o m Issued March 24. 1013< U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN 161. A. D. MELVIN, CHIEF OF BUREAU. STUDY OF THE BACTERIA WHICH SURVIVE PASTEURIZATION. BY S. HENRY AYERS, Bacteriologist^ AND WILLIAM T. JOHNSON, JR., Scientific Assistant, Dairy Division. lifornia ional ility WASHINGTON: GOVERNMENT PRINTING OFFICE. 1913. Issued March 24, 1913. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN 161. A. D. MELVIN, CHIEF OF BUREAU. A STUDY OF THE BACTERIA WHICH SURVIVE PASTEURIZATION. BY S. HENRY AYERS, Bacteriologist, AND WILLIAM T. JOHNSON, JR. Scientific Assistant, Dairy Division. WASHINGTON: GOVERNMENT PRINTING OFFICE. 1913. THE BUREAU OF ANIMAL INDUSTRY. Chief: A. D. MELVIN. Assistant Chief: A. M. FARRINGTON. Chief Clerk: CHARLES C. CARROLL. Animal Husbandry Division: GEORGE M. ROMMEL, chief. Biochemic Division: M. DORSET, chief. Dairy Division: B. H. RAWL, chief. Field Inspection Division: R. A. RAMSAY, chief. Meat Inspection Diinsion: R. P. STEDDOM, chief. Pathological Division: JOHN R. MOHLER, chief. Quarantine Division: RICHARD W. HICKMAN, chief. Zoological Division: B. H. RANSOM, chief. Experiment Station: E. C. SCHROEDER, superintendent. Editor: JAMES M. PICKENS. DAIRY DIVISION. B. H. RAWL, chief. HELMER RABILD, in charge of Dairy Farming Investigations. S. C. THOMPSON, TO charge of Dairy Manufacturing Investigations. L. A. ROGERS, in charge of Research Laboratories. ERNEST KELLY, in charge of Market Milk Investigations. ROBERT McADAM, in charge of Renovated Butter Inspection. 2 ADDITIONAL COPIES of this publication -tx may be procured from the SUTEKINTENIV KNT or DOCUMENTS, Government Printing Office, Washington, D. C., at 10 cents per copy LETTER OF TRANSMITTAL. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY, Washington, D. C., October 7, 1912. SIR: I have the honor to transmit herewith for publication in the bulletin series of this bureau a manuscript entitled "A Study of the Bacteria Which Survive Pasteurization," by Messrs. S. Henry Ayers and William T. Johnson, jr., of the Dairy Division. Respectfully, A. D. MELVIN, Chief of Bureau. Hon. JAMES WILSON, Secretary of Agriculture. 3 CONTENTS. Page. Introduction 9 Objects of this investigation 10 Methods 11 Temperatures used throughout the country 12 Bacterial reductions by laboratory pasteurization, using the ''holder " process . . 13 Bacterial reductions at 60 C. (140 F.) and 65.6 C. (150 F.) 13 Bacterial reductions at 62.8 C. (145 F.) 15 The control of pasteurization 16 Effect of long heating on bacteria in milk 17 Three hours' pasteurization 17 Six hours' pasteurization 18 Effect of sudden cooling on the bacteria in pasteurized milk 20 Groups of bacteria which survive pasteurization 21 Grades of milk studied 21 Methods .... 21 The bacterial groups in grade A milk before and after pasteurization for 30 minutes at 62.8 C. (145 F.) 24 The bacterial groups in grade A milk before and after pasteurization for 30 minutes at 7 1.1 C. (160 F.) 25 The bacterial groups in grade B milk before and after pasteurization for 30 minutes at 62.8 C. (145 F.) 25 The bacterial groups in grade C milk before and after pasteurization for 30 minutes at 62.8 C. (145 F.) 26 The bacterial groups in milk pasteurized at high temperatures 27 The activity of bacteria of the various groups isolated from raw and pasteurized milk measured by their ability to produce changes in litmus milk after dif- ferent lengths of incubation 28 Grade A milk before and after pasteurization for 30 minutes at 62.8 C. (145 F.) ,. 28 Grade A milk before and after pasteurization for 30 minutes at 71.1 C. (160 F) 30 Grade B milk before and after pasteurization for 30 minutes at 62.8 C. (145 F.) 31 Grade C milk before and after pasteurization for 30 minutes at 62.8 C. (145 F.) 32 Comparison of the percentage of acid-forming bacteria in raw and pasteurized milk 33 Comparison of the percentage of peptonizing bacteria in raw and pasteurized milk 34 Average percentages of the bacterial groups of raw milk which survive pasteuri- zation 34 5 6 CONTENTS. The effect of pasteurization on the Dumber of bacteria of different groups in milk 35 The bacterial development in milk pasteurized in the laboratory and held at different temperatures 37 Qualitative study of the groups of bacteria which survive pasteurization 43 The acid-forming group of bacteria 43 The inert group of bacteria 45 The alkali-forming group of bacteria 45 The peptonizing group of bacteria 47 The gas-forming bacteria 47 Thermal death points of bacteria which survive pasteurization 51 The possible use in pasteurization of acid-forming bacteria of high thermal death point 52 Qualitative study of the complete bacterial flora of one sample of pasteurized milk 53 Summary 58 Conclusions. 61 Appendix. Tables 65 ILLUSTRATIONS. Page. FIGURE 1. Bacterial reduction during pasteurization for three hours at 54.4 C. (130 F.), 57.2 C. (135 F.), and 60 C. (140 F.) 17 2. Bacterial reduction during pasteurization for six hours at 62.8 C, (145 F.). Plotted after one-half, three, and six hours. . . ... 19 3. Bacterial reduction during pasteurization for six hours at 62.8 C. (145 F.). Plotted every half hour 20 4. Comparison of the milk-tube and plate method for the determination of the bacterial groups in milk 23 5. Bacterial groups of grade A milk before and after pasteurization for 30 minutes at 62.8 C. (145 F.) 24 6. Bacterial groups in grade A milk before and after pasteurization for 30 minutes at 71.1 C. (160 F.) 25 7. Bacterial groups in grade B milk before and after pasteurization for 30 minutes at 62.8 C. (145 F.) - 26 8. Bacterial groups in grade C milk before and after pasteurization for 30 minutes at 62.8 C. (145 F.) 27 9. Bacterial groups which survive pasteurization for 30 minutes at 76.7 C. (170 F.), 82.2 C. (180 F.), 87.8 C. (190 F.), and 93.3 C (200 F.) 28 10. Changes in the bacterial group relations in grade A milk when de- termined by litmus milk reactions after different lengths of incu- bation. Milk pasteurized at 62.8 C. (145 F.) for 30 minutes. . . 29 11. Changes in the bacterial group relations in grade A milk when de- termined by litmus milk reactions after different lengths of incu- bation. Milk pasteurized at 71.1 C. (160 F.) for 30 minutes. . . 30 12. Changes in the bacterial group relations in grade B milk when de- termined by litmus milk reactions after different lengths of incu- bation 31 13. Changes in the bacterial group relations in grade C milk when de- termined by litmus milk reactions after different lengths of incu- bation 33 14. Comparison of the percentage of the acid-forming bacteria in raw and pasteurized milk 33 15. Comparison of the percentage of the peptonizing bacteria in raw and pasteurized milk 34 16. Daily changes in the bacterial group relations in pasteurized milk. Sample G held at room temperature " 39 17. Daily changes in the bacterial group relations in pasteurized milk. Sample G held in the ice box 39 18. Daily changes in the bacterial group relations in pasteurized milk. Sample H held at room temperature 40 7 8 ILLUSTRATIONS. Page. FIGURE 19. Daily changes in the bacterial group relations in pasteurized milk. Sample II held in ice box 41 20. Daily changes in the bacterial group relations in pasteurized milk. Sample K held at room temperature 42 21. Daily changes in the bacterial group relations in pasteurized milk. Sample K held in the ice box 42 22. The acid-forming group of bacteria 44 23. The alkali-forming group of bacteria 45 24. The peptonizing group of bacteria 46 25. Apparatus for the determination of gas production in milk 48 26. Gas produced by culture Z 49 27. A series. Cultures from. milk pasteurized at 60 C. (140 F.) for 30 minutes 54 28. B series. Cultures from milk pasteurized at 65.6 C. (150 F.) for 30 minutes 56 29. The hypothetical relation of the bacterial groups to raw and pas- teurized milk 62 30. The hypothetical relation of the bacterial groups in raw and pas- teurized milk. ... 63 A STUDY OF BACTERIA WHICH SURVIVE PASTEURIZATION. INTRODUCTION. The bacteria which survive the process of pasteurization are of great importance, since they form the foundation for the subsequent bac- terial development. It is well known that the most efficient pasteu- rization will not destroy all of the microorganisms in milk. The process must, then, leave a certain number of bacteria which it is impossible to destroy at the temperatures employed. The prevailing view has been that the organisms left after heating were largely of the peptonizing spore-forming type, with some few inert forms. This idea was based on the belief that all vegetative cells were destroyed at temperatures below the minimum pasteurizing temperature of 60 C. (140 F.). Theoretically, then, only spore-forming organisms should survive pasteurization. This view was strengthened by numerous investi- gations of the bacterial flora of boiled milk or milk pasteurized at extremely high temperatures. As lower temperatures came gradu- ally into use investigations indicated that vegetative cells might have higher thermal death points than was generally believed, Russell and Hastings 1 found a micrococcus which was capable of standing a temperature of 76 C. (168.8 F.) for 10 minutes. Other investigators pointed out that lactic acid bacteria occasionally ap- peared in pasteurized milk and yet the thermal death point of the lactic acid bacteria was stated to be in the neighborhood of 57.2 to 60 C. (135-140 F.). Rogers 3 found that in milk pasteurized at 85 C. (185 F.) by the "flash" process lactic acid bacteria occasionally survived. Marshall 3 states "It is largely supposed that in pasteur- ized milk the lactic acid bacteria are killed. This is largely true but not universally." Maz4* found that the ordinary lactic acid bacteria i Russell, IT. L., and Hastings, E. G. A micrococcus, the thermal death limit of which is 76 C. Cen- tralblatt fiir BrJrtorioiogie, Parasitenkunde und Inlektionskrankheiten, Abteilung 2, vol. 8, No. 11, pp. 339-342. Jena, Mnr. 13, 1902. *Rogers, I/. A. The bacteria of pasteurized and unpasteurized milk under laboratory conditions. United States Department of Agriculture, Bureau of Animal Industry, Bulletin 73. Washington, 1905. 'Marshall, C. E. Pasteurization of milk. Michigan Agricultural Experiment Station, Bulletin 147. Agricultural College, August, 1897. 4 Ma//, P. Pasteurization du lait destin6 Ma consommation. I/Industric Laitiferc, vol. 32, No. 8, pp. 121-127. Paris, Feb. 24, 1907. 9 67796 Bull. 16113 2 10 BACTERIA WHICH SURVIVE PASTEURIZATION. were destroyed by five minutes' heating at temperatures between 55 and 65 C. (131 and 149 F.), but that lactic acid bacteria existed which resist five minutes' heating at 75 C. (167 F.). When milk was pasteurized in bottles for 60 minutes at 65 C. (149 F.), Gerber l and Weiske found that the bacteria which resisted belonged for the most part to the inoffensive lactic bacilli. An investigation of com- mercially pasteurized milk in this country carried on by the authors 2 showed the presence of high-temperature resisting lactic-acid bacteria, the thermal death point of one of which was 77.8 C. (172 F.) with a 10 minutes' exposure and 75.6 C. (168 F.) with a 30 minutes' heating. It was further found that commercially pasteurized milk always soured. Kohler 3 and Tonney, after studying pasteurized milk in Chicago, concluded that reinfection was the cause of the sour- ing of heated milk. It is evident that the knowledge of the bacteria which actually survive pasteurization is very limited. OBJECTS OF THIS INVESTIGATION. The general object of this investigation was to study quantitatively and qualitatively the bacteria which survive pasteurization under conditions which excluded any possibility of recontamination of the milk after pasteurization. The special objects were as foUows: 1. To ascertain the average temperature of pasteurization in both the "holder" and "flash" process used throughout the country. 2. To determine the effect of various pasteurizing temperatures for one-half hour's exposure upon the bacteria in different grades of milk. 3. To study the effect of holding periods longer than one-half hour during pasteurization. 4. To determine the effect of sudden cooling on bacteria after pasteurization. 5. To study quantitatively the bacterial groups which survive pasteurization at various temperatures. 6. To trace the development of the various bacterial groups in pasteurized milk held at different temperatures. 7. To study qualitatively the bacteria which survive pasteurization. i Gerber, N., and Weiske, P. Pasteurisation des flacons dans la grande Industrie (pasteurisation avec agitation). Revue Gen6raledu Lait, vol. 2, No. 8, pp. 169-177. Lierre, Jan. 30, 1903. * Ayers, S. Henry, and Johnson, W. T., jr. The bacteriology of commercially pasteurized and raw market milk. United States Department of Agriculture, Bureau of Animal Industry, Bulletin 126. Washington, 1910. * Kohler, Gottfried and Tonney, F. O. The control of pasteurization. Journal of the American Medical Association, vol. 5, No. 10, pp. 713-718. Chicago, Mar. 11, 1911. METHODS EMPLOYED. 11 METHODS. In this investigation the technique of plating and the preparation of beef infusion agar with a reaction of 1.5 (Fuller's scale) was carried out according to the recommendations of the committee of standard methods for the bacteriological analyses of milk. 1 In the prepara- tion of fermentation bouillons and litmus lactose gelatin, Liebig's beef extract was used as a basis. Fermentation bouillons were prepared by dissolving 4 grams of Liebig's beef extract and 10 grams of Witte's peptone in a liter of water and correcting reaction to 0.0 (Fuller's scale). The broth was then brought up to the sterilizing temperature for five minutes and filtered. When a clear filtrate was obtained, 0.5 per cent potas- sium dibasic phosphate and 1 per cent of the substance to be fer- mented was dissolved in it. The following substances were used to test the fermenting ability of the bacteria: Sugars: Dextrose, galactose, lactose, saccharose, raffinose. Alco- hols: Mannite, glycerin. Starches: Inulin, wheat starch. Glucoside: Salicin. The ability of the bacteria to reduce nitrates to nitrites was shown by growing the organisms in a bouillon composed of 0.1 per cent Witte's peptone and 0.02 per cent potassium nitrate (nitrite-free) for 14 days at 30 C. (86 F.) and then testing for the presence of nitrites. The liquefaction of gelatin by an organism was deter- mined by inoculating with a drop of milk culture on the surface of the tube of medium and incubating for 30 days at 18 C. (64.4 F.) and then measuring the depth of liquefaction. Special media. For ordinary routine plating of a milk sample, and especially for the determination of the peptonizing bacteria, a casein agar was devised, 2 consisting of dissolved chemically pure casein and agar, with a final reaction slightly acid. In isolating gas formers, Smith tubes containing lactose peptone bile 3 and dextrose liver broth * were used. All samples of milk were pasteurized in sterile flasks in the labora- tory. At all the temperatures except 60 C. (140 F.) and 65.6 C. (150 F.), 800 cubic centimeters of milk were heated. During the few experiments at those temperatures only 100 cubic centim- eters of milk were used. In all the experiments the period of heating was 30 minutes from the time the pasteurizing temperature 1 Report of the Committee on Standard Methods of Bacterial Milk Analysis. American Journal of Public Hygiene, new series, vol. 6, No. 2, pp. 315-345. Columbus, Ohio, May, 1910. * Ayers, S. Henry. Casein media adapted to determining bacteria In milk. United States Department of Agriculture, Bureau of Animal Industry, Twenty-eighth Annual Report, 1911 (in press). ' Jackson, Daniel D. A new solution for the presumptive test for Bacillus coll. Biological Studies of Pupils of W. T. Sedgwick, pp. 292-299. Boston, 1906. 1 Jackson, D.D., and Muer.T.C. Liverbroth. A medium for the determination of gas-forming bacteria in water and sewage. Journal of the American Public Health Association, vol. 1, No. 12, pp. 927-929. Urbana, 111., December, 1911. 12 BACTERIA WHICH SURVIVE PASTEURIZATION. was reached. The methods employed throughout this investiga- tion make the results comparable with those obtained by pasteuri- zation in sealed bottles. TEMPERATURES USED THROUGHOUT THE COUNTRY. It was necessary to ascertain the temperatures most universally used at milk plants in this country in order that an average tempera- ture might be employed in these experiments. Circular letters were therefore sent to pasteurizing plants in nearly aU cities with a population of over 25,000. Replies showed that the average tem- perature with the "holder" process was 62.8 C. (145 F.). With the "flash" process 71.1 C. (160 F.) was about the average. The reports from 219 milk plants which pasteurized showed that 75 used the "holder" process and 144 the "flash" process. Assuming that the correct temperatures for pasteurization are from 60 to 65.6 C. (140 to 150 F.) with the "holder" process, then 62 of the 75 pasteurized at the proper temperature, one used a tem- perature too low, 54.4 to 57.2 C. (130 to 135 F.), and 12 too high, ranging from 66.7 to 76.7 C. (152 to 170 F.). With the "flash * process the correct temperature was assumed to be 71.1C. (160 F.). As there is usually a variation in the range of temperature during pasteurization, the maximum and the minimum given by each plant have been averaged. When the average was 71.1 C. (160 F.) it was assumed that the proper temperature was used. It was found that of 144 plants using the "flash" process only 61 used the correct temperature, 61 pasteurized too low and 22 too high. The low tem- peratures ran down to 60 C. (140 F.), the high temperatures up to 82.2 C. (180 F.). These figures show a decided lack of uniformity of temperatures used in pasteurization. The temperatures below 60 C. (140 F.) with the "holder" process render no protection as far as the destruction of pathogenic organisms is concerned, while those above 65.6 C. (150 F.) only increase the cost of pasteurization and tend to reduce the cream line. The same is true when using the "flash" process below or above temperatures ranging from 71.1 to 73.9 C. (160 to 165 F.). As pasteurization is practiced, the milk might have been heated from 1 minute at 60 C. (140 F.) to 30 minutes at 76.7 C. (170 F.) and it would all be known as pasteurized milk. This lack of uni- formity is undoubtedly due to a misunderstanding of the effects of heat on the bacterial flora of milk. BACTEKIAL REDUCTIONS. 13 BACTERIAL REDUCTIONS BY LABORATORY PASTEURIZATION, USING THE "HOLDER" PROCESS. Since regulations often require a definite percentage bacterial reduction and as the efficiency of pasteurizers is based on the same figures, it seemed advisable to study the quantitative bacterial reduc- tions under exact laboratory conditions. BACTERIAL REDUCTIONS AT 60 C.(l40 F.) AND 65.6 C. (150 F.). In these experiments the milk was heated in sterile flasks in a water bath at a temperature of 60 C. (140 F.) and 65.6 C. (150 F.) for a period of 30 minutes. The temperature recorded by a ther- mometer in the milk was maintained for the full 30 minutes. Twelve samples of milk from dairy X were used. Each was divided and a portion pasteurized at both temperatures. The counts were made on litmus lactose gelatin incubated at 18 C. (64.4 F.) for six days. Table 1 shows the total counts and efficiency of the process as deter- mined by the percentage bacterial reduction. An examination of the table shows that the raw milk was of poor quality, having a high bacterial count. The samples pasteurized at 60 C. (140 F.) showed a high count, while the same samples at 65.6 C. (150 F.) showed a low count, as a rule. The effect of the few degrees of heat may be plainly seen. At the lower temperature 9 out of the 12 samples showed a reduction of less than 99 per cent, while at the higher temperature only 4 showed less than a 99 per cent reduction. TABLE 1. Percentage reduction of bacteria by laboratory pasteurization, using the "holder" process Raw milk obtained from dairy X. Pasteurized at 60 C. Pasteurized at Raw milk. (140 F.) for 30 65.6 C. (150 F.) minutes. for 30 minutes. Sample No. Total count. Total count. Percent- age re- duction. Total count. Percent- age re- duction. 1 33,900,000 200,000 99.41 9,800 99.97 2 4,900,000 118,000 97.59 12,300 99.74 3 5,050,000 130, 000 97.45 25,100 99.50 4 850,000 32,000 96.23 25,500 97.00 5 6, 100, 000 20,200 99. 06 12,100 99.80 6 8,400,000 23,000 99.72 12,600 99.85 7 1,670,000 284,000 82.99 72,000 95.68 8 4,100,000 74,000 98.19 28,500 99.30 9 1,120,000 66,000 95.00 19,900 98.22 10 4,800,000 153,000 96.81 19,800 99.58 11 4,400.000 200, 000 95.31 09,000 98.43 12 12,300,000 19(1,000 98.41 40,000 99.67 The effect of pasteurization on the bacterial content of another series of 12 samples from dairy Y was studied in the same way. The results are shown in Table 2. It may be seen that the milk was of far better quality than that from dairy X. The counts of 14 BACTEKIA WHICH SURVIVE PASTEURIZATION. milk pasteurized at 60 C. (140 F.) were low and still lower at the higher temperature. In spite of the low counts the percentage bacterial reductions were not so great as when a poor grade of milk was pasteurized as shown in Table 1. Eleven out of twelve samples showed a reduction of less than 99 per cent at 60 C. (140 F.). At 65.6 C. (150 F.) 7 out of 12 showed a bacterial reduction of less than 99 per cent. A comparison of Tables 1 and 2 show that the bacterial content after pasteurization of the better grade of milk was far lower than that of milk from dairy X, which contained a large number of bacteria before heating. If, however, the efficiency of the process is studied, the higher percentage bacterial reductions will be found in Table 1, where the milk was of poor quality. It is very evident that the percentage bacterial reduction is of no value in determining the quality of pasteurized milk. Compare, for example, sample No. 1 in Table 1 with sample No. 3 in Table 2. In the first case, the raw milk contained 33,900,000 bacteria per cubic centimeter and after pasteurization 200,000 per cubic centimeter, which is a percentage reduction of 99.41 per cent. In the second case, raw milk, 128,000 bacteria per cubic centimeter and after pasteurization 13,900 per cubic centimeter, which is a reduction of only 89.14 per cent. A 99.41 per cent reduction against 89.14 per cent, and yet the pasteurized milk in which only 89.14 per cent of the bacteria were destroyed was far superior in its bacterial content. TABLE 2. Percentage reduction of bacteria by laboratory pasteurization, using the ' ' holder " process Raw milk obtained from dairy Y. Pasteurized at 60 C. Pasteurized at Raw milk. (140 F.) for 30 65.6 C. (150 J F.) minutes. for 30 minutes. Sample No. Total count. Total count. Percent- age re- duction. Total count. Percent- age re- duction. 1 2,500,000 27,600 98.89 4,210 99.83 2 1,090,000 13,100 98.79 2,320 99.78 3 128,000 13,900 89.14 5,670 95. 57.-. 4 149,000 7,500 94.96 3,100 97.91 5 COO, 000 16,400 97.26 11,100 98.15 6 119,000 2,530 97.87 1,020 99.14 7 168,000 1,890 98.87 3,230 98.07 8 84,000 2,600 96.90 2,720 96.76 9 ICO, 000 5,110 96.80 2,010 98.74 10 590,000 13,200 97.76 7,100 98.79 11 2,900,000 28,700 99.01 20,400 99.29 12 1,170,000 34,100 97.08 3,560 99.69 The uselessness of figures in expressing percentage reductions is further shown in Table 3. The bacterial content of 12 samples of milk before and after pasteurization has been averaged, two grades of milk having been used. While the percentage reduction is approxi- mately the same at 60 C. (140 F.) the average bacterial counts vary BACTEKIAL REDUCTIONS. 15 widely, one being 124,000 per cubic centimeter, the other only 13,900 per cubic centimeter. At 65.6 C. (150 F.) while the percentage reduction is increased to 98 per cent, the bacterial content holds the same relation as at the lower temperature. TABLE 3. Comparison of the average bacterial content of two grades of milk and percentage reduction in bacteria by pasteurization at 60 C. (140 F.) and 65.6 C. (150 F.). AVERAGE OF 12 SAMPLES IN EACH GRADE. Grade. Raw milk. Pasteurized at60C. (140 F.) 30 minutes. Pasteurized at 65.6 C. (150 F.) 30 minutes. Bacteria per cubic centimeter. Bacteria per cubic centimeter. Percent- age re- duction. Bacteria per cubic centimeter. Percent- age re- duction. Grade 1 Grade 2 7,300,000 804,000 124,000 13,900 96.39 96.95 28,900 5,500 98.72 98.40 BACTEKIAL REDUCTION AT 62.8 C. (l45 F.). Another series of 28 samples of milk was studied in a similar man- ner. The results are shown in Table 4. In this series a tempera- ture of 62.8 C. (145 F.) was used for a period of 30 minutes. All of the samples of raw milk contained over 1 ,000,000 bacteria per cubic centimeter. The bacterial counts were made on infusion agar, incu- bated at 30 C. (86 F.) for six days. It may be seen from the table that the percentage reductions were high, only 3 out of 28 being less than 99 per cent; however, 13 of the pasteurized samples contained over 20,000 bacteria per cubic centimeter. TABLE 4. Efficiency of the' 1 holder" process of pasteurization under laboratory conditions, using raw milk with a bacterial content over 1,000,000 per cubic centimeter. Pasteurized at 62.8 Pasteurized at 62.8 Raw milk. C. (145 F.) for 30 Raw milk. C. (145 F.) for 30 minutes. minutes. >;ITTI !(!' No Bacteria per cubic centimeter. Bacteria per cu- bic cen- timeter. Percent- age reduc- tion. Surnplc No. Bacteria per cubic centimeter. Bacteria per cu- bic centi- meter. Percent- age reduc- tion. 1 16,800,000 85,000 99.49 15 12,200,000 33,300 99.72 2 91,000,000 14,400 99.98 10 28, 200, 000 127,000 99.54 3 15,900,000 18,200 99.88 17 6,480,000 2,400 99.% 4 90,000,000 5,800 99.99 18 4,540,000 9,500 99.97 6 19,800,000 41,000 99.79 19 2,460,000 13,600 99.44 6 105,000,000 17,500 99.98 20 4,900,000 9,900 99.79 7 47,300,000 8,900 99.98 21 1,060,000 1,010 99.90 8 12,400,000 9,700 99.92 22 2,790,000 8,800 99.68 9 8,600,000 15,000 99.82 23 5,400,000 36,600 99.32 10 17,800,000 8,900 99.95 24 6,060,000 30,100 99.50 11 18,900,000 41,800 99.97 25 1,140,000 1,560 99.86 12 15,100,000 39,700 99.74 26 14,600,000 468,000 96.79 13 22,000,000 20,400 99.90 27 1,440,000 22,900 98.41 14 98,000,000 28,200 99.97 28 1,780,000 26,500 98.51 BACTERIA WHICH SURVIVE PASTEURIZATION. The results of similar experiments using a series of 24 samples of clean milk are shown in Table 5. The bacterial counts of the pasteur- ized milk were very low as a rule and yet 14 of the 24 samples showed a percentage bacterial reduction of less than 99 per cent. Sample 17 contained, before heating, only 119,000 bacteria per cubic centimeter; after the process the bacterial content was 20,800, a reduction of only 82.52 per cent. This sample evidently contained a high percentage of resistant bacteria which were able to withstand the process of pasteur- ization. From these results it is apparent that regulations of boards of health calling for a 99 per cent reduction of bacteria in commercial pas- teurization are of no value. As a general rule, it may be said that the higher the bacteria hi raw milk the greater the percentage reduction through pasteurization. When the bacterial content of milk is low it is almost always impossible to obtain a 99 per cent reduction of the bacte- ria. It is also evident that bacterial standards for pasteurized milk must not be set below the limit which it is theoretically possible to obtain. When pasteurizing in the laboratory for a period of 30 minutes at 62.8 C. (145 F.) under conditions which permitted no reinfection of the milk, the bacterial content of pasteurized milk was by no means uniform. Sometimes the counts were high and sometimes low and yet the maintenance of the proper temperature of pasteurization would have assured in all cases the same degree of protection from pathogenic organisms. TABLE 5. Efficiency of the ' ' holder " process of pasteurization under laboratory conditions, using raw milk with a bacterial content less than 1,000,000 per cubic centimeter. Pasteurized at 62.8 Pasteurized at 62.8 Raw milk. C (145 F.) for 30 Raw milk. C. (145 F.) for 30 minutes. minutes. Sample Sample No. Bacteria per cubic centimeter. Bacteria per cu- bic cen- timeter. Percent- age reduc- tion. No. Bacteria per cubic centimeter. Bacteria per cu- bic cen- timeter. Percent- age reduc- tion. 1 372,000 910 99.75 13 540,000 11,800 97.81 2 434,000 2,330 99.46 14 970,000 46,000 95.25 3 540,000 4,890 99.09 15 162,000 8,400 94.81 4 410,000 1,170 99.71 16 118,000 9,000 92.37 5 127,000 80 99.93 17 119,000 20.800 82.52 6 143,000 765 99.46 18 137,000 3,100 97.73 7 9,300 90 99.03 19 15,600 300 98.07 8 18,100 180 99.00 20 9,800 100 98.97 9 19,900 62 99.68 21 18,700 1,340 92.83 10 5,300 34 99.35 22 25,900 830 96.79 11 134,000 7,500 94.40 23 15,300 334 97.81 12 300.000 7,600 97.47 24 9,900 320 96.76 THE CONTROL OF PASTEURIZATION. The control of the process of pasteurization should be by bacterial limits for the milk which is to be used, together with supervision for the maintenance of the proper pasteurizing temperature and protec- EFFECT OF LONG HEATING ON BACTEEIA. 17 7S000 70,000 tion against reinfection. The bacterial content of the milk will then consist only of those organisms which are able to withstand the tem- perature in use. EFFECT OF LONG HEATING ON THE BACTERIA IN MILK. In some milk plants a holding period longer than half an hour is employed. It seems to be believed that an increased holding period produces a greater bacterial reduction. Several experiments were made at different tem- peratures and for dif- ferent periods in order to gain more information on this point. THREE HOURS' PAS- TEURIZATION. In order to determine the effect of a long pe- riod of pasteurization, milk was heated in ster- ile flasks at54.4C.(130 F.), 57.2 C. (135 F.), and 60 C. (140 F.), and plated every half hour for three hours. The bacterial counts are shown in Table 6. One sample of milk was di- vided and a portion heated at the two lower temperatures, so the bacterial content of the raw milk was the same. The sample of milk heated at 60 C. (140 F.) contained approximately the same number of bacteria per cubic centimeter and was from the same dairy, so it is probably fair to compare the results. After one-half hour's heat- ing a much greater reduction was observed where the milk was heated at 60 C. (140 F.). At the end of one hour's heating the bacterial content of the milk were more nearly alike and continued to show a gradual reduction throughout the three hours of heating. The final examination showed that the bacterial content was approximately the same in all the samples of milk. The bacterial reductions are shown better by the curves in figure 1 . It is evident that long heating 67796 Bull. 16113 3 FIG. 1. Bacterial reduction during pasteurization for three hours at 54.4 C. (130 F.), 57.2 C. (135 F.), and 60 C. (140 F.). 18 BACTERIA WHICH SURVIVE PASTEURIZATION. at 54.4 C (130 F.) and 57.2 C. (135 F.) is of more value than at 60 C. (140 F.), as shown by curves A, B, and C, respectively. From a comparison of curves A and C it seems that one and a half hours' heating at 54.4 C. (130 F.) will produce as great a bacterial reduc- tion as one-half hour's heating at 60 C. (140 F.). It must not be assumed, however, that one and one-half hours' heating at 54.4 C. (130 F.) will destroy pathogenic organisms as surely as will one- half hour's heating at 60 C. (140 F.). That point can only be determined by experiment. TABLE 6. Bacterial reduction during pasteurization for 3 hours at 54.4 C. (130 F.), 57.2 C. (135 F.), and 60 C. (140 F.). Temperature of pasteuriza- tion. Bacteria per cubic centi- meter in raw milk. Bacteria per cubic centimeter in milk pasteurized for 1 hour. 1 hour. 1J hours. 2 hours. 2J hours. 3 hours. C. F. 54.4 130 57.2 135 60.0 140 2,530,000 2,530,000 2,330,000 75,000 63,000 20,800 27,600 15,400 19,400 19,400 12,600 14,100 13,200 8,400 12,400 12,300 7,900 10,300 9,200 5,600 8,700 SIX HOURS PASTEURIZATION. A similar experiment was made, using a six hours' period of heating at 62.8 C. (145 F.). In order to meet extreme conditions where the effect of long heating might show, a milk containing 27,000,000 bacteria per cubic centimeter was selected. Plates were made every half hour during the period of heating. Table 7 shows the results. TABLE 7. Bacterial reductions during pasteurization for 6 hours at 62.8 C. (145 F.) Length of pasteur- ization (hours). Bacteria per cubic centi- meter. Length of pasteur- ization (hours). Bacteria per cubic centi- meter. Raw 27,000,000 8} 32,900 i 46,000 4 31,200 1 46,000 41 44,000 IJ 45,000 5 35,700 2 65,000 B4 39,000 2V 62,000 6 34,500 3" 54,000 It may be seen that there was little difference in the bacterial reduc- tion produced by a half hour's heating and by six hours' heating. There were numerous variations in the numbers of bacteria as deter- mined, but they were within the experimental error, as the flask of heated milk could not be thoroughly shaken. In this experiment the groups of bacteria which withstood the heating were determined. The plating was done on infusion agar plates, which were incubated at EFFECT OF LONG HEATING ON BACTERIA. 19 73 70 60 Z \ 30 0. (86 F.) for six days. The plates were counted and then each colony picked from a plate and inoculated into litmus milk tubes. After 14 days' incubation the milk cultures were examined and as 'a result of the reactions shown the bacteria picked from the plates were divided into five groups, the acid forming and coagulating, acid- forming, inert, alkali-forming, and peptonizing groups. The value of this method of determining bacterial groups will be discussed later in this paper. Figure 2 shows the relations of the groups after heat- ing one-half hour, three hours, and six hours. The acid-coagulating and simple acid-forming groups composed the largest portion of the bacteria after a half hour's heating and the same after six hours' pasteurization. It may be seen, however, that the percentage of the acid-coagulating group was reduced by the heating for six hours. It seems probable that the heating weakened the activity of the acid- forming bacteria so that in the litmus milk tubes not enough acid was produced to coagulate the milk in the 14 days' incubation period. That would result in a lowering of the percent- age of the acid-coagulating group, while increasing at the same time the simple acid-forming group. The percentage of the alkali and peptonizing groups was extremely low throughout the experiment. In figure 3 the group percentages have been plotted at every half hour's sampling and gives a more complete picture of the changes in the bacterial groups. The changes in the group percentages between tliree and one-half and four hours is undoubtedly due to the errors in sampling and inaccuracy of the method of the bacterial group differentiation. 25 /s \ FIG. 2. Bacterial reduction during pasteurization for six hours at 62.8 C. (145 F.). Plotted after one-half , three, and six hours. 20 BACTERIA WHICH SURVIVE PASTEURIZATION. EFFECT OF SUDDEN COOLING ON THE BACTERIA IN PASTEURIZED MILK. The sudden cooling of the hot milk after pasteurization has been considered by many to be an essential part of the process. It was* believed that the sudden change from hot to cold aided in the destruc- tion of the bacteria. In the recent develop- ments of the process of pasteurization, how- ever, this opinion lost ground, although at the present time there are IS those who still believe in its value. In order to throw light on this point a few experi- ments were made under laboratory conditions. pasteurized in sterile fe flasks, then cooled by running through a sterile copper coil sur- rounded by brine into another sterile flask. Table 8 shows the results of the experiments. Two samples of milk were pasteurized at 62.8 C. (145 F.) for 30 minutes, then cooled in 15 seconds to from 1.7- 3.9 C. (35-39 F.). FIG. 3. Bacterial reduction during pasteurization for six hours at The experiment Was T6- 62.8 C. (145 F.). Plotted every half hour. . . , peated, using a period of 30 minutes' heating at 71.1 C. (160 F.). The bacterial content of the milk before and after cooling was approximately the same, the differences always being within the limits of experimental error. It is evident from the results shown in the table that sudden cooling is of no value in causing a destruction of bacteria, at least not at the temperatures used in the experiments. BACTERIAL GROUPS. 21 TABLE 8. Effect of sudden cooling on the bacterial content of pasteurized milk. Pasteurized at 62.8 C. Pasteurized at 71.1 C. (145 F.) 30 minutes. (160 F.) 30 minutes. Sample No. Bacteria per cubic centimeter in raw milk. Sample No. Bacteria per cubic centi- meter in raw milk. Bacteria per cubic centimeter Bacteria per cubic centimeter Bacteria per cubic centimeter Bacteria per cubic centimeter in milk in cooled in milk in cooled not cooled. milk. not cooled. milk. 1 186.000 8,600 ^ 8,500 3 400.000 1,880 1,950 2 233,000 1,470 2,160 4 1,350,000 1,750 1,700 The value of sudden cooling then, lies in the fact that milk after pasteurization is not allowed to cool slowly through temperatures below 37.8 C. (100 F.) where a rapid development of bacteria might occur. GROUPS OF BACTERIA WHICH SURVIVE PASTEURIZATION. The most important feature of this investigation has been the determination of the various groups of bacteria which survive the process of pasteurization. These bacteria form the basis of the bacterial growth in pasteurized milk. GRADES OF MILK STUDIED. Since the bacterial groups in various grades of milk vary with the quality of the milk, it was decided to examine three grades one of poor quality, one of fair quality, and one of good quality. The bacterial content of each of the samples of the three grades has been averaged as shown in Table 9. With poor milk designated grade A, the average of 20 samples showed a bacterial content of 32,950,000 per cubic centimeter. An average of 24 samples of grade B showed 3,451 ,000 per cubic centimeter, while the average bacterial count of 12 samples of grade C milk showed 24,700 per cubic centimeter. TABLE 9. Grades of milk studied. Average number of Number Grade. bacteria per cubic centimeter. samples averaged. A 32.9.50.000 20 B 3.451.000 24 C 24,700 12 METHODS. In the determination of the bacterial groups a method was employed which may be designated as the milk-tube method. This method, as described earlier in this paper, consists in picking off each colony on the plate and inoculating it into litmus milk tubes. The milk tubes 22 BACTERIA WHICH SURVIVE PASTEURIZATION. were incubated for 14 days at 30 C. (86 F.), and the bacteria from the plate were then divided into groups according to the reactions which they produced in the litmus milk tubes. By using this method it was possible to divide the bacteria developing on a plate into five groups, namely, the acid-forming and coagulating, the acid-forming, the inert which produce no change in milk, the alkali-forming, and the peptonizing groups. The acid-coagulating and the acid-forming groups are not necessarily composed of distinct organisms, although the acid-coagulating group probably included organisms which always coagulate milk, and in the acid group there are undoubtedly bacteria which never coagulate milk. The bacteria were divided by the reaction in litmus milk after incubation for 14 days at 30 C. (86 F.). It is impossible by the ordinary method of plating on litmus lactose gelatin to separate the alkali-forming bacteria from those whiqji are inert on gelatin on account of the fact that colonies of alkali-forming bacteria on litmus lactose gelatin plates do not form enough alkali to produce a change of the litmus. The alkali and inert groups must then be classed together and also include acid-form- ing bacteria which do not produce enough acid to give a red coloration on the plate. In order to show the value of the tube method a comparison with the plate method was made. Four different samples of milk were used, a portion of each being heated to 60 C. (140 F.) and also 65.6 C. (150 F.). The pasteurized milk was then plated on litmus lactose gelatin plates which were incubated at 18 C. (64.4 F.) for six days. A differential count was then made of the acid-forming, alkali or inert, and the peptonizing colonies. After counting, the colonies were picked off and inoculated into litmus milk in order to complete the milk-tube method. The results of the experiments are shown in Table 10. It may be plainly seen that the plate method was very inaccurate. The percentage of the acid group of bacteria was always increased by the milk-tube method of differentiation, and the alkali or inert groups were decreased. TABLE 10. Comparison of the milk-tube and plate methods for the determination of the bacterial groups in milk. Sample No. Temperature of heating. Acid group. Alkali or inert group. Peptonizing group. Milk- tube method. Plate method. Milk- tube method. Plate method. Milk- tube method. Plate method. 1 1 2 2 3 3 4 4 "C. F. 60.0 140 65.6 150 60.0 140 65.6 150 60.0 140 65.6 150 60.0 140 65.6 150 Per cent. 73.21 75.68 62.29 77.89 58.39 94.36 92.17 90.64 Per cent. 58.73 56.96 25.15 12.93 34.35 45.68 56.37 49.41 Per cent. 16.07 11.01 35.16 18.95 32.83 4.83 7.25 7.20 Per cent. 40.21 41.79 72.32 86.20 58.77 52.15 42.64 50.58 Per cent. 10.71 13.30 2.54 3.15 8.75 .80 .56 2.15 Per cent. 1.05 1.24 2.52 .86 6.87 2.15 .98 BACTERIAL GROUPS. 23 57.68 Usually the peptonizing group of bacteria was also increased. The differences in the differentiation of the bacterial groups is shown graphically in figure 4. The bacterial flora of four samples of milk pasteurized at 60 C. (140 F.) as shown by averages and deter- mined by the plate method consisted of 43.65 per cent of the acid group, 53.48 per cent of alkali or inert group, and 2.85 per cent of the peptonizing group. When determined by the milk- tube method the group percentages were changed to 71.51 per cent of the acid group, 22.83 per cent of the alkali or inert group, and 5.64 per cent of the peptonizing group. When milk was pas- teurizedat65.6C. (150 F.) there were marked differences in the bacte- rial flora when determ- ined by the two methods (see fig. 4). The re- sults from four samples were averaged. As de- termined by the plate method the bacterial flora consisted of 41.24 per cent of the acid group, 57.68 per cent of the alkali or inert group, and 1 .06 per cent of the . . peptonizing group. When determined by the milk-tube method the percentages were changed to 84.64 per cent of the acid group, 10.49 per cent of the alkali or inert group, and 4.85 per cent of the peptonizing group. It is evident from these experiments that the milk- tube method was much superior to the litmus lactose gelatin plate method of differentiation. Its advantages may be summarized as follows: 1. The milk-tube method is more accurate than the plate method for the differentiation of bacteria into groups. 2. It is possible by this method to separate the alkali-forming group of bacteria from the inert group, which is not possible on lit- mus lactose gelatin plates. 3. The activity of the bacteria in the various groups can to some extent be determined by their ability to produce changes in litmus milk after various lengths of incubation. FIG. 4. Comparison of the milk-tube and plate method for the determination of the bacterial groups in milk. 24 BACTERIA WHICH SURVIVE PASTEURIZATION. THE BACTERIAL GROUPS IN GRADE A MILK BEFORE AND AFTER PAS- TEURIZATION FOR 30 MINUTES AT 62.8 C. (145 F.). Throughout the study of the bacterial groups in raw and pasteur- ized milk the milk-tube method of differentiation has been used. The plating was on infusion agar, which was incubated for six days at 30 C. (86 F.). The milk used in the first experiments was of poor quality and constitutes grade A milk. Pasteurization was per- formed in sterile flasks and heated in a water bath for 30 minutes at 62.8C. (145F.). The percentage of the differ- ent bacterial groups was determined before and after pasteurization of the milk. In order to show the results in the most simple manner the group percentages of 10 samples of raw milk and 12 of pasteurized milk have been averaged and are shown graphically in figure 5. The aver- age of the. total acid group in raw milk was 46.88 per cent of the AC/0 /0.7/ ACJD GOAGULA77MG 3GJ7 \G/.07 2S.-3/ ALKAU 6.47 0EP7OM/Z/N6 /7.3/ /7.S/ 9.06 9.77 FIG. 5. Bacterial groups of grade A milk before and after pasteuri- zation for 30 minutes at 62.8 C. (145 F.). total bacteria. After pasteurization the per- centage was increased to 79.78 per cent. The acid coagulating group was decreased from 36.17 percent to 17.91 per cent. The inert group was also decreased and the alkali group slightly increased, from 6.47 per cent to 9.77 per cent, which was due to the averaging of one sample of pasteurized milk which was exceptionally high in the percentage of the alkali formers. The peptonizmg group was decreased from 17.31 per cent in the raw milk to 1 .39 per cent in the pasteurized milk. These results are most striking in so far that they are contrary to the most generally accepted ideas of the effect of pasteurization on the bacterial flora of milk. BACTERIAL GROUPS. 25 THE BACTERIAL GROUPS IN GRADE A MILK BEFORE AND AFTER PAS- TEURIZATION FOR 30 MINUTES AT 71.1 C. (160 F.). A second set of experiments was made, using grade A milk which Was pasteurized at 71.1 C. (160 F.) for 30 minutes and examined in the same manner as before. The averaged results of five samples of raw and six samples of pasteurized milk are shown in figure 6. The changes in the bacterial groups were about the same as at 62.8 C. (145 F.). "While the percentage AC/D COAGVLAT/A/G 24.O7 AC/D //.36 of the total acid group was higher than in the milk pasteurized at the lower tempera- tures, it will be seen that the percentage of the acid-coagulating group was smaller. The acid group was made up largely of slow acid-forming or- ganisms, as will be shown later. The alkali group was re- duced and the pepton- izing group reduced from 16.26 per cent in the raw to 0.53 per cent in the pasteurized milk. \B439 FIG. 6. Bacterial groups in grade A milk before and after pasteuriza- tion for 30 minutes at 71.1 C. (160 F.). THE BACTERIAL GROUPS IN GRADE B MILK BEFORE AND AFTER PAS- TEURIZATION FOR 30 MINUTES AT 62.8 C. (145 F.). The same experiments were repeated, pasteurizing a better quality of milk, grade B, at 62.8 C. (145 F.). The averaged results of 17 samples of raw and 20 samples of pasteurized milk are shown in figure 7. The total acid group was increased from 22.72 per cent in the raw to 66.76 per cent in the pasteurized milk. It will be noticed that the acid-coagulating group was increased from 12.98 per cent to 31.89 per cent in the pasteurized milk. The alkali and peptonizing groups were reduced from 19.66 per cent to 5.63 per cent and from 14.10 to 3.59 per cent, respectively. 67796 Bull. 16113 4 26 BACTERIA WHICH SURVIVE PASTEURIZATION. AC/D AC/D ( COAGULAT/A/G{ /2.98 34.87 3A&9 THE BACTERIAL GROUPS IN GRADE C MILK BEFORE AND AFTER PAS- TEURIZATION FOR 30 MINUTES AT 62.8 C. (145 F.). A third grade of milk of good quality, grade C, was studied in the same manner as the other grades. The percentage of the various bacterial groups changed in a similar manner as in the other experiments. The milk was pasteurized for 30 minutes at 62.8 C. (145 F.). Figure 8 shows the averaged results. It may be seen that again the total acid group was increased in percentage from 40.70 per cent to 73.10 per cent of the total bacteria. The acid- coagulating group, however, decreased in percentage from 33.85 per cent to 11.85 per cent. All the other groups were decreased in their percentage by pasteurization. These results show that in- stead of the acid-form- ing bacteria being destroyed by pasteuri- zation they were ac- tually increased in their percentage of the total bacteria sur- 24.OO viving the process. This does not mean that there were greater numbers of acid-form- ing bacteria after pasteurization but merely that a greater percentage of the bac- teria in pasteurized milk were of the acid-forming group than in the milk before heat- ing. The percentage of the other groups was lowered by pasteuri- zation. This is of particular interest in the case of the peptonizing group, which has been believed to constitute the majority of the bacteria which survive the heating process. It must be remembered that these results apply for the most part to milk pasteurized at 62.8 C. (145 F.), although they hold for the few samples studied which were heated at 71.1 C. (160 F.). So far as the practical side is concerned, only the results at 62.8 C. (140 F.) are of value, for the higher temperatures are seldom used in commercial work. FIG. 7. Bacterial groups in grade B milk before and after pasteuri- zation for 30 minutes at 62.8 C. (145 F.). BACTERIAL GROUPS. 27 THE BACTERIAL GROUPS IN MILK PASTEURIZED AT HIGH TEMPERATURES . In order to determine the bacteria which survive pasteurization at high temperatures, the bacterial groups were determined by the milk-tube method in seven samples of milk. Two samples were pasteurized at 76.7 C, (170 F.), three at 82.2 C. (180 F.), one at 87.8 C. (190 F.), and two at 93.3 C. (200 F.). All were heated in sterile flasks and the temperatures held for 30 minutes. The per- centages of the bacterial groups have been averaged at each temper- ature and are shown in figure 9. It will be seen that even at 76.7 C. (170 F.) a large percentage of the acid- forming group was found, the percentage 33.85 of the total acid-form- ing group, including both the acid-coagulat- ing and the simple acid- forming group, being 80.91 per cent. The bacteria of this group, however, produced acid very slowly, which differentiates them from those which sur- vive at lower tempera- tures. An increase in the percentage of the peptonizing group over that at the lower temperatures is notice- able. The average per- centage of the pepton- izing group at 62.8 C. (145 F.) ranged from 1.39 per cent to 4.47 per cent in three grades of milk. At 71 .1 C. (1 60 F.) the percentage was 0.53 per cent, while at 76.7 C. (1 70 F.) it was increased to 7.25 percent. The increase is of course produced by the greater destruction of the other groups. At 82. 2 C. (1 80 F.) a distinct change took place in percentage of the groups which survived the heating. The acid group was reduced greatly, while the peptonizing group was increased to 68.12 per cent of the total bacteria. The alkali group comprised 16.08 per cent of the bacteria, which is evidently a very high average. If a large number of samples had been studied, the average percentage of the PEPTOWZ/MG ( FIG. 8. Bacterial groups in grade C milk before and after pasteuriza- tion for 30 minutes at 62.8 C. (145 F.). 28 BACTERIA WHICH SURVIVE PASTEURIZATION. ACJO COA6ULAT/NC group would have probably been very low, since at 71.1 C. (160 F.) and 76.7 C. (170 F.) only a very small percentage survived. The bacterial groups at the higher temperatures were similar to those at 82.2 C. (180 F.). The almost total absence of the acid-coagulating group and the low percentage of the acid group, together with the high percentage of the peptonizing group, is very noticeable. THE ACTIVITY OF BACTERIA OF THE VARIOUS GROUPS ISOLATED FROM RAW AND PASTEURIZED MILK MEASURED BY THEIR ABILITY TO PRODUCE CHANGES IN LITMUS MILK AFTER DIF- FERENT LENGTHS OF INCUBATION. The activity of the bacteria found in raw and pasteurized milk is important since acid-forming bacteria which coagulates milk in 48 hours produce a more normal souring iij pasteurized milk than the Kxyra. .^^^^k s l w a id - producing forms which may be overgrown by the pep- tonizing bacteria. Or- ganisms of the pep- tonizing group which cause peptonization rapidly are of far greater importance than those which re- quire long incubation to produce changes in milk. During the de- termination of the bac- terial groups by the milk-tube method the reactions produced by the different cultures of bacteria were recorded after 2, 5, and 14 days' incubation. The length of time required to produce changes in litmus milk served as a measure of the activity of the culture. It then fol- lows that the changes in the group relations as determined by the litmus milk tube reactions gave a general indication of the activity of the bacteria which composed the group. The activity of bacteria of the various groups isolated from three grades of milk has been studied. ffPTOMZ/fiiG FIG. 9. Bacterial groups which survive pasteurization for 30 min- utes at 76.7 C. (170 F.), 82.2 C. (180F.), 87.8 C. (190 F.), and 93.3 C. (200 F.). GRADE A MILK BEFORE AND AFTER PASTEURIZATION AT 62. 8 C (145 F.) FOR 30 MINUTES. 0. The results shown in Table 1 1 indicate the rapidity of the growth of the bacteria of the various groups isolated from milk before and after pasteurization at 62.8 C. (147> F.) as measured by their ability to produce a change in litmus milk. As may be noted from the table, BACTERIAL GROUPS. 29 plates were made of infusion and casein agar and the colonies develop- ing on each, medium were picked off and differentiated by the tube method. The group percentages in the table represent the average of a number of samples. In general it may be said that casein agar seemed to favor the growth of the alkali forming and the peptonizing bacteria so 'that their group percentages were usuaUy higher when determined on that medium. In order to show the results more plainly the averages have been plotted as shown in figure 10. The blocks in the columns represent the bacterial group percentages determined by the tube method after 2, 5, and 14 days' incubation in milk. The column at 2 days, for example, shows the group percent, ages determined by the reactions of the colonies picked from an infusion agar plate. The percentages were determined by the re- actions of the milk tubes after 2 days' incubation at 30 C. (86 F.). The tubes were then incu- bated for 3 days more and the group percent- AC/D COAGULATWG 3OM/NUTES FIG. 10. Changes in the bacterial group relations in grade A milk when determined by litmus milk reactions after different lengths of incubation. Milk pasteurised at 62.8 C. (145 F.) for 30 minutes. ages again determined by the reactions. Incu- bation was then contin- ued for 14 days, when the final reactions were recorded. The figure shows that the percent- age of the total acid group was greater in raw milk after two days' incubation than in pas- teurized. After five days, however, the total acid group was higher in percentage in pasteurized milk. After 14 days the total acid group in pasteurized milk was much larger. This shows that the bacteria of the acid group in pasteurized milk develop slower, or at least produce acid slower, than those in raw milk. Since the differentiation of the groups was based on the change produced in litmus milk by the bac- teria, it may be seen from the figure that as a rule the alkali-forming bacteria did not develop enough alkali to change the color of litmus, which differentiates them as belonging to the alkali group, until the fifth day of incubation. While the figure may seem rather compli- cated, a careful study clearly shows the difference in the bacterial groups in raw and pasteurized milk based on the rapidity with which they produce changes in litmus milk. 30 BACTERTA WHICH SURVIVE PASTEURIZATION. TABLE 11. Changes in the bacterial group relations in grade A milk when determined by litmus milk reactions after different lengths of incubation Milk pasteurized at 62.8 C. (145 F.)for 80 minutes. Bacterial groups. Infusion agar plates. Casein agar plates. Raw milk. Pasteurized milk. Raw milk. Pasteurized milk. 2 days. days. 14 days. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. Acid coagulating Acid . Perot. 24. IS 18.08 42.94 1.29 13.47 99.96 Per ct. 32.72 14.64 33.23 4.02 15.37 Perot. 36.17 10.71 29.31 6.47 17.31 Perct. 6.01 19.01 74.69 .02 .26 Perct. 9.52 48.01 35.13 6.55 .77 Perct. 17.91 61.87 9.06 9.77 1.39 Perct. 7.89 15.13 62.26 .70 13.99 Perct. 16.96 14.34 45.95 5.69 17.04 Per ct. 19.51 12.12 32.99 17.58 17.77 Perct. 2.42 11.63 85.80 Perct. 4.34 42.17 48.67 4.17 .62 Perct. 15.12 51.95 15.98 15.73 1.20 Inert Alkali Peptonizing Total Number of sam- ples averaged. . .12 99.98 99.97 99.99 99.98 100.00 99.97 99. 9S 99.97 99.97 99.97 99.98 10 10 10 12 12 12 7 7 7 10 10 10 GRADE A MILK BEFORE AND AFTER PASTEURIZATION FOR 30 MINUTES AT 71.1 C. (160 F.). Earlier in this paper it was shown that the percentage of the total acid group was higher in grade A milk pasteurized at 71.1 C. (160 F.) than at 62.8 C. (145 F.). It is natural to suppose, then, that milk pasteurized at the higher temperatures would sour more quickly than when heated at the lower temperatures. Such is not the case, however; first, because the total number of bacteria are less in the milk pasteurized at 71.1C.(160F.),and, second, because the acid group of bacteria is less active. This last reason is shown by the changes in the bacterial group rela- tions as determined in litmus milk by differ- ent lengths of incuba- tion. Table 12 shows the averaged group percentages deter- mined from infusion and casein agar plates. The results are shown to better advantage in figure 11. It will be noticed that after two days' incubation only a small percentage of acid-forming bacteria were recorded in the pasteurized milk and none of the acid-coagulat- FIG. 1 1 . Changes in the bacterial group relations in grade A milk when determined by litmus milk reactions after different lengths of incu- bation. Milk pasteurized at 71.1 C. (160 F.) for 30 minutes. BACTERIAL GEOUPS. 31 ing group. After five days the acid group was increased greatly, but there was only an extremely small percentage of the acid-coagulating group. Even after 14 days' incubation the reactions showed only a small percentage of the acid-coagulating group. In both raw and pasteurized milk the slow growth of the alkali group is shown by the fact that only a small percentage was found in the raw milk after five days' incubation of the litmus milk tubes, while none was found in the pasteurized milk until after 14 days' incubation. TABLE 12. Changes in the bacterial group relations in grade A milk when determined by litmus milk reactions after different lengths of incubation Milk pasteurized at 71.1 C. (160 F.)for 30 minutes. Bacterial groups. Infusion agar plates. Casein agar plates. Raw milk. Pasteurized milk. Raw milk. Pasteurized milk. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. Acid coagulat- ine . Perct. 16.58 Perct. 20.22 Perct. 24.07 Perct. Perct. 0.17 85.29 14.14 Perct. 5.49 84.59 8.99 .40 Perct. 12.47 10.65 64.30 .13 Perct. 16.49 6.44 58.85 2.43 Perct. 17.50 7.22 47.28 10.06 Perct. Perct. Perct. 10.88 63.44 24.93 Acid 16.35 52.95 14.60 49.51 .89 11.36 43.51 4.77 12.03 87.56 7.67 92.26 70.10 29.53 Inert Alkali Peptonizing Total Number of sam- ples averaged. 14.11 14.76 16.26 .39 .39 .53 12.43 15.77 17.92 .06 .36 .70 99.99 99.98 99.97 99.98 99.99 100.00 99.98 99.98 99.98 99.99 99.99 99.95 5 5 5 6 6 6 9 9 9 11 11 11 GRADE B MILK BEFORE AND AFTER PASTEURIZATION FOR 30 MINUTES AT 62.8 C. (145 F.) The changes in the bacterial group relations determined by the milk-tube method after various lengths of incu- bation showed a differ- ence from those in grade A milk. Table 13 shows the averages of the group percentages which are plotted in figure 12. A study of the plot shows that the percentage of the total acid group and the acid - coagulating group itself was greater in the pasteurized milk fvfmw/zwei after the two days' in- OAKS cubation period than in the raw milk. In other words, the activity of the acid-producing or- ganisms which survived the heating was as great as those in raw milk. The reason for the difference in the two grades of milk is not apparent. a*rs-*z 5 /# TUffGSEQfi'f/l.tr 'C (/tStj 7&S Fio. 12. Changes In the bacterial group relations in grade B milk when determined by litmus milk reactions after different lengths of incubation. 32 BACTERIA WHICH SURVIVE PASTEURIZATION. TABLE 13. Changes in the bacterial group relations in grade B milk when determined by litmus milk reactions after different lengths of incubation Milk pasteurized at 62.8 C. (145 F.)for 30 minutes. Bacterial groups. Infusion agar plates. Casein agar plates. Raw milk. Pasteurized milk. Raw milk. Pasteurized milk. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. Acid coagulat- ing Perct. 6.59 11.39 73 45 Perct. 9.54 12.55 58.04 Per ct. 12.98 9.74 43.51 Per ct. 20.05 11.27 67.46 Per ct. 23.12 27.48 45.02 Per ct. 31.89 34.87 24.00 Perct. 6.14 4.67 81.37 Per ct. 7.66 5.17 66.89 Per ct. 8.45 7.18 50.09 Perct. 10.58 6.72 82.30 Perct. 12.86 27.98 55.84 Perct. 18.73 29.72 40.63 Acid Inert Alkali Peptonizing Total Number of sam- ples averaged .21 8 35 7.86 11 99 19.66 14.10 "Lai 2.39 1.98 5.63 3.59 .09 7.69 6.53 13.73 17.79 16. 47 .03 .35 2.43 .87 7.92 2.99 99.99 99.98 99.99 99.99 99.99 99.98 99.96 99.98 99.98 i 99.98 99.98 99.99 16 17 17 19 20 20 17 18 18 21 22 22 GRADE C MILK BEFORE AND AFTER PASTEURIZATION FOR 30 MINUTES AT 62.8 C. (145 F.) When grade C milk was studied in the same manner, the per- centage of the acid-coagulating group in pasteurized milk was found to be small and the group inactive. The averaged results in Table 14^ and shown graphically in figure 13, bring out this point. It will be seen that the acid-coagulating group percentage was very small up to the 14 days' incubation period. The acid group also was rather inactive as indicated by the percentages. A large part of the pepton- izing group developed slowly, as indicated by the increase in the percentage of groups shown in the various plots. TABLE 14. Changes in the bacterial group relations in grade C milk when determined by litmus milk reactions after different lengths of incubation Milk pasteurized at 62.8 C. (146 F.)for 30 minutes. Bacterial groups. Infusion agar plates. Casein agar plates. Raw milk. Pasteurized milk. Raw milk. Pasteurized milk. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. 2 days. 5 days. 14 days. Acid coagulat- ing Perct. 18.10 15.18 64.54 .20 1.96 Ptra,. 31.33 9.42 49 44 1.66 8.13 Perct. 33.85 6.85 43.13 3.33 12.81 Perct. 0.06 6.82 91.21 1.89 Per ct. 0.35 43.02 53.46 .06 3.09 Per ct. 11.85 61.25 22.06 .36 4.47 Perct. 7.25 5.59 84.53 2.61 Per ct. 9.96 7.17 76.09 1.11 5.65 Per ct. 9.90 7.94 63.79 5.90 12.44 Perct. 0.36 1.96 93.02 4.65 Perct. 0.36 19.84 71.53 .90 7.35 Perct. 3.19 34.57 49.74 2.99 9.49 Acid Inert Alkali Peptonizing Total Number of sam- ples averaged 99.98 99.98 99.97 99.98 99.98 99.99 99.98 99.98 99.97 99.99 99.98 99.98 12 12 12 11 11 11 9 9 9 11 11 11 It is interesting to note the activity of the total acid group in pasteurized milk of grade B, and although the average bacterial count was high this grade of milk more nearly represents the ordinary quality of milk which is pasteurized commercially than either grades A or C. PEECENTAGE OF ACID FOEMING BACTEEIA. 33 AC/O COAGULAT/HG} AC/D COMPARISON OF THE PERCENTAGE OF ACID-FORMING BACTERIA IN RAW AND PASTEURIZED MILK. As has already been shown, the acid bacteria instead of being destroyed by pasteurization for 30 minutes at 62.8 C. (145 F.) are actually increased in their percentage of the total bacteria. The large percentage of the / acid group which survive AC/O } the process are shown in the frequency curve in figure 14. The number of samples were plotted as ordinates and the per- centage of the acid-form- ing bacteria as abscissae, the ranges in percent- ages from 0-1, 1-10, 10-20, etc., being taken as points. Thirty-nine samples of raw and 42 of pasteurized milk were ex- amined. The samples of milk were pasteurized for 30 minutes at 62.8 C. ft A n T? '\ T* '11 K FlG> 13 '~~ chan S es in the bacterial group relations in grade Cmilk ,145 r.}. It Will De Seen wnen determined by litmusmilk reactions after different lengths from the figure that with of incubation. the raw milk the peak of the curve is at 1-10, while one peak of the curve of pasteurized milk is at 10-20 and another at 50-60. In other 20^30 30-40 40-50 SO-SO 6O-7O TV'SO BO-OO 9O-VO ffff CNT Of AC/O FQf?/*f/A/G BAGTFf?/* FIG. 14. Comparison of the percentage of the acid-forming bacteria in raw and pasteurized milk. words, the majority of the samples of raw milk contained from 1 to 10 per cent of bacteria of the acid group. The number of samples con- 67796 BulU61 13 5 34 BACTEEIA WHICH SURVIVE PASTEURIZATION. taming higher percentages gradually diminished until no sample contained over 80 per cent. Among the samples of the pasteurized milk, it may be seen from the curve that the majority of them contained from 10 to 60 per cent of the acid group and some samples as high as 90 per cent. .-' COMPARISON OF THE PERCENTAGE OF PEPTONIZING BACTERIA IN RAW AND PASTEURIZED MILK. In the same manner 39 samples of raw milk and 42 samples of milk pasteurized for 30 minutes at 62. 8 C. (145 F.) have been grouped according to the percentage of peptonizing bacteria which they con- tained. A comparison of the frequency curves in figure 15 shows that the pasteurized milk contained a lower percentage of the pepton- izing group than did the raw milk. A large number of samples of pasteurized milk contained between and 1 per cent of peptonizing bacteria, while only one sample of raw milk con- tained that low percent- age. The same number of samples of both raw and pasteurized milk contained from 1 to 10 per cent. It will also be seen from the curve that a few samples of i-/o /o-zo 20-30 30-40 *o-5o &>-&> V^f}, raw anf j r>ast,PUr- f>fff Cf/VT Of PFP7T3M/Z/MG BACTfff/A FIG. 15. Comparison of the percentage of the peptonizing bacteria ized milk Contained in raw and pasteurized milk. ^^ ^^ j t() 1Q ^ cent of the peptonizing group. However, in a comparison of the raw and pasteurized milk it was found that a larger number of samples of raw milk contained the high percentages of peptonizers. AVERAGE PERCENTAGES OF THE BACTERIAL GROUPS OF RAW MTT/KT WHICH SURVIVE PASTEURIZATION. The effect of pasteurization upon the bacteria in milk may be shown by a determination of the percentage of the bacterial groups of raw milk which are found after heating. This percentage was obtained by subtracting the percentage reduction by pasteurization of the various groups from 100 per cent. The samples of raw and pasteurized milk were plated on infusion and casein agar and the percentage reduction of the various groups determined by the milk tube method. Table 15 shows the averaged results from infusion and casein agar plates, using three grades of milk for examination. EFFECT OF PASTEURIZATION ON NUMBER OF BACTERIA. 35 It may be seen that a greater percentage of the acid group of bacteria in the raw milk remained after heating than any of the other groups. As a rule a smaller percentage of the peptonizing group survived than either of the other groups. In general the result when casein agar was used was higher, although the reason is not apparent. TABLE 15. Average percentages of the bacterial groups of raw milk which survive pasteurization by the "holder" process. Pasteurization temper- ature. Media. Bacterial groups. Acid. Inert. Alkali. Pepton- izing. Grade A, 62.8 C. (145 F.).. Grade A, 71.1 C. (160 F.). . Grade B, 62.8 C. (145 F.). . Grade C, 62.8 C. (145 F.).. (Infusion agar 1.27 4.26 1.71 2.34 6.98 6.50 4.55 13.09 0.37 .31 .12 .39 1.48 2.06 1.59 4.49 0.05 1.55 .10 1.2T 2.05 .19 1.13 0.05 .08 .01 .03 1.86 .51 1.24 1.25 \Casein agar (Infusion agar .... \Casein agar \Casein agar /Infusion agar \Casein agar THE EFFECT OF PASTEURIZATION ON THE NUMBER, OF BACTERIA OF DIFFERENT GROUPS IN MILK. In order to show the effect of pasteurization on the numbers of the bacteria of different groups, Table 16 has been arranged. This shows the calculated number of bacteria in each group determined from the average total counts on infusion agar and the group per- centages as shown by the milk-tube method. To illustrate this fur- ther take the samples from dairy A, as shown in Table 16. The bacterial count of 12 samples of raw milk averaged was 21,100. The averaged group percentages of the 12 samples was 33.85 per cent of the acid-coagulating group, 6.85 per cent of the acid group, and so on. The acid-coagulating group contained on an average 33.85 per cent of the average total count, or approximately 7,160 bacteria per cubic centimeter. The other bacterial numbers were calculated in the same way. 36 BACTERIA WHICH SURVIVE PASTEURIZATION. TABLE 16. Comparison of the number of bacteria of different groups in milk before and after pasteurization. Average total count per cubic centimeter. Average group per- centage. Group. Calculated number of each group per cubic centimeter. 33.85 Acid-coagulating 7,160- 6.85 Acid 1,450 Raw milk, 21, 100. Average of 12 samples. Dairy A. 43.13 Inert 9,120 3.33 Alkali 705 12.81 Peptonizing 2,700 11.85 Acid-coagulating 50 61.25 Acid 257 Pasteurized mflk, 62.8 C. (145 F.), 30 minutes, 420. Average of 11 samples Dairy A 22.06 Inert 92 .36 4.47 Alkali Peptonizing 2 19 12.98 Acid-coagulating 368,800 9.74 Acid 276,700 Raw mflk, 2,840,000. Average of 17 samples. Dairies B and C 43.51 Inert 1,236,000 19.66 14 10 Alkali Peptonizing 558,000 400,000 31.89 Acid-coagulating 3,760 34.87 Acid 4,110 24,00 Inert 2.830 5.63 Alkali 660 3.59 Peptonizing 420 36.17 Acid-coagulating 8,200,000 10.71 Acid 2,430,000 Dairy D 29.31 Inert 6, 700, 000 6.47 17.31 Alkali Peptonizing 1, 480, 000 3,900,000 17 91 Acid-coagulating 14.900 61.87 Acid 51,500 9.06 Inert 7.500 9.77 Alkali 8,130 1.39 Peptonizing 1,150 The most striking features of the table is the high number of acid- forming bacteria in proportion to the low number of peptonizers in the pasteurized milk and the marked difference in the numbers of peptonizing bacteria before and after pasteurization. The milk from dairy A was exceptionally good, the average total count of the raw milk being 21,100 and after pasteurization for 30 minutes at 62.8 C. (145 F.) only 420 per cubic centimeter. The peptonizers were reduced from 2,700 to 19 per cubic centimeter. The milk from dairies B and C was of poorer quality, the average count of the raw milk being 2,840,000. After pasteurization the count was 1 1 ,700. The peptonizers were reduced by the process from 400,000 to 420 per cubic centimeter. In the last part of the table the results of the examination of poor milk is shown. The average count was 22,800,000 bacteria per cubic centimeter before and 83,000 after pasteurization for 30 minutes at 62.8 C. (145 F.). The raw milk contained an average of 3,900,000 peptonizers per cubic centimeter and after pasteurization only 1,150 per cubic centimeter. Through- out the whole series may be noticed the low number of alk ah' -forming bacteria in the pasteurized milk in proportion to those in the raw milk. After a comparison is made of the kinds of bacteria in the raw milk BACTEKIAL DEVELOPMENT IN MILK. 37 from dairies B and C and in the extremely poor milk from dairy D with the same milk after pasteurization there should be no question as to the beneficial effect of the process. THE BACTERIAL DEVELOPMENT IN MILK PASTEURIZED IN THE LABORATORY AND HELD AT DIFFERENT TEMPERATURES. After having determined the groups of bacteria which survived pasteurization, their subsequent development was traced in a few samples of milk. The changes in the bacterial flora of milk pasteurized for 30 minutes at 62.8 C. (145 F.) during storage at different temperatures has been studied in a poor, medium, and good grade of milk. One sample of each grade of milk was divided and each part pasteurized in a sterile flask. After pasteurization each sample was held in the ice box at approximately 7.23 C. (45 F.) for 18 hours; then one flask was held at room temperature, the other was kept in the ice box. After pas- teurization and on each successive day each sample was plated to determine the number of bacteria which were then differentiated by the milk-tube method. On account of the great amount of work involved and time consumed in determining the bacterial flora by the milk-tube method only one sample of each grade was studied. 38 BACTERIA WHICH SURVIVE PASTEURIZATION. - e S 4 sS i : : i i S5 SKg b- CO C 1 ! o : : : : : SSSSg ij.il -^^Ot> i i i : : S *O COO 3 00 : : : : : 283S" : : : : : 1 1 - c? o C^OiTt*cOOO ... 8SS8 ^r 5. ~3 : : : : : -8*-. . . . . , -H- I- SS : ^ sss 8 i 1 <-> a o 1-1 W to en S!S : "e *w 3 "t ^rH?tM N O b- .2 ^ milkh *t O CO OC to CN to CO CO CO i ^ | o || I -l| ssSHS JH Of to rHCN tO * : : : : : 88 : SS : i P< S, . t^. CC CC CC r~S ccS 88 i * *"*>' o m 10 a 1 (- tooc tc S- 1 o o 1PI i i i i8 S . i t , O CO CO ' 8 i "S M ^OCOtjO, ggg SSg CO CSI b- W5 C i is :8 88 i i rO o tO W C^l CD T t CO CO i~i r- 1 g^"5;- i i 5 i 8 SS : 5 M N C$ O C^ O C^ CO CS o : S ^^^ 8 ^* " S2g coSS'S . to to r~j P3 H Holding temperatur c 1 I i i i i : i i i : H | : i i i wg-;:::.cj ; : : : : H 1. ; ; : : | w i fe i : SI *" * s *3 - s ' ^ g 5 05 1 "ill f * 1 '^ Icebox, W'C. (50 F. Acid-coagulating Acid Inert Alkali : .3 i i i : i Ss :8 i i i i i" Jf w, ; ; ; ; Ci P o "^ . s 5 "c ft^ & ; := fe i x" s / Flo. 26. Gas produced by culture / . ized milk or of inefficient pasteurization. Several boards of health are at present using such a test. If gas-forming organisms do not survive pasteurization, then of course their presence in pasteurized milk means recontamination or inefficient pasteurization. If, how- ever, certain types of gas-forming bacteria do survive the process, then the test is of no value. In order to determine if gas-forming bacteria survived pasteurization for 30 minutes at 62.8 C. (145 F.), 19 samples of raw milk were heated in sterile flasks. Fermentation 50 BACTERIA WHICH SURVIVE PASTEURIZATION. tubes in sets of four were then inoculated with 1 cubic centimeter and 5 cubic centimeters of both the raw and the pasteurized milk. The results are shown in Table 18. Twelve of the samples of raw milk showed the presence of gas-forming organisms in lactose bile tubes when 1 cubic centimeter of milk was used for inoculation. After pasteurization three samples showed gas when 1 cubic centimeter was used and eight samples when 5 cubic centimeters of milk was used. When dextrose liver broth fermentation tubes were used, a larger number of samples of the pasteurized milk showed the pres- ence of gas-forming bacteria. It may be seen from the table that the number of gas producers in the raw and pasteurized milk was low as determined by the dilution and the number of tubes of each set of four which showed gas. For example, sample 1, four tubes of lactose bile, each inoculated with 1 cubic centimeter of raw milk, showed no gas, which means that no gas-forming organisms were present in 4 cubic centimeters of the raw milk. Four tubes, each inoculated with 5 cubic centimeters of the same milk pasteurized, showed gas in two tubes. That means at least one gas-forming organism in every 10 cubic centimeters of the milk. TABLE 18. Gas production in lactose-bile and dextrose-liver broths inoculated with raw milk and milk pasteurized at 66.8 C. (145 F.) for 30 minutes. Four tubes used in each dilution. Gas produced in Lactose-bile broth. Dextrose-liver broth. Sample num- Inocu- Inocu- Inocu- Inocu- ber. Inocu- lated lated Inocu- lated lated lated withl with 5 lated withl with 5 withl c. c. pas- c. c. pas- with 5 c. c. pas- c. c. pas- c. c. raw teur- teur- c. c. raw teur- teur- milk. ized ized milk. ized ized milk. milk. milk. milk. Tubes. Tubes. Tubes. Tubes. Tubes. Tubes. 1 2 3 2 2 3 2 3 1 4 3 1 1 1 5 3 2 6 3 3 3 2 7 3 1 3 1 8 2 9 2 2 10 2 11 1 3 2 1 12 13 4 3 1 4 14 3 4 15 1 3 1 2 1 1 16 4 2 4 4 2 2 17 4 4 2 18 4 19 3 1 4 1 Milk incubated at 37 C. for 18 hours and then pasteurized. Litmus lactose agar plates were made and inoculated from almost every sample which showed gas in the bile tubes, but no organisms of the colon type were found. The only gas producer which was THEEMAL DEATH POINT. 51 found was a spore-forming anaerobic bacillus of the butyric group. One of these organisms, known as culture T, produced acid in dex- trose, galactose, saccharose, raffinose, and starch broths, and gas in dextrose, galactose, lactose, saccharose, and starch broths. In this work no organisms of the colon group were found to survive pas- teurization, but gas was often produced in lactose bile tubes inocu- lated with pasteurized milk. The gas production, however, was not due to organisms of the colon group, but to those of the butyric-acid group. This anaerobic butyric bacillus produced gas in lactose bile tubes and would appear in the preliminary tests for the colon bacillus. Therefore, if only lactose bile fermentations were used to determine the presence of the colon group in pasteurized milk, the anaerobic butyric types of bacteria would produce fermentations which would be taken as an indication of the colon group. It is evident, then, that the presence of the colon group of organisms in milk must be deter- mined by something more than simply lactose bile fermentation tubes. These results are by no means conclusive, for if a larger number of samples had been examined colon bacilli might have been found which survived pasteurization. De Jong and De Graaff, 1 working on various strains of Bacillus coli, found some forms which required 30 minutes heating at 70-72 C. (158-161.6 F.) to destroy. Zelenski 2 also found some strains of B. coli which resisted high temperatures. Evidently resistant types of the colon group do exist and that fact complicates the value of the colon test for reinfec- tion of pasteurized milk. THERMAL DEATH POINTS OF BACTERIA WHICH SURVIVE PASTEURIZATION. As a matter of purely scientific interest the thermal death points of several bacteria which survived pasteurization were determined. The determinations were made by heating 2 days' old lactose dibasic broth cultures for 30 minutes in Sternberg bulbs. After heating, the contents of the bulbs were inoculated into litmus milk tubes. Cul- tures were selected from the acid, alkali, and peptonizing groups for the experiment. The results are shown in Table 19. Culture 40 of the acid group stood as high as 79.4 C. (175 F.). Numbers 58 and 156 were destroyed at 73.9 C. (165 F.) and culture 81 at 65.6 C. (150 F.). One culture of the alkali group was destroyed at 65.6 C. (150 F.). Culture 450 of the peptonizing group was destroyed at 71.1 C. (160 F.), while the thermal death point of culture 456 was indefinite, although about 79.4 C. (175 F.). i De Jong, D. A., and De Graaff, W. C. Die Coll-Kontrolle der pasteurisierten Milch. [Nederlandsch Weekblad voor Zuivell>ereiding en Veeteelt, vol. 12, Nos. 37 and 38, 1906]. Abstract: Milchwirtschaft- liches Zentralblatt, vol. 3, No. (i, pp. 265-2o ^ ptj s FH S fe 8 h 8 fe S SH S d d d d d d d 00 s o 8 1 ~ Ol M i- 52 * gi ACID GROUP. Culture 58 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ Culture40 + + + + H Culture 81 Culture 15C + + ++ ++ ALKALI GROUP. Culture 311 + + + + + + ++ ++ ++ H FEPTONIZTXG GROUP. Culture 450 ... . . . ++ ++ 4 H ++ H Culture 45ti In one sample of milk heated at 82.2 C. (180 F.) for 30 minutes it was found by the tube method of group differentiation that a very large percentage of the acid group survived. Sixty-four milk cul- tures of the resistant acid bacteria were examined roughly for their thermal death point by inoculating fresh litmus milk tubes with one drop of the milk culture. One set of milk tubes was then heated for 30 minutes at 87.8 C. (190 F.) in a water bath and another set at 93.3 C. (200 F.) for the same length of time. Of the 64 tubes heated at 87.8 C. (190 F.) 27 showed growth, but all were destroyed at 93.3 C. (200 F.). These heat-resistant acid-forming organisms are of great importance in connection with the pasteurization of milk. From a purely scientific standpoint they are of interest, since in general the thermal death point of vegetative cells has been believed to be below 60 C. (140 F.). From the numbers of these heat-resistant organisms it is evident that they are not u-icommon in milk. THE POSSIBLE USE IN PASTEURIZATION OF ACID - FORMING BACTERIA OF HIGH THERMAL DEATH POINT. Since certain strains of typical lactic-acid bacteria are known to be able to withstand pasteurizing temperatures, it seems possible to make use of their resistance. It has been suggested that cultures of lactic-acid bacteria be added to milk after pasteurization in order to insure a normal souring. A process of that nature would necessarily involve a danger of contamination by pathogenic bacteria from employees at the dairy, since the acid-forming bacteria are to be added after the heating process. By the use of a lactic-acid organism of high thermal death point it would be possible to add the culture to the QUALITATIVE STUDY OF BACTERIAL FLORA. 53 raw milk and then pasteurize. One experiment was tried to show this point. As may be seen from Table 20, a sample of certified milk was divided into two parts of 800 cubic centimeters each. Both portions were heated in sterile flasks for 30 minutes at 62.8 C. (145 F.). To one flask 5 cubic centimeters of a high-temperature resistant lactic- acid culture was added before pasteurization. The effect of the addi- tion of the culture can be seen by the change in the milk when held at room temperature. After four days the milk without the culture was curdled with a rennet curd and was partly peptonized, very little acid having developed. The other sample to which the heat resistant cul- ture was added was curdled on the fourth day and showed a smooth firm acid curd with no digestion. By the addition of the culture before pasteurization in sealed bottles subsequent infection would be impossible. This possibility of addition of high temperature resistant lactic-acid bacteria is merely suggested for those who advocate the addition of cultures after pasteurization. TABLE 20. Change in acid development in pasteurized milk produced by the addition of high-temperature resisting lactic-acid bacteria previous to pasteurization. PER CENT LACTIC ACID. [Samples held at room temperature, 21.1-23.9 C. (70 c -75 F.).] Lactic acid after 1 2 3 4 day. day. days. days. days. Perct. Perct. Perct. Perct. Perct. Certified milk pasteurized at (145) C2.8 C. for 30 minutes Some of same milk with 5 c. c. of high-temperature resisting lactic-acid cul- 0.17 0.17 0. 10 10.31 ture added to 800 c.c. of milk; milk then pasteurized at 02.8 C. (145 F.) for 30 minutes .17 .17 . 18 2.50 i Curdled and peptonized. Curdled, acid curd. QUALITATIVE STUDY OF THE COMPLETE BACTERIAL FLORA OF ONE SAMPLE OF PASTEURIZED MILK. Since in the qualitative study of the bacteria surviving pasteuriza- tion a few typical cultures representing each group were selected from each sample, it was thought advisable to make a more thorough quali- tative study of the bacteria in one sample of pasteurized milk. The complete bacterial flora of one sample of milk pasteurized for 30 minutes at 60 C. (140 F.) and another portion of the same milk pasteurized at 65.6 C. (150 F.) were studied qualitatively. Litmus lactose gelatin plates were made, and after 6 days' incuba- tion at 18 C. (64.4 F.) each colony was picked off, purified by plating, and studied. The A series of cultures is made up of 176 cul- tures picked from a plate from milk pasteurized at 60 C. (140 F.). The B series is composed of 92 cultures from a plate from milk pas- teurized at 65.6 (150 F.). Each series has been divided into five BACTERIA WHICH SURVIVE PASTEURIZATION. IK QUALITATIVE STUDY OF BACTERIAL FLORA. 55 divisions according to the differentiation of the bacteria. Table 21 shows the A series. Division 1, figure 27, shows the division of the 176 cultures of bacteria which survive pasteurization at 60 C. (140 F.) into three groups the acid, the alkali, and the peptonizing group. Per cent. Acid group 94. 88 Alkali group 1. 1 3 Peptonizing group 3. 97 In division 2 the acid group has been further divided as shown and the percentages given. Each division shows the complete percent- ages of the various classes. Division 5 shows the final classification and percentages. Each subdivision of division 5 has been given a letter which makes it possible by the use of the following key to deter- mine the complete reactions of the bacteria in each subdivision : KEY TO LETTERS AND SIGNS USED IN FORMULA SHOWING CULTURAL REACTIONS OF THE SUBDIVISIONS. The formula reads in the order of the numerals : 1. B Bacilli. C Cocci. 2. A Gelatin not liquefied. X Gelatin liquefied. Xi-1-10 mm. of liquefaction. X 2 -over 10 mm. of liquefaction. 3. Gram () 4. Nitrate reduced N () 5. Dextrose (sugar broth) D ( ) 6. Lactose (sugar broth) L () 7. Saccharose (sugar broth) C () 8. Galactose (sugar broth) G () 9. Mannite M () 10. Raffinose (sugar broth) R () 11. Glycerin broth Glyc. () 12. Inulin broth I () 13. Starch broth S () 14. Pigment production : - -P () The formulas of the reactions of the subdivisions are given in Table 21. 56 BACTEBIA WHICH SURVIVE PASTEURIZATION. QUALITATIVE STUDY OF BACTERIAL FLORA. 57 TABLE 21. Cultural reactions of organisms in subdivisions cultures of A series from milk pasteurized for 30 minutes at 60 C. (140 F.). Subdivi- sion. Formula of reactions. CA+N-D+L+CG+MR-Glyc.-I-S. CA-fN-D+L+CG+MR + Glyc.-I-S+. CA + N-D+L+CG+MR+Glyc.-I-S-. CA+N-D+L+CG+MR-Glyc.+I-S+. CA+N-D + L+CG + MR-Glyc.4l-S-. CA+N-D+L+CG+MR-Glyc.-I-S-P+. CA+N+D+L+CG+MR + Glyc.-I-S+. CA+N+D-f-L+CG+MR-Glye.-I-S-. CA + N4- D+L+CG+MR+ Glyc.-I-S+. CA+N+D + L+CG f MiR + Glye.-I-S-. CA+N+D+L+CG + M(other reactions ()). CA-N-D+L+CG+-MR-Glyc.-I-S. CA-N-D+L+CG + M(other reactions ()). CA-N+D+L+CG+MR-Glvc.+I-S. CA+N+D-L-C-G-M-R-Giyc.-I-S-P-Kred). CXs-N-D + L+C-f-G + M-R-Glye.-I-S-. CXs+N-D+L+C+G + MR-Glyc.I-S-I'. The formula reads as follows, as shown in table: Subdivision A : C. Coccus. A. Gelatin not liquefied. + . Gram+. N . Nitrate not reduced. D-f-. Acid produced in dextrose broth. L+. Acid produced in lactose broth. C . May or may not produce acid in saccharose broth. G+. Acid produced in galactose broth. M. May or may not produce acid in mannite broth. R . No acid produced in raffinose broth. Gly. . No acid produced in glycerin broth. I . No acid produced in inulin broth. S. May or may not produce acid in starch broth. The B series of cultures from milk pasteurized at 65.6 C. (150 F.) were divided in a similar manner as shown in Fig. 28. The formulas of the reactions of the subdivisions are given in Table 22. TABLE 22. Cultural reactions of organisms in subdivisions cultures of B series from milk pasteurized for SO minutes at 6.5.6 C. (150 F.). Subdivi- sion. Formula of reactions. A It C D B F G H I J K L M CA+N-D+L+CG+MR-Glyc.-I-S. CA+N-D+L+CO4-MR-^Glyc.-I-Sj-. CA+N-I-D+L+CG + MR+ Glyc.-I-S+. CA+N+D+L+CG+MR-t-Glyc.-I-S-. CA+N+D + L+CG + MR-Glyc.-I-S+. CA + N+D+L+CG+MR-Glyc.-I-S-. CA + N+D+L+CG+MR + Glyc. + I-S+. CA+N + D+L+CG+MR+Glyc.-I+S+. CA-N-D-t-L+CG + MR-Glyc.-I-S+. CA-N-D+L+CG + MR + Glvc.-l+S+. CA-N+r>+L+CG + MiR + GIyc.-I-8+. CA-N+D+L+C-tG + M-R-Glye.-I-S+. CA-N-D-L-C-G-M-R-Glvc.-I-S-. 58 BACTERIA WHICH SURVIVE PASTEURIZATION. By this system of division one is able to see at a glance the general groups of bacteria which survived pasteurization in one sample of milk heated at 60 C. (140 F.) and 65.6 C. (150 F.). Or if desired, the complete reactions of the various classes of bacteria can be deter- mined. There is little that need be said regarding the results, for they are plainly shown. The most noticeable features were the high percentage of the acid-forming bacteria and the low percentage of the peptonizing group. It may be seen that a large percentage of the acid group were typical lactic acid bacteria as based on the formula of subdivision A. SUMMARY. 1. The average temperature used throughout the country with the "holder" process is 62.8 C. (145 F.). With the "flash" process, 71.1C. (160 F.). Reports from 219 milk plants show that 75 used the "holder" and 144 the "flash" process. The range in temperatures is from 60 C. (140 F.) to 82.2 C. (180 F.). Of the plants using the "holder" process only 1.3 per cent employ temperatures too low for proper pasteurization. Of those which use the "flash" process approxi- mately 42 per cent use temperatures too low to be effective. 2. Percentage bacterial reduction is of no value in determining the efficiency of the process of pasteurization. As a general rule when the bacterial content of raw milk is high there will be a high per- centage reduction. When the bacterial content is low, then the per- centage reduction is often low. The percentage bacterial reduction may be 99.9 per cent and yet the pasteurized milk may show a count of 100,000 per cubic centimeter. When the pasteurized milk con- tains only 10,000 bacteria per cubic centimeter the percentage reduction may only have been 95 per cent. It is often impossible to obtain a 99 per cent reduction when a good quality of milk is pas- teurized, therefore regulations which require a 99 per cent reduction of bacteria during pasteurization are of no value. 3. A heating period of three hours causes a marked increase in the reduction of bacteria in milk over one-half hour's heating, when a temperature of 54.4 C. (130 F.) and 57.2 C. (135 F.) are used. At 60 C. (140 F.) the increased reduction is much less. According to the results of one experiment in this investigation, a six hours' period of heating at 62.8 C. (145 F.) does not produce any more destruction of bacteria than does one-half hour's heating. 4. Sudden cooling from 62.8 C. (145 F.) or 71.1 C. (160 F.) within 15 seconds to from 1.7-3.9 C. (35-39 F.j does not cause any destruction of bacteria. The long-prevailing idea that sudden cooling is an essential part of the process of pasteurization is errone- ous so far as any increased destruction of bacteria is concerned. Its QUALITATIVE STUDY OF BACTERIAL FLORA. 59 value lies only in the fact that milk suddenly cooled is not allowed to stand at temperatures between 37.8 C. (100 F.) and 10 C. (50 F.), where rapid bacterial development might occur. 5. Four distinct groups of bacteria, the acid-forming, the inert, the alkali-forming, and the peptonizing, survive pasteurization, as differentiated by their reactions in litmus milk after 14 days' incu- bation at 30 C. (86 F.). The percentage of the acid group is increased by pasteurization while the other groups are decreased in their percentage of the total bacteria. The average percentage of lactic acid bacteria in raw milk which survive pasteurization for 30 minutes at 62.8 C. (145 F.) ranges from 1.27 per cent to 4.55 per cent in the various grades of milk. When the temperature of pasteurization is below 76.7 C. (170 F.) the bacterial flora of milk is composed for the most part of organisms of the acid group. At temperatures above 76.7 C. (170 F.) the acid group is largely destroyed and the majority of the bacteria which survive are of the peptonizing type. These bacterial group relations are clearly shown in the figures in the conclusions. 6. When different grades of milk are pasteurized at 62.8 C. (145 F.) in the laboratory and held at room temperature the bac- terial flora may undergo three distinct changes. First, when a fair quality of milk is pasteurized the acid group may develop at once and overgrow all the other groups, forming acid and producing a normal curd. Second, when a poor quality of milk is pasteurized the peptonizing group may grow rapidly at first along with the acid group, which later overgrows them. In this case the milk will first become curdled with a rennet curd due to the peptonizing bacteria, then later will become sour from the development of the lactic-acid group of organisms. Third, when a good grade of milk is pasteur- ized the peptonizing bacteria may overgrow the acid group of organ- isms so that the milk becomes peptonized without the develop- ment of any acid. These same grades of milk, treated in the same manner but held in an ice chest at 10 C. (50 F.), show entirely different changes in their bacterial contents. The growth of the peptonizing group is restrained so that they are of little importance. The percentage of the acid group remains about the same through a long period. Occasionally the percentage of the alkali group may increase after five days, but eventually the acid group forms the major group. These results were obtained from laboratory experi- ments and only indicate possible changes in the bacterial flora of pasteurized milk when held at different temperatures. They show the delicate balance between the bacterial groups, but can not be applied to indicate the bacterial changes in milk pasteurized and handled under commercial conditions. 60 BACTERIA WHICH SURVIVE PASTEURIZATION. 7. A qualitative study of the acid group shows that many organ- isms giving characteristics of the typical lactic-acid bacteria survive pasteurization for 30 minutes at 62.8 C. (145 F.). Other lactic- acid forming bacteria survive which may form pigment or vary from the typical lactic types in the fermentative reactions. The inert group was made up of organisms which produce no change in litmus milk during 14 days' incubation, and therefore include many which did not grow, but which according to the method of differentiation would be included as inert forms. The only truly inert form recognized was a yellow pigment-forming organism. The inert group is probably of little importance. The alkali group which survive pasteurization is of importance. Bacteria of this group can not be differentiated from those of the inert group or from slow acid-forming organisms by plating, but are easily determined by the milk-tube method. They grow at low temperatures and produce a strong alkaline reaction in milk. In pure cultures enough alkali is produced to dissolve the casein. No acid is produced in fermentation broths, but often an alkaline reaction is found. The peptonizing group includes those organisms which peptonize casein. A number of the organisms isolated peptonized casein, but did not liquefy gelatin. Various types of this group are able to sur- vive pasteurization and vary widely in their cultural characteristics. The few gas-forming bacteria in pasteurized milk, so far as the results of this investigation indicate, do not include organisms of the colon-aerogenes group. One gas-forming organism was found which produced gas in milk and peptonized casein. Another culture, known as Z, seems to be an entirely new type, which is characterized by its ability to produce gas continuously in milk through a long period, and by the fact that while gas is formed in milk none is formed in lactose broth. Gas-forming anaerobic bacteria are often found in milk which pro- duce gas hi lactose bile tubes and which might be mistaken in a preliminary test for the colon organism. 8. In view of the fact that gas-forming organisms survive pas- teurization which will give the preliminary test for the colon organ- ism by the lactose bile fermentation-tube method, it seems advisable to suggest that boards of health which depend on the presence of the colon-aerogenes group as an indication of reinfection or inefficient pasteurization make a complete determination by cultural reactions of organisms suspected as being colon forms. 9. The thermal death point of one lactic acid organism isolated during this investigation was 79.4 C. (175 F.) when a broth culture was heated in Sternberg bulbs for 30 minutes. The thermal death points of 64 acid-forming bacteria from one sample of milk which CONCLUSIONS. 61 survived pasteurization for 30 minutes at 82.2 C. (180 F.) deter- mined roughly was between 82.2 C. (180 F.) and 93.3 C. (200 F.). 10. Only a small percentage of spore-forming bacteria are found in milk pasteurized for 30 minutes at 62.8 C. (145 F.). Of 225 cul- tures selected at random which were studied, only 3, or 1.35 per cent, formed spores. 11. For those who advocate the inoculation of pasteurized milk after heating, with a culture of lactic acid bacteria to produce a normal souring, it is suggested that a culture of a high-temperature resisting lactic-acid organism may be added before pasteurization. By such a method there would be no danger of infection as might result accidentally by inoculation after pasteurization. 12. In view of the results of this investigation it seems that the control of pasteurization should be maintained by bacterial limits for the milk to be pasteurized, together with supervision which will insure the use of the proper pasteurizing temperature and guard against reinfection. A bacterial standard then need not be set for pasteurized milk. CONCLUSIONS. The results in this paper are based upon laboratory experiments performed under conditions which made reinfection impossible. The bacteria which survived pasteurization, therefore, represent those bacteria which could not be destroyed at the temperature of pasteuriza- tion. A temperature of 62.8 C. (145 F.) for 30 minutes was used in most of the experiments, since that is the most universally used temperature with the " holder" process in this country. Higher temperatures, however, were also used in order to show the changes in the bacterial groups which survived. From the results of the work it has been possible to show graphically in figure 29 the hypo- thetical relations of the bacterial groups in raw milk and milk pas- teurized by the "holder" process at various temperatures under laboratory conditions. The bacterial flora of the various kinds of milk is represented by columns of equal length divided into sections, which represent in a general way the relative size of the bacterial groups. From the figure it may be seen that raw milk contains four prin- cipal groups of bacteria the acid, the inert, alkali, and peptonizing groups. The acid group is divided into two groups the acid-coagu- lating, which coagulates milk in less than 14 days, and the acid group, which merely produces acid and does not coagulate milk inside of 14 days. In raw milk the inert group is the largest. In milk pasteurized at 62.8 C. (145 F.) the great increase in the pro- portion of the acid-coagulating and acid groups is plainly shown. The percentage of the alkali and peptonizing groups are reduced. At 71.1 C. (160 F.) the total acid group is still the largest, but 62 BACTERIA WHICH SURVIVE PASTEURIZATION. the acid coagulating group is made up of bacteria which coagulate very slowly. At this temperature the alkali group is greatly reduced and the peptonizing reduced to the minimum. At 76.7 C. (170 F.) the total acid group remains about the same, but the organisms pro- duce acid and coagulate very slowly. The alkali group is practically CCMGVL4T//VG &P7VWZWG GffOf/ff 30 I i 767C. 82.2C. 87.8C. _ (/8o/r) (/9o/y (200^) 11 vjr c^ fu i k I I i FIG. 29. The hypothetical relation of the bacterial groups to raw and pasteurized milk. destroyed, although occasionally a sample may show a fairly high percentage. The most important change is in the peptonizing group. At this temperature the percentage of the peptonizing group to the total bacteria begins to increase. The increase when milk is pas- teurized at 82.2 C. (180 F.) is even more striking. At this tem- perature over 75 per cent of the bacteria which survive are pep- CONCLUSIONS. 63 Ionizers. None of the acid-coagulating group are found and only a small percentage of the acid group. Occasionally a few of the alkali group may be found. At 87.8 C. (190 F.) and 93.3 C. /oo FIG. 30. The hypothetical relation of the bacterial groups in raw and pasteurized milk. (200 F.) the bacterial groups which survive are about the same in their relative sizes as at 82.2 C. (180 F.). The hypothetical rela- tions of the bacterial groups are further shown in figure 30. The rela- 64 BACTERIA WHICH SURVIVE PASTEURIZATION. tion of the bacterial groups is also shown, which brings out more distinctly, perhaps, the changes in the bacterial groups. Here the reversal of the percentages of the total acid group and the pep- tonizing group in milk pasteurized above 76.7 C. (170 F.) for 30 minutes is plainly shown. It is very evident that when the bac- terial flora of pasteurized milk is under discussion the temperature of the process is of fundamental importance. From these figures the bacterial groups in various kinds of milk may be seen at a glance, and they summarize in a very general way the most important results of this investigation. It must be remembered, however, that the relations of the bacterial groups only represent average conditions, and the bacterial flora of every sample of milk must not be expected to conform exactly to these averages. Varia- tions in methods and conditions in the production of milk beyond the control of the investigator may widely influence the bacterial group relations of an individual sample. APPENDIX. 65 APPENDIX. TABLE I. Complete characteristics of the different varieties of acid-forming bacteria which survive pasteurization. Acid production in 3 s a i s > Yellow. 66 BACTERIA WHICH SURVIVE PASTEURIZATION. TABLE II. Complete characteristics of the different varieties of alkali-forming bacteria which survive pasteurization. WWWOOOO Morphology. 1 O Gelatin liquefied or nonliquefied. Nitrate reduced. "3 E Acid production in Alkaline reaction in litmus milk. Dextrose. a Saccharose. Galactose. Mannite. d ! 3 Rafflnose. Inulin. Starch. Salicin. + 4 4 - 4- 1 4 - - - - - - - - - - ! - 4 ' 4 - - - - - - - - - - Yellow. TABLE III. Complete characteristics of the different varieties of the peptonizing bacteria which survive pasteurization. Acid production in Yellow. Brown. Dark cream. 1 Spores. O UC SOUTH] .. QQ Q University of California SOUTHERN REGIONAL LIBRARY FACILITY 305 De Neve Drive - Parking Lot 17 Box 951388 LOS ANGELES, CALIFORNIA 90095-1388 Return this material to the library from which it was borrowed. Universi South Libr