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Thermophilic and thermoduric organisms associated with spoilage of evaporated milk Atkinson, Lyle A, 1935

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1 THERMOPHILIC AND THERMODURIC ORGANISMS-- ^ ibj. nps/^ ASSOCIATED WITH SPOILAGE OP EVAPORATE^ MILK, Lyle A, Atkinson, B.S.A., and George J, Okulitch, B.S.A. A Thesis submitted for the Degree of MASTER OP SCIENCE IN'AGRICULTURE In the Department of DAIRYING, The University of British Columbia April, 1935. TABLE OP CONTENTS, ) Page Introduction. 1 Historical .. 1 Exp erimental 5 Checking the Raw Milk 11 Alteration of Plant Practices .. 22 Altering the Salt Balance 27 Reproducing the Spoilage 31 Innoculation Experiments 40 Classification of Organisms Isolated 43 Summary 52 Conclusions 56 References 59 Acknowledgments 61 THERMOPHILIC AND THERMODTJRIC ORGANISMS ASSOCIATBD WITH SPOILAGE OP EVAPORATED MILE* INTRODUCTION. An outbreak of spoilage of evaporated milk was experienced during the summer of 1935 by a factory shipping milk to tropical countries. The trouble was reported chiefly from India and Java and in such proportions as to threaten the loss of this business. Reports indicated that the spoilage was not general throughout any one s t e r i l i z e r batch, but the spoiled cans were scattered throughout the shipment, a few in each case. Furthermore, the cans did not give evidence of spoilage on arrival at the consignee's warehouses, but only after i t had been distributed in smaller parcels to the retailers. A significant fact In this situation was that, while this company exported large quantities of this same milk to points in the British Isles, no similar spoilage was experienced there. It was with a view to determine the oause of the trouble and prevent i t s recurrence that this work was undertaken. HISTORICAL.. The literature on spoilage of evaporated milk i s (2) not extensive, although some types of spoilage have been reported. Gassedebat ( 1 ) , i n studies on blown tins of milk failed to find either aerobio or anaerobic bacteria and concluded that deteriorations in condensed milk are of a chemical or physical character and not of bacterial origin. Dodge (2), was unable to isolate from condensed milk an organism which he believed was responsible for the trouble. From experiments with butyric and lactio acids he concluded that i t was probable, that in the cans of spoiled milk, the gas was not formed by the bacteria directly but was formed by electrolytic action between the metals of which the cans were composed and the acid gener-ated by the growth of bacteria in the milk before the latte was condensed. Hunziker and Wright (3)demonstrated that by scratching the tins and f i l l i n g cans with »4?fc solutions of l a c t i c and acetic acid, then sealing, s t e r i l i z i n g and incubating for some time at 90 degrees F. that the cans containing the dilute acid began to swell but the control cans containing d i s t i l l e d water only did not. Savage and Hunwicke (4) put lactic acid into cans of evaporated milk sufficient to bring the total acidity to .68$. They then sealed, sterilized and incubated the milk for a long time and could detect no gas formation. On the bases of these experiments they concluded that i t i s very unlikely that tins of milk ever become blown through chemical means alone. (3) The Vermont Agricultural Experiment Station (5), has published a short paper on the coagulation of supposedly-sterilized milk in which the milk curdled without souring, after which the curd was apparently digested leaving a whey like liquid above, with thick masses of slimy curd and precipitate in the bottom. B. subtilis was the organism most actively figuring in the change. Kelly (6) reports the spoilage of evaporated milk by sweet curdling types of bacteria which coagulate the milk with a custard like clot and show no change in the acidity, taste or smell of the milk. Gurdling started at the top and proceeded downwards. The causative organisms were identified as atypical forms of B. oereus,  B. simplex and B. megatherium. Hammer and Hussong (7),isolated an organism which they identified as Bacillus oereus from a spoiled can of evaporated milk. They did not attribute the original outbreak of spoilage, in the factory investigated, to this organism but found i t interesting in i t s action on evaporated milk as coagulation took place with'no change in the odor and flavor unless the incubation period was extended. They also found i f a satisfactory air supply was provided by transferring the innoculated milk to test tubes, or removing part of the milk from a can, the organism curdled the milk rapidly, produced an objectionable odor (4) and flavor and could be found in large numbers microscopic ally. Hammer (8) reports an outbreak of spoilage in evaporated milk, involving acid coagulation without gas due to a" heat resistant spore forming rod shaped organism to which he gave the name B, coagulans. Hussong and Hammer (9) isolated B. calidolaotis from sour curdled evaporated milk. This i s a spore formin facultative aerobe whioh curdles the evaporated milk rapidly at from 55 degrees 0. to 60 degrees 0. The milk showed an acidity of .67 percent to .74 percent, calculated as lactic acid. This thermophile failed to grow and did not curdle the milk at 37 degrees 0. or below. Hunziker (10) referring to curdiness caused by bacteria states that, in the majority of cases, single cans appear to be affected rather than the whole batch, suggesting uneven heat i n the s t e r i l i z e r or a limited distribution of the organism. In the majority of cases, the trouble failed to show for many weeks after manufacture suggesting abnormally slow growth, perhaps because of the high solids concentration, or unfavorable temperatures. When the temperature became high enough, spoilage occurred. After quoting several instances of spoilage in evaporated milk, Hunziker makes the following observations: (5) "These several citations of outbreaks of sour curdled evaporated milk suggest that the organisms responsible are highly heat resistant and may therefore withstand s t e r i l i z i n g temperatures that are adequate to destroy the usual germ l i f e present in evaporated milk. These observations emphasize the importance of greater attention to sanitation and scrupulous cleanliness in the factory to the end that the presence of these organisms may be avoided as much as possible. They further emphasize the advisability of using relatively high s t e r i l i z i n g temperatures and the importance of uniform heat distribution in the s t e r i l i z e r in order to insure maximum germ k i l l i n g efficiency." EXPERIMENTAL. It should be mentioned at the outset that this milk conformed to the British Standard for evaporated milk and contained approximately 9.0/2 fat and 31.5$ total solids, which is muoh heavier than Canadian or United States domestic standard. When the original outbreak of spoilage was reported, cans of the affected milk were returned for an examination and experiments were conducted to determine, i f possible, the causes of the spoilage. Practically, a l l of the cans returned showed a definite " f l a t swell", the milk in most instances being clotted with some production (6) of gas. Working on the assumption that the changes had been brought about by bacterial action, efforts were made to isolate causative organisms from this milk. Direct miscroscopio examination failed to show the presence of any organisms. Samples plated directly on Difco Purple Lactose Agar (11), and incubated at 37 degrees G. and 55 degrees G. failed to show any growth. Samples Incubated for two days at both temperatures and then plated and incubated, likewise showed no growth of l i v i n g organisms. These results indicated that i f the spoilage had been caused by microrganisms they had subsequently died out due to the production of aoid, lack of air, or some other oause. Duplicate cans of those returned as spoiled had been retained in the factory and these were subjected to incubation for 17 days at 55 degrees G. None of these cans showed physical evidence of spoilage similar to that experienced in the tropics, nor did they show presence of any li v i n g organisms after plating and incubation. Samples of this spoiled milk were submitted for examination to the Dairy and Gold Storage Branch, Dominion Department of Agriculture, Ottawa, and also to the Laboratories of the American Can Company at Maywood, I l l i n o i s . Both of these laboratories also failed to find l i v i n g organisms in the spoiled milk. The possibility suggested i t s e l f that spoilage (7) may have been due to defective sealing of the cans or to an electrolytic action set up between the metals of the t i n -plate, as suggested by Gassedebat. Examinations by our own engineers and those of the American Can Go. failed to show defective manufacture or sealing. As the tinning on the insides of a l l cans was bright and shiny, no evidences of electrolytic action were discovered which practically eliminated this possibility from our reasoning and investigation. It w i l l thus be apparent that a l l efforts to determine the cause of spoilage from affected cans proved unavailing. It has been the practice for some years, in the laboratory maintained by the Company manufacturing this evaporated milx, to examine representative samples from each s t e r i l i z e r batch for s t e r i l i t y . This was done by incubating the sealed cans at 37 degrees G. for 48 hours and then plating. The technique used was as follows: the tops of incubated cans were sterilized by swabbing with alcohol and then flaming with a gas jet. A hole was punched in the can by means Of an ice picx which had been dipped in alcohol and flamed and the sample drawn out with a sterile pipette. About l/2 c c . sample was used and plat-ing was done using Difco Purple Lactose Agar. The plates were incubated at 37 degrees C. for 48 hours and growth of (8) colonies noted. The purpose of incubating the cans of milk before plating was to encourage the growth of any organisms or spores present in the milk so that an occasional colony due to air contamination when plating would not be interpreted as indicating unsterile milk. The result of this was that the finished plates showed either heavy bacterial growth or no growth at a l l . The routine examinations had sometimes shown unsterile samples, although the percentage was not high. After the investigation was started i t was considered advisable to incubate the routine samples at 55 degrees 0, also, since a l l of the spoilage reported had taken place in tropical countries, there being a complete absence of i t in shipments to the United Kingdom. This practice was started in September 1933, and has since been continued. Incubated at 55 degrees 0. the percentage of unsterile samples increased until in November and December the greater percentage of them appeared unsterile. The great majority of the colonies growing on plates appeared to be non lactose fermenters as the reaction of the media was almost invariably alkaline. Several questions at this point now presented themselves. F i r s t l y , would a l l evaporated milk appear unsterile i f incubated at sufficiently high temperatures and for a sufficient length of time. Secondly, at what point (9) in the operation did these contaminating organisms gain entrance to the milk? Thirdly, would alterations in the method of manufacture permit of effective sterilization? Fourthly, could these apparently nbn-lactose fermenting organisms produce acid in milk, sufficient to cause coagulation? To obtain an answer to the f i r s t question, samples of three other brands of evaporated milk were purchased in the market and two cans of each brand were incubated at 37 degrees C. and 55 degrees 0. respectively, for 48 hours, and then plated. With the exception of one can which bloated during incubation, a l l samples appeared sterile and free from heat resistant organisms. The bloated can showed the presence of acid colonies rather than the al k a l i forming colonies experienced in routine plating. Deming and Davis (12), report an investigation on the bacteriology of evaporated milk in which they found practically a l l of 154 samples of milk purchased in the open market to be efficiently s t e r i l i z e d . Extensive experiments were conducted to determine the point of entrance of these heat resistant organisms. In making these determinations a procedure was adopted which i t was believed would show only the heat resistant organisms. A l l samples were taken in sterilized 16 ounce evaporated milk cans, sealed and sterilized at the usual temperature of manufacture, which varied between 226 degrees (10) F. and 230 degrees F. for a holding period of 35 minutes. The cans were subsequently incubated at 55 degrees 0. for 48 hours and then plated in the same manner as the routine samples. The results of s t e r i l i t y tests are shown in Table No.l. On the f i r s t attempt to determine the point of entrance of these resistant organisms six cans were taken from each of several points in the manufacturing process. A l l samples from the raw milk and after the preheater appeared st e r i l e , indicating that no organisms had survived the s t e r i l i z i n g temperature of 230 degrees F. for 35 minutes. Those taken from the vacuum pan and after the homogenizer, cooler and f i l l e r s a l l appeared unsterile and contained organisms which gave colonies similar to those experienced in the routine examination of the finished product. A second attempt showed a l l samples, even those from the preheater to be unsterile. No samples of raw milk were taken on this occasion. A third attempt showed a l l samples of raw milk to be steril e , while samples from the preheater, hot wells, vacuum pan and drop tank a l l showed some samples sterile and some not st e r i l e . The results of this check up tended to indicate that either the actual contaminating influence was slight or was intermittant during the day's run. A fourth and more systematic check up was made when samples were taken from each point in the TABLE.NO.1. Process Samples Showing Heat Resistant Bacteria  Results of Five Determinations. S - indicates sterile condition. U - indicates unsterile condition. £>ATE SOURCE AND. CONDITION. OF SAMPLE. Pre- Hot. Vacuum Drop Raw Heater . Wells .,. Pan • Tank S . u s U S U ,s U S U 1933. Nov. 16th 8 0 8 0 - - 0 8. 0 30 Nov. 20th - - 0 6 3 3 0 12 0 18 Nov. 26th 10 0 19 13 14 2 6 8 3 15 Dec. 8th 15 0 17 2 19 Z 13 7 12 6 1934. Jan. 19th 28 2 25 1 26 0 56 4 29 1 (11) process at ten minute intervals. The raw milk again appeared free from contamination by heat resistant organisn That from the preheater, hot wells, vacuum pan and drop tank appeared clear unt i l late in the day's run when samples bearing heat resistant organisms appeared in milk from a l l of these sources. A f i f t h similar exhaustive check was made with the same results, except that this time two of the cans from the raw milk late in the run showed heat resistant organisms. This was the f i r s t Occasion on which the raw milk had shown these organisms and indicated the possibility of the rav; milk being the original source of the contamination. CHECKING- THE RAW-MILE, Since in practically a l l cases the heat resistant organisms appeared only late in the day's operation, a preliminary check was made of the last three trucks of milk arriving at the Plant. Six cans from each truck were taken at intervals of five minutes during the dumping prooess. A l l gave negative results. Following this i t was decided that the raw milk from a l l trucks to the plant should be checked. This was done on February 15th and 16th, 1934. The method of taking these samples was as follows. A dipper from each can of shippers' milk was placed in a sterilized 8-gallon milk can. The composite from each load thus obtained was thoroughly mixed and four one pound cans (IS) of each were taken. These cans of mixed milk were sealed and sterilized at 250 degrees F. for thirty-five minutes. The samples were then brought to the laboratory and incubated for two days at 55 degrees G. They were then plated on Purple Lactose Agar and the plates incubated for 48 hours at 55 degrees C. The results of the tests on composite samples from trucks are shown in Table No.2* In this Table i t w i l l be noted that on the f i r s t day's examination a l l samples from a l l trucks appeared free of organisms which would survive the s t e r i l i z i n g temperature of 230 degrees F. for 55 minutes. On the second day one can from Truck #7 showed the presence of heat resistant organisms. On the third day samples from Truck #7 showed their presence i n two cans while samples from Trucks #6 and #15 showed them in one can only, while on the fourth day samples from a l l trucks examined appeared clear of these organisms. It should here be mentioned that on April 3rd a small amount of glucose was added to each can as an enrichment medium. The fact that milk from Truck #7 showed the presence of heat resistant bacteria to a greater extent than any other led us to examine the milk of this truck s t i l l further. Two samples from shipments of each of 49 shippers were examined and only 7 of these showed bacteria surviving the sterilization process. Milk from 6 of these TABLE NO.2. SHOWING PRESENCE OF HEAT RESISTANT BACTERIA IN RAW MILK. S - Sterile Plate. U - Unsterile Plate. Truck Feb. 15 Feb. 16 Apr. 3 Apr. 4 1. SSSS SSSS ssss ssss 2. SSSS ssss ssss ssss 3. SSSS ssss ssss ssss 4. ssss ssss ssss ssss 5. ssss ssss ssss ssss 6. ssss ssss sssu ssss 7. ssss sssu ssuu ssss 8. ssss ssss ssss ssss 9. ssss ssss 10. ssss ssss — 11. ssss ssss ssss ssss 12. ssss ssss --13. ssss ssss — — 14. ssss ssss — — 15. — — sssu ssss 16. — — ssss ssss (15) shippers showed organisms in only one can out of the two examined while the milk from the other shipper showed their presence in both cans. A further check on these seven shippers was deemed desirable and a test was made of six one pound cans of milk from each shipper. These showed a l l six cans sterile from six of the shippers,while from the seventh only one appeared sterile while five contained the resistant bacteria. This latter milk was from the same shipper as that showing both cans unsterile on the f i r s t examination. A v i s i t to this particular farm was deemed advisable and was subsequently made on May 12th. The barn on this farm was very old and rather dark in the cow stable. On the floor of the passageway in front of the. mangers was a layer of old dusty hay. The mangers themselves were dirty with bits of old hay and mash, notwithstanding the fact that the farmer had fed no grain or mash to the cows for some six weeks. Samples were taken from the wash water from udders, the feed bin, the hay in front of the mangers, the residue in the mangers, the piped water and the water in the cooling tank. These samples were a l l put into sterile milk and taken to the laboratory where they were steamed for thirty minutes, then incubated at 55 degrees 0. for 48 hours. By this time the milk had clotted with some digestion and gas production. Plates were made from each (14) on Purple Laotose Agar. After 48 hours incubation of the plates, they were found to be too f u l l of colonies to observe properly. Picked colonies showed both long and short rods with spore formation. The reaction of the medium was alkaline and a strong odor was produced. After successive platings, typical colonies were picked and transferred to one pound cans of evaporated milk, which were sealed and sterilized. On this f i r s t attempt a l l organisms appeared to be k i l l e d off in the s t e r i l i z i n g process of 230 degrees P. for 35 minutes. A second attempt when, a s t e r i l i z i n g temperature of 224 degrees was used showed their presence in some of the cans. Those samples from udders, hay and mash showed typical growth and from these, colonies were picked and purified. Classification later showed them to be the same as strains isolated from the finished milk, indicating dust on hay and mash to be among the sources of these baoteria on farms. Milk from the above farm was excluded from supplies used for the manufacture of this export evaporated milk. Coincidental with the removal of this milk, the routine samples examined showed a marked f a l l i n g off in numbers of those showing the presence of heat resistant organisms. It appeared that possibly this shipper was responsible for the bulk of the heat resistant organisms. (15) This conclusion was later proven to be a fallacy. No further determinations on the raw mille were made for some months following, as during June, July and August, practically a l l of our routine samples examined both at 37 degrees C. and 55 degrees 0. appeared free of organisms surviving the s t e r i l i z i n g temperatures. In September the percentage of samples showing heat resistant organisms started to increase and 15$ of 248 samples appeared unsterile at 55 degrees G. This increased to 49$ in October and further to 85.7$ in December, the Plant being closed from October 15th to December 6th. When these organisms commenced to increase in September i t was deemed advisable to again check the raw milk supplies. Samples were taken of composites of truck loads in the manner previously described. Six cans from each composite were sealed and sterilized at 225 degrees F. for 25 minutes. The results of three examinations are shows in Table No.3. From these examinations i t i s obvious that, at certain periods of the year at least, milk from a l l districts contained bacteria which would survive the st e r i l i z i n g temperature of 225 degrees to 228 degrees F. for twenty-five minutes. In cases where a l l six samples appeared sterile on one day's examination tests made on TABLE NO.3. SHOWING PRESENCE OP HEAT RESISTANT BACTERIA IN .SAW. MILK FROM TRUCKS S- indicates sterile condition. U- indicates unsterile condition. TRUCK Oct.3rd.' Pot.10th. Dec.17th. 1. ssuuuu uuuuuu suuuuu 2. uuuuuu suuuuu ssuuuu 3. ssssss uuuuuu ssssss 4. ssssss suuuuu ssuuuu 5. suusss sssssu _ 6. ssuuuu - _ 7. ssssuu ssssss sssssu 8. sssuuu - -9. ssssss ssssuu -10. uuuuuu uuuuuu -11. - ssssuu 12. - sssssu 13. sssuuu (16) subsequent days showed baoteria surviving the s t e r i l i z i n g process. In many cases only one or two cans of the six taken appeared unsterile which would indicate that these resistant organisms did not appear in large numbers in the original milk. The plates, however, invariably showed large numbers of colonies, indicating their vigorous growth at the temperature of incubation, viz., 55 degrees 0. The problem of eliminating-these'heat resistant organisms from supplies used in export milk now appeared more d i f f i c u l t than ever. If truoks from a l l districts showed their presence the next question which presented i t s e l f was this: would they be confined to a few shippers or would their occurrence be general? It was then decided to check the milk of individual shippers for the presence of these organisms. Accordingly on October 10th, 1.934, Truck #6 was examined. Samples were taken in the described manner, in one pound cans, of the mixed milk of each shipper. On.this truck out of 43 shipments, 8 only showed bacteria surviving s t e r i l i z a t i o n . It was not possible to make further examinations of the milk of individual shippers for some little.time after this as the plant was closed for a period of two months. About the middle of December, however, this work was taken up again as the percentage of samples of finished milk showing the heat resistant bacteria was steadily increasing. (17) This work was started on December 21st and continued to January 11th, 1935, during which time the milk of a l l remaining shippers was examined. Table No.4 shows the results of examination of individual shipper's milk from a l l districts being then received at the plant. The examination showed that out of 570 shippers, 496 shipped milk which appeared free of bacteria which would survive the s t e r i l i z i n g process while that from 74 shippers showed their presence. Previous experience had shown that milk from certain trucks would appear sterilized after being subjected to" heat process while samples from the same truck on subsequent days would sometimes appear to be not sterilized. It is quite possible that the milk of individual shippers would act in the same manner and many of the above shippers, whose milk appeared sterile on the f i r s t examination, would, i f examined further, show the presence of organisms surviving the s t e r i l i z i n g process. However, i t was f e l t that i f these bacteria were present in any considerable numbers they would .have shorn up, due to the size of the sample taken which was in each case one pound of the mixed shipment. The next logical step was to see what would be the- effect of withholding this affected milk from supplies TABLE NO.4. SHOWING NUMBERS OP SHIPPERS WHOSE MILE CONTAINED.. HEAT RESISTANT BACTERIA.. DATE TRUCK STERILE UNSTERI1E Deo.21 . 1. 49 21 tt 2 e 14 9 Jan. 3 3. §3 7 H 4. 33 16 I T 5* 19 4 Jan. 8 6. 153 6 IT 7. 29 1 Jan.11 8. 84 3 I I 9. . 62 7 TOTALS .496 74 (18) used for the manufacture of the export standard milk. A change was accordingly made in the method of handling supplies of shippers' raw milk. A l l that which had appear-ed free of resistant organisms was put through in the regular manner, evaporated to the export standard, packed and steri l i z e d . The balance containing the heat resistant organisms was dumped into separate vats and made into milk for domestic consumption. The results achieved by this segregation were interesting and gratifying. Table No.5 shows the condition of the evaporated milk both before and after the segregation of the unsatisfactory raw milk. It w i l l be noted that before segregation, during the f i r s t half of January, that 60 .7$ of the finished samples appeared unsterile at 37 degrees 0. and 92.5fo unsterile at 55 degrees 0. During the f i r s t week after the contaminated milk was withheld the number of unsterile samples had dropped to 10.0/1 at 37 degrees 0. and 20$ at 55 degrees 0. The table also shows the condition of the milk made from selected shippers at subsequent periods. It i s interesting to note that from January 16th to April 8th, 395 samples were examined at 37 degrees 0. and only 2.8$ appeared unsterile, and during the same time 487 samples were examined at 55 degrees 0. and 27.1$ were unsterile. Thus, at the latter temperature the percentage of finished samples showing heat resistant organisms had TABLE NO.5. SHOWING THE EPPECT ON THE FINISHED . MILK.OP THE SEGREGATION OF SHIPPERS' MILK CONTAINING HEAT RESISTANT BACTERIA. DATE INCUBATED AT 37° C. INCUBATED 55 0 • STERILE UNSTERILE STERILE UNSTERILE 1935 No. No • . No. No. Samples $ Samples $ Samples $ . Samples $ Before Segregation: Jan.1-15 58 39.3$ 89 60.7$ 11 7.5$ 136 92.5$ After Segregation: Jan.16-23* 54 90.0$ 6 10.0$ 92 80.0$ 23" . 20,0$ Feb.15-16 41 100.0$ 0 0.0$ 70 89.7$ 8 10.3$ Mar-. 1-15 117 98.3$ 2 1.7$ 83 69.7$ 36 30.3$ Mar.16-27 124 97.6$ 3 2.4$ 75 59.0$ 52 41.0$ Apr. 3-8 48 100.0$ 0 0.0$ 35 72. 9$ 13 27.1$ Total since Segregation 384 97.2$ 11 2.8$ 355 72.9$ 132 27.1$ Shippers' milk containing heat resistant bacteria, when made into evaporated milk during the period showed: 12 13.1$ 80 86.9$ 2 2.1$ 93 97.9$ (19) been reduced from 92.5/6 to 27.1$. It i s of further interest to note that, during the period of January 16th to 23rd, raw milk containing these troublesome organisms, when made into evaporated milk s t i l l showed 97.9$ unsterile at 55 degrees 0. The practice of holding out affected milk has proven of such value in obtaining sterile finished samples that i t i s being continued. It i s hoped that by periodic examination of a l l raw milk supplies and the segregation of contaminated milk that the percentage of samples of unsterile evaporated milk w i l l be kept at a minimum. The result of this segregation of milk free from, and that containing, the troublesome heat resistant bacteria has been to give the factory a good deal of available milk which appeared eff i c i e n t l y sterile at both temperatures of incubation. This has been of great value to the manufacturers in that they now have an available supply of milk, for export to tropical countries, which can be shipped with confidence in the knowledge that i t w i l l with-stand the conditions of temperature which i t must undergo during transit and storage. The authors believe that evaporated milk, which w i l l withstand the routine periods t of 48 hours insubation at 37 degrees 0. and 55 degrees 0., and show, on subsequent plating, no growth of organisms whatever, would not spoil under any conditions involving (£0) changes in temperature. At the commencement of this investigation and after the 55 degrees 0. incubator had been put into operation, a l l milk which did not appear sterile at both temperatures in the routine analyses was withheld from shipment to hot countries and was confined in i t s distribution to countries within.the Northern Temperate Zones. It i s of significant note that since this practice has been operative there have been no reports ol- recurrent spoilage of the original type from consignees i n any part of the world to which this milk has been shipped. From the ahove discussion i t w i l l be apparent that we have been successful in determining the point of entry into the supplies of heat resistant organisms which w i l l survive the s t e r i l i z i n g temperature and which w i l l grow actively at temperatures in the neighborhood of 55 degrees C. It has been conclusively shown that they gain entrance through the medium of the raw milk from shippers, and that their occurrence i s widely scattered through different di s t r i c t s and further that by segregation of affected milk a product can be produced which is relatively free of organisms which w i l l survive the commercial s t e r i l i z i n g processes. A further interesting point in connection with these heat resistant organisms i s their occurrence (21) incidental to seasons. Table No.6 and attached chart show that the varied conditions of seasons have a good deal to do with their presence or absence in the raw milk supplies. This chart i s based on the result of examinations of 4303 routine samples of export evaporated milk. It shows that during the year 1934 the percentage of samples appear-ing unsterile at 55 degrees 0. was very high during the Pall and Winter months and low during the Spring and Summer. Commencing in January with 72.6$ of the samples appearing unsterile their occurrence dropped markedly each month to a low of 1.6$ in June and then gradually increased to a high point of 85.7$ unsterile samples in December of the same year. This subsequently increased to 96.3$ samples unsterile in January 1935. .The samples incubated at 37 degrees 0. showed the same general tendencies although in every case, the percentages were lower. Commencing in January with 26.'6$ of samples unsterile the numbers decreased to none unsterile in September and increased to 8.4$ i n December, with a sharp rise to 71.3$ in January 1935. These figures point very definitely to the effect of seasonal conditions surrounding the production of milk. During the Pa l l and Winter when milk is produced under barn conditions the incidence of heat resistant types of bacteria in the milk i s greatest while during the Spring TABLE NO.6, SHOWING- SEASONAL INCIDENCE OF HEAT RESISTANT  BACTERIA IN ROUTINE SAMPLES OF EXPORT. EVAPORATED MILK. 1934 37° C. INCUBATION 55°0. INCUBATION MONTH STERILE jo UNSTERILE jo STERILE fo UNSTERILE fo January 69 73.4 25 26.6 31 27.4 82 72.6 February 204 96.7 7 3.3 140 70.0 60 30.0 March 339 97.4 3.6 287 83.2 54 16.8 April 96 96.0 4 4.0 93 91.1 9 . 8.9 May 101 96.2 4 3.8 98 95.1 5 4.9 June 129 98.5 2 129 98.4 2 1.6 July 258 98.5 4 1 e 5 253 96.5 9 3.5 August 294 99.7 1 0.3 279 94.5 16 5.5 September 247 100.0 0 0.0 211 85.0 37 15.0 October 124 89.8 14 10.2 70 50.7 68 49.3 December 198 91.6 18 8.4 32 14.3 191 85.7 Yearly 2059 95.9 88 4.1 1623 75.4 533 24.6 (22) and especially daring the Summer when cows are out on pasture and only brought into barns during the milking period their incidence i s least and constitutes only a negligible factor in the production of this type of milk. ALTERATION Off.PLANT PRACTICES. In the early stages of this investigation various changes were made in plant practices to effect the removal of heat resistant bacteria from equipment or to destroy them in s t e r i l i z i n g . When i n our f i r s t examinations their presence was noted i n a l l milk at various points in the process and not in the raw milk, i t was thought that parts of the equipment may have been retaining these bacteria and adding them to the milk supplies. As mentioned earlier in this report the f i r s t tests made showed bacteria surviving s t e r i l i z i n g temperatures In milk from the preheater, hot wells, vacuum pans and a l l subsequent points. They were particularly noticeable after the milk had passed through the vacuum pans. This equipment was already being reasonably cleaned and sterilized according to the usual plant practices. However a more rigorous routine was proposed as part of the equipment was, at the best, d i f f i c u l t to clean, i t had been d i f f i c u l t to completely prevent the formation of milk stone i n the tubes of the preheater, there being a small amount always present. The pipes from (23) the vacuum pan are not of the s a n i t a r y k i n d which can be taken down and brushed d a i l y . I r o n p i p i n g i s used from the vacuum pan to the pumps. The reason f o r t h i s i s that since these pumps are working against a vacuum at a l l times i t i s e s s e n t i a l to have a l l pipe j o i n t s a i r t i g h t . With the s a n i t a r y type of p i p i n g the vacuum would be destroyed and the e f f i c i e n c y of the pumps impaired, as the j o i n t s soon become s l i g h t l y worn. The usual p r a c t i c e i n c l e a n i n g the i r o n pipes was to run a strong soda s o l u t i o n through them and brush a c c e s s i b l e p a r t s w i t h a brush. Since a l l parts were not a c c e s s i b l e and the mi l k they c a r r i e d was always hot, the formation of a c e r t a i n amount of milk stone was unavoidable. The new p r a c t i c e adopted was to wash a l l parts as usual, then f i l l a l l of the i r o n p i p i n g w i t h a strong c a u s t i c soda s o l u t i o n and allo w i t to soak f o r h a l f an hour. F o l l o w i n g t h i s the vacuum pan was p a r t l y f i l l e d w i t h a s o l u t i o n of " D i v e r s o l " c o n t a i n i n g approximately 300 p.p.m. of c h l o r i n e . This was allowed to run i n t o and to stand i n the pipes overnight. In the morning before., the day's operations were commenced a s o l u t i o n of " D i v e r s o l " c o n t a i n -i n g 200 p.p.m. c h l o r i n e was made up i n the weigh tank and pumped through the whole mi l k l i n e . The i n s i d e of the vacuum pans and the storage tanks were also sprayed w i t h " D i v e r s o l " . Soaking w i t h t r i - s o d i u m phosphate followed by (24) vigorous brushing removed the m i l k stone from the preheaters. These changes i n c l e a n i n g methods appeared to help i n reducing the contamination and the numbers of samples show-in g heat r e s i s t a n t b a c t e r i a i n our examinations of milk at v a r i o u s p o i n t s i n the process. The percentage of f i n i s h e d cans showing i n e f f e c t i v e s t e r i l i z a t i o n , however, continued to i n c r e a s e . In the l i g h t of our present knowledge the reason f o r the persistence of t h i s contamination i s easy to , understand, as we now know that i t was being added d a i l y through the raw m i l k . In our e a r l y i n v e s t i g a t i o n s , however, a good deal of a t t e n t i o n was d i r e c t e d to removing the p o s s i b i l i t y of contamination: from equipment. As a check on the s t e r i l i z i n g e f f i c i e n c y of our r e v i s e d plant c l e a n i n g p r a c t i c e s a s e r i e s of t e s t s were run on m i l k a f t e r i t had passed from the vacuum pan through the i r o n pipe and pumps to the drop tank. Many of these samples appeared s t e r i l e a f t e r the s t e r i l i z i n g process but many others s t i l l contained heat r e s i s t a n t b a c t e r i a . These l a t t e r were more predominant at the beginning and toward the end of the run. On s e v e r a l occasions the f i r s t m i lk released from the vacuum pan appeared u n s t e r i l e . This f i n d i n g was of some s i g n i f i c a n c e i n i n d i c a t i n g t h a t some of these troublesome b a c t e r i a remained i n the equipment and s u r v i v e d even the strong s t e r i l i z i n g s o l u t i o n s used i n c l e a n s i n g the (25) equipment. As a g a l l o n or so of the f i r s t m i l k through • the equipment was l a r g e l y d i l u t e d w i t h the c h l o r i n e s o l u t i o n remaining i n the pipes, i t would appear that the organisms were extremely r e s i s t a n t to both the s t e r i l i z i n g heat and the g e r m i c i d a l a c t i o n of the c h l o r i n e . I t would therefore appear that any p r a c t i c a l method of plant c l e a n s i n g and s t e r i l i z i n g could not be depended upon t o e f f e c t the removal of these types of b a c t e r i a while they were being c o n t i n u a l l y r eintroduced through the raw m i l k . Experiments were also c a r r i e d out to determine temperatures and h o l d i n g times necessary to k i l l a l l r e s i s t a n t b a c t e r i a during the s t e r i l i z i n g process. I t should be r e c a l l e d at t h i s point that t h i s m ilk was packed under vacuum which gave about seven inches vacuum pressure w i t h i n the cans. The s t e r i l i z i n g had been done using a r e l a t i v e l y low temperature of 2S8 to 230 degrees F. f o r a h o l d i n g p e r i o d of 25 to 35 minutes r a t h e r than a higher temperature f o r a sho r t e r period of time. On November 16th, 1935, a number of cans were prepared, some' w i t h vacuum and some without and s t e r i l i z e d at both the high temperature short time and the low temperature long time processes. These cans were incubated at 55 degrees C. f o r three days and then plated w i t h the f o l l o w i n g r e s u l t : 4 cans held at 23C% degrees F. f o r 35 minutes, vacuum pack - a l l u n s t e r i l e • (26) 4 cans held at 23 0§- degrees F. for 35 minutes, no vacuum - 1 steri l e , 3 unsterile. 12 cans held at 241 degrees F. for 15 minutes, vacuum packed - a l l unsterile. 4 cans held at 241 degrees F. for 15 minutes, no vacuum - a l l unsterile. The above results would indicate that the organisms survived whether packed with or without vacuum. They also survived high temperature short time s t e r i l i z i n g . On November 26th following the above test, 48 cans of milk from one batch were taken. Twenty four were steri l i z e d at 230 degrees F. for 35 minutes and the other 24 at 240 degrees F. for 50 minutes. The cans were incubated for four days at 55 degrees G* and plated. The plates were then incubated for 48 hours at the same temperature and then examined. The results showed: 1. 24 cans sterilized at 230 degrees F. for 55 minutes, - 9 steri l e , 15 not st e r i l e . 2. 24 cans sterilized at 240 degrees F. for 30 minutes,- 24 s t e r i l e . This test showed that while the majority of the cans sterilized at 230 degrees F. showed the resistant organisms, thereby indicating their presence in the original milk, the temperature of 240 degrees F. for 30 minutes was effective in destroying them. At the latter temperature the milk i t s e l f , however, was darkened and badly grained, showing the unsuitability of this temperature from the standpoint of physical condition. (27) ALTERING THE SALS BALANCE. When experimental evidence showed the di f f i c u l t y , i f not the impossibility of obtaining effectively sterilized milk by improving the condition of the plant equipment, an attempt was made to find means of raising the st e r i l i z i n g temperature. It was realized that, i f these resistant organisms could not be kept out of the milk, means should be found for destroying them during the st e r i l i z i n g process. This presented certain d i f f i c u l t i e s . As mentioned earlier in this report the plant practice was to st e r i l i z e by holding the milk for a period of 25 to 35 minutes at temperatures ranging from 226 degrees F. to 230 degrees F. Some manufacturers use a s t e r i l i z i n g temperature of 240 degrees F. to 242 degrees F. for a holding period of from 15 to 16 minutes. The temperature range in sterilization of evaporated milk is between very narrow limits. These limits are even more restricted in the manufacture of the type of milk with which we are concerned in this report, viz., "British Standard" milk containing, as i t does, about 31-§- percent of total milk solids. The ever present problem is to heat the milk sufficiently high to destroy l i v i n g organisms and yet hold i t below the temperature at which coagulation with resultant graininess takes place. The st a b i l i t y of milk to heat i s a problem which (28) has called for a good deal of work on the part of many investigators. In summarizing the results of work-by Rogers, Deysher and Evans; Leighton and Deysher; Sommer and Hart; Deysher, Webb and Holm and Webb-and Holm, Hunziker (10) makes the following deductions: 1. "The salt balance is one of the most important known phases in the combination of factors that control the heat stability of evaporated milk. 2. "The casein is most stable when i t is in combination with a definite amount of calcium. Excess or deficiency of calcium available for the oasein-oalcium combination lowers the heat st a b i l i t y of the casein. The calcium and magnesium ions represent the positive charges, and are therefore opposed to the citrates and phosphates which represent the negative charges. The amount of calcium combination is determined by the balance between the calcium and magnesium group and the citrate and phosphate group of salts. An excess or deficiency of either group tends to unstabilize the casein. .5. "In the absence of the proper balance of the salts in milk, heat coagulation d i f f i c u l t i e s can be guarded against by the addition of the proper amount of those salts in which the milk i s deficient. In the great majority of cases of heat coagulation d i f f i c u l t i e s , the trouble is due to an excess of calcium (deficiency of citrates and phosphates). Such milk i s stabilized by the addition of a small amount of sodium citrate or di-sodium phosphate. If the low heat stability i s due to a deficiency of calcium (excess of citrates and phosphates) the addition of a soluble calcium salt such as calcium chloride usually provides the desired improvement. Cases of calcium deficiency are rare. 4. "Addition of salts for the correction of the salt balance are most effective when these salts, or a considerable portion thereof are (29) placed in the milk before condensing, 5. "The effect of the salt balance on heat st a b i l i t y varies with many other factors, such as acid reaction, the presence of ferment-ation products other than acid, albumen and casein content, forewarming temperature, concentrations of solids not fat, etc. In other .-words, as expressed by Benton and Albery, each lot of milk must be regarded as a separate colloidal system with i t s optimum combination of acid reaction and salt balance, for 'maximum heat st a b i l i t y . ,6. "Because of these facts, heat st a b i l i t y ' tests of the fresh milk, such as the alcohol test, the phosphate test, etc., do not . always furnish a dependable index to the heat sta b i l i t y of the evaporated milk. 7, "The above observations emphasize the value of the use of the pilot s t e r i l i z e r for systematic control of the behavior of the evaporated milk in the s t e r i l i z e r . " Experiments, with a view to raising the s t e r i l i z -ing temperatures, were carried out according to the procedure advocated by Sommer and Hart (13), They found that a range of from 2 ounces to 10 ounces of dry salt added to eaoh thousand pounds of evaporated milk could usually be counted upon to correct any d i f f i c u l t i e s in the salt balances. At the time our test was made the plant was getting a s t e r i l i z i n g temperature of 229 degrees F. for 35 minutes, using one and a half ounces of sodium bicarbonat per thousand pounds of milk. Samples were taken of 16 ounces of evaporated milk in each can. To these were added solutions containing the equivalent of two, four, six, (30) eight and ten ounces of di-sodium phosphate and sodium citrate respectively. This milk already contained the 1-|-ounces per thousand of sodium bicarbonate which had been added in the hot wells before condensing. The cans were sealed and sterilized at 232 degrees F. for 35 minutes, or rise of three degrees. Table No.7 shows the effect on the milk of each concentration. These results appeared "to indicate benefioial effects from the addition of from eight to ten ounces of di-sodium phosphate and from six ounces of sodium citrate. -Further tests failed to confirm this, however, and after several t r i a l s in actual manufacture using disodium phosphate, without very marked benefit, the practice was discontinued. The Plant Superintendent found he could get better results by continuing the use of the bicarbonate of soda. As i t did not appear possible.to raise the temperature more than one or two.degrees,which would be ineffective in destroying the troublesome organisms, i t did not seem profitable to pursue this line of investigation further. Sufficient work on this phase was not done, however, to warrant any f i n a l conclusions in the matter and should be carried out further at some future date using these salts and also the calcium salt. Sommer and Hart (13) and the findings of other workers reported by Hunziker (10) also stressed the albumen content of the milk as an important factor in TABLE, NO .7. EFFECT OF ALTERING THE SALT BALANCE• Sample Number Salt Concentration Condition of milk after ster i l i z i n g . A-l 1 o.e. sterile water only added Heavy grain A-2 2 oz. dry di-sodium phosphate per 1000 lbs. milk A-3 3 oz. dry di-sodium phosphate per 1000 lbs. milk A-4 6 oz. dry di-sodium phosphate per 1000 lbs. milk A-5 8 oz. dry di-sodium phosphate per 1000 lbs. milk A-6 10 oz. dry di-sodium phosphate per 1000 lbs. milk B-l 1 c.o. sterile water only B-2 2 oz. dry sodium citrate per 1000 lbs. milk B-3 4 oz. dry sodium citrate per 1000 lbs. milk B-4 6 oz. dry sodium citrate per 1000 lbs. milk B-5 8 oz. dry sodium citrate per 1000 lbs. milk B-6 10 oz. dry sodium citrate per 1000 lbs. milk Heavy grain Slight grain Slight grain 30 second shake 30 second shake Heavy grain Heavy grain Heavy grain 30 second shake 1 minute shake l-J- minute shake (51) heat stabi l i t y . The high albumen content of milk experienced at the beginning and end of the lactation period often causes d i f f i c u l t y i n s t e r i l i z i n g . A l l investigators recommend high preheating temperatures before condensing as a means of precipitating excess albumen. A temperature of over 200 degrees P. and holding as long as possible in the hot wells was recommended as effective. As i t was already the plant practice in the factory concerned to preheat the milk,to a temperature of 210 degrees P. while in the hot wells i t was not f e l t that much improve-ment could be effected here. REPRODUCING THE SPOILAGE. As mentioned earlier in this report, practically a l l of the routine samples appearing unsterile on lactose media showed an alkaline reaction indicating the A b i l i t y of these organisms to ferment this sugar. The question which presented i t s e l f then was: Would these apparently non-lactose fermenters grow in the evaporated milk and bring about a condition of spoilage? To obtain an answer to this disturbing question and to observe the keeping qualities of milk appearing unsterile,: a series of experiments was carried out. A number of cans from s t e r i l i z e r batches, showing the presence of heat resistant organisms, were placed in the 55 degrees 0. inoubator and (32) also in the 37 degrees G. incubator. Observations were made from time to time by taking some of the cans from the incubator, plating them in the manner previously described and then opening the cans and noting the physical condition of the contents,.: as well as testing the milk for acidity. The f i r s t of these experiments was started on January 3rd, 1934. Twenty-four cans from batch No.782-L reported as unsterile In the routine examination were placed in the 55 degrees 0. incubator. At intervals, six of these cans were taken out and examined. The results of these examinations are shown in Table No.8. After one week the condition of the milk in five of the cans was normal with one can appearing partially clotted. The acidity in most cans had increased slightly while that of the clotted can had increased considerably. Plates made from a l l cans appeared unsterile and showed the typical alkaline forming colonies. After two weeks' incubation a l l oans examined were clotted, the two showing a firm clot, also showed the greatest acid development. Four of the cans appeared unsterile with typical colonies while the two with the firm clot and high acidity showed no grov/th of l i v i n g organisms, indicating the possibility of their having been k i l l e d off by the acid produced. After twenty-four days' incubation a l l six cans were clotted with considerable acid production and a l l appeared sterile on TABLE NO. 8. SPOILAGE EXPERIMENT. Experiment on 24 cans of milk from batch 782-L, reported unsterile. Cans incubated at 55 degrees G., Jan.3,1934, DATE OP EXAMINATION. ... CONDITION OF MILK ACIDITY . SIEERILITY Jan. '10th, " 1. Normal .55$ Unsterile after 7 days' Normal .72 M incubation O « Normal .62 I I 4. Normal . 65 H - 5. Normal .75 I ! 6. Partial clot .89 rt Jan. 17th, 1 » soft clot .70$ Unsterile after.14 days' 2. Soft clot . 82 I ! incubation 3. Hard clot .90 Sterile 4. Hard clot . 81 Sterile 5. Soft clot .63 Unsterile 6. Soft Clot .59 Unsterile Jan. 27th, 1 ® Hard clot .85$ Sterile after 24 days' 2. Hard Clot .90 I I incubation 3. Weak, clot .75 I I 4. Weak clot .90 I I 5. Weak clot .82 I I 6. Weak clot .80 I I Peb. 5th, X « Clotted _ _ Unsterile after 33 days' 2 d Clotted __ Sterile incubation 3. Clotted __ I I 4. Clotted „_ I I 5. Clotted —— 6. Clotted ti (33) p l a t i n g . The remaining s i x cans were held f o r a period of t h i r t y - t h r e e days when a l l were o l o t t e d and a l l bat one appeared to have no l i v i n g organisms remaining. The very pronounced tendency of the c l o t t e d milk, to show no l i v i n g organisms would account f o r our i n a b i l i t y to i s o l a t e any b a c t e r i a from cans returned -when the o r i g i n a l outbreak of spoilage was reported. The f a c t was also demonstrated that these apparently non-lactose fermenting b a c t e r i a were capable of producing a c o n d i t i o n of spoilage i n the mi l k i f stored'at a favorable temperature f o r t h e i r growth. Concurrently w i t h t h i s experiment twenty-four oans from the same batch were placed i n the 37 degrees 0. incubator and p e r i o d i c examinations of them made. The r e s u l t s of f i n d i n g s are reported i n Table No.9. The f i r s t t h i r t y - t h r e e days' i n c u b a t i o n gave no change i n the p h y s i c a l c o n d i t i o n of the mi l k and very l i t t l e change i n the t i t r a t a b l e a c i d i t y . Many of the samples appeared u n s t e r i l e showing the presence of the t y p i c a l c o l o n i e s . A f t e r f i f t y - e i g h t days two samples showed an increase i n a c i d production and three of them were p a r t i a l l y c l o t t e d . The m a j o r i t y of them s t i l l showed the presence of l i v i n g organisms. The r e s u l t s obtained w i t h the milk held at the lower temperature of 37 degrees 0. was i n sharp contrast to those found w i t h TABLE NO.9. SPOILAGE EXPERIMENT. Experiments on 24 cans from batch 782-L, reported unsterile. Incubated at 37 degrees 0., Jan.3,1934 BATE OF EXAMINATION CONDITION OF - MILK ACIDITY - STERILITY Jan. 10th, 1. Normal .53$ Sterile after 7 days' 2. Normal . 54 Unsterile incubation 3. Normal • 55 Unsterile Jan. 17th, 1. Normal .55$ Sterile after 14 days' 2. Normal . 54 Sterile incubation 5 e Normal .50 Unsterile Jan. 27th, 1. Normal . 59$ Unsterile after 24 days' 2. Normal .60 Sterile incubation 3. Normal .60 Sterile Feb. 5th, 1. Normal .53$ Sterile after 35 days' £. Normal .59 Unsterile incubation 3. Normal .54 Sterile 4. Normal . 55 Sterile 5, Normal .60 Sterile 6« Normal .57 Sterile March 2nd, X« Partial clot .76 Unsterile after 58 days' 2. Normal .60 Unsterile incubation 5. Normal .52 Sterile 4. Partial clot .64 Unsterile 5. Normal .53 Sterile 6. Normal .58 Unsterile 7. Normal .55 Sterile 8 • Normal © 55 Unsterile 9. Soft clot .95 Unsterile (34) milk, held at 55 degrees 0. In the l a t t e r a c i d production s t a r t e d e a r l y and the milk was soon c l o t t e d while i n the former even a f t e r f i f t y - e i g h t days' h o l d i n g , s i x out of nine cans s t i l l e x h i b i t e d a normal c o n d i t i o n i n the milk. I t would appear that milk held at 37 degrees 0. or l e s s would experience l i t t l e i f any spoilage by these heat r e s i s t a n t b a c t e r i a . The experiments w i t h these two sets of cans of evaporated milk v e r i f i e d what had a c t u a l l y taken place i n the commercial handling of the m i l k . Spoilage had been reported from hot t r o p i c a l c o u n t r i e s where the temperature sometimes runs over 50 degrees 0. while no spoilage whatever had been reported from the B r i t i s h I s l e s where r e l a t i v e l y low temperatures p r e v a i l at a l l seasons of the year. A second experiment w i t h the f i n i s h e d milk was commenced on A p r i l 4 th, 1934, the r e s u l t s of which are shown i n Table No.10. This time eighteen samples from Batch 924-K, reported u n s t e r i l e at both 55 degrees 0. and 37 degrees 0., and an equal number of samples from Batch 930-M, reported s t e r i l e at both temperatures, were incubated at 55 degrees 0. I t was considered!-': d e s i r a b l e to compare the keeping q u a l i t i e s of milk reported s t e r i l e w i t h that shown to be u n s t e r i l e . A f t e r one week's in c u b a t i o n a l l samples showed no apparent change i n p h y s i c a l c o n d i t i o n or a c i d i t y and no l i v i n g b a c t e r i a . A f t e r TABLE HO.10. SPOILAGE .EXP.ERIIIEHT» 36 samples taken from Batch Ho. 924-K, reported "Unsterile" at both 55 degrees G» and 37 degrees G« 36 samples taken from Batch Ho. 930-M, reported "Sterile" at both 55 degrees 0. and 57 degrees C. A l l cans were incubated at 55 degrees 0. on April 4th,1934« DATE OF CONDITION EXAMINATION OF MILK ACIDITY STERILITY April 11, 924-K 1 Normal - Sterile after 1 week 2 Normal - " incubation 3 Normal - " 4 Normal 5 Normal - " 6 Normal - a-930-M 1 Normal - Sterile 2 Normal - " 3 Normal - " 4 Normal - " 5 Normal - " 6 Normal - " April 19th, 924-K 1 Lumpy .64$ Sterile after 15 days' 2 Normal .54 " incubation 3 Normal .52 " 4 Normal .52 " 5 Lumpy .53 " 6 Lumpy .55 " 930-M 1 Normal .55 Sterile 2 Normal .51 " 3 Normal .47 " 4 Normal .50 " 5 Soft Clot .73 " 6 Normal .51 11 Aoril 27th, 924-K 1 Clotted .58 Sterile after 23 days' 2 soft Clot .64 " incubation 3 Lumpy .61 4 Normal 5 Normal - " 6 Normal ' -930-M 1 Normal .56 Sterile 2 Normal - " 3 Normal - " 4 Normal - " 5 Normal 6 Normal - " (35) two weeks the milk in three of the 924-K samples appeared slightly lumpy, but with l i t t l e change in the acidity and apparently no l i v i n g organisms. The six cans of 930-M showed only one with any sign of spoilage, the rest being unchanged in acidity and appearing st e r i l e . After twenty-three days the cans appeared about the same as at two weeks, three of 924-K being clotted and the balance normal while a l l of 930-M were normal in every respect. In this experiment changes were not brought about as quickly as in the test reported above or were they as pronounced. Although a few of the cans clotted there were no marked increases in acidity, while plates showed no colonies in a single instance. Evidently any bacteria which had been present in 924-Z had died out or were too inactive to show growth. The test, however, demonstrated the superior keeping qualities of milk appearing sterile in routine examinations as only one oan of 930-M exhibited signs of spoilage while six of 924-IC showed a definite clot or lumpiness. To determine the effect of long storage on organisms in cans, twelve samples of batch No.767-B were taken. This milk had been manufactured in October 1933, having been stored for about six months. In the original test i t had shown an unsterile condition. These cans were incubated at 55 degrees 0. on April 27th, 1934, and subsequently examined in the usual way after ten and (36) twenty-one days' incubation. The results of the examinations are shown in Table #11. At the end of ten days a l l samples showed evidence of thickening and the presence of organisms. At twenty-one days the six cans examined were a l l definitely clotted, four showing the presence of organisms and two being apparently ster i l e . This test demonstrated that some of these heat resistant organisms are capable of remaining alive in the milk for several months and when conditions of temperature become favorable are capable of spoiling the milk. On February 18th, 1935, a fourth series of cans of finished milk were incubated at 55 degrees 0. This group was made up of 24 samples each of Batch 155-J, reported unsterile after resterilization, Batch 174-D, reported unsterile at both 37 degrees G. and 55 degrees C.s and Batch 185-B, reported sterile at both temperatures. The samples of 155-J had been packed for about a month, those of 174-D were packed in January 1935, when such a large percentage of a l l samples appeared unsterile in routine plates, and those of 185-B were taken from milk packed after raw milk showing the presence of heat resistant bacteria had been kept out of the supply used for the manufacture of this particular standard of milk. The results of this series are shown in Table No.12. SABLE NO,11. SPOILAGE EXPERIMENT, 12 samples from Batch. No. 767-B reported "Unsterile" after double checking i n October, 1933, and held at the warehouse in the interval were incubated at 55 degrees G. on April 27th, 1934. BATE OP EXAMINATION May 6th, after 10 days' incubation CONDITION OF MILK ACIDITY 767-B 1 Soft Clot 2 " " 3 4 5 6 " " it n ti ii ii ti STERILITY Unsterile May 18th, after 21 days' incubation 767-B 1 Clotted 2 3 " 4 •"• 5 '• 6 " ,68'$' .69$ Sterile Unsterile n Sterile Unsterile Unsterile TABLE NO.12 SPOILAGE EXPERIMENT 24 samples each of Baton. No.l55~J, reported "Unsterile" after resterilization, Batch No. 174-D, reported "Unsterile" at both 55 degrees 0. and 57 degrees 0., and Batch No. 185-B reported "Sterile" at both 55 degrees 0. and 37 degrees C , were incubated at 55 degrees C. on February 8th, 1935. LATE OF CONDITION REACTION EXAMINATION. - ..' -OF: MILE ..... ACIDITY STERILITY . OF. MEDIUM Feb. 11. 155-• J 1 Normal .51$ Unsterile Alk. after 2 Normal .51 it I I 72 hours' 174-•D 1 Normal .51 Unsterile Alk. incubation 2 •Normal .51 I I IT 185-•B 1 .Normal .47 Sterile 2 Normal .47 I I Feb. 15th. 155- J 1 Normal .58 Sterile After 1 2 Normal .55 Unsterile Alk. week's 3 Normal .56 I I I T incubation 4 Normal • 52 n Acid 174-•D 1 Normal .66 Unsterile Alk. 2 Normal .55 ti n 3 Normal .54 I I IT 4 Normal .56 I I IT 185-•B 1 Normal .52 Unsterile Acid 2 Normal .50 n it 3 Normal .50 I I IT 4 Normal .60 it Alk. Feb. 22nd. 155- J 1 Soft Clot .80 Sterile After two 2 Normal . 56 Unsterile Alk. we eks' 3 Normal .54 I T IT incubation. 4 Soft Olot .65 I I I I 174-D 1 Normal .59 Unsterile Alk. 2 Normal .58 TT IT 3 Normal .58 tl I I 4 Normal .57 Sterile 185-B T A. Soft Clot .65 Unsterile Alk. 2 Normal .55 I I I I 3 Normal .52 IT I I 4 V.S.Clot ,61 11 IT Mar. 1st. 155-3 1 Soft Olot .75 Sterile After three 2 Normal .63 Unsterile Alk. weeks' 3 V.S.Clot .65 Sterile incubation. 4 Y.S.Clot .63 Sterile (Continued on next page) TABLE NO.12, (Continued) DATE OF CONDITION REACTION EXAMINATION OF MILK , ACIDITY . . STERILITY ..OF MEDIUM Mar oil 1st 174 -D 1 Normal .59 Sterile (Cont'd) 2 Normal .59 Unsterile Alk 5 Normal .59 Sterile 4 Normal .60 Sterile 185 -B 1 Normal .60 Sterile 2 Normal .67 Unsterile Alk 3 V.S.Clot .70 Sterile 4.Soft Clot .82 Sterile Mar.8th. 155 - J 1 Hard Clot .84 Sterile After four 2 Soft Clot .65 IT weeks' 3 Soft Clot . 66 ti incubation. 4 Soft Clot .68 Unsterile Alk . 174 -D 1 Normal .60 Sterile 2 Normal .59 3 Normal .60 IT 4 Normal .61 !T 185 -B 1 Normal . 59 Sterile 2 Normal e 61 ti 3 Firm Clot .84 Unsterile Alk 4 Normal .77 n I I Mar.15th. 155--J 1 S.Thick. .75 Unsterile Alk After five 2 S.Thick. .64 Sterile weeks' 3 Normal • 66 IT incubation 4 7.S.Thick. .75 - I T 5 Soft.Clot .73 II 6 Soft Clot .76 1  174--D 1 Normal .60 Unsterile Alk 2 Normal . 6 4 n I T 3 Normal . 68 I T 11 4 Normal .65 I I IT 6 Normal . 66 Sterile I I 185--B 1 Normal .68 ii 2 Normal »65 1! 3 Soft Clot ,80 IT 4 Normal .68 11 5 Soft Clot .76 11 6 Normal .60 I I (37) Examinations of these cans a f t e r v a rious periods of i n c u b a t i o n showed those of No. 155-J t o be normal i n c o n d i t i o n during the f i r s t week w i t h the presence of the t y p i c a l c o l o n i e s . During the second week c l o t t i n g had commenced and t h i s increased during the t h i r d , f o u r t h and f i f t h weeks. As the period of i n c u b a t i o n increased the number showing the presence of organisms decreased u n t i l at the end of the f i f t h week only one out of s i x samples showed these a l k a l i forming b a c t e r i a . The samples of 174-D maintained a normal p h y s i c a l c o n d i t i o n throughout the whole f i v e weeks although the m a j o r i t y of the samples showed the presence of larg e numbers of organisms. Those of 185-B acted s i m i l a r l y to 155-J although not to such a pronounced extent. While the r e s u l t s of t h i s experiment were not so c l e a r cut as some of those preceding i t , nevertheless the tendency f o r these apparently a l k a l i n e forming b a c t e r i a to s p o i l evaporated milk was again demonstrated. The f a c t that some of both the samples appearing s t e r i l e i n routine examination and those appearing u n s t e r i l e , showed some s p o i l e d cans and some w i t h a normal c o n d i t i o n i s f u r t h e r evidence of the absence or non a c t i v i t y of these b a c t e r i a i n some of the cans from each s t e r i l i z e r batch. The behaviour of cans of batch No.174-D i n d i c a t e d that some b a c t e r i a s u r v i v i n g the s t e r i l i z i n g process have l i t t l e or (38) no a c t i o n i n the 'milk. From the above four experiments the concl u s i o n can s a f e l y be drawn that the heat r e s i s t a n t organisms, showing an a l k a l i n e r e a c t i o n on l a c t o s e p l a t e s , can and do set up a c o n d i t i o n of spoilage i n evaporated m i l k i f stored at s u i t a b l e temperatures and f o r a s u f f i c i e n t l y long time. The chemical changes i n v o l v e d have not been determined. However, we have n o t i c e d i n the c l a s s i f i c a t i o n of organisms i s o l a t e d that p r a c t i c a l l y a l l produce a c i d on glucose and many on g a l a c t o s e . I t i s p o s s i b l e that i n the heating processes or due to the a c t i o n of b a c t e r i a l enzymes some of the l a c t o s e i s broken down to glucose'and g a l a c t o s e , and t h i s i s used by the b a c t e r i a to produce s u f f i c i e n t a c i d to r e s u l t i n a c o n d i t i o n of c l o t t i n g . A f u r t h e r p o s s i b l e cause of the spoilage experienced i n milk c o n t a i n i n g these heat r e s i s t a n t , non l a c t o s e fermenting organisms, has r e c e n t l y come to l i g h t . As mentioned e a r l i e r i n t h i s report the number of samples appearing u n s t e r i l e a f t e r i n c u b a t i o n decreased very markedly a f t e r raw mil k s u p p l i e s , c a r r y i n g heat r e s i s t a n t b a c t e r i a , were withheld from manufacture of t h i s m i lk. Some u n s t e r i l e samples, however, s t i l l appeared. The m a j o r i t y of these d i d not c a r r y the t y p i c a l organisms which gave an a l k a l i n e r e a c t i o n on l a c t o s e media, but a d i f f e r e n t type of colony was n o t i c e d . These l a t t e r gave l i t t l e or no evidence of (39) change in the reaction of the media as indicated by the color. They did, however, produce a distinctly acid smell, showing some production of acid from the lactose. These lactose fermenting colonies were present on the plates in small numbers only. This would point to the fact that they did not grow vigorously in the milk incubated at 55 degrees G. None were noticed in plates from milk incubated at 37 degrees C. The possibility now i s suggested that these acid forming organisms may have been present in the milk before, but were obscured in the plates by the, very vigorous growth of the alkaline forming bacteria. If present, i t is possible that after prolonged periods of incubation they would have produced sufficient acid to cause coagulation. This possibility i s strengthened by the fact that in our spoilage experiments the milk,in many cases, did not coagulate, until after one or two weeks' incubation. While further work w i l l have to be done on these organisms, i t is nevertheless the opinion of the authors that although these lactose fermenters may be contributory factors they cannot account for a l l spoilage experienced, as experiments described below w i l l show that organisms, definitely non lactose fermenting, when innoculated into evaporated milk, set up a condition of spoilage. (40) INN0GULATI0N EXPE5IMBNTS. On February 8th, 1935, an experiment was commenced to determine the effect on allegedly sterile evaporated milk of pure cultures of organisms isolated from routine plates during the course of this investigation* The cultures used were 762-L, the type culture of a group of strong lactose fermenters which has been classified as a sal i c i n fermenting strain of B. ooagulans (Hammer), 786B2, the type culture of a group of non-lactose but strong mannite fermenters, which has been classified as B. calidus, (,Blau. ), and 207, the type culture of a group which show praotically no action in carbohydrate media but which clot and digest litmus milk with a slightly alkaline clot and to which has been given the name B. thermoalkalans» A sterile water suspension of each culture was made. That from 762-L contained 930,000 colonies per c c , 786B2 contained 13,200,000 colonies per c c , and 207 contained 17,400,000 colonies per c c A suspension of a mixture of a l l three cultures was also used. Sealed cans of evaporated milk, from a batch reported sterile at both 37 degrees 0. and 55 degrees 0. were chosen for this experiment. The milk was innoculated in the following manner* The tops of the sealed cans were swabbed with alcohol and flamed. A small area of the t i n (41) was covered with a solution of commercial hydrochloric acid and a bead of solder melted on i t . At the edge of the solder a hole was punched in the can by means of a sterilized ice pick. One cubic centimeter of the bacterial suspension was run into the can and the hole then sealed with a solder-ing iron. Bach can and contents was then shaken Well. One cubic centimeter of sterile ?/ater was added to each of the six control cans in the manner described above. Six cans were thus innoculated with each of the bacterial suspensions. After re-sealing, a l l cans were placed in the incubator and held at 55 degrees 0. Periodic examinations of the contents were made, the results of which appear in Table No.13. From this table i t w i l l be noted that the milk in the control cans appeared normal in s t e r i l i t y , physical condition and acidity for the whole two weeks of incubation. Those cans containing the innoculation of B. coagulans were clotted with some production of acid within seventy-two hours and showed a very hard clot with high acid production at the end of the one and two week intervals. The six cans containing, the 3. .calidus^Blauy,. remained normal in physical condition and acidity throughout, while those containing the B. thermo-alkalans were not thickened at seventy-two hours but had developed a cheesy smell. After one week these latter showed a soft clotting with slight PLATE I. SHOWING EFFECT OF INNOCULATING EVAPORATED MILK JgJ?H JSPJ5C E? IC ORGANISMS. A f t e r one week i n c u b a t i o n a t 55° C. Co n t r o l 762-L 207 786B2 Mixture A f t e r two weeks' i n c u b a t i o n at 55° C. Con t r o l 762-L 207 786B2 Mixture 762-L B. coagulans 786B2 B. c a l i d u s 207 B. thermoalkalans Mixture B. coajgulans and B. thermoalkalana TABLE- NO. 13. EFFECT. ON MILK OF SUSPENSIONS OF ORGANISMS ISOLATED. Oultures Used: 762-L, 786B2, 207 and mixture of a l l three. Six oans innooulat.ed with each culture. Incubated at 55 degrees 0. PERIOD OF INCUBATION CONDITION OF MILK ACIDITY STERILITY REACTION OF .MEDIUM Feb. 11th. After 72 hours' incubation. Feb. 15th. After one week's incubation Feb. 22nd. After two weeks' incubation. Control 762-L 786B2 207 Mixture 1 Normal 2 Normal 1 Clotted 2 Clotted 1 Normal .2 Normal 1 Normal with cheesy small 2 do. 1 Clotted with cheesy smell 2 do. .51 .51 .86 .84 .51 .51 .60 . 60 .92 .92 Control 762-L 786B2 207 Mixture Control 762-L 786B2 207 Mixture 1 Normal .51 2 Normal .52 1 Hard Clot 1.12 2 Hard Clot 1.1 1 Normal .56 2 Normal .52 1 Weak Clot .71 2 Weak Clot .68 1 Hard Clot 1.26 2 Hard Clot 1.26 1 Normal .55 2 Normal .57 1 Hard Clot . 2 Hard Clot 1 Normal .55 2 Normal• .56 1 Soft Clot .70 2 Soft Clot .69 1 Hard Clot cheesy smell 2 do. Sterile Sterile Unsterile Unsterile Unsterile Unsterile Unsterile Unsterile Unsterile Unsterile Sterile Sterile Unsterile Unsterile Unsterile Unsterile Unsterile Unsterile Unsterile Unsterile Sterile Sterile Unsterile (few colonies) do. Unsterile (few colonies) do. Unsterile Unsterile Unsterile Unsterile Acid Acid Alk. Alk. Alk. Alk. Alk.Acid Acid Acid Acid Alk. Alk. Alk. Alk. Alk. Alk. Acid Acid Alk. Alk. Alk. Alk. Alk. Alk. (42) productions of acid and at two weeks a definite soft clot with, no further increase in acidity. The cans containing the mixed culture showed similar results to those with B. coagulans except that the milk had a cheesy smell in addition to the hard acid clot. The control cans were sterile at a l l examinations whereas a l l innoculated cans showed the presence, in large numbers, of the specific organisms used. In no cans was there any sign of gas production. Plate I. gives a graphic picture of the effect of these cultures on evaporated milk after one and two weeks' incubation. In this experiment these type cultures gave reactions similar to those experienced in a r t i f i c i a l media. The culture of B. coagulans, a strong lactose fermenter, quickly clotted the milk with a high acid production. That of B. oalidus.f Blau . a non-lactose fermenting strain, had no apparent effect on the evaporated milk. The culture of B. thermo-alkalans, whioh in the pure culture study did not ferment lactose but did clot milk, liquefied gelatine and reduced nitrates, clotted the evaporated milk and also decomposed the proteins to some extent as evidenced by the strong cheesy odor produced. The results of this experiment give further proof of the a b i l i t y of certain-of the organisms,surviving (43) s t e r i l i z i n g temperatures, to set up a condition of spoilage in evaporated milk. Their action was hastened by the suitability of the temperature employed and the large number of organisms contained in the innoculum. The results obtained with B. oalidus,1Blau_ t give additional evidence that some of the surviving organisms are incapable of spoiling the milk, a fact already demonstrated by experiments with cans from batch number 174-D. As organisms are not l i k e l y to exist in pure culture i n commercial evaporated milk the drastic effect of the mixture of cultures here employed i s of considerable interest. CLASSIFICATION OF THE ORGANISMS ISOLATED.  Methods Used. As a classification reference., Bergey's Manual of Determinative Bacteriology (14) was used. Sugar fermenting a b i l i t i e s were determined by the method devised by S. Orla-Jensen (15^ Employing Casein Digest Broth (16), single strength, as the nitrogen source, sugar broths were prepared. In each case the respective sugar i s added at the rate of 2 percent. The broth is then tubed and plugged, 10 c.o. in each test tube, and sterilized at twelve pounds pressure' for twenty minutes. After sterilization each tube is innoculated with the desired culture. Uniformly a 2 m.rn. loop innoculation from a vigorous growth in milk or casein (44) digest broth is made. After innocillation, the series with controls i s incabated for fourteen days at the appropriate temperature. In this case, seven days' incubation and 55 degrees C. were used. A preliminary test showed that the maximum total titratable acidity was reached before seven days. When incubation is completed, the cultures are titrated with N/4 Sodium hydroxide, using phenolphthalein as indicator, the tit r a t i o n of the controls deducted, and the results worked out and recorded as grams of lactic acid per mille. P. S. Prickett (17) noticed a certain v a r i a b i l i t y among the thermophiles in the production of acid from carbohydrates. Therefore, when placing organisms together in groups, only a slight significance was given to small variations in the fermentation of sugars. Rather, the sum of the characteristics of the organisms was used, following the principle enunciated by S. Orla-Jensen. Glassification. Typical colonies of bacteria which appeared with the greatest frequency in routine examination of condensed milk, during the year of 1954, were pioked from Purple Lactose Agar plates and retained for study. Cultures were also picked from plates prepared from other sources during the course of this investigation. Over one hundred such organisms were isolated and after preliminary tests and (45) microscopic examination forty-one of them were subjected to a detailed study and classification. A l l of the forty-one organisms are aerobic, spore forming, gram positive rods, varying i n length from two to seven microns. In old agar cultures the cells tend to grow out into threads, often strangely c u r l i n g , I n d i v i d u a l threads are made up, however, of several segments. In young cultures and in milk the cells occur for the most part singly, or in very short chains. Most of the organisms are motile. A l l of the organisms grow well on agar slants. Some liquefy gelatine, but most of them do not. A l l strains grow at 37 degrees 0., but grow more vigorously a t 55 degrees 0. In sealed cans of evaporated milk, they withstand a temperature of 110 degrees 0. for 30 minutes. The detailed description of the organisms i s found on Tables No.14 and No.15. The morphology and cultural characteristics stated above suggest that a l l the organisms isolated be placed in the family Bacillaceae, genus Bacillus.- (14) The forty-one organisms may be divided into two main groups: lactose fermenting and non lactose fermenting strains. Group I. - Lactose Fermenting.' Only six cultures were found to ferment lactose (Table No.15). They are: 762-L, 795-Q, 786-Bla, 786-B, (46) D.T.I and D.T.3. As seen from Table No.14, the f i r s t four were isolated from cans of condensed milk, while D.T.I and D.T.5',.were- isolated from milk in the drop tank in the Plant. These strains are motile, gram positive rods, with terminal spores. They do not liquefy gelatine, form a clean hard, acid clot in milk and reduce litmus in 48 hours at 55 degrees 0. Nitrates are not reduced and the cultures do not produce indol. Gas is not formed in any of the carbo-hydrates. Using Casein Digest Broth (16) as a nitrogen source, laevulose, dextrose, mannose, galactose, saccharose, maltose, laotose, dextrin and starch are fermented. It is to be seen (Table No.15) that although a l l six organisms ferment the above sugars, 762-L, 795-Q, 786-BLa and 786-BL produce more acid than do cultures D.T.I and D.T.3. None of the above cultures form acid from mannite or inulin and in some cases an alkaline reaction i s produced. Salicin i s fermented by cultures 762-L and 795-Q, but is not fermented by the other cultures. These organisms seem to bear a marked resemblance to Hammer's Bacillus coagulans. and on the sum of the characteristics, cultures 786-BLa, 786-BL, D.T.I and D.T.'S are classified as Bacillus ooagulans (Hammer) (8) (14). Cultures 762-L and 795-Q are tentatively classified as sa l i c i n fermenting strains of B. coagulansi (Hammer) (8) (14). Group II. - Non Lactose Fermenting. Thirty-five cultures do not ferment lactose. They (47) can be divided into several groups, within'the large main group, CULTURES 786-B2, 795-P, Mash 5, 161-2, 142-1, 782-05, 146-F, 145, Mash 6, Mash 4, 122-C, 143-G 120-0, 161-3. These strains are quite important as they comprise a large percentage of Hie organisms, and as seen from Table No«14, are isolated from a variety of sources. They are a l l strong mannite fermenters. The morphology and cultural characteristics of the above cultures, bear a marked resemblance to Bacillus calidus (Blau) (14) (see Table No.14 and No»15)'. They are gram positive, motile rods, with terminal elongated spores. They do not liquefy gelatine, do not reduce nitrates or produce indole They produce an alkaline reaction i n milk, but do not peptonize or clot i t . In this respect they differ from B. calidus (Blau), as the latter peptonizes and clots milk. In Casein Digest Broth a l l cultures ferment mannite, laevulose, dextrose and maltose. Mannose is fermented by a l l cultures except 122-G, 143-G and 120-C. Galactose i s fermented only by cultures Mash 6 and Mash 4» Saccharose, lactose, inulin, dextrin, starch and sal i c i n are not fermented by any of the cultures (Table No»15). In their i n a b i l i t y to ferment saccharose, they differ from the strain of B. calidus described by P. S. Prickett (17) (48) as the latter ferments saccharose. No tw it hst an cling these variations, cultures 786-B2, 795-P, Mash 5, 161-2, 142-1, 782-03, 146-P, 143, Mash 6, Mash 4, 122-G, 143-G, 120-C and 161-5 are tentatively classified as strains of Bacillus ,oalidus ( Blau ) (14). It i s interesting to note, that i f this group of organisms were not spore formers, they could have been definitely. classified as Thermobacterium cereale, (Orla-Jensen) (15), while the lactose fermenters (Group I.) as Thermobacterium l a c t i s (Orla Jensen) (15). 2. CULTURES YII-D and .236 have a marked resemblance in their morphology and cultural characteristics to Bacillus pepo (Shaw) (14) (Table No.14 and No.15). They are gram positive non-motile rods, with elongated terminal spores. They liquefy gelatine at 20 degrees 0. and at 55 degrees 0. The liquefaction becomes saccate with slight production of scum. It is f a i r l y rapid and is completed in fourteen days. Nitrates are reduced to nitrites and ammonia. Litmus milk clots and starts to peptonize on the third day at 55 degrees 0. With Casein Digest Broth as the nitrogen source, these organisms ferment Mannite, laevulose, dextrose, mannose, galactose, saccharose, maltose, starch and s a l i c i n . They do not ferment lactose inulin and dextrin. No.236 differs from B. pepo in i t s in a b i l i t y to ferment salicin, while VII-D (49) differs by i t s inab i l i t y to ferment galactose. Notwithstanding these variations, on the sum of their characteristics cultures No.236 and VII-D are tentatively classified as strains of Bacillus pepo (Shaw) (14). 3. CULTURES Mash 2, 145-N, 144-F37, 138-0, 782-M2, 782-M3, 795-J, 161-alk., XI-Hunt and 795-0 resemble in their morphological and cultural characteristics Bacillus armarus (Hammer) (14). They are gram positive motile rods with terminal spores. They do not liquefy gelatine and do not reduce nitrates. -Litmus milk i s not changed in any way. There is a marked variation in the sugar fermenting a b i l i t i e s of these organisms. A l l of the cultures ferment laevulose and dextrose. None of the organisms ferments mannite, lactose, dextrin or starch. Cultures 138-0, 795-J, 161-alk., XI-Hunt and 795-G do not ferment mannose, while the remaining five cultures possess the a b i l i t y to ferment this carbohydrate. Galactose is fermented only by Mash 2, 145-N and 782-M2. Four of the cultures do not ferment saccharose; they are Mash 2, 145-N, 138-0 and 782-M3. Maltose is slightly fermented by 144-F37, 138-0, 782-M3, 795-J, XI-Hunt and 795-G. Only one culture, XI-Hunt, ferments s a l i c i n . In many of the carbohydrates an alkaline reaction i s produced. (50) In s p i t e of these v a r i a t i o n s these c u l t u r e s are t e n t a t i v e l y c l a s s i f i e d as s t r a i n s of B a c i l l u s amarus (Hammer) (14). 4. CULTURES Hay I and 782-M resemble i n t h e i r morphology • and c u l t u r a l c h a r a c t e r i s t i c s B a c i l l u s t h e r m o i n d i f f e r e n s ( W e i n z i r l ) (14). They are m o t i l e , gram p o s i t i v e rods, w i t h t e r m i n a l , elongated spores. They do not reduce n i t r a t e s . Litmus m i l k i s l e f t unchanged. They do not l i q u e f y g e l a t i n e and i n t h i s respect d i f f e r from B. thermoindifferens. Both c u l t u r e s ferment glucose and show a s l i g h t i n d i c a t i o n of fermenting s t a r c h . Hay I i s , i n a d d i t i o n , able to ferment l a e v u l o s e . None of the other carbohydrates are fermented. Organisms Hay I and 782-M are t e n t a t i v e l y c l a s s i f i e d as s t r a i n s of B a c i l l u s , t h e r m o i n d i f f erens ( W e i n z i r l ) (14). 5. CULTURES No.31, Truck VI and Truck IV resemble on the whole B a c i l l u s thermoalimentophilus ( W e i n z i r l ) (14). They are gram p o s i t i v e , motile rods, w i t h t e r m i n a l elongated spores. They do not l i q u e f y g e l a t i n e or change l i t m u s m i l k . They reduce n i t r a t e s to n i t r i t e s . As a whole they show no change i n carbohydrate media, but Culture Truck IV ferments mannose and galactose s l i g h t l y , (51) and No.31 mannoae only. Cultures No.31, Truck VI and Truck IV are tentatively classified as strains of Bacillus thermo- alimentophilus (Wenzirl) (14). 6. CULTURE 161 resembles Bacillus, tritus (Batchelor). It is a gram positive motile rod, with rounded ends and terminal spores. Litmus milk is not changed, nitrates are not reduced, gelatine i s not liquefied and carbohydrates are not fermented. The optimum temperature, however, is higher than that for B. t r i t u s , so Culture 161 i s considered as a strain of Bacillus tritus (Batchelor) (14). 7. CULTURES 207, Truck V and Hunt XI could not be classified as any of the strains described by Bergey (14). They are gram positive, motile rods with elongated terminal spores. On Purple Lactose Agar plates, subsurface colonies have a starlike or exploded appearance and a strong alkaline reaction is produced. Turbidity and a pellieire are produced in nutrient broth. Litmus milk i s reduced and an alkaline clot i s formed, which later becomes peptonized. Gelatine Is liquefied quickly and completely at 55 degrees 0. and not at 20 degrees C. Nitrates are reduced to n i t r i t e s . No acid is formed but an alkaline reaction is produced in a l l of the carbohydrate media (52) employed. For d e t a i l e d morphological and c u l t u r a l c h a r a c t e r i s t i c s see Tables No.14 and No.15. I t i s believed that these are undescribed organisms. I s they occur f r e q u e n t l y i n routine pl a t e s and present p e c u l i a r i t i e s of some i n t e r e s t , i t i s proposed that the name of B a c i l l u s thermoalkalans be given.to them. SUMMARY. This i n v e s t i g a t i o n was c a r r i e d out to determine the causes of spoilage i n evaporated milk i n t r o p i c a l c o u n t r i e s during the s p r i n g and summer of 1935. The milk concerned was of " B r i t i s h Standard" bearing the r e l a t i v e l y high s o l i d s content of 9 percent b u t t e r f a t and 51.5 percent t o t a l s o l i d s . Various methods of c u l t u r i n g f a i l e d to show the presence of any l i v i n g organisms i n cans of s p o i l e d milk returned from the t r o p i c s . Defective s e a l i n g of the cans and e l e c t r o l y t i c a c t i o n i n the milk were dismissed, a f t e r i n v e s t i g a t i o n , as pos s i b l e sources of the spo i l a g e . Routine examinations of repre s e n t a t i v e samples from each s t e r i l i z e r batch of t h i s milk had been made f o r some years using an in c u b a t i n g temperature of 57 degrees 0. When the p r a c t i c e of in c u b a t i n g at 55 degrees 0. also was adopted an increase i n the percentage of u n s t e r i l e samples was experienced. In the m a j o r i t y of cases the (53) b a c t e r i a encountered gave an a l k a l i n e r e a c t i o n on Purple Lactose Agar p l a t e s . The p o s s i b i l i t y suggested i t s e l f t hat these organisms s u r v i v i n g s t e r i l i z a t i o n may have been responsible f o r the spoilage of the evaporated milk i n hot c o u n t r i e s . I n any case t h e i r e l i m i n a t i o n was d e s i r a b l e and an attempt was made to determine t h e i r source. A technique was developed f o r d e t e c t i n g t h e i r presence i n samples of m i l k . B a c t e r i a s u r v i v i n g the normal s t e r i l i z i n g temperatures, v i z . , 228 degrees to 230 degrees P. f o r twenty-five t o t h i r t y - f i v e minutes, were detected i n milk at v a r i o u s stages of the manufacturing process, then i n raw m i l k and f i n a l l y i n shipments from c e r t a i n i n d i v i d u a l farms. E v e n t u a l l y raw milk showing the presence of these thermodurio b a c t e r i a was excluded from supplies used f o r t h i s standard of evaporated m i l k . .This p r a c t i c e r e s u l t e d i n o b t a i n i n g a great many more batches, than formerly, of milk which appeared s t e r i l e at both temperatures of i n c u b a t i o n . Hence the f a c t o r y obtained greater supplies of milk which could be shipped w i t h assurance to f o r e i g n c o u n t r i e s r e g a r d l e s s of the p r e v a i l i n g temperatures. During the i n v e s t i g a t i o n a l l m i lk appearing u n s t e r i l e on routine examinations was withheld from shipment to t r o p i c a l c o u n t r i e s and no r e p o r t s of recurrent spoilage have been rec e i v e d . (54) The seasonal incidence of 'these thermoduric bacteria in the finished evaporated milk was definitely established from determinations on 4303 samples. During the Pal l and Winter months their presence was most pronounced, while during the summer there vrere very few. Routine plates showed, after incubation at 55 degrees 0., 85.7 per-cent of samples containing thermoduric bacteria during December, as compared with 1.6 percent in June. Plant practices, such as more rigorous cleaning and s t e r i l i z i n g and attempts to raise the temperatures employed in cooking the milk, reduced only slightly the contaminations of finished supplies of this export evaporated milk by heat resistant bacteria. Spoilage experiments were conducted with representative cans from batches of milk appearing both sterile and unsterile in routine examinations to determine the effect on the evaporated milk of the presence of these thermoduric bacteria. These experiments indicated that evaporated milk showing the presence of the heat resistant organisms, when subjected to incubation temperatures of 55 degrees'0. exhibited a definite tendency to clot and spoil, while those apparently free of them did not. Milk incubated at 37 degrees 0. showed only very alight tendencies in this direction, even after long periods of holding time. In most cases some acid was produced in spite of the fact (55) that most of the organisms experienced gave an alkaline reaction on lactose plates. The phenomenon of bacteria dying out in the milk, after clotting had taken place, gave a clue to the reason for the inability to recover organisms from cans of milk i n the original outbreak of the spoilage. It was also shown that milk packed and stored for as long a period as six months spoiled when subjected to high temperatures, indicating the thermophilic nature of some of these organisms and their a b i l i t y to remain dormant in the milk until conditions became favorable to growth. Some of these thermoduric bacteria, while surviving s t e r i l i z i n g temperatures, are incapable of bringing about a condition of spoilage, as indicated by the results of experiments with cans from batch No.174-D, and by the innoculation experiments with B. oalidus,/ Blau The spoilage experiments carried out by innoculat-ing milk with cultures of representative organisms isolated during the investigation showed the effect on evaporated milk of the predominant types of these thermoduric bacteria. Strains of lactose fermenters like B. ooagulans produced a high acid clot. Non-lactose fermenters like B. thermoalkalans spoiled the milk by a slight production of acidity and a decomposition of the proteins while other non-lactose fermenters like B. oalidusJ Blau jxad no apparent effect on (56) the evaporated milk even at high temperatures. Organisms isolated during theoourse of the investigation have been classified as strains of B. coagulans (Hammer); B. calidus. (JBlau ); B. pepo, (Shaw); B. amarus, (Hammer); B. thermoindiff erens, (Weinzirl); B. thermoalimentophilus. (Weinzirl); B. .tritus. (Batchelor); and B» thermoalkalans (Atkinson and Okulitch). CONCLUSIONS, 1, In this investigation, causes of spoilage i n evaporated milk were impossible to determine from cans of milk returned from the tropics as spoiled* 2. Thermoduric and thermophilic bacteria, capable of withstanding s t e r i l i z i n g temperatures of 110 degrees C, for thirty minutes In sealed cans of evaporated milk, were received In supplies of raw milk. Alteration of plant practices, such as more rigorous cleansing and s t e r i l i z i n g of equipment and attempts to raise the s t e r i l i z i n g temperatures proved ineffective In removing these thermoduric bacteria from finished supplies of evaporated milk. Due to the extremely resistant nature of these bacteria, the authors believe It i s Impossible to use temperatures high (57) enough to k i l l them in the sealed cans of finished milk* There i s a definite seasonal incidence of these thermoduric bacteria in raw milk supplies, the period of greatest occurrence being the Pal l and Winter months and least during Spring and Summer months. The contamination by thermoduric organisms i s not general through the raw milk, but i s evidently due to farm practices on individual farms. Milk from certain shippers has repeatedly sho?m the presence of these bacteria, while that from the majority of shippers has continuously shown their absence. The percentage of ster i l e batches of finished evaporated milk has been greatly increased by withholding from manufacture raw milk supplies containing bacteria which w i l l survive s t e r i l i z i n g temperatures of 225 degrees P. for twenty-five minutes. Some of these thermoduric bacteria are lactose fermenters and at suitable temperatures of incubation are able to produce sufficient acid to clot the evaporated milk. Others are non-lactose fermenting organisms, some of which spoil the milk and some of which do not. (58) Organisms isolated, during the course of the investigation have been classified as B» coagulans, B. calidvis, B. pepo, B. amarus, B, thermoindifferens, B. thermoalimentophllus, B. tritus, and previously unidentified strains to which have been given the name of B. thermoalkalans. Our findings in this investigation agree with conclusions drawn by Hunziker (10), that i n some cases of evaporated milk spoilage, single cans appear to be affected rather than the whole batch. Hunziker suggest! improperly cleansed and sterilized equipment as being the sources of the troublesome bacteria., We found this to be true only i n part, the most important source being the raw milk. Hussong and Hammer (9) reported heat resistant organisms as occurring with the greatest frequency during the summer months, while our findings indicate the greatest occurrence during the F a l l and Winter months« In a l l work herein reported by other Investigations, research proceeded only to the point of isolating, classifying and testing the causative organisms, but did not determine their point of origin. In this investigation so many thousands of samples have been examined that the authors feel that the sources, frequency of occurrence and behavior of the thermoduric bacteria concerned have been quite definitely established* (59) REFERENCES » .!• Cassedebat (See Coutts) "Report of the Local Government Board on Public Health and Medicine"• Subj. Food Report 15, 1911, 2, Dodge. Journal of Infectious Diseases, Supp.1,355. 3* Hunziker, O.F, and Wright, W«R,, "Experimental Study of Bloats Caused by Chemical Action on Tin." Condensed Milk and Milk Powder, Pub. Hunziker, O.F., La Grange, 111,, 1935, 4, Savage, W.G, and. Hunwicke, R.F., "Studies on Unsweetened Condensed Milk", Food Inspection Board, London, Special Report, 1923, 5, Vermont Agricultural Experiment Station, Bulletin 170, 1912. 6, Kelly, CD. "Bacteria Causing Spoilage of Evaporated Milk", Transactions of the Royal Society of Canada, Section V. 1926. 7, Hammer, B.W. and Hussong, R.V. "Action of an Aerobic Spore-forming Organism on Evaporated Milk", Journal of Dairy Science,.Volume XV, Number 3, May 1932. 8, Hammer, B.W. "Bacteriological Studies on the Coagulation of Evaporated Milk", Agricultural Experiment Station, Iowa State College, Research Bulletin #10, 1915. 9, Hussong, R.V. and Hammer, B..W., "Observations on Bacillus calidolactis", Iowa State College, Journal of Science, VI, 89, 1931. 10, Hunziker, O.F. "Condensed Milk and Milk Powder", Published by the author, La Grange, I l l i n o i s , 1935. 11o Digestive Ferments Company, Detroit Michigan, U.S.A. 12, Deming, Jean and Davis, Hilda, "A Bacteriological Investigation of Evaporated Milk", Reprint, Archives of Pediatrics, Vol.XLVIII, No.l. (60) 13. Spmmerj H>H, and Hart, E,B<» "Heat Coagulation of Evaporated Milk". Wisconsin Research Bulletin#67, 1926* 14• Bergey ;s Manual of Determinative Bacteriology, 3rd Ed, 1930, Williams and Wilkins, Baltimore, Md., U.S.A. . 15. S. Orla-Jensen, "The Lactic Acid Bacteria." (In English) D. Kgl. Danske Vidensk. Skrifter, Natur og Mathematisk, Afd.8, Raekke, V.2, Copenhagen, 1919. 16. Eaples, B.A. and Sadler, W.? Canadian Journal of Research, 7, 364-369, 1952. 17. Prickett, P.S. "Thermophilic and Thermoduric Organisms with Special Reference to Species Isolated from Milk". "V. Description of Spore-forming Types", Tech. Bull. No. 147, New York State Agr. Exp, Stn. 1928. (61) ACENOV/LEDGMENTS eWe wish to express our appreciation and thanks to Dr. B, A. Eagles and Miss Olga Oiotlitch, of the Department of Dairying, University of British Colvimbia, for assistance given during the course of this investig-ation. • Thanks are also tendered the Praser Valley Milk Producers Association for f a c i l i t i e s rendered and experimental material supplied© L • A»A» , G • J • 0 T A B L E N 0«, 14, M O R P H O L O G Y Ho. of Source of Culture Agar Slants Milk 72 hrs. 55° C. 0 • 13 • 133? O "til Gr3?Q331 72 hrs. 550 G. Stain Spores Litmus Milk 72 hrs. inc. 55 deg.C« C U L T U R A L C H A R A C T E R I S T I C S Nutrient - Broth 24 hrs.55° 762*L Evap. Milk 795-Q Evap. 1111k 786-Bl.a Evap.Milk 786-B1. Evap. Milk D.T.I Drop Tank D.T.3 Drop Tank 786B2 Evap.Milk 795-P Evap. Milk Mash 5 161-2 142-1 Old Mash Shipper 161D Comp. sample Milk 161D Evap. Milk 782-03 Evap.Milk 146-P Evap. Milk 143 Shipper 143D Mash 6 Old Maeh Shipper 161D Mash 4 Old Mash Shipper 161D 122-G Evap, Milk 143-G Evap. Milk H3 Evap. Milk VII-D Composite of a truck 236 Mash 2 Comp.Sample Shipper's Milk Old Mash Shipper 161D 145-N Evap. Milk 144-P37 Evapo.Milk 138-0 Evap. Milk 782-M2 Evap. Milk 782-M3 Evap. Milk 795-J Evap. Milk 161-A1* ESSTSk XI Hunt Composite of a truck 795-G Evap. Milk Hay-1 Hay dust Shipper 161D 782-M1 Evap, Milk No.31 Comp.sample Shipper 31D Truck VI Composite of a truck Truck IV Composite of a truck 161 207 Comp.sample Shipper 161D Comp.sample Shippers' Milk Truck V Composite of a truck Hunt XI Composite of a truck long rods threads and single, ,5-lM x 4-7M thin and thick long rods,single, chains long rods, single and chains, 4,5-lMx4-6 rods,mostly single ,5-lM x 3-4M thin long rods,single chains,.5-.8MX3-5M ^ n S n ? i ! l ^ - 6 M thick long rods sing-le, long chains 10x5M rods, single and chains ,8-lMx4M. rods,single and chains .8-lmx4M. rods, single and chains !8-lMx4M short rounded rods lx3M, rods, single :8Mx3M single rods .8M-5M rods,single and chains ,8x4M, rods,single and chains .8x4M. long rods ,8M x 5 M. single rods ,8M x 4 M, single rods •8M x 4 M. long single rods lx6M. long rods,single & long chain ,8x6M single rods single rods .5-.8M x 3-5M. single rods .5-.8M x 3-5M single rods .5-.8M x 3-6M single rods •5-.8M x 3-6M single rods few chains, ,8x6M. single rods, short chains, .7x411. single rods and chains, ,7Mx4M. single rods ,7M x 4M. single rods ,7M x 4M.. . rods, single and chains lx5M. c ^ n f ^ l M ^ c S n s l S x t r single rods . ,6Mx3-4M single rods and chains .6Mx3-4M rods single and chains long rods, single 1J&4-7M Long rods,single 1MX4-7M long rods single and chains long rods,single and chains long single rods single rods single rods single rods single rods few chains single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods and chains single rods single rods single rods single rods single rods single rods single rods single rods long single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods rods, single,pairs «• and chains long rods,single and short chains 4 thick rods,single + few pairs single rods + single rods + single rods + thick rods,single + and chains single rods and + chains single rods + single rods * single rods + single rods 4 single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods single rods and pairs single rods single rods, single rods single rods single rods single rods single rods single rods single rods single rods elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal reduced,clean hard clot reduced,clean hard clot reduced, clean hard clot. reduced,clean hard clot Motility i n Broth Gelatine Liquefaction 2 0 D C. 55° C. Reduction of Nitrates H 0 2 NH3 Indol Production Growth on Agar Slants 48 hrs. at 55° G. Turbid Turbid Turbid Turbid reduced, clean Slightly hard clot Turbid reduced,clean hard clot unchanged unchanged unchanged unchanged darkened unchanged unchanged elongated terminal elongated terminal unchanged unchanged elongated unchanged terminal elongated terminal Elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated t erminal elongated terminal elongated terminal elongated terminal unchanged unchanged, unchanged unchanged clotted, , peptonized clotted peptonized unchanged unchanged unchanged unchanged unchanged unchanged unchanged elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal elongated terminal unchanged unchanged unchanged unchanged unchanged unchanged unchanged unchanged unchanged reduced, clotted reduced, clotted reduced, clotted Slightly Turbid Slightly Turbid Slightly Turbid Slightly Turbid Slightly Twrbid Slightly Turbid Slightly Turbid Slightly Turbid I s i i g h t l y ^Turbid !Slightly Turbid Slightly Turbid Slightly !Turbid Slightly Turbid iSlightly Turbid I Slightly Turbid white p e l l c l e 'Turbid white pelicle Turbid Turbid iTurbid Turbid :Turbid Turbid I Turbid 'Turbid Turbid Turbid Turbid jSediment Turbid JSediment iTurbid jTurbid Turbid Turbid Turbid ring on top Turbid ring on top Turbid ring on top + 4 + 4 4 4 4 4 4 + 4 bubble bubble bubble bubble 4 4 abundant,beaded,dull, smooth, opaque, grayish; media unchanged abundant, beaded, dull, smooth, opaque, grayish, media unchanged abundant, beaded, dull, smooth, opaque, grayish, media iinchanged abundant, beaded, d u l l , smooth, opaque, grayish, media unchanged moderate, beaded, glistening, smooth opaque, grayish, media unchanged moderate, beaded, glistening, smooth op a. que, grayish, media unchanged abundant, effuse, dul l , smooth beaded, opaque,grayish;media unchanged moderat e,b eaded,f1at,gray!sh, dull opaque, media unchanged moderate,beaded,glistening,grayish,dark centre", colonies, media unchanged moderate, neaded,glistening,grayish, dark centre colonies, media unchanged moderate, smooth,spreading,opalescent f l a t , media unchanged abundant,filiform,flat,glistening, smooth,opaque, media unchanged abundant,echinulate,flat, dull smooth, opaque,grayish, media unchanged moder a t e,b eaded,gli s t ening,op aque grayish, media unchanged abundant, beaded, glistening, f lat, smocth grayish, media unchanged abundant,beaded,gli stening,f1at, smooth grayish, media unchanged mod er a te,beaded,glist ening,op aque, grayish, media unchanged moderate, beaded,glistening, opaque grayish, media unchanged abundant, filiform,flat,glistening smooth,opaque, media unchanged Abundant,beaded, f l a t , glistening smooth, opaque, media unchanged abundant, spreading, f l a t , glistening,smooth, transluscent, grayish, media unchanged moderate, beaded,flat,glistening,smooth transluscent, grayish, media unchanged moderate, beaded, glistening, grayish,dark centre colonies, media unchanged mod era t e , f i l i f o r m , f l a t , g l i stening, smocfch opaque, grajrish, media unchanged. moderate, smooth,spreading,opalescent, f l a t , media unchanged. moderate,spreading,flat,glistening,smooth, tr anslus c ent,grayi sh, medi a unchang ed moderate,spreading,flat,glistening,smooth, opalescent, media unchanged moderate,spreading,flat,glistening, smooth, opalescent, media unchanged moderate,beaded,grayish,gIistening,dark centre colonies, media unchanged abundant,spreading,raised,glistening,smooth, opaque,grayish, media unchanged moderate,spreading,raised,glistening,smooth, opaque,grayish, media unchanged moderate,spreading,raised,glistening,smooth, opaque,grayish, media unchanged abundant,beaded,gli st ening,opaque, grayish, media unchanged abundant, beaded, glistening, opaque, grayish,, media unchanged moderate,filiform,flat,glistening, opalescent,smooth,grayish,media unchanged moderate,spreading,flat,glistening, opalescent, smooth^ grayish, media tmehang ed moderate,beaded,echinulate, glistening, opalescent, smooth, grayish, media unehanged abundant, spreading, f l a t , dull, opaque, smooth, grayish, media unchanged abundant,spreading,flat,dull,opaque,smooth, grayish, media unchanged abundant,spreading,flat,glistening,opqque, smooth, grayish, media unchanged abundant,spreading,flat,glistening,opalescent, smooth, grayish, media unchanged 4 4 4 + T A B L E N Q . 1 5 ^ NO. OP W EH H rULOSE ?R0SE o LCT0SE 3HAR0SE ?0SE ?0SE H £ 3 H STARCH H O •a* MILK W CULTURE a < 1—1 R Si a o CO hi a O % H EH X m P STARCH MILK a ^ 762 - L -0.9 5.2 4.5 4.7 3.8 5.9 4.5 4.3 -0.9 _ 1,8 3.4 1.4 5.9 clot 6.1 clot 795-Q -1.4 4.7 5.9 5.2 4,3 5.4 4.7 3,6 -1.1 2.9 3.8 3.6 6.1 clot 5.2 clot 786Bla -0.9 4.7 4.5 5.0 4.1 5.2 4.7 3,2 -1.1 2.9 3.2 0,0 6.1 clot 5.4 clot 786B1 -0.2 5.2 4.1 4.7 4.7 4.3 4.9 4,1 0.0 0.7 2.3 0.0 5.9 clot 6.1 clot D.Tel 0.0 0.8 2.0 1.4 1.4 0.0 2.0 2.0 0.0 1,6 2.3 0.0 1.4 clot 1.6 clot D.T.3 0.0 1.8 1.4 1.1 1.4 0.7 1 . 6 - 0.0 0.0 0.5 0.0 1.8 clot 1.8 clot 786B2 4.1 3 , 6 4.0 0.9 - a . 7 0.0 2.7 -0.9 -0*9 -1.4 -0.5 -0.5 0.0 0.7 795-P 3.6 3.8 4.1 3.4 -0.9 0.0 2.2 -1.4 -0.9 ,0.0 -1.4 0.0 0.0 0.7 Mash 5 3.6 4.3 4,3 3.8 -0.0 -0.7 1.3 -1,4 -0.9 -0,9 -0.9 -0.9 -1.1 0.0 161-2 2.3 3.8 3.6 2.0 0.0 -0.5 0.0 -1.4 -1.1 -0.9 -0.9 0.9 -0.9 -1,4 142-1 2.0 3.2 4.1 1.1 0.0 G . O 2.2 -0.0 -0.7 0.0 0.0 0.0 -1.4 -1.4 782-03 1.8 4.1 3.6 0.5 -0.7 0.5 2.5 -1.1 -0.5 -0.5 0.0 0.0 0.0 -0.5 146-P 1.1 2.9 1.8 1.1 0.0 0.0 2.0 -0.7 0.0 0.0 0.0 0.0 0.0 -0,5 143 0.9 3.6 3.6 3 . 6 0,0 0.0 -1.2 0.9 -0.9 0.0 0.0 0.0 -1.4 -1.4 Mash 6 3.8 3.4 3,4 2.9 2.9 0.0 1.3 -1,1 -1.4 -0.9 -0.9 -0.7 0.0 0.0 Mash 4 4.3 4.3 3.8 3 . 6 3.2 0.0 1.1 -0.9 . -1.1 -0.7 -0.9 -0.7 0.0 0,0 122-G 3.4 3.4 3.8 0.0 -0.9 0.0 2.7 -0.9 -0.7 0.0 0.0 0.0 -0.7 -1.4 143-0 2.3 2.0 3 . 6 0.0 0,0 0.0 2.0 -0.5 -0,0 0.0 0.0 -0.5 0.0 -1.1 120-C 1.4 2.9 3.2 0.0 -0,7 0.0 2.2 -0.7 0.0 0.0 0.0 0.0 -1.4 -1.4 161-3 4.3 3.4 3.4 4.1 -1.4 0,5 0.9 -1.1 -1,4 -1.1 -0,0 2.9 0.0 -1.4 VII-D 2.9 2.3 1.1 - -1,6 2.7 1.6 -1.4 -1.1 0.9 1.4 2.0 -0.9 clot -0.5 clot 236 2.9 2.7 2,3 1.8 1.8 2.7 1.6 -0.7 -1.4 0,9 1,1 0.0 0.0 -0.2 clot Mash 2 0.0 3.2 3.8 2.3 1.8 0.0 0.0 0.0 -0.9 0.0 -0,5 0.0. 0.0 -0.5 clot 145-N 0.0 0.9 3 , 6 3.8 2.3 0.0 -1.2 -0.5 0.0 0.0 0.0 0.0 -0.5 clot -0.5 144P37 -0.9 3,2 4.1 2.9 0.0 4.1 2.0 -0.9 -0.9 -0.9 -0.7 -0.5 0.0 -1.4 138-0 0.0 3.6 3,8 0.0 -1.5 0.0 2.0 -1.4 -0.5 0,0 0.0 0.0 0.0 -1.4 782-M2 - 1 . 6 3.2 4.1 2.9 2.0 2.9 0.0 -1,4 -0.9 - i . i -1.4 1.6 0.0 0.0 782-M3 0.0 3.2 4.1 3.4 -1.1 0.0 0.7 -1.1 0.0 0.7 0,0 0.0 0.0 -1.4 795-J -1.4 3,2 3.2 -0.9 -1.4 4.1 2.5 -1.1 -0,9 -1.1 -1.4 -0,5 0,0 0.0 161-alk. 0.5 1.4 1 . 6 1.1 0,0 1.4 0,0 0.0 0,0 0.0 0.0 0.0 0,0 0.0 XI Hunt 0.7 2.3 1.4 0,0 -1.4 2.0 1.3 -0.9 0.7 -0.9 2.0 0,0 0,0 795-G - 4 - - - + f - - - - - - -Hay 1 0.0 2.9 5.9 -0.7 -0.5 - 0.0 0.0 0.0 0,0 0,5 0.5 0,0 0,0 -0,5 782-Ml - - + - - - - - - - - - - -No. 31 0.0 0.5 0,0 1.8 0.0 0.0 0.0 0.0 0.0 0.5 0,0 0.0 0.0 -0.7 Truck VI -0.7 -0.9 -0,7 -1.4 -1.6 0.5 -1.2 -1.4 -1.4 -1,1 -0.9 -0.9 0,0 0.0 Truck IV 0.0 0.0 0.0 2.5 2.0 0.0 0.0 0.0 0.0 0,0 0.0 0.0 0.0 -0.7 161 -0,7 -0.9 -1,4 -1.4 -1.6 -0.7 0.0 -1,1 -1.1 -0.9 -1.1 -0.7 0.0 -0.7 clot 207 -0.9 1.8 -1.4 -1.4 -1.8 -0.7 -1.2 -0.9 -1,1 -0,9 -0,7 -0.9 0.0 clot digested 0dlge S?ed -0.5 clot Truck V 0.0 0.0 -1.1 -1.6 -1.4 0.9 0,0 - -1.4 -1.1 -1.1 -0,9 -0.9 -0.5 clot Hunt XI -1.6 -1.8 -2.0 -2.0 -2.3 -0.9 0.0, 0,0 -0,9 0.0 0.5 0.0 0.0 clot -0.7 clot Casein Digest Broth . -Sugars added at the rate of 2 per cent. Results recorded as grain l a c t i c acid per mille. digested # Yeast Extract added at the rate of .15 per cent. 

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