made from each sample. For the first dilution 5 cc. of milk was added to 45 cc. of water, and for the second dilution, 1 cc. of milk was added to 99 cc. of water. Every bottle was then shaken violently, receiving 30 double shakes in such a manner that with each single stroke the bottle passed thru a distance of ten inches. From each dilution two plates were seeded, each one with 1 cc. of the bacterial suspension. 1917] GERM CONTENT OF MILK AS INFLUENCED BY BARN FACTORS 31 It is well known that in the quantitative bacteriological examina- tion of milk by the plate method, plates seeded with the same milk will rarely develop the same number of colonies, even when the plating is done with care and accuracy. In order to ascertain the extent of variation due to the laboratory methods employed in this study, ten experiments were undertaken in each of which 100 plates were seeded with the same milk. The same dilution was used for all the plates in each experiment. The results from one of these experiments are shown in Table 1. TABLE 1. FREQUENCY DISTRIBUTION FOR NINETY-SIX PLATES* MADE FROM THE SAME BACTERIAL SUSPENSION Class Number of colonies per plate Frequency distribution 1 2 3 4 5 6 110-120 121-130 131-140 141-150 151-160 161-170 111 ItH IHi IHrl 1W1 liH IHri 1 1 Utl 1111 im UH 111 li LHl liH 1111 'Four of the 100 plates were spoiled. The mean number of colonies for all the plates in this experiment was 142, while the lowest count was 110 and the highest count was 170. In order to show the frequency distribution of the plates, they were divided into six classes, each of which had a class range of ten units. It will be seen from Table 1 that of the 96 plates counted, 62 fell into the third and fourth classes, having more than 130 and less than 151 colonies. With respect to variability, it is important to note that these individual counts showed very moderate variation. Indeed, no individual count deviated as much as 25 percent from the average. The other nine experiments on this subject showed approximately the same variation. According to the theory of statistics, the average of the counts for sets of four plates would tend to be about half as variable as the counts of the individual plates. Since, with but few exceptions, every determination in Tables 2 to 7 was an average of four plates it seems reasonable to conclude that the wide variations in the germ con- tent of the samples shown in these tables were due primarily to varia- tions in the number of bacteria actually present in the samples of milk and not to inaccuracies of the laboratory methods. Medium. The following medium was used thruout the entire study. Agar shreds 15 grams Liebig 's meat extract 3 " Witte 's dry peptone 10 " Lactose 10 " Distilled water 1000 cc. The reaction of the medium was adjusted to 1.0 percent normal acid to phenolphthalein. 32 BULLETIN No. 199 [May, Incubation and Counting. All the plates were incubated for five days at 20 C., and for two days at 37 C. This length of time and the two temperatures of incubation were used in order to induce a larger number of the bacteria present in the milk to form visible colonies. According to Harding and Wilson, 1 the bacteria that form colonies at 37 C. but not at 20 C. may occasionally be present in the freshly drawn milk. As stated above, two dilutions were made from each sample of milk and two plates were seeded from each dilution. All four plates from each sample were counted regardless of the number of bacteria on them, unless they showed some evidence of contamination. In Tables 2 to 7, therefore, the number of bacteria given for each sample of milk is an average based upon four plates. As stated before, the samples were taken from the milk of indi- vidual cows. In Barn I, 511 samples were taken from 35 cows in 1914, and 349 samples from 37 cows in 1915. In Barn II, 360 samples were taken from 26 cows in 1914, and 207 samples from 21 cows in 1915. Of the 238 samples in Barn III, 161 were taken from 10 cows in 1914, and 77 from 9 cows in 1915. The data from the analyses of these samples are given in Tables 2 to 7. ^Harding, H. A., and Wilson, J. K. A Study of the Udder Flora of Cows. N. Y. (Geneva) Agr. Exp. Sta. Tech. Bui. 27. 1913. 1917] GERM CONTENT OF MILK AS INFLUENCED BY BARN FACTORS 33 c * Q rH fc eft K M ^ fc 53 2 a w t (MrHr-lr-lrHCOCOCO OOi IOOOiOO(MlOCOi II ^ CO rH 1C rH OlOrHrHOOtOlOCOOl fto 02 tOtOtDrHO 10 in t~ _ lOtOrHCOOlOOOOCOtOt^ 36 BULLETIN No. 199 [May, m CI>.IOOOCCCO(>.O(>C1OQONO ^tit-oiotot-og locst^t^cooioo rH tOrHCOO rH -*lOO5rH 1917] GERM CONTENT OF MILK AS INFLUENCED BY BARN FACTORS 37 e i i o t o o o T-H >OfOOO 1 00 IO 00 CO Ot->00>Ol-OOCMO CC CO i-H t-OO ICt-CdCOO5COO i-H 25 172 647 96 287 000 557 25 j j 26. .. : 195 197 202 127 100 1 035 25 j ) 27 168 340 125 835 100 747 23 ) > 30 195 943 435 882 000 2224 23 April 2 201217 347 905 800 1720 23 > > 3 200 426 45 504 300 227 23 > t 6 203 634 95 930 600 401 23 > } 8 203 283 55 541 600 272 23 9 166 099 63 860 400 384 23 ) ) 15 201 745 204 035 300 1011 23 ) j 18 202 228 189 717 900 938 23 22 211 326 225 902 200 1 068 23 i y 24 206976 212 762 400 1 027 22 > > 27.. 200 558 371 639 600 1853 21 TABLE 13. GERM CONTENT OF TOTAL DAILY MILK PRODUCTION: BAKN II, 1915 Date Total milk production incc. Total germ content of milk Aver, germ content per cc. of milk Number of cows milked March 30 171 250 183 603 000 1072 21 ) > 31 174 680 103 339 000 591 21 April 1 170 420 74 382 000 436 21 j > 2 169 360 340 440 000 2010 20 ) > 5 167 670 107 045 000 638 21 > > 6 170 720 130 837 000 766 21 7 172 660 127 417 000 737 21 t j 8 172 140 116 521 000 676 21 it 9 172 480 151 763 000 879 21 > ) 12.. 172 780 87 308 000 505 21 1917] GERM CONTENT OF MILK AS INFLUENCED BY BARN FACTORS 45 TABLE 14. GERM CONTENT OF TOTAL DAILY MILK PRODUCTION : BARN III, 1914 Date Total milk production incc. Total germ content of milk Aver, germ content per ec. of milk Number of cows milked April 15 73 929 384 652 800 5 203 10 18 72566 385 029 700 5305 10 " 28 54502 502 218 700 9214 10 30 50854 200 743 700 3947 10 May 1 60523 252 388 900 4180 10 " 2 50 106 340 778 300 6801 10 " 4 57974 382 213 700 6592 10 " 5 53271 238 415 500 4475 10 " 6am . 55029 244 532 300 4443 10 " 6 p. m 48041 317 808 600 6615 10 " 11 50 326 361 966 203 7 192 9 " 12 a. m 48 964 282 436 100 5 768 9 " 12 p. m 38 811 589 285 700 15 183 9 " 13 50634 199 918 900 3948 9 " 14 50898 432 363 200 8612 9 " 16 48480 200 273 700 4131 9 " 18 .. 48172 336 195 700 6979 7 TABLE 15. GERM CONTENT OF TOTAL DAILY MILK PRODUCTION: BARN III, 1915 Date Total milk production incc. Total germ content of milk Aver, germ content per cc. of milk Number of cows milked March 23 49884 291 376 000 5842 9 > ) 24 55298 287 836 000 5202 8 26 59025 225417000 3819 8 27 62855 312 854 000 4977 8 > > 29 55525 258 690 000 4659 8 April 1 49713 498 719 000 10031 8 > > 2 47 780 129 098 000 2 702 g j j 5 49010 162 435 000 3314 7 > 7 54110 308 700 000 5705 8 ) > 21.. 33625 135 038 000 4541 5 An examination of Tables 10 to 15 shows that the variation in the number of bacteria in the milk at the different milkings was sur- prisingly small in each barn. This is particularly true of the results from Barn I for 1915 and from Barn II for 1914 and 1915. The lowest average daily count in Barn I during 1915 was 2,224 and the highest count was 3,840 bacteria per cubic centimeter. In Barn II for 1914 the lowest count was 227 and the highest count was 2,224, and for 1915 the lowest count was 436 and the highest count was 2,010 bacteria per cubic centimeter. A somewhat more pronounced variation in the average daily germ content was obtained from Barn I for 1914. During that year, how- ever, the samples at that barn were taken from only a part of the herd and, furthermore, not from the same cows at each milking, and it is 46 BULLETIN No. 199 [May, probable that the greater variation was partly due to this procedure. It is of interest to note that the few exceptionally high average daily counts, particularly the counts of May 22 and June 8, 1914, in Barn I, and those of May 12, 1914, and April 1, 1915, in Barn III, were due to exceptionally high counts in the milk of one or two cows. For example, in Barn I, on June 8, 1914, the total number of bacteria in the milk of the 19 cows from which the samples were taken was 1,043,284,410, of which number 958,814,000 were in the milk of Cow 55 and only 84,470,410 were in the milk of the remaining 18 cows. If this cow's milk had been excluded, the average germ content of the milk of the remaining 18 cows would have been about 1,000 bacteria per cubic centimeter ; but with the milk of Cow 55 included, the germ content was 10,498 bacteria per cubic centimeter. COMPARISON OF THE RESULTS FOR 1914 AND FOR 1915 As previously noted, in 1914 the udders of all the cows in the three barns were wiped .with a damp cloth previous to each milking, but in 1915 this practice was discontinued. In all other respects, so far as pos- sible, the same conditions and operations were maintained during both years. However, in a study of this nature certain factors which may affect the germ content of the milk are often beyond the control of the investigator. For example, in 1914 the study extended from March 10 to June 20, while in 1915 it was necessary to confine the study to March and April. According to Stocking, the different milkers may decidedly influence the germ content of the milk. In this study only two of the sixteen milkers employed in the three barns during the two years remained thruout the entire period of the experiment. More- over, not all the cows milked in 19] 4 were milked in 1915. Some of those milked in 1914 were sold, and some new ones were added during the period between the experiments of 1914 and 1915. Thus 71 cows were milked in 1914 and 67 in 1915, and only 49 of these were milked during both years. The difference between the data obtained in 1914 and in 1915 may be emphasized by a comparison based on the average counts of the different cows grouped as shown in Table 16. It is seen from Table 16 that there was no appreciable difference in the grouping of the animals in Barn II and Barn III for the two respective years. On the other hand, in Barn I there were 22 cows in the first group and 10 in the second group in 1914 and only 8 cows in the first group and 25 cows in the second group in 1915. If the results are expressed in percentage, it will be found that in Barn I, 62.9 percent of the 35 cows milked in 1914 and only 21.6 percent of the 37 cows milked in 1915 were in the first group, while the second group contained only 28.6 percent of the cows in 1914 and 67.6 percent of the cows in 1915. 1917] GERM CONTENT OP MILK AS INFLUENCED BY BARN FACTORS 47 TABLE 16. COMPARISON OF EESULTS IN 1914 AND 1915 BASED ON AVERAGE GERM CONTENT OF MILK OF THE INDIVIDUAL Cows Number of cows having average germ content of milk Below 1,000 per cc. Between 1,000 and 5,00'0 per cc. Over 5,000 per cc. Bam I: 1914 22 8 15 15 10 . 25 11 6 6 5 3 4 4 4 1915 Barn II: 1914 1915 Barn III: 1914 1915.. When the data for each of the two years are compared on the basis of the average germ content, per cubic centimeter, of all the milk produced in each barn during the entire study, the relation shown in Table 17 is obtained. TABLE 17. COMPARISON OF RESULTS OF 1914 AND 1915 BASED ON THE AVERAGE GERM CONTENT OF MILK FOR THE THREE BARNS Average germ content per cc. of milk 1914 1915 Barn I 2140 973 6189 3260 830 5050 Barn II Barn III Average.. 2188 2552 Table 17 shows that the milk produced in Barn I had a germ con- tent of 1,120 bacteria per cubic centimeter less in 1914 than in 1915, while the milk from Barn II had a germ content in 1914 of 143 bacteria more than in 1915, and the milk from Barn III had a germ content of 1,139 more in 1914 than in 1915. The data for the two years may also be compared on the basis of the average germ content of all the milk produced in all three barns in 1914 and 1915, respectively. Such calculations show that the milk produced in 1914 during the course of the experiment had an average germ content of 2,188 bacteria per cubic centimeter and in 1915 an average germ content of 2,552 bacteria per cubic centimeter. In other words, every cubic centimeter of milk in 1914 contained 364 bacteria less than in 1915. In all three barns the only operation that was intentionally altered during the two years was that of wiping the cow 's udder with a damp cloth previous to each milking. The data for the two years show that in Barn I there was an appreciable increase and in Barn III an appre- 48 BULLETIN No. 199 [May, ciable decrease in the germ content of the milk in 1915, and in Barn II the germ content of the milk was approximately the same for both years. It is evident that no conclusion can be drawn from the data concerning the relative importance of the practice of wiping the ud- ders as compared with the other sources of contamination in these barns. The data, however, do point to the conclusion that the wiping of the udders under the conditions obtaining in these barns did not affect the germ content of the milk to any appreciable extent. It is also to be noted that .altho pronounced fluctuations in the numbers of bacteria do occur in the individual samples and in the aver- ages of the different cows, the collective influence of all the sources of contamination on the germ content of the total daily milk production was remarkably uniform for both years in each of the three barns. The results obtained from the 1,665 samples of milk from the three barns show pronounced variation. Accordingly, any attempt to estimate the combined influence of the various sources of contamina- tion in any barn on the basis of a single set or a small number of sets of analyses gives no dependable results. On the other hand, the mass- ing of the results from a large number of samples should give figures which are fairly representative. The data are therefore brought to- gether in Table 18 so as to show the total milk production in each barn during the study, the total number of bacteria in the milk, and the average germ content per cubic centimeter of milk. TABLE 18. GERM CONTENT OF THE TOTAL MILK PRODUCTION FROM EACH BARN Total milk production in cc. Total germ content of milk Average germ content per cc. of milk Barn I: 1914 2 909 880 6227838000 2 140 1915 2 343 540 7 640 708 000 3260 Total 5 253 420 13 868 546 000 2639 Barn II: 1914 2 895 718 2 819 813 000 973 1915 1 714 160 1 422 655 000 830 Total 4 609 878 4 242 468 000 920 Barn III: 1914 913 080 5 651 213 00-0 6 189 1915 516 825 2 610 163 000 5050 Total 1 429 905 8261376000 5777 1917] GERM CONTENT OF MILK AS INFLUENCED BY BARN FACTORS 49 According to these calculations, all the sources of contamination in these barns contributed, as an average for the entire investigation, 2,639 bacteria per cubic centimeter to the milk from Barn I, 920 bac- teria to the milk from Barn II, and 5,777 bacteria to the milk from Barn III. The purpose of this investigation was, as stated before, to measure the collective influence of all the barn factors upon the germ content of the milk, and not to measure their influences separately. Neverthe- less, the data obtained point to certain conclusions concerning the rela- tive importance of some of the separate factors. The influence of the udder of a given cow is confined to her own milk, and when her udder is a large factor, numerically, her milk will have a large germ content regardless of the degree of cleanliness of the barn and the cow. An examination of Table 9, page 42, brings out the fact that the number of bacteria added to the milk by the udder was small in the case of most of the animals. In Barns I and II, 61 cows were milked in 1914 and 58 cows in 1915. The average germ content of 60 of these 119 cows milked during the two years was less than 1,000 bacteria per cubic centimeter, and of 32 cows the aver- age germ content was between 1,000 and 2,000 bacteria. It is evident, therefore, that since the small average counts of these 92 cows were due to all the barn factors, their udders could not have been numeri- cally a large factor. Of the remaining 27 cows, 20 had average counts between 2,000 and 5,000; 4 averaged between 5,000 and 10,000; one averaged 12,168 ; and one had an average of 35,131 for 1914 and 26,840 for 1915. In the case of the last animal, Cow 55, additional study showed that, altho apparently healthy, she persistently gave milk with a high germ content, the source of which was her udder. The average germ content of the milk from Barn I for the entire investigation was 2,639 bacteria per cubic centimeter. If Cow 55 were omitted _from the calculations, the average would be reduced approximately 1,000 bacteria per cubic centimeter. In other words, the udder of Cow 55 alone contributed about two-fifths of all the bac- teria that were found in all the milk produced in Barn I during the entire investigation. These results point to the conclusion that in the production of milk of low germ content, the udder of some cows may become the principal source of contamination. This conclusion is supported by the studies of Hastings and Hoff- man and of Harding and "Wilson. Hastings and Hoffman 1 con- cluded that "there is no reason to believe that the average bacterial count of milk as it is drawn from the udders of healthy cows is over 1,000 bacteria per cubic centimeter." These authors, however, found that the milk from two cows in the herd studied averaged 30,700 and 38,800 bacteria per cubic centimeter, respectively. In a more 'Hastings, E. G., and Hoffman, C. Bacterial Content of the Milk of Individual Animals. Wis. Agr. Exp. Sta. Ees. Bui. 6, pp. 189-196. 1907. 50 BULLETIN No. 199 [May, extensive study, Harding and Wilson 1 examined 1,230 samples of milk taken directly from the udders of 78 cows. This examination showed that, on the average, only 428 bacteria per cubic centimeter were added to the milk by the udders of these cows, but that 8 percent of the samples contained more than 1,000 bacteria per cubic centimeter, and the highest count was 16,610. The 2,639 bacteria per cubic centimeter in the milk from Barn I may be considered to have been derived from three separate sources ; namely, the udder of Cow 55, the udders of the remaining cows, and the barn factors. Since none of the cows in Barn II gave uniformly high counts, the 920 bacteria per cubic centimeter of the milk from this barn may be considered as having been derived from two sources the udders of the cows and the barn factors. If it is assumed that approximately 500. bacteria per cubic centimeter were added by the cow's udders, it will be seen that the conditions and operations at Barn I, omitting Cow 55 from consideration, contributed approxi- mately 1,100 bacteria per cubic centimeter of milk, and at Barn II about 400. The general appearances of Barns I and II would seem to indi- cate that Barn I was the cleaner ; and yet from the above deductions it is seen that more bacteria were added to the milk at Barn I than at Bam II. It might be argued from the results obtained at these two barns that a dirty barn does not contribute more bacteria to the milk than a clean barn. Such conclusion, however, would be against a well established fact. This apparent discrepancy is only a side issue to the general problem, and it would be a mere conjecture to attempt to explain it. The real significance of the results from these two barns lies in the fact that the number of bacteria in the milk from both barns was remarkably small, and that the difference in the conditions and the operations in the two barns exerted practically negligible in- fluence upon the germ content of the milk. Even more significant are the results from Barn III. The average contamination here was 5,777 bacteria per cubic centimeter. This milk, so far as the germ content was concerned, would meet the requirements for certified milk, and yet the conditions of the barn as to cleanliness were such that it is doubtful whether the milk produced here would have been admitted to the milk supply of some cities. These results must not be construed as a defense of dirty barns. They simply point to the fact that the large numbers of bacteria com- monly found in milk do not have their origin in the barn. 'Harding, H. A., and Wilson, J. K. A Study of the Udder Flora of Cows. N. Y. (Geneva) Agr. Exp. Sta. Tech. Bui. 27. 1913. 1917} GERM CONTENT OF MILK AS INFLUENCED BY BARN FACTORS 51 SUMMAEY This study was conducted in three dairy barns, differing widely in the degree of cleanliness. Samples were taken from the milk of individual cows when the milk was brought from the barn to the milk room and the germ content of each sample was then determined. A total of 1,665 samples were taken from 138 cows. The samples were collected during March, April, May, and June in 1914 and again during March and April in 1915. While the germ content of the individual samples varied from 3 to 218,250 bacteria per cubic centimeter, the large majority of the samples in all three barns had a low germ content. The average germ content of the milk of individual cows was low in most cases. Cow 55 had the highest average of 35,131 bacteria per cubic centimeter of milk, but the udder of this animal was the source of this high average. The average germ content of all the milk produced at each milking was over 10,000 only once in Barn I and only twice in Barn III, and in Barn II the highest average was only 2,224. The milk produced in 1914 and in 1915 had approximately the same germ content. The average germ content of all the milk produced during the entire study was 2,639 bacteria per cubic centimeter in Barn I, 920 in Barn II, and 5,777 in Barn III. CONCLUSIONS The study of these three barns shows that even under wide ex- tremes in barn conditions it is possible to produce milk with a germ content of less than 10,000 bacteria per cubic centimeter when the utensils are properly prepared. These intensive studies made at the Illinois and at the New York Agricultural Experiment Stations, together with accordant observa- tions upon about twenty-five ordinary dairy barns by the former insti- tution 1 and upon thirty-four dairy barns by the latter institution, 2 make it plain that when the influence of utensils is excluded, the dairy barns exert little measurable influence upon the germ content of the milk. J In connection with other studies not included in this bulletin, samples of milk from about one hundred different barns have been recently examined for germ content, and in no case did the varied conditions in the barn have any marked effect upon the germ content of the milk. "Brew, James D. Milk Quality as Determined by Present Dairy Score Card. N. Y. (Geneva) Agr. Exp. Sta. Bui. 398. 1915. Whenever attempts are made to measure barn activities, the attitude of the workmen becomes an important element in the success of the study. Because such measurements add something to their labor, the men may become antagonistic ; or because such measurements may be taken as an index of the care with which they do their work, they may modify their actions during such tests so as to lead to ab- normal results. Either of these attitudes may modify the results and endanger the conclusions. Accordingly, the colleague who has im- mediate charge of the barn workmen becomes a vital part of the investi- gation and his influence in keeping barn conditions normal during the progress of the study is a large factor in the success of the work. Both because of the harmonious relations which have existed and on account of numerous check experiments, we believe that the results here given are representative of the conditions regularly obtaining in the three barns in which this investigation was made. These barns were under the supervision of Professor W. J. Fraser, and Messrs. R. S. Hulce and W. T. Crandall. The authors are greatly indebted to these colleagues for their hearty cooperation, without which the investiga- tion could not have been performed successfully, UNIVERSITY OF ILLINOIS-URBANA