'■■*rr"\^ ^^ %:^<.^ m^ "m-. %W ■,M1 m #^'^-,""fE;," m ■'V ':.^H-^ .^>'*!*»^.: 1^ CORNELL UNIVERSITY. THE THE GIFT OF ,^ ROSWELL P. FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE. 1897 Cornell University Library QR 121.H22 , the numbers of bacteria in mi 3 1924 000 890 404 The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000890404 STUDIES ON THE NUMBERS OF BACTERIA IN MILK SHOWING VARIOUS CHANGES BY B. W. HAMMER AGRICULTURAL EXPERIMENT STATION IOWA STATE COLLEGE OF AGRICULTURE AND MECHANIC ARTS DAIRY SECTION RESEARCH BULLETIN NO. 29 JANUARY, 1916 AMES, IOWA January, 1916 Research Bulletin No. 29 STUDIES ON THE NUMBERS OF BACTERIA IN MILK SHOWING VARIOUS CHANGES BY B. W. HAMMER AND R. H. HIX AGRICULTURAL AND EXPERIMENT STATION IOWA STATE COLLEGE OF AGRICULTURE AND MECHANIC ARTS DAIRY SECTION AMES, IOWA Studies on the Numbers of Bacteria Pres- ent in Milk which has Undergone Various Changes By B. W. Hammer and R. H. Hix. The relationship between bacteria and the change^! thai occur in milk has been recognized since the middle of the last century. Certain of the milk abnormalities are almost characteristic of micro-organisms while others, such as abnormal flavors and odors, quite commonly result from causes other than bacterial. Recently the dairy section of the Iowa Agricultural Experiment Station in working with samples of milk from various parts of the state found that milk sometimes received low flavor scores when the number of contained bacteria was exceedingly small, while samples with high flavor scores sometimes contained large numbers of bacteria. This suggested an investigation of the number of organisms required to produce changes in the flavor and odor of milk. The investigation of the odors and flavors produced in milk by bacteria, however, presents certain difficulties. The ordinary sterilization of milk so influences the flavor and odor that those which may be subsequently developed are largely overshadowed. Samples of aseptic milk sometimes have an undesirable flavor and odor that tends to mask other flavors that may be developed. Moreover, the accurate detection of changes in odor and taste presents difficulties because of the varying acuteness of the senses involved. Because of these difficulties much of the work herein reported deals with changes other than those in flavor and odor, such as the production of ropiness, the coagulation of milk and the reduction of litmus milk. EI8T0BIGAL. Changes in milk have been studied by many investigators and a large number of types of organisms that can cause pronounced changes have been described. The type of change in milk de- pends on the type or types or organisms developing, which, in raw milk, is determined very largely by the temperature of holding. Wide variations have been encountered in the changes in the numbers of bacteria in milk under apparently identical condi- 36 tions. Conn and Esten^ have pointed out that "the number of bacteria which are present in fresh milk gives no indication as to the number that may be present in later hours. It frequently happens that milk which at the outset has small numbers will in later hours have numbers considerably larger than those in other samples which at the outset contained more bacteria, even tho the two samples of milk are kept under identical conditions. The relationship between the numbers of bacteria present and the changes produced in milk has been considered with the lactic acid producers more than with any other group. The old idea regarding this relationship is stated by Conn^ as follows: "Now while there is a general parallelism between the growth of these organisms and the production of lactic acid, the parallel is by no means a close one. For a considerable time after milk is inoculated with lactic bacteria, there is no increase in the amount of lactic acid, altho the bacteria are themselves multiply- ing very rapidly and becoming very numerous. Then there is a rapid increase in the development of lactic acid, accompanying a still further growth in the bacteria." Rahn^, after quoting the above statement by Conn as well as an analogous one by Knosel* which refers to a "short multipli- cation" preceding the sugar-destroying power of yeasts, con- tends that "there is no experiment on record to prove that in the first stage of development the multiplication takes place without fermentation. As soon as a determination of fermen- tation products is possible, it shows the fermentation per cell to be the faster the younger the culture." This author found that the fermenting capacity decreases with the age of the cul- ture. Heinemann^ found the amount of acid produced by the original strain of Streptococcus lacticus progressively decreased by animal passage ; repeated transfers thru litmus milk did not change materially the fermentative ability. A decreased acid production after a culture has been transferred a number of times is a condition commonly met with in the laboratory. MarshalP and his associates have pointed out that the other organisms present may influence the acid as well as the flavor and odor developed by Bad. lactis acidtl. Buchanan and Ham- mer' have found that the inoculation, along with Bact. lactis acidi, of an organism isolated by them decreased the acid formed. 1. The Comparative Growth of Different Species of Bacteria in Normal Milk. 14th Annual Rpt. Storrs Apr. Expt. Sta. 76. 1901. 2. Bacteria in Milk and its Products. 74. 1903. 3. The Fermenting Capacity of the Average Single Cell of Bacterium lactis acidi. 24th Annual Rpt. Mich. Agr. Expt. Sta. 443-480. 1911. 4. Centbl. f. Bakt. Abt. 2, 8: 272. 5. The Variability of two Strains of Streptococcus lacticus. Jour of Inf. Dis. 16: 221-239. Mr. 1915. 6. Various Bulletins of the Mich. Agr. Expt. Sta. 7. Slimy and Eopy Milk. Res. Bull. la. Agr. Expt. Sta. 22. 37 Heinemann^ found that "The amount of acid formed in the souring process of milk or cream is not dependent solely on a definite number of bacteria of the Streptococcus lacticus group. Temperature and the presence of other bacteria may influence the result." He also found that coagulation is not dependent solely on definite numbers of bacteria or a definite amount of acid and suggests that acid forming and coagulative enzymes ex- creted by bacteria or liberated after disintegration of the cells, play an important role in bringing about coagulation. In ster- ilized milk, this author found that coagulation was complete with a smaller amount of acid than inoculated raw milk, but that more time was required for completion of the process. Heinemann^ also states that "we did some work in trying to determine the number of lactic acid streptococci that are nec- essary in order to change milk and cream in such a way that you could tell by taste whether it was sour or not. In a general way, the temperature influences this. The higher the tempera- ture the fewer the bacteria (that) are necessary to sour milk." A number of investigators have found wide variations between the acidities and bacterial contents of samples of milk showing acidities above the normal and Hammer^" found these variations in samples of sour cream delivered to one of the Iowa creameries. Heinemann^'^ found in sterilized milk inoculated with Strep- tococcus lacticus that at 37° C. the milk was coagulated after about 36 hours and contained 540,000,000 per c. c. while at room temperature coagulation took place after 4 days with 350,000,000 per c. c. Rahn'^^ has observed an acidity increase with Bad. lactis acidi where the number per c. c. was as low as 4,800,000 ; the rise in acidity, however, was only .005%. It is evident that wide variations apparently exist in the action of a given number of a certain type of organism ; evidently the variation is much greater in impure culture than in pure cul- ture, altho it undoubtedly exists in the latter case. Certain factors, such as temperature and character of the milk, un- doubtedly play a part in causing this variation. Marshall and Farrand^^ write as follows regarding another possible cause: 'We are disposed from our studies to attribute these irregularities to associative action . . " 8. Relation of the Number of Streptococcus lacticus to the Amount of Acid Formed in Mills and Cream. Jour, of Inf. Pis. 16: 285-291. Mr. 191B. 9. Rn't of the Hearing on Ice Cream Before Dr. C. A. Alsberg. 120. Je. 1914. (Published by the Nat'l Assn. of Ice Cream Mfrs.) 10. Pasteurization of Cream for Pnttermaking. Part II Bacteriolcgical Studies. Bull. la. Agr. Expt. Sta. 156. 11. I. c. ref. 8. I?.. ). c. 13. Bacterial Associations in the Souring ol Milk Centbl. f. Bakt. 2. 21-B8 1908. 38 GENERAL METHODS. In general, the method of procedure was to inoculate milk, either sterile or aseptic, with a pure culture of some organism producing a change in milk and, when this change was detected, to determine the number of bacteria present by the plate method. The sterile milk used, both litmus and plain, was sterilized in the autoclave; the use of intermittent sterilization, even with temperature as low as 0° C, failed to show any advantage over autoclaving, from the standpoint of the production of a heated flavor and odor. The aseptic milk used was secured at the Iowa State College dairy farm. Previous work on the bacterial content of the milk of some of the animals in the herd made possible the selection of animals whose milk ordinarily contained but few bacteria. When plates were poured from this milk after inocu- lating various organisms and allowing time for development, the colonies developing were ordinarily practically all of the type inoculated. Most of the data were secured by using tubes of milk altho a few tests were run with Petri dishes. The amount of milk used varied, altho some tests were run with 10 c. c. quantities. The numbers of organisms used for inoculation were not deter- mined; where comparative inoculations were desired equal vol- umnes of a suspension were employed. The agar of the American Public Health association for bac- terial milk analysis was used in all of the plates, which were all incubated at 37° C. for 48 hours with the exception of those poured with Bad. lactis viscoswm, which were inoculated at room temperature for 48 hours. After a change had taken place in inoculated milk, the whole volume was thoroly agitated before the sample for plating was taken. The inoculated milk was com- monly held at room temperature, altho a few tests were run at 37° C. Koom temperature with its customary variation seemed to present more practical conditions than any constant tempera- ture, altho a constant temperature might have given more uni- form results. The classification of the changes produced in milk by the various organisms presented considerable difficulty ; the changes recognized for each organism are given in the methods used with that type. The methods of detecting changes, such as slight ropiness (touching the milk with a platinum wire) or slight coagulation (by tipping the tube) are quite unsatisfactory. Moreover, it does not seem desirable to apply these methods too frequently because of the effect of the agitation involved on the ropiness and on the tendency of the curd to cling to the bottom of the test tube. Accordingly, in the work on the 39 changes in the consistency of milk, examinations were only made at from 2 to 4 hour intervals; in the work on odor and flavor the intervals were ordinarily shorter. On account of the difficulties involved, any classification of the changes that is adopted can be only approximately adhered to. Because of the wide variations in the results, the averages for the various sets of data are given in general rather than in exact figures. The cultures of Bad. lactis acidi and the sweet curdlers used were isolated for the work herein reported while the cultures of the other organisms employed were cultures that had been held for varying lengths of time. In many of the tables pre- sented, the determinations were all, or nearly all, made on dif- ferent days so that a considerable period of time and various conditions are represented. The results obtained on Bad. ladis acidi and the sweet curdlers shortly after isolation are essen- tially the same as those obtained later, so it seems probable that the holding of the organisms in pure culture for considerable periods did not exert an appreciable influence on the results ; nevertheless the history of the cultures must be taken into con- sideration in drawing conclusions from the data given. The problem herein considered is being investigated at the present time by studying the numbers of organisms in milk that has undergone various changes without artificial inoculation. "With certain changes, the depth of the milk layer involved will un- doubtedly be of some influence. RESULTS OBTAINED Bad. ladis aerogenes The culture of Bad. ladis aerogenes that was used was iso- lated from a sample of ropy milk. It produced an extremely ropy condition in milk and retained this ability after being carried in sterile milk for a number of years. The organism was studied simply from the standpoint of its production of ropiness. SPECIAL METHODS All the work with Bad. ladis aerogenes was done with sterile milk. Generally tubes containing from 8 to 15 c. c. of milk were used, but in a few instances Petri dishes (with from 8 to 15 c. c. of milk) or 150 c. c. Erlenmeyer flasks (with 100 c. c. milk) were employed; unless otherwise stated, tubes were used. Eoom temperature was always used as a holding temper- ature for the inoculated milk. Ropiness in the inoculated milk was detected by touching a flamed and cooled platinum needle to the surface, the container being so held that the surface of the milk made an acute angle with the needle, and then carefully 40 withdrawing it. The fermented milk was classified as follows: (A) Slightly Ropy: Milk showing only a slight tendency to follow the platinum needle when it was with- drawn from the surface. (Bj Medium Ropy: Milk which could be drawn out into threads extending 2 or 3 inches from the surface. ( C ) Very Ropy : Milk which could be drawn out into threads, extending beyond the mouth of the tube, but which could not be included in the following class. (D) Extremely Ropy : Milk which could be drawn out into threads several feet long. RESULTS OBTAINED The counts obtained on milk which was classed as slightly ropy are shown in tables I and II, the data having been divided because of the very wide variations existing. Table I gives the results of seven determinations which vary from 11,000,000 to 49,000,000 per c. c. and average 32,200,000; the ages of the cultures range from 8 to 36 hours. Table II presents the results of 37 determinations which vary from 79,000,000 to 156,000,000 and average 115,700,000 per c. c, the ages of the cultures rang- ing from 9 to 36 hours. Table III presents data obtained on samples of milk classed as medium ropy; the 36 counts range from 160,000,000 to 285,000,000 per c. c. and average 223,000,- 000 while the ages of the cultures vary from 9 to 72 hours. The counts obtained on the samples of milk classed as very ropy are presented in table IV; the 37 counts vary from 290,000,000 to 495,000,000 per c. c. and average 373,000,000 while the ages of the cultures range from 8 to 72 hours. Table V presents the data secured on milk (in tubes) that was classed as extremely ropy. The nine counts range from 500,000,000 to 830,000,000 per c. c. and average 650,000,000, while the ages of the cultures vary from 12 to 24 hours. The data obtained on extremely ropy milk fermented in thin layers (in Petri dishes) is presented in table VI ; the average for the 5 counts is 3,800,000,000 per c. c. and the individual counts vary from 2,870,000,000 to 4,960,000,- 000, while the ages of the cultures range from 8 to 12 hours. Table VII presents data relative to the numbers of bacteria present in inoculated milk at 2 hour periods, the determination having been made on milk held in flasks. Organisms were present up to 86,000,000 per c. c. without ropiness being de- tected. There was considerable variation in the numbers of organisms present at the time the milk was slightly ropy and some of the figures are somewhat higher than those reported in tables I to VII. The number of organisms which pro- duced a slightly ropy condition in trial 5 is smaller than the 41 TABLE I— Bact. lactis acrotrcnes. Number of Organisms in Slightly Ropy Milk. TABLE III— Continued. Age of No. Bact. Date Ciilture per in Hours c. c. Nov. 11. 1914.. 24 22,000,000 Jan. 10, 1916.. 12 11,000,000 Jan. 30, 1916.. 10 49,000,000 Feb. 1, 1915.. 12 38,000,000 Feb. 16, 1916. . 36 49,000,000 March 18, 1916 9 42,000,000 March 30, 1916 8 14,400,000 Average 32,200,000 TABLE II — Bact. lactis aerogenes. Number of Organisms in Slightly Ropy Milk. Age of No. Bact. Date Culture per in Hours c. c. Nov. 2, 1914.. 24 166.000.000 Nov. 2, 1914.. 24 80.000.000 Nov. 13, 1914.. 24 114.000.000 Nov. 13, 1914.. 24 130,000.000 Nov. 16. 1914.. 16 81,000,000 Nov. 19, 1914.. 24 90,000,000 Nov. 21, 1914.. 24 105,000,000 Nov. 21, 1914.. 24 106,000,000 Nov. 22, 1914.. 24 166,000.000 Nov. 23. 1914.. 24 111,000,000 Dec. 12. 1914.. 24 80,000,000 Dec, 13. 1914.. 24 84,000,000 Dec. 14. 1914.. 24 129,000,000 Jan. 18, 1916.. 12 150,000,000 Jan, 19. 1915.. 12 151,000,000 Jan. 21. 1915.. 12 109,000,000 Jan. 22. 1915.. 12 95,000,000 Jan. 22. 1915.. 12 166,000.000 Jan. 22. 1916.. 12 142.000.000 Jan. 23. 1915.. 12 79.000,000 Jan. 26. 1915.. 12 137.000.000 Jan. 28. 1915.. 12 136,000.000 Jan. 31. 1915.. 24 132.500.000 Jt'eb. 3. 1915.. 12 161.000.000 i'eb. 6, 1915.. 12 110,000,000 i'eb. 6. 1915.. 12 87,000,000 feb. 15. 1915.. 36 113,000,000 i'eb. 17. 1915.. 12 84,000.000 i'eh. 21. 1915.. 12 104.000.000 i'eb. 26. 1915.. 12 134.000,000 March 2. 1916 12 93,000.000 March 3. 1916 12 126.000.000 March 8. 1915 12 117.000,000 March 20, 1915 9 100,000,000 March 24, 1916 9 99,000,000 March 26, 1916 9 160,000,000 March 26, 1915 9 106,000,000 Average 116,700,000 TABLE III — Bact. lactis aerogenes. Number of Organism in Medium Ropy Milk. Age of No. Bact. Date Culture per in Hours c.-c. Oct. 26, 1914.. 24 280.000.000 Oct.. 29, 1914.. 24 208,000,000 Oct. 30. 1914.. 24 280,000.000 Oct. 31, 1914.. 24 180.000.000 Nov. 7, 1914.. 18 230.000.000 Nov. 6, 1914.. 18 186,000.000 Nov. R, 1914.. 72 285.000,000 Nov. 25, 1914.. 24 166.000.000 Nov. 27. 1914.. 24 226,000.000 Nov. 2S. 1914.. 48 166,000,000 Nov. 29. 1914.. 24 160,000,000 Age of No. Bact. Date Culture per in Hours c. c. Dec. 10, 1914.. 24 267.000.000 Dec. 11, 1914.. 24 204.000.000 Dec. 7, 1914.. 18 188.000.000 Dec. 18. 1914.. 18 246.000.000 Dec. 21. 1914.. 18 176.000.000 Jan. 1. 1916. . 18 270.000,000 Jan. 11. 1916.. 24 250,000,000 Jan, 14. 1915.. 12 160,000,000 Jan. lb. 1916.. 12 233,000,000 Jan. lb. 1915.. 12 233.000,000 Jan. 16. 1915.. 12 260,000,000 Jan. 16. 1916.. 12 260,000,000 Jan. 16. 1915.. 12 183,000,000 Jan. 17. 1916.. 12 190,000,000 Jan. 18. 1915.. 12 225,000.000 i'eb. 2. 1916.. 12 228.000.000 feb. 9. 1916.. 12 181.000.000 J;'eb. 11. 1916.. 12 269.000,000 i'eb. 23. 1916.. 12 228,000,000 i'eb. 24. 1915.. 12 262,000,000 h'eb. 27. 1916. . 12 230,000,000 March 4. 1916 12 272,000,000 March 6. 1916 12 272,000,000 March 16, 1916 12 210,000,000 March 22, 1916 9 187,000,000 Average 223,000,000 TABLE IV — Bact. lactis aerogenes. Number of Organisms in Very Ropy Milk. Age of No. Bact. Date Culture per in Hours c. c. Oct. 17. 1914. . 18 440,000.000 Oct. 18. 1914.. 16 320,000,000 Oct. 19. 1914.. 18 400,000,000 Oct. 22. 1914.. 18 300,000.000 Oct. 23. 1914.. 24 310.000.000 Oct. 25. 1914.. 24 370,000,000 Oct. 26. 1914.. 24 370,000,000 Oct. 28. 1914. . 24 300,000,000 Nov. 1. 1914.. 24 320,000,000 Nov. 4, 1914.. 24 370,000,000 Nov. 6, 1914.. IS 370,000,000 Nov. 8, 1914.. 72 425,000,000 Nov. 9, 1914.. 24 315,000,000 Nov. 12, 1914.. 24 324,600,000 Nov. 14, 1914. . 24 367,000,000 Nov. 20, 1914.. 24 330,000,000 Nov. 26, 1914.. 24 339,000,000 Nov. 29, 1914.. 18 450,000,000 Dec. 2. 1914.. 24 406,000,000 Dec. 3. 1914.. 24 416,000,000 Dec. b. 1914.. 24 465,000,000 Dec. 6. 1914.. 24 370,000,000 Dec. 7. 1914.. 24 430,000.000 Jan. 10. 1915.. 24 450,000.000 Jan. 13. 1915.. 18 495.000.000 Jan. 17. 1915.. 12 390.000.000 Jan. 18. 1916.. 12 360,000,000 Jan. 20. 1915.. 12 360,000,000 J^'eb. 6. 1915.. 24 420,000,000 Jj'eb. 12. 1915. . 36 360,000,000 i'eb. 13. 1916.. 18 366,000,000 ^'eb. 21. 1916.. 12 400,000,000 l''eb. 22. 1915.. 12 338.000,000 Feb. 25. 1915.. 12 315,000,000 March 17, 1916 8 290,000,000 March 27. 1916 24 428,000,000 March 30. 1916 8 320,000,000 Averaare 373.000,000 42 TABLE V — Bact. lactis aerogenes. Number of Organisms in Extremely Ropy Milk. Age of No. Bact. Date Culture per in Hours c. c. Nov. 30, r9i4.. 24 565,000,000 Dec. », 1914.. 24 710,000.000 Dec. 2H, 1914.. 24 830,000,000 Dec. 31. 1914.. 24 695,000,000 Jan. 2, 1915.. 24 580,000,000 Jan. 7, 1915.. 24 690,000,000 Jan. », 1915.. 24 760,000,000 Jan. n«, 1915.. 15 530,000,000 March 6, 1915 12 500,000,000 Average 660,000,000 TABLE VI — Bact. lactis aerogenes. Number of Organisms in Extremely Ropy Milk in Petri Dishes. Age of No. Bact. Date Culture per in Hours c. c. March 8, 1915 12 3,880,000,000 March 10, 1916 12 3,290,000,000 March 11, 1915 12 4,000,000,000 March 15, 1916 12 2,870,000,000 March 30. 1915 8 4,960,000,000 Average 3,800,000,000 TABLE VII— BACT. LACTIS AEROGENES. TRIAL 1. TRIAL 2. TRIAL 3. Culture in houre Bacteria per c. c. Cond. of Milk Bacteria per c. c. Cond. of Milk Bacteria per c. c. 1 Cond of 1 Milk 2 4 6 8 10 4,400,000 26,300,000 80,000,000 186,000,000 630,000,000 no change no change no change slightly ropy extremely ropy 12,350,000 42.400,000 86,000,000 202,600,000 no change no change no change slightly ropy 14.600,000 22,500,000 44,000,000 248,000,000 no change no change no change slightly ropy TRIAL 5. Bacteria 1 Cond. of Bacteria Cond. of per c. c. 1 Milk per <=. c. Milk no no 8,900,000 change 7,500,000 change no no 60,000.000 change 12.900,000 change med. no 348,000,000 ropy 25,000,000 change very slightly 500,000,000 ropy 70,000,000 ropy med. 125,000,000 ropy numbers present in trials 1 and 2 when there was no ropiness present. The results obtained on Bact. lactis aerogenes indicate that under the conditions employed, a very large number of organ- isms must be present in milk before a ropy condition results. The smallest number of organisms encountered in milk that was at all ropy was 11,000,000 per c. c. and this was an exceptional case. In many instances over 100,000,000 organisms per c. c. were present in the milk when ropiness was first detected. On the other hand numbers up to 86,000,000 per c. c. were found present in milk without ropiness having developed and since the data on this point are not extensive, the figures probably by no means represent extremes. For the development of an extremely ropy condition in milk exceedingly large numbers of organisms, ordinarily in the hundreds of millions, must be present. Wide 43 variations regarding tlie numbers of organisms present in milk classed as showing the same stage of ropiness were constantly encountered ; while this is undoubtedly due in part to the diffi- culty and uncertainty of the method used for the classification, it may be in part due to the effect of different temperatures on the organism. High temperatures commonly limit or prevent the development of ropiness by certain bacteria altho growth goes on rapidly ; the more rapid development of acid at higher temperatures tends to reduce the ropiness, but there is likely some direct effect on the organisms also. BACT. LACTIS VISCOSUM The culture of Bact. lactis viscoswn employed was isloted from an outbreak of ropy milk in one of the smaller towns of the state. Altho the culture had been carried in litmus milk for several months at the time it was selected, it produced a very ropy condition in milk altho not so ropy as the culture of Bact. lactis aerogenes used. Bact. lactis viscosum was studied only from the standpoint of its production of ropiness in milk. SPECIAL METHODS Tubes containing from 8 to 15 c. c. of sterilized milk were used for all of the work with this organism. The cultures (and plates also as already stated) were incubated at room temper- ature; ropiness was tested for in the same manner as with Bact. lactis aerogenes. The fermented milk was classified as follows 1 (A) Slightly Ropy: Milk which showed only a slight tendency to follow a platinum needle withdrawn from the surface of the milk. (B) Very Ropy: Milk which could be drawn out into threads several inches long. RESULTS OBTAINED The counts obtained on the milk classed as slightly ropy are presented in table VIII ; the 19 determinations range from 15,- 500,000 to 44,000,000 per c. c. and average 26,100,000, while the ages of the cultures range from 9 to 24 hours. Table IX gives the data obtained on very ropy milk; the four counts, which were all made on the same day, range from 450,000,000 to 786,- 000,000 and average 556,000,000 per e. c, while the age of all of the culture was 24 hours. Table X gives the data obtained when cultures were plated at two hour periods from the time of inoculation until ropiness was detected. The numbers of organisms present at the time a Blight ropiness was detected were in general the same as those 44 given in table VIII, altho the range was somewhat greater than the range encountered there. The lowest number of organisms found in slightly ropy milk was 12,300,000 per e. e. while num- bers larger than that (up to 19,500,000 per e. e.) were found in milk that showed no evidence of ropiness. The results obtained with Bad. lactis viscosum indicate that under the conditions employed, a large number of organisms must be present in milk before a ropy condition is produced. Slightly ropy milk showed a smaller variation in the numbers of organisms present with Bad. ladis viscosum than withBact. lactis aerog&nes. The smallest numie^ in ropy milk was 12,300,00 per c. c. when Bad. ladis viscosum was used for inoculation; this value agrees very closely with the corresponding value of 11,000,000 per c. c. obtained with Bad. laciHs aerognes. With TABLE VIII. — Bact. lactis viscosum. Number of Organisms in Slightly Ropy Milk. Age of No. Bact. Date Culture per in Hours c. c. Nov. 30, 1914. . 24 21,000,000 Dec. 10, 1914.. 24 29,600,000 Dec. 2, 1914.. 24 44,000,000 March 23, 1916 9 21,600,000 March 24, 1916 9 38,000,000 March 26, 1916 9 39,000,000 March 27, 1915 9 21,000,000 March 27, 1915 9 20,000,000 March 31, 1916 24 37,600,000 April 16, 1916. 12 18,760,000 April 16, 1916. 12 16,600,000 April 16, 1916. 12 20,200,000 April 16, 1916. 12 28,300,000 April 21. 1916. 9 18,160,000 TABLE VIII— Continued. Date Age of Culture in Hours No. Bact. ptr c. c. April April April April April 21, 1916. 21, 1915. 21, 1916. 21, 1916. 21, 1916. 9 9 9 12 9 Average 26,000,000 23,750,000 41,200,000 17,600,000 16,800,000 26,100.000 TABLE IX — Bact. lactis viscosum. Number of Organisms in Very Ropy Milk. June 26, 1916. 24 620,000,000 June 26, 1916. 24 470,000,000 June 26, 1915. 24 460,000,000 June 25, 1916. 24 Average 786,000,000 556,000,000 TABLE X— BACT. LACTIS VISCOSUM. Age of TRIAL 1. TRIAL 2. TRIAL 3. Culture Bacteria Cond. of Bacteria Cond. of Bacteria Cond. of in hours per e- c. Milk per e- c. Milk per c- c. MHk no no no 2 1,860,000 change no 2,200,000 change no 2,780,000 change no 4 6,400,000 change slightly 6,480,000 change 7,600,000 change no 6 12,300,000 ropy slightly slighily 10,950.000 change slightly 8 14,000,000 ropy 24,000,000 ropy 30,600,000 ropy TRIAL 4. TRIAL 6. Bacteria per e- c. ICond. of 1 Milk Bacteria ICond. of per ^- c. 1 Milk 8,800,000 16,600,000 62,600,000 no change no change slightly ropy 6.840,000 16,800,000 19,600,000 21,090,000 I no change no change no change slightly ropy 45 Bad. lactis viscosum very ropy milk contained large numbers of organisms in the few cases studied, 450,000,000 per c. c. repre- senting the minimum and 786,000,000, the maximum. BITTER ORGANISM The bitter organism employed was isolated in the student dairy bacteriology laboratory. It causes a coagulation of milk, begining at the bottom and gradually extending upward, a bitter tasre. an increase in the acidity and eventually a digestion of the curd. Acidity determinations made at the time of coagula- tion indicate that acid is not responsible for the precipitation of the casein. The organism was encountered only once but an apparently identical organism has been supplied to this labor- atory by Prof. B. Gr. Hastings. A considerable portion of the work on this organism dealt with the numbers of organisms present at the various stages of coagulation ; the number of organisms present in aseptic milk at the time bitterness is first evident was, however, determined in a number of trials. SPECIAL METHODS The work on coagulation was done with tubes of sterile milk (8 to 15 c. e. amounts). The aseptic milk used was drawn directly into large test tubes in amounts varying from 30 to 50 c. c. All the inoculated milk was held at room temperature. The coagulation of the milk was observed by tipping the tubes and allowing the uncoagulated milk to flow against the stopper ; the stages of coagulation were classed as follows : (A) Very Slight Coagulation: Milk in which coagula- tion had just definitely taken place, a very small amount of curd being present. (B) Slight Coagulation: Milk showing a stage of coagulation somewhere between very slight coagulation and the following stage. (C) Base Coagulation: Milk that had curdled up as far as sides of the tube. (D) Nearly Complete Coagulation: Milk which was curdled for about one inch up from the bottom of the tube. (E) Freshly Coagulated Cultures : Milk in which coag- ulation had been completed with a few hours. (F) Old Coagulated Cultures: Milk held from 24 to 48 hours after complete coagulation. RESULTS OBTAINED Table XI gives the data obtained on the milk showing very slight coagulation. The average of the 18 determinations is 46 TABLE XI.— Bitter Organisms. Numbers of Organisms in Very Slight- ly Coagulated Milk. Agecf No. Bact Date Culture per in Hours c. c. Dec. 6, 1914. . 24 68,000,000 Jan. 1, 1916.. 18 46,000,000 Jan. 31, 191B. . 12 30,000,000 Feb. 1, 1915.. 12 51,000,000 Feb. 2, 1915.. 12 43,000,000 Feb. 3, 1915. . 12 41,000,000 Feb. 11, 1915.. 36 69,000,000 Feb. 23, 1916.. 12 47,000,000 March 4, 1916 12 47,000,000 March 8, 1916 12 22,000,000 March 18, 1915 9 66,000,000 March 18, 1916 9 39,000,000 March 19, 1915 9 41,000,000 March 20, 1916 9 68,000,000 March 22, 1916 9 29,000,000 March 26, 1916 9 34,000,000 March 26, 1915 9 60,000,000 March 28, 1916 12 22,000,000 Average 44,000,000 TABLE XII — Bitter Organism. Number of Organisms in Slightly Coagulated Milk. Ageo£ No. Bact. Date Culture per in Hours c. c. Oct. 29, 1914.. 24 110,000,000 Oct. 30, 1914.. 24 90,500,000 Mov. 20, 1914.. 24 152,000,000 Dec. 11, 1914.. 24 131,000,000 Dec. 12, 1914.. 24 138,000,000 Dec. 14, 1914.. 24 136,000,000 Dec, 17. 1914.. 18 84,000,000 Dec. 19, 1914.. 18 162,000,000 Jan. 8, 1916.. 18 116,000,000 Jan. 11, 1916.. 24 113,000,000 Jan. 17, 1915.. 12 141,000,000 Jan. 18, 1916.. 12 87,000,000 Jan. 19, 1915.. 12 166,000,000 Jan. 21, 1-916.. 12 118,000,000 Jan. 22, 1916.. 12 119,000,000 Jan. 26, 1915.. 12 86,000,000 Jb'eb. 5, 1916.. 10 107,000,000 i'eb. 5, 1916.. 12 125.000,000 J<'eb. 16, 1916.. 36 117,000,000 t'eb. 16, 1915.. 12 113,000,000 t'eb. 21, 1915.. 12 105,000,000 i'eb. 26, 1916.. 12 157,000,000 t'eh. 26, 1916.. 12 167,000,000 l''eb. 26, 1916.. 12 89,000,000 March 3, 1915 12 100,000,000 March 6, 1915 12 85,000,000 March 31, 1915 24 114,000,000 Average 119,000.000 TABLE XIII — Bitter Organism. Number of Organisms in Base Coag- ulated Milk. Age of No. Bact. Date Culture per in Hours c. c. Oct. 31, 1914.. 24 267,000,000 Nov. 2, 1914.. 24 260,000,000 Nov. 6, 1914.. 24 360,000,000 Nov. 6, 1914.. 18 181,000,000 Nov. 7, 1914.. 18 266,000,000 Nov. 12, 1914.. 48 280,000,000 Nov. 26, 1914.. 24 320,000,000 Nov. 30, 1914.. 24 350,000,000 Dec. 2, 1914.. 24 296,000,000 TABLE XIII— Continued 1 Age of 1 No. Bact . Date Culture 1 Strain per in Hours] c. c. Dec. 3. 1914.. 24 260,000,000 Dec. 3, 1914.. 24 373,000,000 Dec. 7, 1914.. 24 200,000,000 Dec. 13, 1914.. 24 285,000,000 Dec. 18. 1914.. 18 260,000,000 Dec. 21, 1914.. 18 340,000.000 Dec. 23, 1914.. 24 375,000,000 Jan. 9, 1915.. 18 270,000,000 Jan. 13, 1915.. 18 370,000,000 Jan. 26, 1916.. 18 280,000,000 Jan. 28, 1916.. 12 300,000,000 Jb'eb. 10, 1915.. 24 310,000,000 Jb'eb. 13, 1915.. 18 210,000,000 i'eb. IV. 1915.. 12 310,000,000 i'eb. 18, 1915.. 12 283,000,000 I'eb. 22. 1916.. 12 280,000,000 March 10, 1915 12 270,000,000 Average 289,000,000 TABLE XIV— Bitter Organism. Number of Organisms in Nearly Com- pletely Coagulated Milk. Age of No. Bact. Date Culture per in Hours c. c. Oct. 17, 1914.. 24 445,000,000 Oct. 18, 1914.. 24 650,000,000 Oct 19, 1914.. 18 660,000,000 Oct. 28, 1914.. 24 470,000,000 Nov. 9, 1914.. 24 610,000,000 Nov. 26, 1914.. 24 690,000,000 Nov. 29, 1914.. 24 600,000,000 Dec. 9, 1914.. 24 630,000,000 Dec. 10, 1914.. 24 600,000,000 Jan. 2, 1916.. 24 416,000,000 l''eb. 9, 1916.. 24 610,000,000 March 6, 1916 12 450,000,000 March 8, 1915 12 470,000,000 Average 546,000,000 TABLE XV^Bitter Organism. Number of Organisms in Freshly Coagulated Milk. Agecf No. Bact. Date Culture per in Hours c. c. Oct. 23, 1914.. 24 1,250,000,000 Oct. 25. 1914.. 24 1,600,000,000 Oct. 26, 1914.. 24 950,000,000 Nov. 1, 1914.. 24 720,000,000 Nov. 4, 1914.. 24 710,000,000 Nov. 13, 1914.. 48 1,116,000,000 Dec. 31, 1914.. 24 790,000,000 Jan. 7, 1916.. 24 1,110,000,000 i'eb. 12, 1916.. 36 890,000,000 March 27, 1915 24 1,000,000,000 Average 1,000,000,000 TABLE XVI^Bitter Organism. Number of Organisms in Old Coagu- ulated Milk. Age of No. Bact. Date Culture per in Hours c. c. Nov. 8, 1914.. 72 2,615,000,000 Nov. 8, 1914.. 48 2,060,000,000 Nov. 28, 1914.. 48 2,366,000,000 Dec. 2, 1914.. 48 2,290,000,000 Jan. 7, 1915.. 48 2,400,000,000 Average |2,300,000,009 47 44,000,000 per c. c, the individual counts varying from 22,000,- 000 to 68,000,000 ; the ages of the cultures range from 9 to 36 hours. The results obtained on milk showing slight coagulation are shown in table XII where the 27 counts vary from 84,000,000 to 167,000,000 per c. c. and average 119,000,000. The ages of the cultures vary from 10 to 36 hours, the majority of them being 12 hours. The counts for the stage recognized as base coagulation are given in table XIII. The average of the 26 counts is 289,000.000 per c. c, while the individual counts range from 181,000,000 to 875,000,000. The ages of the cultures vary from 12 to 48 houis. The milk classed as nearly completely coagulated is represented in table XIV where the average for the 13 determinations is 545,000,000 per c. c. while the individual counts vary from 415,000,000 to 690,000,000 ; the ages of the cultures range from 12 to 24 hours. Table XVI gives the data obtained on the milk that was freshly coagulated. The average for the 10 de- terminations is 1,000,000,000 per c. c. while the individual counts vary from 710,000,000 to 1,500,000,000. The ages of the cul- tures represented vary from 24 to 48 hours. The data dealing mth the old coagulated milk is shown in table XVI. The average for the five determinations is 2,300,000,000 per c. c. while the in- dividual counts vary from 2,060,000,000 to 2,515,000,000. All of the cultures except one were 48 hours old and that was 72 hours old. Table XVII gives the data obtained on inoculated cul- tures at two hour intervals. In general the counts agree fairly well with those obtained in the preceding tables. The lowest count recorded in table XVII as causing very slight coagulation is 31,000,000 per c. c. (22,000,000 per c. c. is recorded in table X) ; in another trial 39,000,000 organisms per c. c. were found without any change being evident in the milk. The data obtained relative to the development of a bitter- TABLE XVII— BITTER ORGANISM Number of Organisms at two-hour Periods. Ace of Trial 1 Trial 2 Trial 3 Bacteria Cond. Bacteria Cond. Bacteria Cond. per of Der of per of c. c. Milk c. c. Milk c. c. Milk Z no no no 313.000 change 860.000 change 350.000 change d no no no 3,500.000 change 2,100.000 change 7,800.000 change 6 no no no ,39,000.000 change very 13,000.000 change very 10,200,000 change very !i slizht 31,000.000 slight 44,000.000 slight 61,000.000 coag. coag. coag. 10 200,000.000 slight coag. 87,000.000 slight coag. 12 241,000.000 base coag. 4S TABLE XVIII— BITTER ORGANISM— ASEPTIC MILK Number of Organisms in Slightly Bitter Milk. Cow Aire Bacteria Date Number of Cuitut'e per c. c. June 23, 1916. 69 16 Slightly Bitter Very Slight Coae. 63,000,000 June 23, 1915. 87 16 Slightly Bitter Very Slight Coag. 60,t00,000 June 23, 1916. 207 18 Slightly Bitter Very Slight Coag. 43,000,000 June 23, 1916. 143 15 SlSghtly Bitter Very Slight Coag. 59,000,000 June 24, 1916. 207 18 Slightly Bitter no Coagulation 19,000,000 June 24, 1916. 143 18 Slightly Bitter no Coagulation 10,000,000 June 24, 1915. 213 18 Slightly Bitter no Coagulation 66,000,000 June 24, 1915. 69 18 Slightly Bitter no Coagulation 23,000,000 June 24, 1915. 87 16 Slightly Bitter no Coagulation 11,000,000 June 24, 1916. 162 16 Slightly Bitter no Coagulation Average 35,000,000 38,900,000 ness in aseptic milk is shown in table XVIII ; the 10 counts vary from 10,000,000 to 66,000,000 and average 38,900,000 per c. c. The samples studied on the first day all showed a very slight coag- ulation while those studied on the second dayjiid not; most of the counts obtained on the second day were lower than those obtained on the first day, but one of those of the second day was a little higher than any of those of the first day. The results obtained with the bitter organism indicate that large numbers of organisms must be present in milk before coagu- lation or bitterness are produced. The smallest number of organ- isms found in a culture showing very slight coagulation was 22,000,000 per c. c. while 10,000,000 per c. c. was the smallest number found in aseptic milk in which bitterness could be de- tected. In general, with an increase in the extent of coagula- tion, there is an increase in the number of organisms present, altho in some cultures having no coagulation numbers of organ- ganisms are found which are greater than the number found in other cultures which do show coagulation. "Wide variations are en- cultures which do show coagulation. Wide variations are en- countered in the numbers of organisms present in cultures show- ing the same stage of coagulation. In freshly coagulated cul- tures, exceedingly large numbers of organisms are found — com- monly over 1,000,000,000 per c. c. After coagulation the develop- ment of the organisms still goes on, as in the old coagulated cultures over 2,000,000,000 per c. c. are ordinarily found. Sweet Gurdlers The three sweet curdling organisms used, which were classed as B. subtilis after a rather brief study of their characteristics, were isolated from three samples of milk. The three strains (A, B, and C), all of which sweet curdled milk rapidly, were studied simply from the stand point of the numbers of organ- isms present in milk in different stages of coagulation. 49 SPECIAL METHODS The work was all done with tubes of sterile milk contain- ing from 8 to 15 o. c. The inoculated tubes were held at 37' C. in the majority of the cases, altho a few trials were made at room temperature. The following stages in the coagulation of the milk were recognized : (A) Slight Coagulation : Milk which was just thickening. (B) Medium Coagulation: Milk which was well curdled but not yet firmly set. (C) Firm Coagulation: Milk which was quite firmly set. RESULTS OBTAINED Table XIX shows the data obtained on the milk classed as showing slight coagulation. Four of the tests were run at room temperature and the remaining 17 at 37° C, but the uniformity of the counts apparently justifies the combining of the data. The average for the 21 counts is 3,100,000 per c. c. while the individ- ual counts vary from 1,250,000 to 4,900,000. The ages of the room temperature cultures vary from 24 to 48 hours and those of the 37° C. cultures from 9 to 24 hours. The numbers of organ- isms present in milk classed as showing medium coagulation are presented in table XX. Seven of the counts were on tubes held at room temperature, while the remaining 15 were on tubes held at 37° C. ; here again the uniformity of the counts makes it seem advisable to combine the data. The 22 counts average 9,300,000 per c. c. and vary from 6,200,000 to 14,600,00. The ages of the room temperature cultures range from 12 to 48 hours and of the 37° C. cultures from 9 to 24 hours. Table XXI presents the TABLE XIX— SWEET CUEDLERS Number of Organisms in Slightly Coagulated Milk. Date Age of Temperature Bact. per Culture Strain in Hours Held c. c. 24 A 37° 4,900,000 24 B 87° 2,600,000 24 A 37° 3,600,000 18 C 37° 1,400,000 18 A 37° 1,400,000 24 37° 3,800,000 24 C 37° 3,800,000 12 A 37° 1,900,000 12 B 37° 4,000,000 12 C 37° 4,600,000 12 C 37° 3,860,000 24 A 37° 1,700,000 12 A 37° 3,700,000 24 A 37° 4,800,000 48 A Room 3,000,000 48 C Room 1,250,000 24 A Room 3,000,000 12 C 37° 4,200,000 12 37° 3,700,000 9 A 37° 1,400,000 24 A Room 3,190,000 Average 3.100,0'00 December December January January January January January January January January January January January January February February February February February March April 31, 1914. 31, 1914. 7, 1915. 8, 1915. 9, 1915. 16, 1915. 16. 1915. 19, 1916. 19, 1916. 21, 1915. 23, 1915. 26, 1914- 28, 1915. 31. 1915. 1, 1915. 9, 1915. 10, 1915. 11. 1915. 20, 1915. 18, 1915., 19, 1915. 50 TABLE XX— SWEET CUEDLERS— NUMBER OF ORGANISMS IN MEDIUM COAGULATED MILK. TABLE XXI— SWEET CUEDLERS- NUMBERS OF ORGANISMS IN FIRMLY COAGULATED MILK. Date [ Age of Culture in Hours Temperature Held No. Bact. per January January February February February February February, February, February March March March March March March March March March March March March March March March March March 1915.. 1915.. 1915.. 1915.. 1915.. 1916.. 1916. 1916. 1915.. 1915.. 1916.. 1916.. 1916.. 1916.. 1916.. 1915.. 1915.. 1915.. 1915.. 1916.. 1916.. 1916.. 1915.. 1916.. 1916.. 1916.. 18 24 12 12 12 12 12 12 12 12 12 12 12 9 9 9 9 9 24 24 24 24 24 9 9 18 B B C C A C C C C B A A A C C A A C B A C B B C 37° 37° 37° 87° 37° 37° 37° 37° 37° 37° 37° 37° 37° 37° 37° 37° 37° 37° 37° Room Room Room Room Room 37° 37° Average 20,000,000 60,000,000 29,000,000 36,000,000 18,900,000 42,400,000 45,000,000 29,200,000 56,000,000 28,400,000 23,900,000 36,200,000 33,700,000 18,400,000 18,200,000 20,200,000 16,950,000 23,400,000 16,960,000 26,600,000 22,200,000 25,600,000 33,000,000 41,200,000 17,000,000 16,700,000 28 ,600,000 TABLE XXII— SWET CURDLERS- -NUMBER OF ORGANISMS AT TWO-HOUR PERIODS. Age of Strain A. Strain B. Strain C. Culture in Hours Bacteria per c. e. Condition of Milk Bacteria per c. c. Condition of Milk Bacteria per c. <■■ Condition of Milk 2 4 6 8 10 1,000.000 4.350.000 7,700.000 8,400.000 9,600.000 no chanice chancre no chancre slightly coapr. medium coat:. 950.000 1,900.000 9,600.000 10,200.000 15,000.000 no chanire no chancre no chanfire slightls coag. medium coacr. 101.000 6,800.000 24,300.000 30,000.000 no chance no chancre med. coae. firmly curdled 51 data obtained on the milk classed as firmly coagulated. Five of the counts were made on milk held at room temperature while the other 21 were made on milk held at 37° C. The average of the 26 counts is 28,500,000 per c. c. while the individual counts range from 15,950,000 to 56,000,000. All of the room temperature cul- tures were 24 hours old while the ages of the 37° C. cultures range from 9 to 24 hours. Table XXII shows the number of organisms found in inocu- lated milk at two hour intervals up until the time of coagulation. Counts were secured on unchanged milk (up to 9,600,000 per c. c. ) which are much larger than the counts obtained on slightly coagulated milk as reported in table XIV. The counts reported in table XXII are in general fairly high for the various stages of coagulation; these high counts both in unchanged and coagulated milk were secured on cultures consid- erably younger than the cultures dealt with in tables XIX, XX, and XXI and it seems probable that the age of the culture is im- portant from the standpoint of the changes produced by a given number of organisms. The amounts of the various products se- creted by a given organism may depend on the time that elapses before that organism divides and thus the changes produced by a given number of cells may be materially influenced by the rapid- ity with which division is taking place. The results obtained on the sweet curdlers indicate that many organisms (in the cases studied always over 1,000,000 per c. c.) are present in milk before there is any evidence of coagulation. The numbers of sweet curdling organisms required to produce changes in mOk, however, are considerably smaller than the numbers of organisms required to produce changes in the cases of the other types considered thus far. Moreover the number of sweet curdlers required to bring about marked changes in milk is smaller than in the cases of the other types studied. The com- paratively small number of sweet curdlers required to produce changes in milk may be due to the comparatively large size of the type; there is at least the possibility that the larger cells may produce products responsible for the changes in milk in larger amounts than the smaller cells. Organism 736 Organism 736 was isolated from a sample of milk brot to the laboratory from one of the smaller cities of the state. It causes a disagreeable putrefactive odor in milk, later a clearing of the milk, beginning at the surface, and finally practically com- plete digestion. The organism grows very well at room tempera- ture. Since the original isolation, the organism has been en- 52 countered a number of times and its description is soon to be published. SPECIAL METHODS The inoculated milk was always held at room temperature. Both sterile milk (8 to 15 c. c. in tubes) and aseptic milk (30 to 50 c. c. in tiibes) were employed; the aseptic milk was used only for the work on odor while the sterile milk was used for the work on odor and the different stages of digestion. The stages in the development of the cultures have been classed as follows : (A) Faint Odor : Milk showing a distinct but faint odor. (B) Marked Odor: Milk showing a pronounced odor. ( C ) Beginning Digestion : Milk showing a definite clear- ing at the surface. ( D ) Considerable Digestion : Milk showing a clear layer from 1-8 to 3-8 inches thick at the surface. (E) Complete Digestion : Milk showing practically com- plete digestion. RESULTS OBTAINED Table XXIII shows the results obtained on milk classed as showing a faint odor. The average for the 20 determinations is 32,000,000 per e. c. and the individual counts vary from 14,000,- 000 to 57,000,000 while the ages vary from 9 to 42 hours. The data obtained on the milk classed as showing a marked odor is presented in table XXIV ; the average of the 37 determinations is 95,000,000 per c. c. while the individual counts range from 66,000,000 to 150,000,000 ; the ages of the cultures vary from 9 to 24 hours. The data secured on the milk classed as showing beginning digestion is shown in table XXV ; the average for the 16 determinations is 202,000,000 per c. c. while the range is from 160,000,000 to 240,000,000. The ages of the cultures vary from 10 to 24 hours. Table XXVI presents the results obtained on the milk classed as showing considerable digestion; the 16 counts range from 250,000,000 to 445,000,000 per c. c. and averages 312,000,000. Most of the cultures were 24 hours old altho one culture of 18 and one of 72 hours are included. The data ob- tained on milk showing complete digestion is given in table XXVII where the 7 counts range from 470,000,000 to 750,000,000 per c. c. and average 646,000,000 ; the ages of the cultures vary from 18 to 48 hours. The data obtained relative to the development of an odor in aseptic milk is shown in table XXVIII. The cultmres were not grouped on the basis of the extent of the odor b|||t all were plated when the characteristic odor was definitely pi-esent. The 31 counts obtained vary from 19,000,000 to 98,000,000 per c. c. and average 48,000,000. The determinations made on aseptic milk 53 TABLE XXIII— Organism 736. Number of Organisms in Milk Showing Faint Odor. TABLE XXIV— Continued. 1 Age of No. Bact. Date Culture per in Hours c. c. Nov. 2 1914.. 24 42,000,000 Jan. 16, 1915.. 12 39,000,000 Jan. 28, 1915.. 10 22,000,000 Feb. 3, 1915.. 12 55,000,000 Feb. 6 1915.. 12 33,000,000 Feb. 6, 1915., 12 39,000,000 Feb. 12 1916.. 36 14,000,000 Feb. 12 1915.. 12 28,000.000 Feb. 16 1915.. 42 29.000.000 Feb. 17 1915,. 12 18.000.000 Feb. 18 1915.. 12 46,000.000 Feb. 26 1916.. 12 24,000,000 March 6, 1916 12 41,000,000 March 6. 1915 12 49.000,000 March 8, 1916 9 25,000,000 March 23, 1916 ■J 23,000,000 March 24. 1915 9 21,000,000 March 24, 1915 19,000,000 March 27. 1915 24 57,000,000 March 28. 1916 12 22,000,000 Average 32,000,000 L , TABLE XXIV— Organism 736. Number of Organisms in Milk Showing Marked Odor. Oct. 23, 1914.. 24 136,000,000 Nov. 1, 1914.. 24 82,000.000 Nov. 11. 1914.. 24 103,000.000 Nov. 23, 1914.. 24 75,000,000 Dec. 2, 1914.. 24 120,000,000 Dec. 17, 1914.. 18 79,000,000 Dec. IS, 1914.. 18 92,000,000 Dec. 19, 1914.. 18 135,000,000 Dec. 21, 1914.. 18 87,000,000 Dec. 23, 1914.. ii 101,000.000 Jan. », 1916.. 18 104,000,000 Jan. 11. 1915.. 24 70,000,000 Jan. 13, 1915.. 18 130,000,000 Jan. 16, 1915.. 12 120,000,000 Jan. 15, 1915.. 12 90,000,000 Oct. 31, 1914.. 24 228,000,000 9. 1914.. 24 190,000,000 Nov. 11, 1914.. 24 210,000,000 Nov, 13, 1914.. 24 220,000,000 Nov. 25, 1914.. 24 186,000,000 Dec 7 1914.. 24 210,000,000 Dec. 11, 1914.. 24 210,000,000 Dec. 31, 1914.. 24 230,000,000 Jan. 1, 1915.. 18 180,000.000 Jan, s. 1915.. 18 180,000,000 Jan. 28, 1916,.. 10 210,000,000 Jan. 31, 1916.. 12 170,000,000 Feb. 10, 1915.. 24 231,000,000 Feb. 21, 1916.. 12 240,000,000 Feb, 22, 1915.. 12 160,000,000 Feb. 24, 1916.. 12 Average 172,000,000 202,000,000 TABLE XXV— Organism 736. Number of Organisms in Milk Showing Beginning Digestion. Age of No. Bact. Date Culture per in Hours c. c. Jan. 17, 1915.. 12 107,000,000 Jan. 18, 1916.. 12 150,000,000 Jan. 19, 1916.. 12 76,000,000 Jan. 20, 1914.. 12 76,000,000 Jan. 21, 1915.. 12 78.000,000 Jan. 22, 1916.. 12 77.000,000 Jan. 23, 1916.. 12 77,000,000 Jan. 26, 1915.. 12 99,000,000 Feb. 2, 1916.. 12 72,000,00t) Feb. 13, 1916.. 12 76,000,000 Feb. 22, 1915.. 12 126,000,000 Feb. 26, 1915. 12 66,000,000 Feb. 27, 1915.. 12 98,000,000 March 2, 1916 12 88,000,000 March 3, 1915 12 90,000,000 March 8, 1915 9 66,000,000 March 11, 1916 9 79,000,000 March 16, 1916 9 100,000,000 March 18, 1916 10 76,000,000 March 19, 1916 9 116,000,000 March 20, 1916 9 108,000,000 March 26, 1915 9 107,000,000 Average 95,000,000 show approximately the same numbers as the determinations made on sterile milk showing either a slight odor or a marked odor. As already stated, cows were selected for the production of the aseptic milk on the basis of the low bacterial content of their milk ; some of these samples of aseptic milk had, however, a pronounced odor that of course tended to mask the odor pro- duced by the organism inoculated. If the data obtained with the milk from each animal is con- sidered, a wide variation is noticed ; with the milk from cow 69 the 7 determinations range from 25,000,000 to 98,000,000 per c. c, with the milk from cow 207 the 5 counts range from 19,000,000 to 82,000,000 per c. c, with the milk from cow 87 the 6 counts range from 33,000,000 to 70,000,000 per c. c, with the milk from cow 217 the 7 counts range from 30,000,000,to 77,000,000 per c.c, with the milk from cow 143 the 4 counts range from 20,000,000 to 44,000,000 per c. c. and the two counts on the milk from cow 162 are 53,500,000 and 42,500,000 per c. c. Three trials made on 54 TABLE XXVI— Organism 736. Number of Organisms in Milk Showing Medixmi Digestion. Age of No. Bact Date Culture per in Hours c. c. Oct. 10. 1914.. 24 283,000,000 Oct. iv, 1914. . 24 340,000,000 Oct 2a, 1914.. 24 446,000,000 Nov. 7, 1914.. 24 285,000,000 Mov. 6. 1914.. 18 250,500,000 Mov. 18, 1914.. 72 440,000,000 JSIov. 19. 1914.. 24 265,000,000 Mov. 20, 1914.. 24 276,000,000 Nov. 22, 1914.. 24 290,000,000 Nov. 27, 1914.. 24 300,000,000 Dec. S), 1914.. 24 260,000,009 Dec. IS. 1914.. 24 250,000,000 Jan. 2, 1915.. 24 416,000,000 Jan. 7, 1915.. 24 290,000,000 i'eb. 3, 1915.. 24 290,000,000 J-'eb. 9, 1915.. 24 1 Average 310,000,000 312,000,000 TABLE XXVII—Organism 736. Number of Organisms in Milk Showing Complete Digestion. Age of No. Bact Date Culture per in Hours c. c. Oct 12, 1914.. 24 750,000,000 Oct 14, 1914.. 48 710,000,000 Oct. 30, 1914.. 24 670,000,000 Nov. 5, 1914.. 18 690,000,000 Nov. 12, 1914.. 48 490,000,000 Nov. 26, 1914.. 24 470,000,000 Nov. 28, 1914.. 48 Average 740.000,000 646.000.000 TABLE XXVIII— Organism 736. Number of Organisms in Milk Showing Characteristic Odor. Age of No. Bact. Date Cow Culture per Number in Hours c. c. 1914 June 10. 69 18 82,000,000 June 10. 69 18 92.600,000 June 10. 69 22 98.000.000 June 10. 213 18 31.600.000 June 10. 213 22 43.000.000 June 10. 87 22 70.000,000 June 16. 87 12 64,000.000 June 15. 207 12 61.000.000 June 15. 213 12 30.000.000 June 15. 69 12 47,000,000 June 15. 87 12 50.000.000 June 15. 207 12 26.000.000 June 16. 213 12 31,000,000 June 16. 69 12 26,000,000 June 16. 162 12 63.500,000 June 16. 143 12 30.000,000 June 16. 207 15 22.000.000 June 17. 87 15 61.500,000 June 17. 143 15 22.000.000 June 17. 213 16 55,000,000 June 17 . 162 16 42.600.000 June 19. 69 16 30.000,00fl June 19. 87 15 33,000.000 June 19. 207 15 19,000,000 June 19. 213 15 30,000,000 June 19. 143 16 20,000,000 June 22. 143 16 44,000.000 June 22. 213 16 77.000.000 June 22. 207 18 82.000.000 June 22. 87 IB 33,000,000 June 22. 69 16 95,000,000 Average 48,000.000 the same day with milk from cow 69 range from 82,000,000 to 98,000,000 per c. c, a reasonably close agreement. The odor of the freshly drawn aseptic milk varied from day to day and this is undoubtedly in part the cause of the variations in the counts obtained on milk from the same animal. The original odor of the milk is likewise responsible for some of the variation in the data presented in table XXVIII. "With Organism 736 a large number of organisms were present in milk before a change in the odor could be detected. The smallest number of organisms producing an odor in sterile milk was 14,000,000 per c. c. and in aseptic milk 19,000,000 per c. c. From these comparatively low values, there was an increase in the number of organisms with an increase in the changes pro- duced in the milk, until in milk which had recently become com- pletely digested numbers as high as 750,000,000 per c. c. were present. As in the case of the other organisms studied, large variations were encountered in the numbers of organisms pres- ent in milk which was classed as showing the same change. Bact. lactis acidi. Three strains of Bacti. lactis acidi were isolated for use in the 55 work herein reported ; A, came from a good starter ; B, from an old discarded starter, and C, from milk that was allowed to sour at room temperature. All the strains curdled milk rapidly and produced the usual color changes in litmus milk. The work with Bad. lactis acidi dealt with the numbers of organisms present at various stages in the reduction of litmus milk, the number pres- ent in coagulated milk, and the number present in aseptic milk at the time an acid taste was first detected. SPECIAL METHODS The work on the reduction of litmus milk and the coagulEftion of milk was done with tubes (8 to 15 c. c. of milk) for the most part. In the work on the numbers of organisms present in inocu- lated aseptic milk when an acid taste could first be detected, large test tubes were employed, the milk being drawn directly into sterilized tubes in amounts varying from 30 to 50 c. c. It was cus- tomary to compare the flavor of the inoculated aseptic milk, after the various periods of holding, with the flavor of the unin- oeulated controls. With this method, it was a common occur- rence to find a distinct rise in acidity in a sample which could not be classed as sour; accordingly in the tables dealing with the development of an acid taste in aseptic milk, the change in flavor if given. All the inoculated milk was held at room temperature. The following classification of the changes was employed : (A) Slight Eeduction: Litmus milk showing a slight but definite decrease in the intensity of the color. (B) Medium Reduction : Litmus milk from which the color of the litmus had largely disappeared. (C) Complete Reduction : Litmus milk from which the color of the litmus had entirely disappeared, except at the very top of the tube. (D) Coagulation: Milk showing a complete coagula- tion. RESULTS OBTAINED The data obtained on the samples of milk classed as slightly reduced are presented in table XXIX. The average for the 15 determinations is 117,000,000 per c. c. and the individual deter- minations range from 62,000,000 to 181,000,000 while the ages of the cultures vary from 9 to 24 hours. Table XXX shows the data obtained on milk that was classed as showing medium reduc- tion; the range of the 29 determinations is from 107,000,000 to 300,000,000 per e. c. and the average is 212,000,000 while the ages of the cultures vary from 10 to 24 hours. The results on litmus milk showing complete reduction are shown in table XXXI. The 21 individual determinations range from 340,000,000 to 950,000,- 56 000 per c. c. and average 560,000,000 while the ages of the cul- tures vary from 12 to 24 hours. Table XXXII presents the data obtained on tubes showing coagulation. The six determinations range from 1,060,000,000 to 2,890,000,000 per e. c. and average 1,800,000,000, while the ages of the cultures vary from 24 to 48 hours. Table XXXIII shows the data obtained at two hour intervals from inoculation until complete reduction. From this data, it is evident that large numbers of organisms (as high as 26,900,000 per c. c. ) may be present without any change in the litmus. Con- siderable variation is evident in the data secured in the different trials ; inasmuch however as there are no consistent differences be- tween the strains in the preceding tables dealing wtih Bact. lactis acidi, it seems probable that this variation cannot be ascribed to the strains used. The data obtained on aseptic milk are shown in tables XXXIV TABLE XXIX— Bact. lactis acidi. Number of Organisms in Slightly Reduced Litmus Milk. 1 Age of No. Bact . Date Culture strain per in Hours! c. c. Nov. 23-14 24 A 88,000,00» Jan. 30-16 10 C 62,000,000 Jan. 31-16 12 B 100,000,000 i^'eb. 9-16 12 B 86,01)0,000 Mar. 18-16 9 C 142,000,000 Mar. 19-16 9 B 166,000,000 Mar. 20-16 8 C 99,000,000 Mar. 21-15 9 A 128,000,000 Mar. 22-lB 9 B 143,000,000 Mar. 23-16 9 B 133,000,000 Mar. 24-16 9 C 90,000,050 Mar 26-15 9 A i8i;ooo,ooo Mar. 30-15 9 C 100,000,000 April 2-16 9 A 130,000,000 April 3-16 9 Av A erage 116,000,000 117,000,000 TABLE XXXI— Bact. lactis acidi. Number of Orsanisms in Completely Reduced Litmus Milk. TABLE XXX— Bact. lactis acidi. Number of Organisms in Medium Re- duced Litmus Milk. Nov. Dec. Dec. Dec. Dec. Dec. Dec. Jan. Dec. Dec. Dec. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Feb. Feb. 21-14 24 1 C 23-14 18 B 19-14 18 B 19-14 18 C 21-14 18 B 31-14 18 B 31-14 18 A 9-16 18 A 3-14 16 B 3-14 16 C 6-14 16 C 16-16 12 18-16 12 A 19-16 12 A 20-15 12 A 22-16 12 A 22-16 12 B 23-15 12 C 25-15 12 B 2.5-15 12 C 28-16 12 A 4-16 12 C 12-16 12 B 240,000,000 199,000,000 280,000,000 290,000,000 230,000,000 156,000,000 230,000,000 107,000,000 300,000,000 146,000,000 116,000,0^0 296,000,000 290,000,000 260,000,000 226,000,000 240,000,000 240,000,00* 208,000,000 128,000,000 247,000,000 130,000,000 265,000,000 144,000,000 1 Age of No. Bact . Date 1 Culture strain per n Hours c. c. Feb. 18-15 12 A 187,000.000 i^'eb. 25-15 12 A 110,000,000 l''eb. 26-16 12 C 242,000,000 Mar. 4-16 12 A 240,000,000 Mar. 6-16 12 A 186,000,00T> Mar. 18-16 10 Ave A rage 239,000,000 212,000,000 Nov. Nov. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Jan. Jan. Jan. Feb. Feb. Feb. Feb. Feb. Feb. 27-14 24 27-14 24 2-14 24 6-14 15 7-14 16 11-14 18 12-14 18 14-14 16 17-14 18 lS-14 18 19-14 18 21-14 18 2-16 24 11-16 24 19-16 12 5-16 12 10-16 12 17-15 12 18-16 12 21-16 12 22-16 12 A B A A B A B A A B A A A A C A B A C A A Average 640,000,000 800,000,000 960,000,000 685,000,000 680,000,000 670,000,000 490,000,000 388,000,000 340,000,000 528,000,000 376,000,000 630,000,000 680,000,000 615,000,000 410,000,000 580,000,000 460,000,000 430,000,000 430,000,000 740,000,000 610,000,000 560,000,000 TABLE XXXII— Bact. lactis acidi. Numbers of Organisms in Litmus Milk Showing Coagulation. Nov. 26-14 24 A 1.650,000,000 Nov. 29-14 48 A 1,060,000,000 Nov. 30-14 24 A 2,630,000,000 Dec. 6-14 24 A 2,890,000,000 Jan. 7-16 24 A 1,180,000,000 Mar. 3-16 36 A 1,420,000,000 Average 1,800,000.000 57 TABLE XXXIII— BACT. LACTIS Number of Organisms and Acidities at ACIDI. 2 Hour Periods Age of Culture STRAIN A. STRAIN B. in Bacteria Cond of 1 Bacteria Cond of Hours per c. c. Milk 1 per f. c. Milk 2 .16 1,750,000 no change .10 830,000 no change 4 .16 8,200,000 no change .16 3,900,000 no change 6 .16 15,500,000 no change slight .16 26,900,000 no change slight 8 .18 130,000,000 reduction medium .18 116.000,000 reduction medium 10 .19 216,000,000 reduction complete .20 176,000,000 reduction complete 12 .21 365,000.000 reduction complete .22 250,000,000 reduction complete 14 .25 600,000,000 reduction .24 460.000,000 reduction STRAIN C. Cond of- Acidity per c. c. Milk .19 460,000 no change .19 920.000 no change .22 8,600,000 no change slight .30 62,000.000 reduction medium .33 125,000.000 reduction complete .36 300.000.000 reduction to XXIX. The tables show the acidities, and whether there was a sour taste or simply a distinct rise in acidity, as well as the numbers of bacteria present. Table XXXIV deals with milk from cow 213. The seven samples showing a definite rise in acidity showed bacterial counts ranging from 30,000,000 to 90,000,000 per c. e. and averaging 56,- 000,000 while the four samples classed as sour showed counts ranging from 53,000,000 to 166,500,000 per c. c. and averaging 104,000,000. Table XXXV presents the data obtained on milk from cow 69. The 5 samples showing a distinct rise in acidity showed counts varying from 49,000,000 to 112,000,000 per c. c. and averaging 70,000,000 while the seven samples called sour showed counts ranging from 93,000,000 to 198,500,000 per c. c. and averaging 152,000,000. Wilh milk from cow 207 (table XXXVI) the seven samples showing a definite rise in acidity showed counts ranging from 37,500,000 to 127,000,000 and averaging 70,000,000 per c. c. while the counts on the 4 sour samples ranged from 91,000,000 to 235,000,000 and averaged 156,000,000 per e. c. Table XXXVII presents the data obtained with milk from cow 87. The counts on the seven samples classed as showing a distinct rise in acidity vary from 57,000,000 to 243,000,000 per c. c. and average 132,000,000, while the 3 samples classed as sour varv from 176,000,000 to 251,000,000 per c. c. and aver- age 219,000,000 per c. c. 58 In table XXXVIII which deals with milk from cow 162, all the seven samples were classed as showing a distinct rise in acidity ; the range was from 67,000,000 to 209,000,000 per c. c. and the average was 121,000,000. The milk from cow 143 is considered in table XXXIX. The four samples classed as showing a distint rise in acidity showed counts ranging from 146,000,000 to 330,000,000 per c. c. and averaging 227,000,000 while the 3 sour samples showed counts ranging from 203,000,000 to 460,000,000 and averaging 305,000- 000 per c. c. Considering tables XXXIV to XXXIX inclusive, there seems to be no very definite relationship between the development of a sour taste and the increase in acidity. In a number of in- stances milk showing increases of .04 or .05% in the acidity was called sour, while in one instance a sample of milk showing an increase of only .03% was considered sour. On the other hand, certain samples of milk showing acidity increases of .04 or .05% or even more were classed as showing a distinct rise in acidity only, instead of being definitely sour. One sample of milk show- ing a rise of only .02% was classed as showing a distinct rise in acidity. These variations in the acid taste of samples of milk showing the same general acidity increases are, in part, undoubt- edly due to the masking effect of the other flavors in milk as well as to the masking effect of considerable amounts of fat. It is evident, however, that comparatively small amounts of acid may be responsible for an acid flavor in milk, and this tends to explain the association of what are apparently normal acidities with acid flavors. There seemed to be only a very general relationship between the rise in acidity and the numbers of organisms present. In the case of cow 69, for example, more organisms were present, in a sample of milk showing a rise of only .02% in acidity than in another sample showing a rise of .07% and this same general situation was observed with the milk from the other cows. With Bad. lactis acidi large numbers of organisms were present in milk before there was a reduction of the litmus (if litmus was I)resent) or any other detectable change. The smallest number of organisms present in litmus milk showing a slight reduction of the litmus was 62,000,000 per c. c. ; in general, with an increase in the extent of the reduction there is an increase in the number of organisms present. The smallest number of organisms present in milk showing coagulation was 1,060,000,000 per c. c. In aseptic milk showing, to the taste, a definite rise in acidity or a sour taste there were always large numbers of organisms present. In aseptic milk as in sterile milk, wide variations were observed in the result producted by the same general number of organisms. 59 TABLE XXXIV— COW 213. Bact, lactis acidi. Date A« of Culture Orie. Acidity Acidity of Culture Bacteria per c. c. Condition of MUk June 9. 1916 14 20 15 22 12 12 15 16 15 15 16 .20 .21 .22 .23 .22 .25 .28 .19 .20 .19 .22 44,000,000 61,000.000 116.000.000 166.500.000 68.000.000 63.000.000 82,500,000 90,000,000 57,500,000 30,000,000 62,000,000 d r. June 9, 1916 d r 1916 .18 .18 .18 .19 .21 .16 .13 .16 .15 1916 1916 d, r. June 1 6, 1915 ]915 June 22, 1916 d. r. 1915 d r. 1916 d. r. 1915 d. r. d. r. — distinct rise in acidity. TABLE XXXV— COW 69. Date Ago of Cultui;e Orip. Acidity Acidity of Culture Bacteria per c. c. Condition of Milk 1915 14 20 22 12 12 15 15 16 15 16 18 18 16 .19 .21 .23 .20 .20 .21 .19 .20 .20 .19 .21 .20 .21 66.000.000 93,000,000 190,000,000 71,000,000 62,000,000 169,(100,000 137,000,000 150,000,000 137,000,000 112,000,000 137,600,000 49,000,000 198.600.000 d. r. 1915 1915 .15 .18 .13 .18 .14 .16 .14 .13 .16 .15 .15 1916 d. r. June 16, 1915 d. r. 1915 1915 June 19, 1916 sour June 19, 1915 June 22 1915 . . d r. 1915 1915 d. r. June 26, 1915 sour d. r. — distinct rise in acidity. TABLE XXXVI— COW 207. Bact. lactis acidi. Date Age Acidity Bacteria Condition of of per of Culture Acidity Culture C. P- Milk 20 .21 37.500.000 d. r. 15 .16 .21 66.500.000 d. r. 22 .16 .24 138,.500,000 sour 12 .19 .23 • 60,000.000 d. r. 12 .18 .22 67.000.000 d. r. 16 .18 .26 159,500,000 sour 16 .14 .22 91.000.000 sour 15 .18 .22 66.000.000 d. r. 16 .18 .23 127.000.000 d.r. 15 .18 .22 66.onO.000 d. r. 15 .18 .27 236.000.000 sour June 9. 1916 June 10. 1916 June !('. 1916 June 15, 1915 June 16, 1915 June V!, 1916 June 19, 1915 June 22, 1915 June 23, 1915 June 24, 1916 June 26, 1916 d. r. — distinct rise in acidity. TABLE XXXVII— COW 87. Bact. lactis acidi. Date Age of Culture Orig. Acidity Acidity of Culture Bacteria per c. c. Condition of Milk 15 22 12 12 15 15 15 16 16 16 .19 .19 .18 .16 .18 .14 .18 16 16 16 .22 .23 .22 .21 .24 .22 .21 .22 .22 .21 243.000.000 261,000.000 100.600.000 67.000.000 230.nno.000 176.n00.000 98,000,000 106,000,000 91,000,000 226,000,000 d.r. June 15 1915 d. r. .Tune 16 1915 d. r. d. r. Tune 23 1916 d. r. June 23, 1916 d r. June 26, 1916 d.r. d. r. — distinct rise in acidity. 60 TABLE XXXVIII— COW 162. Bact. lactis acidi. Date Age of Culture Orig. Acidity Acidity of Culture Bacteria per c. c. Condition of MUk June 16, 1915 12 16 16 16 16 15 16 dlty. .17 .16 .16 .16 .16 .14 .15 .23 .21 .20 .20 .20 .20 .20 76,000,000 145,000,000 162,000,000 96,600,000 92,000,000 67,000,000 209,000,000 d. r. June 17, 1915 a. r. June 19, 1915 d. r. June 22, 1915 d. r. June 23, 1916 d. r. June 24, 1915 d. r. June 26, 1916 d. r. — distinct rise in ac d. r. TABLE XXXIX— COW Bact. lactis acidi; 143. Date Afre of Culture OriK. Acidity Acirtll^' of Culture Bacteria per c. c. Condition of MUk June 16, 1916 12 15 16 18 16 15 16 .16 .16 .14 .13 .13 .13 .13 .21 .23 .20 .19 .20 .20 .20 203,000,000 460,000,000 252,000,000 146,000,000 173,000,000 330,000,000 260,000,000 sour June 17, 1916 1916 June 22, 1916 d. r. .June 23, 1915 . d. r. June 24, 1916 d. r. June 26, 1916 d.r. d. r. — distinct rise in acidity. SUMMARY In the work reported, an effort was made to secure information regarding the numbers of bacteria required to produce various changes in milk. While changes in the flavor and odor first attracted attention, other changes were considered because of the difficulties presented by all work dealing with changes in flavor and odor. Sterile milk was used for most of the work, altho some experiments were carried out with aseptic milk. A summary of the results obtained is presented in table XL. From the data presented, it appears that changes in milk due to the growth of bacteria therein occur only after large numbers of bacteria are present ; the samples of milk which showed changes of one kind or another always contained over 1,000,000 bacteria per c. c. and ordinarily much larger numbers. The sweet curdlers produced changes in milk with the samllest num- bers of organisms and here the smallest number observed with slight coagulation was 1,250,000 per c. c. With some organisms, pronounced changes required approximately 1,000,000,000 per c. c. and between this value and the minimum already mentioned wide variations were encountered. Wide variations apparently exist in the numbers of organisms present in milk showing the same condition. This is evident from the percentage variation between the minimum and maximum and also by the results obtained when freshly inoculated cultures were plated at two hour interv^.s for considerable periods ; the difficulty of classifying the conditions observed in milk are, in part, responsible for the variations obtained. 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