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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-ASSOCIATED WITH SPOILAGE OP EVAPORATE^ MILK,  Lyle A, Atkinson, B.S.A., and George J , Okulitch, B.S.A.  A Thesis submitted f o r the Degree of MASTER OP SCIENCE IN'AGRICULTURE In the Department of DAIRYING,  The University of B r i t i s h Columbia A p r i l , 1935.  ^  ibj. nps/^  TABLE OP CONTENTS, Page  )  Introduction. Historical  1 ..  Exp erimental Checking the Raw Milk A l t e r a t i o n of Plant Practices  1 5 11 .. 22  A l t e r i n g the Salt Balance  27  Reproducing the Spoilage  31  Innoculation Experiments  40  C l a s s i f i c a t i o n 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 t r o p i c a l countries.  The trouble was reported  c h i e f l y from India and Java and i n such proportions as to threaten the loss of t h i s 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 i n each case.  Furthermore, the cans did not give  evidence  of spoilage on a r r i v a l at the consignee's warehouses, but only after i t had been distributed  i n smaller parcels to the  retailers. A s i g n i f i c a n t fact In t h i s s i t u a t i o n was that, while t h i s company exported large quantities of t h i s same milk to points i n the B r i t i s h 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 l i t e r a t u r e 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 t i n s of  milk f a i l e d to f i n d either aerobio or anaerobic bacteria and concluded that deteriorations i n condensed milk are of a chemical or physical character and not of b a c t e r i a l origin.  Dodge (2), was unable to i s o l a t e from  condensed  milk an organism which he believed was responsible f o r the trouble.  From experiments with butyric and l a c t i o acids  he concluded that i t was probable, that i n the cans of spoiled milk, the gas was not formed by the bacteria d i r e c t l y but was formed by e l e c t r o l y t i c action between the metals of which the cans were composed and the acid generated by the growth of bacteria i n the milk before the l a t t e was condensed.  Hunziker and Wright (3)demonstrated that  by scratching the t i n s 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 f o r 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 l a c t i c acid into cans of evaporated milk s u f f i c i e n t to bring the t o t a l a c i d i t y to .68$.  They then  sealed, s t e r i l i z e d and incubated the milk f o r 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 t i n s of milk ever become blown through chemical means alone.  (3)  The Vermont A g r i c u l t u r a l Experiment Station (5), has published a short paper on the coagulation of supposedlys t e r i l i z e d milk i n which the milk curdled without after which the curd was  souring,  apparently digested leaving a  whey l i k e l i q u i d above, with thick masses of slimy curd and precipitate i n the bottom.  B. s u b t i l i s was  the  organism most a c t i v e l y f i g u r i n g i n the change. K e l l y (6) reports the spoilage of evaporated milk by sweet curdling types of bacteria which coagulate the milk with a custard l i k e c l o t and show no change i n the a c i d i t y , taste or smell of the milk.  Gurdling  started at the top and proceeded downwards.  The  causative  organisms were i d e n t i f i e d as atypical forms of B. oereus, B. simplex  and B. megatherium. Hammer and Hussong ( 7 ) , i s o l a t e d an organism  which they i d e n t i f i e d as B a c i l l u s oereus from a spoiled can of evaporated milk.  They did not attribute the o r i g i n a l  outbreak of spoilage, i n the factory investigated, to t h i s organism but found i t i n t e r e s t i n g i n i t s action on evaporated milk as coagulation took place with'no change i n the odor and f l a v o r unless the incubation period extended. was  was  They also found i f a s a t i s f a c t o r y a i r supply  provided by t r a n s f e r r i n g 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 f l a v o r and could be found i n large numbers microscopic ally. Hammer (8) reports an outbreak of spoilage i n 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. c a l i d o l a o t i s from sour curdled evaporated milk.  This i s a spore formin  f a c u l t a t i v e aerobe whioh curdles the evaporated milk rapidly at from 55 degrees 0. to 60 degrees 0.  The milk  showed an a c i d i t y of .67 percent to .74 percent, calculated as l a c t i c acid.  This thermophile f a i l e d to  grow and did not curdle the milk at 37 degrees 0. or below. Hunziker (10) r e f e r r i n g to curdiness caused by bacteria states that, i n 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 l i m i t e d d i s t r i b u t i o n of the organism.  In the majority of cases,  the trouble f a i l e d to show f o r 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 i n evaporated milk, Hunziker makes the following observations:  (5) "These several c i t a t i o n s 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 i n evaporated milk.  These  observations emphasize the importance of greater attention to sanitation and scrupulous cleanliness i n the factory to the end that the presence of these organisms may be avoided as much as possible.  They further emphasize the  a d v i s a b i l i t y of using r e l a t i v e l y high  sterilizing  temperatures and the importance of uniform heat i n the s t e r i l i z e r  distribution  i n order to insure maximum germ k i l l i n g  efficiency." EXPERIMENTAL. It should be mentioned at the outset that t h i s milk conformed to the B r i t i s h Standard f o r evaporated and contained approximately  milk  9.0/2 f a t and 31.5$ t o t a l s o l i d s ,  which i s muoh heavier than Canadian or United States domestic standard. When the o r i g i n a l outbreak of spoilage was reported, cans of the affected milk were returned f o r 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 i n most instances being clotted with some production  (6) of gas.  Working on the assumption that the changes had  been brought about by b a c t e r i a l action, e f f o r t s were made to isolate causative organisms from t h i s milk.  Direct  miscroscopio examination f a i l e d to show the presence of any organisms.  Samples plated d i r e c t l y on Difco Purple Lactose  Agar (11), and incubated at 37 degrees G. and 55 degrees G. f a i l e d to show any growth.  Samples Incubated f o r 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 a i r , or some other oause. Duplicate cans of those returned as spoiled had been retained i n the factory and these were subjected to incubation f o r 17 days at 55 degrees G.  None of these cans  showed physical evidence of spoilage similar to that experienced i n the t r o p i c s , nor did they show presence of any l i v i n g organisms after plating and incubation. Samples of t h i s spoiled milk were submitted f o r 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 f a i l e d to f i n d l i v i n g organisms i n the spoiled milk. The p o s s i b i l i t y suggested i t s e l f that spoilage  (7) may have been due to defective sealing of the cans or to an e l e c t r o l y t i c 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. f a i l e d to show defective manufacture or sealing.  As the tinning on the  insides of a l l cans was bright and shiny, no evidences of e l e c t r o l y t i c action were discovered which p r a c t i c a l l y eliminated t h i s p o s s i b i l i t y from our reasoning and investigation. It w i l l thus be apparent that a l l e f f o r t s to determine the cause of spoilage from affected cans proved unavailing. It has been the practice f o r some years, i n the laboratory maintained by the Company manufacturing t h i s evaporated milx, to examine representative samples from each s t e r i l i z e r batch f o r s t e r i l i t y .  This was done by  incubating the sealed cans at 37 degrees G. f o r 48 hours and then p l a t i n g .  The technique used was as follows: the  tops of incubated cans were s t e r i l i z e d by swabbing with alcohol and then flaming with a gas j e t . A hole was punched i n the can by means Of an ice picx which had been dipped i n alcohol and flamed and the sample drawn out with a s t e r i l e 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. f o r 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 i n the milk so that an occasional colony due to a i r contamination  when plating  would not be interpreted as indicating unsterile milk.  The  result of t h i s was that the finished plates showed either heavy b a c t e r i a l growth or no growth at a l l . The routine examinations had sometimes shown unsterile samples, although the percentage was not high. After the i n v e s t i g a t i o n 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 i n t r o p i c a l countries, there being a complete absence of i t i n shipments to the United Kingdom. practice was continued.  started i n September 1933,  This  and has since been  Incubated at 55 degrees 0. the percentage of  unsterile samples increased u n t i l i n November and December the greater percentage of them appeared u n s t e r i l e .  The  great majority of the colonies growing on plates appeared to be non lactose fermenters was  as the reaction of the media  almost i n v a r i a b l y a l k a l i n e . Several questions at t h i s point now  themselves.  presented  F i r s t l y , would a l l evaporated milk appear  unsterile i f incubated at s u f f i c i e n t l y high temperatures and f o r a s u f f i c i e n t length of time.  Secondly, at what point  (9) i n the operation did these contaminating organisms gain entrance to the milk? method of manufacture  Thirdly, would alterations i n the permit of e f f e c t i v e  sterilization?  Fourthly, could these apparently nbn-lactose fermenting organisms produce acid i n milk, s u f f i c i e n t  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 i n 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 s t e r i l e and free from heat resistant organisms.  The bloated  can showed the presence of acid colonies rather than the a l k a l i forming colonies experienced i n routine plating. Deming and Davis (12), report an investigation on the bacteriology of evaporated milk i n which they found p r a c t i c a l l y a l l of 154 samples of milk purchased i n the open market to be e f f i c i e n t l y  sterilized.  Extensive experiments were conducted to determine the point of entrance of these heat resistant In making these determinations a procedure was  organisms. adopted  which i t was believed would show only the heat resistant organisms.  A l l samples were taken i n s t e r i l i z e d 16 ounce  evaporated milk cans, sealed and s t e r i l i z e d at the usual temperature of manufacture, which varied between 226 degrees  (10) F. and 230 degrees F. f o r a holding period of 35 minutes. The cans were subsequently incubated at 55 degrees 0. f o r 48 hours and then plated i n the same manner as the routine samples.  The results of s t e r i l i t y tests are shown  i n Table No.l. On the f i r s t attempt to determine the point of entrance of these resistant organisms s i x cans were taken from each of several points i n the manufacturing process. A l l samples from the raw milk and after the preheater appeared s t e r i l e , i n d i c a t i n g that no organisms had survived the  s t e r i l i z i n g temperature of 230 degrees F. f o r 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 i n the routine examination of the f i n i s h e d product.  A second attempt showed a l l samples, even those  from the preheater to be u n s t e r i l e . were taken on t h i s occasion.  No samples of raw milk  A t h i r d attempt showed a l l  samples of raw milk to be s t e r i l e , while samples from the preheater, hot wells, vacuum pan and drop tank a l l showed some samples s t e r i l e and some not s t e r i l e .  The results of  t h i s check up tended to indicate that either the actual contaminating influence was s l i g h t or was intermittant during the day's run. A fourth and more systematic check up was made when samples were taken from each point i n the  TABLE.NO.1. Process Samples Showing Heat Resistant Bacteria Results of Five Determinations.  S - indicates s t e r i l e condition. U - indicates unsterile condition.  £>ATE Raw S .u  SOURCE AND. CONDITION. OF SAMPLE. PreHot. Vacuum Drop Heater . Wells .,. Pan • Tank s U S U ,s U S U  1933. Nov. 16th  8  0  8  0  -  -  0  Nov. 20th  -  -  0  6  3  3  0  Nov. 26th  10  0  19  13  14  2  Dec. 8th  15  0  17  2  19  28  2  25  1  26  8.  0  30  12  0  18  6  8  3  15  Z  13  7  12  6  0  56  4  29  1  1934. Jan. 19th  (11) process at ten minute i n t e r v a l s .  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 u n t i l late i n the day's run when samples bearing heat resistant from a l l of these sources.  organisms appeared  i n milk  A f i f t h similar exhaustive  check was made with the same results, except that t h i s time two of the cans from the raw milk late i n 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 p o s s i b i l i t y of the rav; milk being the o r i g i n a l source of the contamination. CHECKING- THE RAW-MILE, Since i n p r a c t i c a l l y a l l cases the heat  resistant  organisms appeared only late i n the day's operation, a preliminary check was made of the l a s t three trucks of milk a r r i v i n g at the Plant.  Six cans from each truck were  taken at i n t e r v a l s of f i v e minutes during the dumping prooess.  A l l gave negative r e s u l t s .  Following t h i s i t was  decided that the raw milk from a l l trucks to the plant should be checked. 1934.  This was done on February 15th and 16th,  The method of taking these samples was as follows.  A dipper from each can of shippers' milk was placed i n a s t e r i l i z e d 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 s t e r i l i z e d at 250 degrees F. f o r t h i r t y - f i v e minutes. The samples were then brought to the laboratory and incubated f o r two days at 55 degrees G.  They were then  plated on Purple Lactose Agar and the plates incubated f o r 48 hours at 55 degrees C. The results of the tests on composite samples from trucks are shown i n Table No.2* In t h i s 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. f o r 55 minutes.  On the second  day one can from Truck #7 showed the presence of heat resistant organisms.  On the t h i r d day samples from Truck  #7 showed t h e i r presence i n two cans while samples from Trucks #6 and #15 showed them i n 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  A p r i l 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 l e d us to examine the milk of t h i s 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 s t e r i l i z a t i o n process. Milk from 6 of these  TABLE NO.2. SHOWING PRESENCE OF HEAT RESISTANT BACTERIA IN RAW MILK. S - S t e r i l e Plate. U - Unsterile Plate. Truck  Feb. 15  1.  SSSS  2.  Feb. 16  Apr. 3  Apr. 4  SSSS  ssss  ssss  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  12.  ssss  ssss  13.  ssss  ssss  —  —  14.  ssss  ssss  —  —  15.  —  16.  —  —  —  —  ssss  ssss  --  sssu  ssss  ssss  ssss  (15) shippers showed organisms i n only one can out of the two examined while the milk from the other shipper showed t h e i r presence i n both cans.  A further check on these seven  shippers was deemed desirable and a test was made of s i x one pound cans of milk from each shipper.  These showed a l l  six cans s t e r i l e from s i x of the shippers,while from the seventh only one appeared s t e r i l e while f i v e contained the resistant bacteria.  This l a t t e r 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 t h i s p a r t i c u l a r farm was deemed advisable and was subsequently made on May 12th.  The barn  on t h i s farm was very old and rather dark i n the cow stable. On the f l o o r of the passageway i n front of the. mangers was a layer o f old dusty hay.  The mangers themselves were d i r t y  with b i t s of old hay and mash, notwithstanding the fact that the farmer had fed no grain or mash to the cows f o r some s i x weeks.  Samples were taken from the wash water  from udders, the feed bin, the hay i n front of the mangers, the residue i n the mangers, the piped water and the water i n the cooling tank.  These samples were a l l put into s t e r i l e  milk and taken to the laboratory where they were steamed for  t h i r t y minutes, then incubated at 55 degrees 0. f o r  48 hours.  By t h i s 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, t y p i c a l colonies were picked and transferred to one pound cans of evaporated milk, which were sealed and s t e r i l i z e d .  On t h i s f i r s t  attempt a l l organisms  appeared to be k i l l e d o f f i n the s t e r i l i z i n g process of 230 degrees P. f o r 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 t h e i r presence i n some of the cans.  Those samples from udders, hay and mash showed  t y p i c a l growth and from these, colonies were picked and purified.  C l a s s i f i c a t i o n l a t e r showed them to be the same  as strains isolated from the finished milk, i n d i c a t i n g 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 f o r the manufacture of this export evaporated milk. Coincidental with the removal of t h i s milk, the routine samples examined showed a marked f a l l i n g o f f i n numbers of those showing the presence of heat resistant organisms.  It appeared that possibly t h i s shipper was  responsible f o r the bulk of the heat resistant organisms.  (15) This conclusion was l a t e r proven to be a f a l l a c y . No further determinations on the raw mille were made f o r some months following, as during June, July and August, p r a c t i c a l l y 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$ i n October and further to 85.7$ i n December, the Plant being closed from October 15th to December 6th. When these organisms commenced to increase i n September i t was deemed advisable to again check the raw milk supplies.  Samples were taken of composites of truck  loads i n the manner previously described.  Six cans from  each composite were sealed and s t e r i l i z e d at 225 degrees F. f o r 25 minutes.  The r e s u l t s of three examinations are  shows i n 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 d i s t r i c t s contained bacteria which would survive the s t 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 s i x samples  appeared s t e r i l e on one day's examination tests made on  TABLE NO.3. SHOWING PRESENCE OP HEAT RESISTANT BACTERIA IN .SAW. MILK FROM TRUCKS S- indicates s t e r i l e 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  8.  sssuuu  9.  ssssss  ssssuu  10.  uuuuuu  uuuuuu  11. 12. 13.  ssssss  -  -  _ _  sssssu  ssssuu sssssu 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 s i x  taken appeared unsterile which would indicate that these resistant organisms did not appear i n large numbers i n the o r i g i n a l milk.  The plates, however, invariably showed  large numbers of colonies, indicating t h e i r vigorous growth at the temperature of incubation, v i z . , 55 degrees 0. The problem of eliminating-these'heat resistant organisms from supplies used i n export milk now appeared more d i f f i c u l t than ever.  If truoks from a l l  d i s t r i c t s showed t h e i r presence the next question which presented i t s e l f was t h i s :  would they be confined to a  few shippers or would t h e i r occurrence be general?  It was  then decided to check the milk of i n d i v i d u a l shippers f o r the presence of these organisms.  Accordingly on October  10th, 1.934, Truck #6 was examined.  Samples were taken i n  the described manner, i n 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 f o r some l i t t l e . t i m e after t h i s as the plant was closed f o r a period of two months.  About the  middle of December, however, t h i s work was taken up again as the percentage of samples of f i n i s h e d 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  i n d i v i d u a l shipper's milk from a l l d i s t r i c t s 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 t h e i r presence. Previous experience had shown that milk from certain trucks would appear s t e r i l i z e d after being subjected to" heat process while samples from the same truck on subsequent days would sometimes appear to be not  sterilized.  It i s quite possible that the milk of individual shippers would act i n the same manner and many of the above shippers, whose milk appeared  s t e r i l e 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 i n any considerable numbers they would .have shorn up, due to the size of the sample taken which was i n each case one pound of the mixed shipment. The next l o g i c a l step was to see what would be the- effect of withholding t h i s affected milk from supplies  TABLE NO.4. SHOWING NUMBERS OP SHIPPERS WHOSE MILE CONTAINED.. HEAT RESISTANT BACTERIA..  DATE  TRUCK  STERILE  Deo.21  . 1.  49  21  2e  14  9  3.  §3  7  4.  33  16  5*  19  4  6.  153  6  7.  29  1  8.  84  3  9.  . 62  7  tt  Jan. 3 H  IT  Jan. 8 IT  Jan.11 II  TOTALS  .496  UNSTERI1E  74  (18) used f o r the manufacture of the export standard milk.  A  change was accordingly made i n the method of handling supplies of shippers' raw milk.  A l l that which had appear-  ed free of resistant organisms was put through i n the regular manner, evaporated to the export standard, packed and s t e r i 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 r e s u l t s achieved by t h i s  segregation were i n t e r e s t i n g and g r a t i f y i n g . 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 6 0 . 7 $ of the f i n i s h e d 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 i n t e r e s t i n g to note that from January 16th  to A p r i l 8th, 3 9 5 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 l a t t e r 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 1935  INCUBATED AT 37° C. STERILE No. Samples $  UNSTERILE No • . Samples $  INCUBATED  55  0•  STERILE UNSTERILE No. No. Samples $ . Samples $  Before Segregation: Jan.1-15  58  39.3$  89  60.7$  11  7.5$  Jan.16-23*  54  90.0$  6  10.0$  92  80.0$  Feb.15-16  41 100.0$  0  0.0$  70  89.7$  8  10.3$  136  92.5$  After Segregation: 23" . 20,0$  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$  48 100.0$  0  0.0$  35  72. 9$  13  27.1$  11  2.8$  355  72.9$ 132  27.1$  Apr. 3-8 Total since Segregation  384  97.2$  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 i n obtaining s t e r i l e 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 e f f i c i e n t l y s t e r i l e at both temperatures incubation. manufacturers  of  This has been of great value to the i n that they now have an available supply  of milk, f o r export to t r o p i c a l countries, which can be shipped with confidence i n the knowledge that i t w i l l withstand the conditions of temperature during t r a n s i t and storage.  which i t must undergo  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 s p o i l under any conditions involving  (£0) changes i n temperature. At the commencement of t h i s investigation and after the 55 degrees 0. incubator had been put into operation, a l l milk which did not appear s t e r i l e at both temperatures i n the routine analyses was withheld from shipment to hot countries and was confined i n i t s d i s t r i b u t i o n to countries within.the Northern Temperate Zones.  It i s of s i g n i f i c a n t note that since t h i s practice  has been operative there have been no reports ol- recurrent spoilage of the o r i g i n a l type from consignees i n any part of the world to which t h i s milk has been shipped. From the ahove discussion i t w i l l be apparent that we have been successful i n 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 a c t i v e l y at temperatures i n 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 t h e i r occurrence i s widely scattered through different d i s t r i c t s and further that by segregation of affected milk a product can be produced which i s r e l a t i v e l y 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 i n connection with these heat resistant organisms i s t h e i r 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 t h e i r presence or absence i n 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 appearing unsterile at 55 degrees 0. was very high during the P a l l and Winter months and low during the Spring and Summer. Commencing i n January with 72.6$ of the samples appearing unsterile t h e i r occurrence dropped markedly each month to a low of 1.6$ i n June and then gradually increased to a high point of 85.7$ unsterile samples i n December of the same year.  This subsequently increased to 96.3$ samples  unsterile i n January 1935. .The samples incubated at 37 degrees 0. showed the same general tendencies although i n every case, the percentages were lower.  Commencing i n  January with 26.'6$ of samples unsterile the numbers decreased to none unsterile i n September and increased to 8.4$ i n December, with a sharp r i s e to 71.3$ i n January 1935. These figures point very d e f i n i t e l y to the effect of seasonal conditions surrounding the production of milk.  During the P a l l and Winter when milk i s produced  under barn conditions the incidence of heat resistant types of bacteria i n 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 MONTH January  37° C. INCUBATION STERILE  55°0. INCUBATION  jo UNSTERILE  jo STERILE  fo UNSTERILE fo  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  1e 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  2059  95.9  88  4.1  1623  75.4  533  24.6  Yearly  (22) and e s p e c i a l l y daring the Summer when cows are out on pasture and only brought into barns during the milking period t h e i r incidence i s least and constitutes only a negligible factor i n the production of this type of milk. ALTERATION Off.PLANT PRACTICES. In the early stages of t h i s investigation various changes were made i n plant practices to effect the removal of heat resistant bacteria from equipment or to destroy them i n s t e r i l i z i n g .  When i n our f i r s t  examinations  t h e i r presence was noted i n a l l milk at various points i n the process and not i n 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 e a r l i e r i n t h i s report the f i r s t tests made showed bacteria surviving s t e r i l i z i n g temperatures  I n milk from  the preheater, hot wells, vacuum pans and a l l subsequent points.  They were p a r t i c u l a r l y noticeable after the milk  had passed through the vacuum pans. This equipment was already being reasonably cleaned and s t e r i l i z e d 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 o f the s a n i t a r y k i n d which can be t a k e n down and brushed d a i l y .  I r o n p i p i n g i s used f r o m  the vacuum pan t o t h e pumps.  The r e a s o n f o r t h i s i s t h a t  s i n c e these pumps are w o r k i n g a g a i n s t a vacuum a t a l l times i t i s e s s e n t i a l t o have a l l pipe j o i n t s a i r t i g h t . the s a n i t a r y type of p i p i n g t h e vacuum would be  With  destroyed  and t h e e f f i c i e n c y of t h e pumps i m p a i r e d , as t h e j o i n t s soon become s l i g h t l y worn.  The u s u a l p r a c t i c e i n c l e a n i n g  the i r o n p i p e s was t o r u n a s t r o n g soda s o l u t i o n t h r o u g h them and b r u s h a c c e s s i b l e p a r t s w i t h a b r u s h .  Since a l l  p a r t s were not a c c e s s i b l e and the m i l k t h e y c a r r i e d was always h o t , the f o r m a t i o n of a c e r t a i n amount of m i l k was  stone  unavoidable. The new p r a c t i c e adopted was t o wash a l l p a r t s  as u s u a l , t h e n f i l l  a l l of the i r o n p i p i n g w i t h a s t r o n g  c a u s t i c soda s o l u t i o n and a l l o w i t t o soak f o r h a l f an hour.  F o l l o w i n g t h i s t h e vacuum pan was p a r t l y f i l l e d  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 a p p r o x i m a t e l y of c h l o r i n e .  with  300 p.p.m.  T h i s was a l l o w e d t o r u n i n t o and t o stand i n  the p i p e s o v e r n i g h t .  I n the morning before., the day's  o p e r a t i o n s were commenced a s o l u t i o n o f " 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 t h e weigh tank and pumped t h r o u g h the whole m i l k l i n e .  The i n s i d e o f t h e  vacuum pans and t h e storage t a n k s were a l s o sprayed "Diversol".  with  S o a k i n g w i t h t r i - s o d i u m phosphate f o l l o w e d by  (24) v i g o r o u s b r u s h i n g removed the m i l k stone from the p r e h e a t e r s . These changes i n c l e a n i n g methods appeared t o h e l p i n r e d u c i n g the c o n t a m i n a t i o n and the numbers of samples showing  heat r e s i s t a n t b a c t e r i a i n our e x a m i n a t i o n s of m i l k a t  v a r i o u s p o i n t s i n the p r o c e s s .  The percentage  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 ,  of f i n i s h e d  however, c o n t i n u e d  to i n c r e a s e . I n the l i g h t  of our p r e s e n t knowledge the  reason  f o r the p e r s i s t e n c e of t h i s c o n t a m i n a t i o n i s easy t o , understand, as we now know t h a t i t was through the raw m i l k .  I n our e a r l y i n v e s t i g a t i o n s , however,  a good d e a l of a t t e n t i o n was possibility  b e i n g added d a i l y  d i r e c t e d t o removing the  o f c o n t a m i n a t i o n : f r o m 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 p l a n t 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 f r o m the vacuum pan t h r o u g h the i r o n p i p e and pumps to the drop t a n k .  Many of these samples appeared  a f t e r the s t e r i l i z i n g p r o c e s s but many o t h e r s c o n t a i n e d heat r e s i s t a n t b a c t e r i a .  sterile  still  These l a t t e r were more  predominant a t the b e g i n n i n g and toward the end o f the run.  On s e v e r a l o c c a s i o n s the f i r s t m i l k r e l e a s e d from  the vacuum pan appeared u n s t e r i l e .  T h i s f i n d i n g was  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 o f these  of some  troublesome  b a c t e r i a remained i n t h e equipment and s u r v i v e d even the s t r o n g 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. the  As a g a l l o n or so o f the f i r s t m i l k t h r o u g h •  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 r e m a i n i n g i n the p i p e s , i t would appear t h a t t h e organisms were e x t r e m e l y r e s i s t a n t t o both t h e s t e r i l i z i n g heat and t h e g e r m i c i d a l a c t i o n o f t h e c h l o r i n e .  I t would  t h e r e f o r e appear t h a t any p r a c t i c a l method o f p l a n t c l e a n s i n g and s t e r i l i z i n g c o u l d not be depended upon t o e f f e c t t h e removal of t h e s e t y p e s o f b a c t e r i a w h i l e t h e y were b e i n g c o n t i n u a l l y r e i n t r o d u c e d t h r o u g h t h e raw m i l k . E x p e r i m e n t s were a l s o c a r r i e d out t o determine t e m p e r a t u r e s and h o l d i n g t i m e s n e c e s s a r y t o 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 s h o u l d be r e c a l l e d a t t h i s p o i n t t h a t t h i s m i l k was  packed  under vacuum w h i c h gave about seven i n c h e s vacuum p r e s s u r e w i t h i n the cans.  The s t e r i l i z i n g had been done u s i n g a  r e l a t i v e l y low temperature of 2S8 t o 230 degrees F. f o r a h o l d i n g p e r i o d o f 25 t o 35 minutes r a t h e r t h a n a h i g h e r temperature f o r a s h o r t e r p e r i o d of t i m e . On November 1 6 t h , 1935, a number o f cans were p r e p a r e d , some' w i t h vacuum and some w i t h o u t and s t e r i l i z e d at  both the h i g h temperature s h o r t time and the low  temperature l o n g time p r o c e s s e s . at  These cans were i n c u b a t e d  55 degrees C. f o r t h r e e days and t h e n p l a t e d w i t h the  following result: 4 cans h e l d 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. f o r 35 minutes, no vacuum - 1 s t e r i l e , 3 u n s t e r i l e . 12 cans held at 241 degrees F. f o r 15 minutes, vacuum packed - a l l u n s t e r i l e . 4 cans held at 241 degrees F. f o r 15 minutes, no vacuum - a l l u n s t e r i l e . 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. sterilized  Twenty four were  at 230 degrees F. f o r 35 minutes and the other  24 at 240 degrees F. f o r 50 minutes.  The cans were  incubated f o r four days at 55 degrees G*  and plated.  The  plates were then incubated f o r 48 hours at the same temperature and then examined.  The results showed:  1. 24 cans s t e r i l i z e d at 230 degrees F. f o r 55 minutes, - 9 s t e r i l e , 15 not s t e r i l e . 2. 24 cans s t e r i l i z e d at 240 degrees F. f o r 30 minutes,-  24 s t e r i l e .  This test showed that while the majority of the cans s t e r i l i z e d  at 230 degrees F. showed the resistant  organisms, thereby indicating t h e i r presence i n the o r i g i n a l milk, the temperature of 240 degrees F. f o r 30 minutes was effective i n destroying them.  At the l a t t e r  temperature the milk i t s e l f , however, was darkened and badly grained, showing the u n s u i t a b i l i t y of this temperature from the standpoint of physical condition.  (27) ALTERING THE SALS BALANCE. When experimental evidence showed the d i f f i c u l t y , i f not the i m p o s s i b i l i t y  of obtaining  effectively  s t e r i l i z e d milk by improving the condition of the plant equipment, an attempt was made to f i n d means of raising the s t e r i l i z i n g temperature. resistant  It was realized  that, i f these  organisms could not be kept out of the milk,  means should be found f o r destroying them during the s t 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 e a r l i e r i n t h i s report the plant practice was to s t e r i l i z e by holding the milk f o r 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. f o r a holding period of from 15 to 16 minutes.  The temperature  range i n s t e r i l i z a t i o n of evaporated milk i s between very narrow l i m i t s .  These l i m i t s are even more r e s t r i c t e d i n  the manufacture of the type of milk with which we are concerned i n t h i s report, v i z . , " B r i t i s h Standard" milk containing, as i t does, about 31-§- percent of t o t a l milk solids.  The ever present problem i s to heat the milk  s u f f i c i e n t l y 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 s t a b i l i t y of milk to heat i s a problem which  (28) has c a l l e d f o r 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 i s one of the most important known phases i n the combination of factors that control the heat s t a b i l i t y of evaporated milk. 2. "The casein i s most stable when i t i s i n combination with a definite amount of calcium. Excess or deficiency of calcium available f o r the oasein-oalcium combination lowers the heat s t 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 c i t r a t e s and phosphates which represent the negative charges. The amount of calcium combination i s determined by the balance between the calcium and magnesium group and the c i t r a t e and phosphate group of s a l t s . An excess or deficiency of either group tends to unstabilize the casein. .5. "In the absence of the proper balance of the s a l t s i n 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 i n which the milk i s d e f i c i e n t . In the great majority of cases of heat coagulation d i f f i c u l t i e s , the trouble i s due to an excess of calcium (deficiency of c i t r a t e s and phosphates). Such milk i s s t a b i l i z e d by the addition of a small amount of sodium c i t r a t e or di-sodium phosphate. If the low heat s t a b i l i t y i s due to a deficiency of calcium (excess of c i t r a t e s 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 f o r the correction of the salt balance are most effective when these s a l t s , or a considerable portion thereof are  (29) placed i n the milk before condensing, 5. "The effect of the salt balance on heat s t a b i l i t y varies with many other factors, such as acid reaction, the presence of fermentation products other than acid, albumen and casein content, forewarming temperature, concentrations of solids not f a t , etc. In other .-words, as expressed by Benton and Albery, each l o t of milk must be regarded as a separate c o l l o i d a l system with i t s optimum combination of acid reaction and salt balance, f o r 'maximum heat s t a b i l i t y . ,6. "Because of these f a c t s , heat s t a b i l i t y ' tests of the fresh milk, such as the alcohol t e s t , the phosphate test, etc., do not . always furnish a dependable index to the heat s t a b i l i t y of the evaporated milk. 7, "The above of the use systematic evaporated  observations emphasize the value of the p i l o t s t e r i l i z e r f o r control of the behavior of the milk i n the s t e r i l i z e r . "  Experiments, with a view to r a i s i n g 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 i n 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. f o r  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 i n 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 c i t r a t e respectively.  This milk already contained the 1-|-  ounces per thousand of sodium bicarbonate which had been added i n the hot wells before condensing.  The cans were  sealed and s t e r i l i z e d at 232 degrees F. f o r 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 s i x ounces of sodium c i t r a t e . -Further tests f a i l e d to confirm t h i s , however, and after several t r i a l s i n 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 i n e f f e c t i v e i n destroying the troublesome organisms, i t did not seem profitable to pursue t h i s l i n e of investigation further.  Sufficient work on t h i s phase was not done,  however, to warrant any f i n a l conclusions i n the matter and should be carried out further at some future date using these s a l t s and also the calcium s a l t . 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 i n  TABLE, NO .7. EFFECT OF ALTERING THE SALT BALANCE• Sample Number  Salt Concentration  Condition of milk after s t e r i l i z i n g .  A-l  1 o.e. s t e r i l e water only added  Heavy grain  A-2  2 oz. dry di-sodium phosphate per 1000 l b s . milk  Heavy grain  A-3  3 oz. dry di-sodium phosphate per 1000 l b s . milk  Slight grain  A-4  6 oz. dry di-sodium phosphate per 1000 l b s . milk  Slight grain  A-5  8 oz. dry di-sodium phosphate per 1000 l b s . milk  30 second shake  A-6  10 oz. dry di-sodium phosphate per 1000 l b s . milk  30 second shake  B-l  1 c.o. s t e r i l e water only  Heavy grain  B-2  2 oz. dry sodium c i t r a t e per 1000 l b s . milk  Heavy grain  B-3  4 oz. dry sodium c i t r a t e per 1000 l b s . milk  Heavy grain  B-4  6 oz. dry sodium c i t r a t e per 1000 l b s . milk  30 second shake  B-5  8 oz. dry sodium c i t r a t e per 1000 l b s . milk  1 minute shake  B-6  10 oz. dry sodium c i t r a t e per 1000 l b s . milk  l-J- minute shake  (51) heat s t a b i l i t y .  The high albumen content of milk  experienced at the beginning and end of the l a c t a t i o n 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 p r e c i p i t a t i n g excess albumen. A  temperature of over 200 degrees P. and holding as long as possible i n the hot wells was recommended as e f f e c t i v e .  As  i t was already the plant practice i n the factory concerned to preheat the milk,to a temperature  of 210 degrees P.  while i n the hot wells i t was not f e l t that much improvement could be effected here.  REPRODUCING THE SPOILAGE. As mentioned  e a r l i e r i n t h i s report, p r a c t i c a l l y  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 t h i s sugar. which presented i t s e l f then was:  The question  Would these apparently  non-lactose fermenters grow i n the evaporated milk and bring about a condition of spoilage?  To obtain an answer  to t h i s disturbing question and to observe the keeping q u a l i t i e s 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 i n the 55 degrees 0. inoubator and  (32) also i n the 37 degrees G. incubator.  Observations were  made from time to time by taking some of the cans from the incubator, plating them i n the manner previously described and then opening the cans and noting the physical condition of the contents,.: as well as t e s t i n g the milk f o r a c i d i t y . 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 i n the 55 degrees 0. incubator.  At intervals, s i x  of these cans were taken out and examined. these examinations are shown i n Table No.8.  The results of After one  week the condition of the milk i n f i v e of the cans was normal with one can appearing p a r t i a l l y c l o t t e d .  The  a c i d i t y i n most cans had increased s l i g h t l y while that of the clotted can had increased considerably.  Plates made  from a l l cans appeared unsterile and showed the t y p i c a l alkaline forming colonies.  After two weeks' incubation  a l l oans examined were c l o t t e d , the two showing a f i r m c l o t , also showed the greatest acid development.  Four of  the cans appeared unsterile with t y p i c a l colonies while the two with the f i r m clot and high a c i d i t y showed no grov/th of l i v i n g organisms, indicating the p o s s i b i l i t y of their having been k i l l e d o f f by the acid produced.  After  twenty-four days' incubation a l l s i x cans were clotted with considerable acid production and a l l appeared s t e r i l e on  TABLE NO. 8. SPOILAGE EXPERIMENT. Experiment on 24 cans of milk from batch 782-L, reported u n s t e r i l e . Cans incubated at 55 degrees G., Jan.3,1934, DATE OP EXAMINATION. ... Jan. '10th, " after 7 days' incubation -  CONDITION OF MILK 1. Normal Normal O « Normal 4. Normal 5. Normal 6. P a r t i a l  Jan. 17th, after.14 days' incubation  1» 2. 3. 4. 5. 6.  Jan. 27th, after 24 days' incubation  1 ® Hard 2. Hard 3. Weak, 4. Weak 5. Weak 6. Weak  Peb. 5th, after 33 days' incubation  soft Soft Hard Hard Soft Soft  SIEERILITY  .55$ .72 .62 . 65 .75 .89  Unsterile  clot clot clot clot clot Clot  .70$ . 82 .90 . 81 .63 .59  Unsterile  clot Clot clot clot clot clot  .85$ .90 .75 .90 .82 .80  Sterile  X « Clotted 2 d Clotted  3. 4. 5. 6.  ACIDITY .  Clotted Clotted Clotted Clotted  clot  __ __ __ „_ ——  M  II  H I!  rt I!  Sterile Sterile Unsterile Unsterile II  II II II II  Unsterile Sterile II  II  ti  (33) plating.  The r e m a i n i n g s i x cans were h e l d f o r a p e r i o d o f  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 t o have no l i v i n g organisms r e m a i n i n g . The v e r y pronounced tendency o f t h e c l o t t e d milk, t o show no l i v i n g organisms would account f o r our i n a b i l i t y t o i s o l a t e any b a c t e r i a f r o m cans r e t u r n e d -when the o r i g i n a l outbreak o f s p o i l a g e was r e p o r t e d .  The f a c t was  a l s o demonstrated t h a t t h e s e a p p a r e n t l y n o n - l a c t o s e f e r m e n t i n g b a c t e r i a were c a p a b l e o f p r o d u c i n g a c o n d i t i o n of s p o i l a g e i n t h e m i l k i f s t o r e d ' a t a f a v o r a b l e temperature f o r t h e i r growth. C o n c u r r e n t l y w i t h t h i s experiment t w e n t y - f o u r oans from t h e same b a t c h were p l a c e d i n t h e 37 degrees 0. i n c u b a t o r and p e r i o d i c e x a m i n a t i o n s o f them made.  The  r e s u l t s of f i n d i n g s are r e p o r t e d 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 t h e p h y s i c a l c o n d i t i o n o f t h e m i l k and v e r y l i t t l e change i n t h e t i t r a t a b l e a c i d i t y .  Many o f t h e  samples appeared u n s t e r i l e showing t h e presence o f the typical colonies.  A f t e r f i f t y - e i g h t days two samples  showed an i n c r e a s e i n a c i d p r o d u c t i o n and t h r e e o f 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 o f them s t i l l  showed t h e presence o f l i v i n g organisms.  The r e s u l t s  o b t a i n e d w i t h t h e m i l k h e l d a t the l o w e r temperature o f 37 degrees 0. was i n sharp c o n t r a s t t o those found w i t h  TABLE NO.9. SPOILAGE EXPERIMENT. Experiments on 24 cans from batch 782-L, reported u n s t e r i l e . Incubated at 37 degrees 0., Jan.3,1934 BATE OF EXAMINATION  CONDITION OF - MILK  ACIDITY  - STERILITY  Jan. 10th, after 7 days' incubation  1. Normal 2. Normal 3. Normal  .53$ . 54 • 55  Sterile Unsterile Unsterile  Jan. 17th, after 14 days' incubation  1. Normal 2. Normal 5 e Normal  .55$ . 54 .50  Sterile Sterile Unsterile  Jan. 27th, after 24 days' incubation  1. Normal 2. Normal 3. Normal  . 59$ .60 .60  Unsterile Sterile Sterile  Feb. 5th, after 35 days' incubation  1. Normal £. Normal 3. Normal 4. Normal 5, Normal 6« Normal  .53$ .59 .54 . 55 .60 .57  Sterile Unsterile Sterile Sterile Sterile Sterile  March 2nd, after 58 days' incubation  X« P a r t i a l clot 2. Normal 5. Normal 4. P a r t i a l clot 5. Normal 6. Normal 7. Normal 8 • Normal 9. Soft clot  .76 .60 .52 .64 .53 .58 .55 © 55 .95  Unsterile Unsterile Sterile Unsterile Sterile Unsterile Sterile Unsterile Unsterile  (34) milk, h e l d at 55 degrees 0.  I n the l a t t e r a c i d p r o d u c t i o n  s t a r t e d e a r l y and the m i l k was soon c l o t t e d w h i l e 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 m i l k . I t would appear t h a t m i l k h e l d at 37 degrees 0. or l e s s would e x p e r i e n c e l i t t l e resistant  i f any s p o i l a g e by these heat  bacteria. The e x p e r i m e n t s w i t h t h e s e two s e t s o f cans of  e v a p o r a t e d m i l k v e r i f i e d what had a c t u a l l y t a k e n p l a c e i n the commercial h a n d l i n g of the m i l k .  S p o i l a g e had been  r e p o r t e d f r o m 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. w h i l e no s p o i l a g e  whatever  had been r e p o r t e d f r o m 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 t e m p e r a t u r e s p r e v a i l at a l l seasons of the y e a r . A second experiment w i t h the f i n i s h e d m i l k  was  commenced on A p r i l 4 t h , 1934, the r e s u l t s of which are shown i n Table No.10.  T h i s time e i g h t e e n samples f r o m  B a t c h 924-K, r e p o r t e d u n s t e r i l e at both 55 degrees 0. and 37 degrees 0., and an e q u a l number of samples f r o m B a t c h 930-M, r e p o r t e d s t e r i l e at b o t h t e m p e r a t u r e s , were i n c u b a t e d at 55 degrees 0.  I t was considered!-': d e s i r a b l e  t o compare t h e k e e p i n g q u a l i t i e s of m i l k r e p o r t e d w i t h t h a t shown t o be u n s t e r i l e .  sterile  A f t e r one week's  i n 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 .  After  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 " S t e r i l e " at both 55 degrees 0. and 57 degrees C. A l l cans were incubated at 55 degrees 0. on A p r i l 4th,1934« DATE OF EXAMINATION A p r i l 11, after 1 week incubation  CONDITION OF MILK 924-K  930-M  A p r i l 19th, after 15 days' incubation  924-K  930-M  A o r i l 27th, after 23 days' incubation  924-K  930-M  ACIDITY  STERILITY  -  Sterile " "  -  " Sterile " " " " "  1 2 3 4 5 6 1 2 3 4 5 6  Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal  1 2 3 4 5 6 1 2 3 4 5 6  Lumpy Normal Normal Normal Lumpy Lumpy Normal Normal Normal Normal Soft Clot Normal  .64$ .54 .52 .52 .53 .55 .55 .51 .47 .50 .73 .51  Sterile " " " " " Sterile " " " "  1 2 3 4 5 6 1 2 3 4 5 6  Clotted soft Clot Lumpy Normal Normal Normal Normal Normal Normal Normal Normal Normal  .58 .64 .61  Sterile "  ' .56 -  a  11  " Sterile " " " "  (35) two weeks the milk i n three of the 924-K samples appeared s l i g h t l y lumpy, but with l i t t l e change i n the a c i d i t y and apparently no l i v i n g organisms.  The s i x cans of 930-M  showed only one with any sign of spoilage, the rest being unchanged i n a c i d i t y and appearing s t 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 i n every respect.  In t h i s  experiment changes were not brought about as quickly as i n the test reported above or were they as pronounced.  Although  a few of the cans clotted there were no marked increases i n a c i d i t y , while plates showed no colonies i n a single instance.  Evidently any bacteria which had been present i n  924-Z had died out or were too inactive to show growth. The test, however, demonstrated the superior keeping q u a l i t i e s of milk appearing s t e r i l e i n 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 i n cans, twelve samples of batch No.767-B were taken.  This milk had been manufactured i n October 1933,  having been stored f o r about s i x months. test i t had shown an unsterile condition.  In the o r i g i n a l These cans were  incubated at 55 degrees 0. on A p r i l 27th, 1934, and subsequently examined i n the usual way after ten and  (36) twenty-one days' incubation. are  The results of the examinations  shown i n 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 s i x cans examined were a l l d e f i n i t e l y clotted, four showing the presence of organisms and two being apparently sterile. This test demonstrated that some of these heat resistant organisms are capable of remaining alive i n the milk f o r several months and when conditions of temperature become favorable are capable of s p o i l i n g 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 r e s t e r i l i z a t i o n , Batch 174-D, reported unsterile at both 37 degrees G. and 55 degrees C. and Batch 185-B, reported s t e r i l e at both temperatures. samples of 155-J had been packed f o r about a month, those of 174-D were packed i n January 1935, when such a large percentage of a l l samples appeared unsterile i n 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 f o r the manufacture  of t h i s particular standard of milk.  results of t h i s series are shown i n Table No.12.  The  s  The  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 i n the i n t e r v a l were incubated at 55 degrees G. on A p r i l 27th, 1934. BATE OP EXAMINATION May 6th, after 10 days' incubation  May 18th, after 21 days' incubation  CONDITION OF MILK  ACIDITY  767-B 1 Soft Clot 2  "  "  3  it  n  4  ti  ii  5  ii  ti  6  "  "  767-B 1 Clotted  STERILITY Unsterile  ,68'$'  2  Sterile Unsterile n  3  "  4  •"•  Sterile  5  '•  Unsterile  6  "  .69$  Unsterile  TABLE NO.12 SPOILAGE EXPERIMENT 24 samples each of Baton. No.l55~J, reported "Unsterile" after r e s t e r i l i z a t i o n , Batch No. 174-D, reported "Unsterile" at both 55 degrees 0. and 57 degrees 0., and Batch No. 185-B reported " S t e r i l e " at both 55 degrees 0. and 37 degrees C , were incubated at 55 degrees C. on February 8th, 1935. LATE OF CONDITION EXAMINATION. - ..' -OF: MILE ..... Feb. 11. after 72 hours' incubation  Feb. 15th. After 1 week's incubation  ACIDITY  REACTION STERILITY . OF. MEDIUM  155-• J 1 2 174-•D 1 2 185-•B 1 2  Normal Normal Normal •Normal .Normal Normal  .51$ .51 .51 .51 .47 .47  Unsterile  Alk.  Unsterile  Alk.  155- J  Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal  .58 .55 .56 • 52 .66 .55 .54 .56 .52 .50 .50 .60  Sterile Unsterile  Soft Clot Normal Normal Soft Olot Normal Normal Normal Normal Soft Clot Normal Normal V.S.Clot  .80 . 56 .54 .65 .59 .58 .58 .57 .65 .55 .52 ,61  Soft Olot Normal V.S.Clot Y.S.Clot  .75 .63 .65 .63  1 2 3 4 174-•D 1 2 3 4 185-•B 1 2 3 4  Feb. 22nd. 155- J After two we eks' incubation. 174-D  1 2 3 4 1 2 3 4 185-B T A. 2 3 4  Mar. 1st. 155- 3 After three weeks' incubation.  1 2 3 4  it II  II  IT  Sterile II  II  n  Unsterile ti  Alk. IT  Acid Alk. n  II  IT  II  IT  Unsterile n II  it Sterile Unsterile IT II  Unsterile TT  tl Sterile Unsterile II  Acid it IT  Alk. Alk. IT  II  Alk. IT II  Alk. II  IT  II  11  IT  Sterile Unsterile Sterile Sterile  (Continued on next page)  Alk.  TABLE NO.12, (Continued) DATE OF EXAMINATION Mar oil 1st (Cont'd)  CONDITION OF MILK  174 -D 1 Normal 2 Normal 5 Normal 4 Normal 185 -B 1 Normal 2 Normal 3 V.S.Clot 4.Soft Clot  155 - J Mar.8th. After four weeks' incubation. . 174-D  Mar.15th. After f i v e weeks' incubation  ,  REACTION ACIDITY . . STERILITY ..OF MEDIUM .59 .59 .59 .60 .60 .67 .70 .82  Sterile Unsterile Sterile Sterile Sterile Unsterile Sterile Sterile Sterile  1 2 3 4 1 2 3 4 185 -B 1 2 3 4  Hard Clot Soft Clot Soft Clot Soft Clot Normal Normal Normal Normal Normal Normal Firm Clot Normal  .84 .65 . 66 .68 .60 .59 .60 .61 . 59 e 61 .84 .77  155--J 1 2 3 4 5 6 174--D 1 2 3 4 6 185--B 1 2 3 4 5 6  S.Thick. S.Thick. Normal 7.S.Thick. Soft.Clot Soft Clot Normal Normal Normal Normal Normal Normal Normal Soft Clot Normal Soft Clot Normal  .75 .64 • 66 .75 .73 .76 .60 .64  . 68 .65 . 66 .68 »65 ,80 .68 .76 .60  Alk  Alk  IT  ti  Unsterile Sterile  Alk  IT !T  Sterile ti  Unsterile n  Unsterile Sterile  Alk II  Alk  IT  -IT  II 11 Unsterile n  IT  Alk IT  11  II  IT  Sterile  II  ii  1! IT 11 11 II  (37) E x a m i n a t i o n s o f these cans a f t e r v a r i o u s p e r i o d s of  i n c u b a t i o n showed those o f No. 1 5 5 - J t o be normal i n  c o n d i t i o n d u r i n g t h e f i r s t week w i t h t h e presence of the typical colonies.  D u r i n g t h e second week c l o t t i n g had  commenced and t h i s i n c r e a s e d d u r i n g t h e t h i r d , f o u r t h and f i f t h weeks.  As t h e p e r i o d o f i n c u b a t i o n i n c r e a s e d t h e  number showing t h e presence o f organisms decreased u n t i l a t the end o f the f i f t h week o n l y one out of s i x samples showed these a l k a l i f o r m i n g b a c t e r i a .  The samples of 174-D  m a i n t a i n e d 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 a l t h o u g h t h e m a j o r i t y o f t h e samples showed t h e presence o f l a r g e numbers o f organisms.  Those o f 185-B  a c t e d s i m i l a r l y t o 1 5 5 - J a l t h o u g h not t o such a  pronounced  extent. While t h e r e s u l t s o f t h i s experiment were not so c l e a r c u t as some o f those p r e c e d i n g i t , n e v e r t h e l e s s the tendency f o r these a p p a r e n t l y a l k a l i n e f o r m i n g b a c t e r i a t o s p o i l e v a p o r a t e d m i l k was a g a i n demonstrated.  The f a c t t h a t  some o f both t h e samples a p p e a r i n g s t e r i l e i n r o u t i n e e x a m i n a t i o n and those a p p e a r i n g 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 t h e absence o r non a c t i v i t y o f these b a c t e r i a i n some o f t h e cans from each s t e r i l i z e r b a t c h .  The  b e h a v i o u r of cans o f b a t c h No.174-D i n d i c a t e d t h a t some b a c t e r i a s u r v i v i n g t h e s t e r i l i z i n g p r o c e s s have l i t t l e or  (38) no a c t i o n i n t h e 'milk. From t h e above f o u r experiments t h e c o n c l u s i o n can s a f e l y be drawn t h a t t h e 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 o f s p o i l a g e  i n evaporated m i l k i f stored  at s u i t a b l e t e m p e r a t u r e s and f o r a s u f f i c i e n t l y l o n g t i m e . The c h e m i c a l changes i n v o l v e d have not been d e t e r m i n e d . However, we have n o t i c e d i n t h e c l a s s i f i c a t i o n o f organisms i s o l a t e d t h a t p r a c t i c a l l y a l l produce a c i d on g l u c o s e 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  p r o c e s s e s or due t o the a c t i o n o f b a c t e r i a l enzymes some of t h e l a c t o s e i s broken down t o g l u c o s e ' a n d g a l a c t o s e , and t h i s i s used by t h e b a c t e r i a t o produce s u f f i c i e n t a c i d t o result i n a condition of clotting. A f u r t h e r p o s s i b l e cause o f t h e s p o i l a g e e x p e r i e n c e d i n m i l k 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 f e r m e n t i n g organisms, has r e c e n t l y come t o l i g h t . As mentioned e a r l i e r i n t h i s r e p o r t t h e number o f samples a p p e a r i n g 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 v e r y markedly a f t e r raw m i l 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 w i t h h e l d  f r o m manufacture o f t h i s m i l k .  samples, however, s t i l l  appeared.  Some u n s t e r i l e  The m a j o r i t y o f these  d i d not c a r r y t h e 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 o f c o l o n y was n o t i c e d .  These l a t t e r gave l i t t l e  o r no evidence o f  (39) change i n the reaction of the media as indicated by the color.  They did, however, produce a d i s t i n c t l y acid smell,  showing some production of acid from the lactose. lactose fermenting  These  colonies were present on the plates i n  small numbers only.  This would point to the fact that  they did not grow vigorously i n the milk incubated at 55 degrees G. incubated  None were noticed i n plates from milk  at 37 degrees C. The p o s s i b i l i t y now  forming organisms may  i s suggested that these acid  have been present i n the milk before,  but were obscured i n the plates by the, very growth of the alkaline forming bacteria.  vigorous  If present, i t  i s possible that after prolonged periods of incubation they would have produced s u f f i c i e n t acid to cause coagulation.  This p o s s i b i l i t y i s strengthened by the fact  that i n our spoilage experiments the milk,in many cases, did not coagulate, u n t i l after one or two weeks' incubation. While further work w i l l have to be done on these organisms, i t i s nevertheless the opinion of the authors that although these lactose fermenters may  be contributory factors they  cannot account f o r a l l spoilage experienced,  as experiments  described below w i l l show that organisms, d e f i n i t e l y 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 s t e r i l e evaporated milk of pure cultures of organisms isolated from routine plates during the course of t h i s investigation*  The  cultures used were 762-L, the type culture of a group of strong lactose fermenters which has been c l a s s i f i e d as a s a l i c i n fermenting s t r a i n of B. ooagulans (Hammer), 786B2, the type culture of a group of non-lactose but strong mannite fermenters, which has been c l a s s i f i e d as B. calidus, (,Blau. ), and 207, the type culture of a group which show p r a o t i c a l l y no action i n carbohydrate media but which c l o t and digest litmus milk with a s l i g h t l y alkaline clot and to which has been given the name B. thermoalkalans» A s t e r i l e 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 s t e r i l e at both 37 degrees 0. and 55 degrees 0. were chosen f o r t h i s experiment. i n the following manner*  The milk was innoculated  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 i n the can by means of a s t e r i l i z e d ice pick.  One cubic centimeter of the b a c t e r i a l suspension  was run into the can and the hole then sealed with a soldering iron.  Bach can and contents was then shaken Well.  One  cubic centimeter of s t e r i l e ?/ater was added to each of the six control cans i n the manner described above.  Six cans  were thus innoculated with each of the bacterial suspensions.  After re-sealing, a l l cans were placed i n  the incubator and held at 55 degrees 0.  Periodic  examinations of the contents were made, the results of which appear i n Table No.13. From t h i s table i t w i l l be noted that the milk i n the control cans appeared normal i n s t e r i l i t y , physical condition and a c i d i t y f o r the whole two weeks of incubation. Those cans containing the innoculation of B. coagulans were clotted with some production of acid within seventytwo hours and showed a very hard clot with high acid production at the end of the one and two week intervals. The s i x cans containing, the 3. .calidus^Blauy,. remained normal i n physical condition and a c i d i t y throughout, while those containing the B. thermo-alkalans were not thickened at seventy-two hours but had developed a cheesy smell. one week these l a t t e r showed a soft c l o t t i n g with slight  After  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.  Control  762-L  207  786B2  Mixture  A f t e r two weeks' i n c u b a t i o n a t 55° C.  Control 762-L 207  762-L  B. coagulans B. t h e r m o a l k a l a n s  207  786B2 786B2 Mixture  Mixture  B. c a l i d u s 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  Feb. 11th. Control 1 Normal .51 After 2 Normal .51 72 hours' 762-L 1 Clotted .86 incubation. 2 Clotted .84 786B2 1 Normal .51 .2 Normal .51 207 1 Normal with cheesy small .60 . 60 2 do. Mixture 1 Clotted with cheesy smell .92 .92 2 do. Feb. 15th. After one week's incubation  Control 1 2 762-L 1 2 786B2 1 2 207 1 2 Mixture 1 2  Normal Normal Hard Clot Hard Clot Normal Normal Weak Clot Weak Clot Hard Clot Hard Clot  .51 .52 1.12 1.1 .56 .52 .71 .68 1.26 1.26  Feb. 22nd. Control 1 Normal .55 After two 2 Normal .57 weeks' 762-L 1 Hard Clot . incubation. 2 Hard Clot 786B2 1 Normal .55 2 1 2 Mixture 1 207  Normal• .56 Soft Clot .70 Soft Clot .69 Hard Clot cheesy smell 2 do.  STERILITY Sterile Sterile Unsterile Unsterile Unsterile Unsterile  REACTION OF .MEDIUM  Unsterile Unsterile  Acid Acid Alk. Alk. Alk. Alk. Alk.Acid Acid  Sterile Sterile Unsterile Unsterile Unsterile Unsterile Unsterile Unsterile Unsterile Unsterile  Acid Acid Alk. Alk. Alk. Alk. Alk. Alk.  Unsterile Unsterile  Sterile Sterile Unsterile Acid (few colonies) do. Acid Unsterile Alk. (few colonies) do. Alk. Unsterile Alk. Unsterile Alk. Unsterile Alk. Unsterile  Alk.  (42) productions of acid and at two weeks a definite soft clot with, no further increase i n a c i d i t y .  The cans containing  the mixed culture showed similar results to those with B. coagulans except that the milk had a cheesy smell i n addition to the hard acid c l o t .  The control cans were  s t e r i l e at a l l examinations whereas a l l innoculated cans showed the presence, i n 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 t h i s experiment these type cultures gave reactions similar to those experienced i n 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 i n the pure culture study did not ferment lactose but did clot milk, l i q u e f i e d gelatine and reduced n i t r a t e s , clotted the evaporated milk and also decomposed the proteins to some extent as evidenced by the strong cheesy odor produced. The results of t h i s 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 i n evaporated milk.  Their action was hastened by the  s u i t a b i l i t y of the temperature employed and the large number of organisms contained i n the innoculum.  The results  obtained with B. oalidus,1Blau_ give additional evidence t  that some of the surviving organisms are incapable of s p o i l i n g the milk, a fact already demonstrated by experiments with cans from batch number 174-D. are  As organisms  not l i k e l y to exist i n pure culture i n commercial  evaporated milk the drastic effect of the mixture of cultures here employed i s of considerable i n t e r e s t . CLASSIFICATION OF THE ORGANISMS ISOLATED. Methods Used. As a c l a s s i f i c a t i o n  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 i s then tubed and plugged,  10 c.o. i n each test tube, and s t e r i l i z e d at twelve pounds pressure' f o r twenty minutes.  After s t e r i l i z a t i o n each tube  i s innoculated with the desired culture.  Uniformly a 2 m.rn.  loop innoculation from a vigorous growth i n milk or casein  (44) digest broth i s made.  After innocillation, the series  with controls i s incabated f o r fourteen days at the appropriate temperature.  In t h i s case, seven days'  incubation and 55 degrees C. were used.  A preliminary  test showed that the maximum t o t a l t i t r a t a b l e a c i d i t y was reached before seven days.  When incubation i s completed,  the cultures are t i t r a t e d with N/4 Sodium hydroxide, using phenolphthalein as indicator, the t i t r a t i o n of the controls deducted, and the results worked out and recorded as grams of l a c t i c acid per m i l l e . P. S. Prickett (17) noticed a certain v a r i a b i l i t y among the thermophiles i n the production of acid from carbohydrates.  Therefore, when placing organisms together  i n groups, only a s l i g h t significance was given to small variations i n the fermentation of sugars.  Rather, the sum  of the c h a r a c t e r i s t i c s of the organisms was used, following the principle enunciated by S. Orla-Jensen. Glassification. Typical colonies of bacteria which appeared with the greatest frequency i n routine examination of condensed milk, during the year of 1954, were pioked from Purple Lactose Agar plates and retained f o r study.  Cultures were  also picked from plates prepared from other sources during the course of t h i s 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 c l a s s i f i c a t i o n . A l l of the forty-one organisms are aerobic, spore forming, gram positive rods, varying i n length from two to seven microns. out  In old agar cultures the c e l l s tend to grow  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 i n milk the c e l l s occur f o r the most part singly, or i n very short chains. are  motile.  slants.  Most of the organisms  A l l of the organisms grow well on agar  Some l i q u e f y 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. f o r 30 minutes. The detailed description of the organisms i s found on Tables No.14  and No.15. The morphology and c u l t u r a l characteristics stated  above suggest that a l l the organisms isolated be placed i n the family Bacillaceae, genus B a c i l l u s . - (14) The forty-one organisms may be divided into two main groups:  lactose fermenting and non lactose fermenting  strains. Group I. - Lactose Fermenting.' Only s i x 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 i n the drop tank i n the Plant. These strains are motile, gram positive rods, with terminal spores.  They do not liquefy gelatine, form a clean  hard, acid clot i n milk and reduce litmus i n 48 hours at 55 degrees 0.  Nitrates are not reduced and the cultures do  not produce i n d o l . hydrates.  Gas i s not formed i n any of the carbo-  Using Casein Digest Broth (16) as a nitrogen  source, laevulose, dextrose, mannose, galactose, saccharose, maltose, laotose, dextrin and starch are fermented. It i s 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 i n u l i n and i n some cases an alkaline reaction i s produced. S a l i c i n i s fermented by cultures 762-L and 795-Q, but i s not fermented by the other cultures. These organisms seem to bear a marked resemblance to Hammer's B a c i l l u s coagulans. and on the sum of the c h a r a c t e r i s t i c s , cultures 786-BLa, 786-BL, D.T.I and D.T.'S are c l a s s i f i e d as B a c i l l u s ooagulans (Hammer) (8) (14). Cultures 762-L and 795-Q are t e n t a t i v e l y c l a s s i f i e d as s a l i c i n fermenting strains of B. coagulansi (Hammer) (8) (14). Group I I . - Non Lactose Fermenting. T h i r t y - f i v e 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 H i e organisms, and as seen from Table No«14, are i s o l a t e d from a variety of sources. They are a l l strong mannite fermenters.  The 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 of the above cultures, bear a marked resemblance to B a c i l l u s calidus (Blau) (14) (see Table No.14  and No»15)'.  They are gram p o s i t i v e , motile rods, with terminal elongated spores.  They do not l i q u e f y gelatine, do not reduce  n i t r a t e s or produce indole  They produce an a l k a l i n e reaction  i n milk, but do not peptonize or c l o t i t .  In t h i s respect  they d i f f e r from B. calidus (Blau), as the l a t t e r peptonizes and clots milk. In Casein Digest Broth a l l cultures ferment mannite, laevulose, dextrose and maltose.  Mannose i s  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, i n u l i n , dextrin, starch and s a l i c i n are not fermented by any of the cultures (Table No»15). their i n a b i l i t y to ferment saccharose, they d i f f e r from the s t r a i n of B. calidus described by P. S. Prickett (17)  In  (48) as the l a t t e r ferments saccharose.  No tw i t 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 c l a s s i f i e d as strains of  B a c i l l u s ,oalidus ( Blau ) (14). It i s interesting to note, that i f t h i s group of organisms were not spore formers, they could have been d e f i n i t e l y . c l a s s i f i e d as Thermobacterium cereale, (OrlaJensen) (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 i n t h e i r morphology and c u l t u r a l characteristics to B a c i l l u s pepo (Shaw) (14) (Table No.14  and No.15).  They are gram positive non-motile rods, with elongated terminal spores.  They l i q u e f y gelatine at 20 degrees  and at 55 degrees 0.  The l i q u e f a c t i o n becomes saccate  with slight production of scum. completed  i n fourteen days.  n i t r i t e s and ammonia.  0.  It i s f a i r l y rapid and i s  Nitrates are reduced to  Litmus milk clots and starts to  peptonize on the t h i r d 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 . ferment lactose i n u l i n and dextrin.  They do not  No.236 d i f f e r s from  B. pepo i n i t s i n a b i l i t y to ferment s a l i c i n , while VII-D  (49) d i f f e r s by i t s i n a b i l i t y to ferment galactose. Notwithstanding these variations, on the sum of their c h a r a c t e r i s t i c s cultures No.236 and VII-D are t e n t a t i v e l y c l a s s i f i e d as strains of B a c i l l u s 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  i n t h e i r 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 B a c i l l u s armarus (Hammer) (14). They are gram positive motile rods with terminal spores.  They do not l i q u e f y gelatine and do not reduce  n i t r a t e s . -Litmus milk i s not changed i n any way.  There i s  a marked v a r i a t i o n i n the sugar fermenting a b i l i t i e s of these organisms. dextrose.  A l l of the cultures ferment laevulose and  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 f i v e cultures possess the a b i l i t y to ferment t h i s carbohydrate. Galactose i s 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 i s s l i g h t l y fermented by 144-F37,  138-0, 782-M3, 795-J, XI-Hunt and 795-G. XI-Hunt, ferments s a l i c i n .  Only one culture,  In many of the carbohydrates an  alkaline reaction i s produced.  (50) In s p i t e o f t h e s e 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) ( 1 4 ) . 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  Bacillus  thermoindifferens (Weinzirl) (14). They a r e m o t i l e , terminal,  elongated spores.  gram p o s i t i v e r o d s , w i t h They do not reduce n i t r a t e s .  L i t m u s m i l k i s l e f t unchanged. gelatine  They do n o t l i q u e f y  and i n t h i s r e s p e c t d i f f e r f r o m B. t h e r m o i n d i f f e r e n s .  Both c u l t u r e s  ferment g l u c o s e and show a s l i g h t  of f e r m e n t i n g s t a r c h . ferment l a e v u l o s e .  Hay I i s , i n a d d i t i o n ,  indication  able t o  None o f the o t h e r c a r b o h y d r a t e s 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 o f B a c i l l u s , t h e r m o i n d i f f erens (Weinzirl) (14). 5.  CULTURES  No.31, Truck V I and Truck IV resemble on the whole B a c i l l u s t h e r m o a l i m e n t o p h i l u s  (Weinzirl) (14). They are gram p o s i t i v e , m o t i l e r o d s , w i t h t e r m i n a l elongated spores. or change l i t m u s m i l k .  They do not l i q u e f y  gelatine  They reduce n i t r a t e s t o n i t r i t e s .  As a whole t h e y show no change i n carbohydrate media, but C u l t u r e Truck I V f e r m e n t s mannose and g a l a c t o s e  slightly,  (51) and No.31  mannoae only. Cultures No.31, Truck VI and Truck IV are  t e n t a t i v e l y c l a s s i f i e d as strains of B a c i l l u s thermoalimentophilus (Wenzirl) (14). 6.  CULTURE 161 resembles Bacillus, t r i t u s (Batchelor). It i s a gram positive motile rod, with rounded  ends and terminal spores.  Litmus milk i s not changed,  n i t r a t e s are not reduced, gelatine i s not l i q u e f i e d and carbohydrates are not  fermented.  The optimum temperature,  however, i s higher than  that f o r B. t r i t u s , so Culture 161 i s considered as a s t r a i n of B a c i l l u s t r i t u s (Batchelor) (14). 7.  CULTURES 207, Truck V and Hunt XI could not be c l a s s i f i e d 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 s t a r l i k e or exploded appearance and a strong alkaline reaction i s produced.  Turbidity and a  pellieire are produced i n nutrient broth.  Litmus milk i s  reduced and an alkaline c l o t i s formed, which l a t e r becomes peptonized.  Gelatine Is l i q u e f i e d quickly and completely  at 55 degrees 0. and not at 20 degrees C. reduced to n i t r i t e s .  Nitrates are  No acid i s formed but an alkaline  reaction i s produced i n a l l of the carbohydrate media  (52) employed.  F o r d e t a i l e d m o r p h o l o g i c a l 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 T a b l e s No.14 and No.15. I t i s b e l i e v e d t h a t these a r e u n d e s c r i b e d organisms.  I s t h e y occur f r e q u e n t l y i n r o u t i n e p l a t e s and  present p e c u l i a r i t i e s o f some i n t e r e s t , i t i s proposed t h a t the name o f B a c i l l u s t h e r m o a l k a l a n s be g i v e n . t o them.  SUMMARY. T h i s i n v e s t i g a t i o n was c a r r i e d out t o determine the causes o f s p o i l a g e i n e v a p o r a t e d m i l k i n t r o p i c a l c o u n t r i e s d u r i n g the s p r i n g and summer o f 1935.  The m i l k  concerned was o f " B r i t i s h S t a n d a r d " b e a r i n g the r e l a t i v e l y high s o l i d s content percent t o t a l  o f 9 percent b u t t e r f a t and 51.5  solids.  V a r i o u s methods o f c u l t u r i n g f a i l e d t o show the presence  o f any l i v i n g organisms  returned from the t r o p i c s .  i n cans of s p o i l e d m i l k  D e f e c t i v e s e a l i n g o f the cans  and e l e c t r o l y t i c a c t i o n i n t h e m i l k were d i s m i s s e d , a f t e r i n v e s t i g a t i o n , as p o s s i b l e sources of t h e s p o i l a g e . R o u t i n e e x a m i n a t i o n s o f r e p r e s e n t a t i v e samples f r o m each s t e r i l i z e r b a t c h o f t h i s m i l k had been made f o r some y e a r s u s i n g an i n c u b a t i n g temperature 0.  of 57 degrees  When t h e p r a c t i c e o f i n c u b a t i n g a t 55 degrees 0. a l s o  was adopted an i n c r e a s e i n t h e percentage samples was e x p e r i e n c e d .  of u n s t e r i l e  I n t h e m a j o r i t y of cases t h e  (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 P u r p l e L a c t o s e 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 h a t 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  r e s p o n s i b l e f o r the s p o i l a g e of the evaporated m i l k 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  desirable  and an attempt was made t o determine t h e i r s o u r c e . t e c h n i q u e was  A  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  sterilizing  t e m p e r a t u r e s , v i z . , 228 degrees t o 230 degrees P. f o r t w e n t y - f i v e t o t h i r t y - f i v e minutes, were d e t e c t e d i n m i l k at v a r i o u s stages of the m a n u f a c t u r i n g p r o c e s s , t h e n 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 m i l k showing the presence of these  thermodurio b a c t e r i a was e x c l u d e d from s u p p l i e s used f o r t h i s s t a n d a r d of evaporated m i l k . . T h i s 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 g r e a t many more b a t c h e s , t h a n f o r m e r l y , of m i l k which appeared s t e r i l e at both temperatures of incubation.  Hence the f a c t o r y o b t a i n e d g r e a t e r s u p p l i e s  of m i l k which c o u l d be shipped w i t h assurance t o 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 t e m p e r a t u r e s .  During  the i n v e s t i g a t i o n a l l m i l k a p p e a r i n g u n s t e r i l e on r o u t i n e e x a m i n a t i o n s was w i t h h e l d f r o m shipment t o 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 r e c u r r e n t s p o i l a g e have been received.  (54) The seasonal incidence of 'these thermoduric bacteria i n the finished evaporated milk was d e f i n i t e l y established from determinations on 4303 samples. P a l l and Winter months t h e i r presence was most while during the summer there vrere very few.  During the  pronounced, Routine  plates showed, after incubation at 55 degrees 0., 85.7 percent of samples containing thermoduric bacteria during December, as compared with 1.6 percent i n 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 i n cooking the milk, reduced only s l i g h t l y the contaminations of f i n i s h e d supplies of t h i s export evaporated milk by heat resistant bacteria. Spoilage experiments were conducted with representative cans from batches of milk appearing both s t e r i l e and unsterile i n 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 s p o i l , while those apparently free of them did not. Milk incubated at 37 degrees 0. showed only very alight tendencies i n t h i s direction, even after long periods of holding time. In most cases some acid was produced i n spite of the fact  (55) that most of the organisms experienced gave an alkaline reaction on lactose plates.  The phenomenon of bacteria  dying out i n the milk, after c l o t t i n g had taken place, gave a clue to the reason f o r the i n a b i l i t y to recover organisms from cans of milk i n the o r i g i n a l outbreak of the spoilage. It was also shown that milk packed and stored f o r as long a period as s i x months spoiled when subjected to high temperatures, indicating the thermophilic nature of some of these organisms and t h e i r a b i l i t y to remain dormant i n the milk u n t i l 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 innoculating milk with cultures of representative organisms  isolated  during the investigation showed the effect on evaporated milk of the predominant bacteria.  types of these thermoduric  Strains of lactose fermenters l i k e B. ooagulans  produced a high acid c l o t . Non-lactose fermenters l i k e B. thermoalkalans spoiled the milk by a slight production of a c i d i t y and a decomposition of the proteins while other non-lactose fermenters l i k e 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 c l a s s i f i e d  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 t h i s investigation, causes of spoilage i n evaporated milk were impossible to determine from cans of milk returned from the t r o p i c s 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 t h i r t y minutes In sealed cans of evaporated milk, were received In supplies of raw milk.  A l t e r a t i o n of plant p r a c t i c e s , such as more rigorous cleansing and s t e r i l i z i n g of equipment and attempts to r a i s e the s t e r i l i z i n g temperatures proved i n e f f e c t i v e In removing these thermoduric bacteria from f i n i s h e d supplies of evaporated milk.  Due to the extremely  r e s i s t a n t nature of these bacteria, the authors believe I t i s Impossible to use temperatures high  (57) enough to k i l l them i n the sealed cans of finished milk*  There i s a d e f i n i t e seasonal incidence of these thermoduric bacteria i n raw milk supplies, the period of greatest occurrence being the P a l l and Winter months and l e a s t 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 i n d i v i d u a l 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 s t e r i l e batches of f i n i s h e d 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 s u f f i c i e n t acid to clot the evaporated milk. Others are non-lactose fermenting organisms, some of which s p o i l the milk and some of which do not.  (58) Organisms isolated, during the course of the investigation have been c l a s s i f i e d as B» coagulans, B. calidvis, B. pepo, B. amarus, B, thermoindifferens, B. thermoalimentophllus, B. t r i t u s , and previously u n i d e n t i f i e d strains to which have been given the name of B. thermoalkalans.  Our findings i n t h i s 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 s t e r i l i z e d 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 i s o l a t i n g , c l a s s i f y i n g and testing the causative organisms, but did not determine their point of o r i g i n .  In t h i s investigation so many thousands  of samples have been examined that the authors f e e l that the sources, frequency of occurrence and behavior of the thermoduric bacteria concerned have been quite d e f i n i t e l y 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 A g r i c u l t u r a l Experiment Station, B u l l e t i n 170, 1912.  6,  K e l l y , 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. " B a c t e r i o l o g i c a l Studies on the Coagulation of Evaporated Milk", A g r i c u l t u r a l Experiment Station, Iowa State College, Research B u l l e t i n #10, 1915.  9,  Hussong, R.V. and Hammer, B..W., "Observations on B a c i l l u s c a l i d o l a c t i s " , 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 B a c t e r i o l o g i c a l Investigation of Evaporated Milk", Reprint, Archives of P e d i a t r i c s , 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 L a c t i c Acid Bacteria." (In English) D. Kgl. Danske Vidensk. S k r i f t e r , Natur og Mathematisk, Afd.8, Raekke, V.2, Copenhagen, 1919.  16.  Eaples, B.A. and Sadler, W. 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. B u l l . No. 147, New York State Agr. Exp, Stn. 1928.  ;  ?  Canadian Journal of  (61) ACENOV/LEDGMENTS  e  We wish to express our appreciation and thanks to Dr. B, A. Eagles and Miss Olga Oiotlitch, of the Department of Dairying, University of B r i t i s h  Colvimbia,  f o r assistance given during the course of t h i s i n v e s t i g 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,  Source of Culture  Ho. of  Agar Slants  762*L  Evap. Milk  long rods threads and single, ,5-lM x 4-7M  795-Q  Evap. 1111k  t h i n and thick long rods,single, chains  C H A R A C T E R I S T I C S  C U L T U R A L  MORPHOLOGY  Stain  Spores  Litmus Milk 72 h r s . i n c . 55 deg.C«  rods, single,pairs and chains  «•  elongated terminal  reduced,clean hard clot  Turbid  +  long rods,single and short chains  4  elongated terminal  reduced,clean hard clot  Turbid  4  thick rods,single few pairs  +  elongated terminal  reduced, clean hard clot.  Turbid  +  abundant, beaded, d u l l , smooth, opaque, grayish, media iinchanged  single rods  +  elongated terminal  reduced,clean hard c l o t  Turbid  4  abundant, beaded, d u l l , smooth, opaque, grayish, media unchanged  single rods  +  elongated terminal  reduced, clean S l i g h t l y hard c l o t Turbid  single rods  +  elongated terminal  reduced,clean hard c l o t  Slightly Turbid  Milk 72 h r s . 55° C.  72 hrs. 550 G.  long rods single and chains  0  • 13 • 133?  O "til  Gr3?Q331  Nutrient - Broth 24 hrs.55°  Motility in Broth  Gelatine Liquefaction 20 C. 55° C. D  Reduction of Nitrates H0  2  NH3  Indol Production  Growth on Agar Slants 48 hrs. at 55° G. abundant,beaded,dull, smooth, opaque, grayish; media unchanged abundant, beaded, d u l l , smooth, opaque, grayish, media unchanged  786-Bl.a  Evap.Milk  long rods, single  and chains, 4,5-lMx4-6  long rods,single and chains  786-B1.  Evap. Milk  rods,mostly single ,5-lM x 3-4M  long single rods  D.T.I  Drop Tank  thin long rods,single chains,.5-.8MX3-5M  single rods  D.T.3  Drop Tank  ^nS ?i!l^-6M  single rods  786B2  Evap.Milk  thick long rods singl e , long chains 10x5M  single rods  thick rods,single and chains  +  elongated terminal  unchanged  Slightly Turbid  795-P  Evap. Milk  single rods few chains  single rods and chains  +  elongated terminal  unchanged  Slightly Turbid  Mash 5  Old Mash Shipper 161D  rods, single and chains ,8-lMx4M.  single rods  single rods  +  elongated terminal  unchanged  Slightly Turbid  161-2  Comp. sample Milk 161D  rods,single and chains .8-lmx4M.  single rods  single rods  *  elongated terminal  unchanged  Slightly Twrbid  142-1  Evap. Milk  rods, single and chains !8-lMx4M  single rods  single rods  +  elongated terminal  darkened  Slightly Turbid  single rods  elongated terminal  unchanged  Slightly Turbid  abundant,filiform,flat,glistening, smooth,opaque, media unchanged abundant,echinulate,flat, d u l l smooth, opaque,grayish, media unchanged  n  782-03  Evap.Milk  short rounded rods lx3M,  146-P  Evap. Milk  rods, single :8Mx3M  143  Shipper 143D  single rods  single rods .8M-5M  4  elongated terminal  unchanged  Slightly Turbid  elongated terminal  unchanged  Isiightly  ^Turbid  moderate, beaded, g l i s t e n i n g , smooth opaque, grayish, media unchanged moderate, beaded, g l i s t e n i n g , smooth op a. que, grayish, media unchanged  4  abundant, effuse, d u l l , smooth beaded, opaque,grayish;media unchanged  4  moderat e,b eaded,f1at,gray!sh, opaque, media unchanged  4  dull  moderate,beaded,glistening,grayish,dark centre", colonies, media unchanged moderate, neaded,glistening,grayish, dark centre colonies, media unchanged  4  moderate, smooth,spreading,opalescent f l a t , media unchanged  4  moder a t e,b eaded,gli s t ening,op aque grayish, media unchanged  4  Mash 6  Old Maeh Shipper 161D  rods,single and chains ,8x4M,  single rods  single rods  elongated terminal  unchanged  !Slightly Turbid  Mash 4  Old Mash Shipper 161D  rods,single and chains .8x4M.  single rods  single rods  elongated terminal  unchanged  Slightly Turbid  122-G  Evap, Milk  long rods ,8M x 5 M.  single rods  single rods  elongated terminal  unchanged  Slightly !Turbid  mod er a te,beaded,glist ening,op aque, grayish, media unchanged  143-G  Evap. Milk  single rods ,8M x 4 M,  single rods  single rods  Elongated terminal  unchanged,  Slightly Turbid  moderate, beaded,glistening, opaque grayish, media unchanged  H3  Evap. Milk  single rods •8M x 4 M.  single rods  single rods  elongated terminal  unchanged  iSlightly Turbid  +  long single rods lx6M.  single rods  single rods  elongated terminal  unchanged  I Slightly  4  Composite of a truck  long rods,single & long chain ,8x6M  single rods  single rods  elongated terminal  clotted, , peptonized  Turbid white p e l l c l e  236  Comp.Sample Shipper's Milk  single rods  single rods and chains  single rods  elongated terminal  clotted peptonized  'Turbid white p e l i c l e  Mash 2  Old Mash Shipper 161D  single rods .5-.8M x 3-5M.  single rods  single rods  elongated terminal  unchanged  Turbid  moderate, beaded, g l i s t e n i n g , grayish,dark centre colonies, media unchanged  145-N  Evap. Milk  single rods .5-.8M x 3-5M  single rods  single rods  elongated terminal  unchanged  Turbid  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.  144-P37 Evapo.Milk  single rods .5-.8M x 3-6M  single rods  single rods  elongated terminal  unchanged  iTurbid  138-0  Evap. Milk  single rods •5-.8M x 3-6M  single rods  single rods  elongated t erminal  unchanged  Turbid  moderate,spreading,flat,glistening,smooth, tr anslus c ent,grayi sh, medi a unchang ed  782-M2  Evap. Milk  single rods few chains, ,8x6M.  single rods  single rods  elongated terminal  unchanged  :Turbid  moderate,spreading,flat,glistening,smooth, opalescent, media unchanged  782-M3  Evap. Milk  single rods, short chains, .7x411.  single rods  single rods  elongated terminal  unchanged  Turbid  moderate,spreading,flat,glistening, smooth, opalescent, media unchanged  795-J  Evap. Milk  single rods  single rods  elongated terminal  unchanged  I Turbid  moderate,beaded,grayish,gIistening,dark centre colonies, media unchanged  single rods and chains, ,7Mx4M.  single rods  single rods  elongated terminal  unchanged  'Turbid  abundant,spreading,raised,glistening,smooth, opaque,grayish, media unchanged  XI Hunt Composite of a truck  single rods ,7M x 4M.  long single rods  single rods and pairs  elongated terminal  unchanged  Turbid  moderate,spreading,raised,glistening,smooth, opaque,grayish, media unchanged  795-G  Evap. Milk  single rods ,7M x 4M.. .  single rods  single rods  elongated terminal  unchanged  Turbid  moderate,spreading,raised,glistening,smooth, opaque,grayish, media unchanged  Hay-1  Hay dust Shipper 161D  rods, single and chains lx5M.  single rods  single rods,  elongated terminal  unchanged  Turbid jSediment  782-M1  Evap, Milk c ^ n f ^ l M ^  single rods  single rods  elongated terminal  unchanged  Turbid JSediment  c S n s l S x t r  single rods  single rods  elongated terminal  unchanged  iTurbid  moderate,filiform,flat,glistening, opalescent,smooth,grayish,media unchanged  Truck VI Composite of a truck  single rods . ,6Mx3-4M  single rods  single rods  elongated terminal  unchanged  jTurbid  moderate,spreading,flat,glistening, opalescent, smooth^ grayish, media tmehang ed  Truck IV Composite of a truck  single rods and chains .6Mx3-4M  single rods  single rods  elongated terminal  unchanged  Turbid  moderate,beaded,echinulate, g l i s t e n i n g , opalescent, smooth, grayish, media unehanged  161  Comp.sample Shipper 161D  rods single and chains  single rods  single rods  elongated terminal  unchanged  Turbid  207  Comp.sample Shippers' Milk  single rods  single rods  elongated terminal  reduced, clotted  VII-D  - * ESSTSk  161  A1  No.31  Comp.sample Shipper 31D  abundant, beaded, glistening, f l a t , smocth grayish, media unchanged  4  abundant,beaded,gli stening,f1at, smooth grayish, media unchanged  abundant, f i l i f o r m , f l a t , g l i s t e n i n g 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, smooth,spreading,opalescent, f l a t , media unchanged.  abundant,beaded,gli st ening,opaque, grayish, media unchanged  +  abundant, beaded, g l i s t e n i n g , opaque, grayish,, media unchanged  4  bubble  abundant, spreading, f l a t , d u l l , opaque, smooth, grayish, media unchanged  Turbid ring on top  bubble  abundant,spreading,flat,dull,opaque,smooth, grayish, media unchanged  Truck V Composite of a truck  long rods, single 1J&4-7M  single rods  single rods  elongated terminal  reduced, clotted  Turbid ring on top  bubble  4  Hunt XI Composite of a truck  Long rods,single 1MX4-7M  single rods  single rods  elongated terminal  reduced, clotted  Turbid r i n g on top  bubble  4  abundant,spreading,flat,glistening,opqque, smooth, grayish, media unchanged abundant,spreading,flat,glistening,opalescent, smooth, grayish, media unchanged  1—1  o  hi  O  Si  CO  H  H  EH  a  %  H  X m  H O  •a*  W  MILK  a  £3  STARCH  R  ?0SE  <  a  o  ?0SE  H  3HAR0SE  EH  NQ.15^  LCT0SE  CULTURE  W  ?R0SE  NO. OP  rULOSE  T A B L E  a  ^  762-L  -0.9  5.2  4.5  4.7  3.8  5.9  4.5  4.3  -0.9  P _ 1,8  3.4  1.4  5.9 clot  6.1 c l o t  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 c l o t  5.2 c l o t  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 c l o t  5.4 c l o t  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 c l o t  6.1 c l o t  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 c l o t  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 c l o t  1.8 c l o t  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  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  G . O  2.2  -0.0  -0.7  0.0  0.0  0.0  -1.4  -1.4  161-2  2.3  -  142-1  2.0  3.2  4.1  1.1  0.0  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  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 c l o t  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 c l o t  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  -0.9  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  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  3.2  4.1  2.9  2.0  2.9  0.0  -1,4  -0.9  -1.4  1.6  0.0  0.0  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  -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  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  -  -  -  +  f  5.9  -0.7  -0.5 -  0.0  0.0  -  -  144P37 138-0 782-M2 782-M3 795-J  -1.6  795-G  -  Hay 1  0.0  4  2.9  -  -  -  -  -  0,0  0,5  0.5  0,0  0,0  -0,5  -  -  -  -  0.0  0.5  0,0  0.0  0.0  -0.7  -1.2  -1.4  -1.4  -1,1  -0.9  -0.9  0,0  0.0  0.0  0.0  0.0  0.0  0,0  0.0  0.0  0.0  -0.7  +  -  No. 31  0.0  0.5  0,0  1.8  0.0  0.0  0.0  Truck VI -0.7  -0.9  -0,7  -1.4  -1.6  0.5  0.0  2.5  2.0  0.0  -  0.0  -  0.0  0.0  -  -  Truck IV  0.0  -0.5 c l o t -0.5  -  782-Ml  -  -  -i.i  -0.9 c l o t -0.5 c l o t  -  -0.7 c l o t  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  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  Truck V  0.0  0.0  -1.1  -1.6  -1.4  0.9  -1.4  -1.1  -1.1  -0,9  -0.9  Hunt XI  -1.6  -1.8  -2.0  -2.0  -2.3  -0.9  0,0  -0,9  0.0  0.5  0.0  0.0 c l o t -0.5 clot digested -0.5 c l o t dlge ?ed 0.0 clot -0.7 c l o t digested  0,0 0.0,  Casein Digest Broth . -Sugars added at the rate of 2 per cent. Results recorded as grain l a c t i c acid per m i l l e .  0  S  # Yeast Extract added at the rate of .15 per cent.  

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