STUDIES ON THE ELECTROPURE PROCESS OF MILK "PURIFICATION" THESIS FOR THE DEGREE OF M. S. A. J Gelpi 1931 ‘.t":" .‘v 5f}??? ’5' .14. ' '1 . ‘-:.r"." "‘ U; " 8-9;? u w’ ' 1. ~ . . ‘ - Ikr ‘1 v ‘v J . . v " la " ,' “ ‘ . ,‘ . a , r‘ -‘.’ I I ‘ . . _ r ,‘ . 0. " ~y ' ‘ -. 1. ‘ ‘ . .4 I‘ .‘ l 1 —" v .uu ‘ I 4\" "I g ‘ ,',' -'.‘ I‘l . .‘l' H "r 'A’ I 'l’ H i» l L l . . I. -- ' . f ‘ ‘ , . r '. ' <,:v ' l I ." . ‘. I ‘ t1 y. - «i. . . . 1t ,‘ " é " "I‘ . ' . f' _ .~. 0‘ - .1..‘, > > _3 ,_. .r,.‘-“. . . _.'. ' . , —-n ' ,. ‘ ' ‘1 . , '. 'r."‘.', 4' r‘~"-‘( ‘—.. ~. ‘.o - ' ‘ . L .3 .. .l> . .:. A“ ,:l ‘ .n I. ~'! . v - , z vvrl .1.) ; ‘1‘ . 11 ... .- .~ ‘. W... _~ . . w .- .» ..| .1 ., , " . ‘ x V . l o ‘ I .. -. , ‘- r , ‘ f ' ,~ . . ‘ .I ‘. ,3. 5 .‘« u " ’ ‘ ', ) I’; >' ' “ h . v '1' 'l _ ‘ ‘ _' ', . 'v I r ‘ _‘. - ' ‘ . ' - .’ ' ' ' — .'.“- - .u » 3 . r - V} 5" " ,- .'— '4 ' ' ' ' \‘ , \V , ., ‘- ‘ g ' ~‘ ' ~ ‘ ‘ ' " : g}, . ..|. _..' . .-1. ' .-, 0‘ o > . . 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A531, . ‘9 1;)! awfigfifi, 2&1”. £3315} Lyme ‘ _1’ . r . ‘ ‘_ , 51% 1%" a: a? -+ a. ._.;;'i 1.5 ' > ‘ 1, . 4mm 1 , 'J} ‘J )1 \v)( “1.;él t); (“ "H?“rqt,‘ r '7 I h l' xtfv‘ :9 --'§"r-‘t‘ “ ‘1'"? 9".{; s.“ i‘ ”1%“ igx «43911231 $‘-°S’;fl'-.‘h i. . 4- . - 1.‘ fr: . ~ 5‘ .1! «Mr I, ““Y STUDIES ON THE ELECTROPURE PROCESS OF MILK "PURIFICATION” THESIS STUDIES ON THE ELECTROPUQE BROCESS OF MILK "PURIFICATION" Thesis Submitted to the Faculty of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the Degree of Master of Science. AL J; Gelpi h—s June 1951' TABLE OF CONTENTS I. Introduction 11 . Historical III. Purpose of Work IV. Experiment s1 A. Effect of the Electropure process on bacterial numbers Be 0. D. E. in narket milk. Effect of the Electropure process of some milk-borne pathogens. Holding method versus Electropure process with respect to some thermodm'io organisms. Holding process versus Electropure process with respect to some thermophilic organisms. The effect of the Electropure process upon some common spore-forming organisms. V. Summary 8111 conclusions I. INTRODUCTION The process of applying heat to human foodstuff in order to make it more palatable, digestible, and otherwise Safe for human consumption most probably dates back to very early primeval times. It has only been within comparatively recent years, however, that heat has been applied to foods for the purpose of preservation. The discovery that animal and vegetable materials could be preserved by the applica- tion of heat began or resulted properly with the researches and subsequent discoveries of Anthony Leeuwenhoek in 1670, followed and confirmed by Spallanzani in 1765. These early investigators boiled such materials as neat and vegetable extracts for varying periods of time in hermetically sealed flasks. By so doing they observed that no decomposition of the material took place thereafter. In 1782, scheele ad- vised the exposing of vinegar to the temperature of boiling water for the purpose of preserving it. In 1804, Appert of Paris discovered the process of canning, and in 1811, pub- lished a treatise on "The Art of Preserving Animal and Vegetable Substances". In 1810, Durand in England patented a process for preserving certain kinds of foods in tins and glass Jars. In 1819, the pioneer canners'of,America, Daggett, Kensett, Underwood, and Mitchell began on a small scale what is now one of the greatest industries in the world. Up to this time, however, the preservation of foods by heat involved many bacteriological principles which were not clearly and definitely understood, and it was not until 4 the results of the researches of Louis Pasteur carried on from 1860 to 1870 on the abnormal fermentations of wines were brought to light that these principles became intelli- gible. He found that by heating wine to a temperature of 62° 0. undesirable fermentations could be prevented. This process ultimately became known as 'Pasteurization" in honor of the discoverer. The process of pasteurization then.may be simply defined as a heating process in.which practically all of the disease-producing nonnspore-forming bacteria as well as most other vegetative bacterial cells are killed. "The temperatures used, however, are not sufficiently high to destroy or otherwise alter the food value of any of the milk censtituents.) By such treatment the substance treated is not only rendered safe for human consumption but bac- terial decomposition is checked or delayed as well. The application of the principle of pasteurization to milk dates back to the time of Soxhlet in 1886, who, according to Roseneau, was the first to prepose the application of heat to milk for the purpose of infant feeding. In 1889, Jacobi, a well known.pediatrician, strongly recommended the use of heated a- pasteurized milk. The develolxment of commercial pasteurization was rather slow due to the great public pre- Judice which had to be gradually overcome. Pregress in the art of efficient pasteurization of:milk was also slow, but in spite of the many difficulties encountered, three stan- dard methods of milk pasteurization have been perfected and 'are in commercial use at the present time. The three generally accepted commercial methods of pasteurization are as follows: 1. the flash, instantaneous, or continuous; 2. the holder, holding, or held; 5. in the bottle or final package method. Besides the above named methods, various modifications of each have from time to time been devised, patented and adapted. Flash pasteurization consists essentially of rapidly heating the milk to a temperature of about 710 C. for the brief period of a few seconds or more followed by an immedi- ate cooling to 10° C. or lower. In this process the heating is usually effected with live steam. Holding method - This method consists essentially of heating the milk to 620 C. and holding at that temperature for a definite length of time, usually for 30 minutes, then cooling to 100 C. or lower. In this process the heat is supplied either by hot water, live steam,or exhaust steam. In the bottle pasteurization - This method consists, as the name implies, of heating the milk after it has been bottled by subjecting the bottles to fine sprays of hot water until the milk reaches the desired pasteurization temperature . In recent years, however, a fourth method - if it may properly be called a method - has been devised and is steadily being developed and perfected, namely, the electro- pure process of milk "purification". The theory and mechanics of the electropure process have been thoroughly described in the literature and will, therefore, not be discussed in detail in this paper. Briefly, the process consists of passing a column or rather a continuous stream of milk through a narrow gage rectangular chamber between two car- bon electrodes. The resistance offered by the various electrolytes in solution in the milk immediately causes sufficient heat to affect a reduction of approximately 99% in the bacterial count of the milk without in any way altering the quality of the milk. The apparently valuable ‘ and interesting feature of this process is that every particle of the milk undergoing treatment is Inactically instantaneously heated to the desired temperature for a brief period of time and cooled immediately thereafter. The electropure process is very often referred to as flash pasteurization, but the above mentioned feature of the electrOpure process serves beyond the shadow of a doubt to differentiate it from the "Flash" method. The electropure process of treating milk, because of the fact that comparatively little work has been done to prove its efficiency (economi-r cally and bacteriologically) thus far, and also because of the fact that the process fails to conform to the definitions of "Pasteurization" as set forth by the various Federal, state, and municipal laws, has not as yet been generally accepted. II. HIETCRI“AL The literature bearing on the subject of the electrOpure treatment of milk is surprisingly scarce. A few articles, mainly of scientific interest, and directly or indirectly pertaining to the subject, will be briefly reviewed here. Stone (1) in va9, studied the influence of electricity on microorganisms. His studies were primarily to determine the possibility of obtaining pure water by means of elec- tricity. His experiments were later applied to the purifi- cation of milk. In brief, his results show that currents of low voltage had a decided stimulating effect upon bacterial growth and reproduction, whereas on the other hand strong currents produced decided decreases in the numbers of microorganisms present. Similar results were obtained with milk, and, where static electricity was used, a positive charge was found to favor the development of bacteria to a very considerable extent. Again, where strong electric charges were used, the number of organisms decreased greatly in numbers, but feeble electric currents and small static charees had stimulating effects upon bacteria in uilk, increasirg their numbers perceptibly. I Beattie (2) in his investigations on the electric treat- ment of milk gives the following interesting report: "Thus it will be seen that miln was only in rare in- stances absolutely sterile, but that it was free from dan- gerous or disease producing bacteria and from the milk souring bacteria and that there was an enormous reduction in numbers of all other forms of bacterial life. The actual reduction was over 993. The chemical composition is un- altered (so far as chemical arblysis can ascertain); chemists' reports also give evidence of non-increase of acidity (which of course is due to the destruction of acid- producing bacteria) and increased keeping cuality of milk. The lactalbumin which is coagulated in ordinary sterilized milk is not coagulated in.the electrically treated milk. In addition the enzymes are not destroyed." Anderson and Finkelstein (5) conducted a series of ex- ;exdnents on the electropure process of treating milk with the following points in view: 1. To determine the efficiency of the electropure process in destroying bacteria together with the keeping quality of the milk so treated. 2. To determine whether the bactericidal action of the process is a result of the current alone, the heat produced by the current, or by a combination of these two factors. 3. To study the cheuucal changes, if any, which are produced by the treatment. 4. To study the effect of the process on sore of the enzymes in milk, the time renuired for coagulation with rennin, the cream line, acidity, etc. 5. To determine if there is any change in nutritive value, such as the destruction of the so-called "vitamins". These investigators found that the electropure process brought about a reduction of 98.73 of the total count of milk which had a count of 161,500 for an average of sixteen samples. A small percentage of lactose-ferventirg organisms were found, however, to escape destruction in the process for some reason or other. As regards the effect of the electric current on bacteria, the results of these workers show that the heat factor was mainly responsible for the destruction of bac- teria. In respect to the keeping Qualities of the electro treated milk as well as the effects on the cream line of the milk and the effect on its food value, the conclusions reached by them agree closely with the conclusions reached by others in this field. As regards the effect of the electric current on bacteria Christian.(4) states that the electric current - both continuous and alternating - is said to have a slight retarding effect on bacterial development, but not suffi- cient to kill bacteria when the auxiliary influences, heat, electrolysis, and the production of ozone are disregarded. Srannon (5) in his studies on the effect of pasteuri- zation on the individual organisms found in milk states that only two but of forty-seven non-spore-forming organisms sur- vived pasteurization. Ayers and Johnson (6) found that of twenty-two typical streptococci, none survived a temperature of 62° C. for thirty minutes. Tanner and Dubois (7) found 10 that, when members of the colon-typhoid group were present in milk in numbers enual to which they would occur naturally, they were destroyed by heating for thirty minutes at 600 C. C. M. Carpenter (8) reports complete destruction of tubercle bacilli in milk at 62.50 0., the heat being obtained by passing the milk between electrodes using a 220 volt 6O cycle alternating current. Guinea pigs injected with the milk treated at 55° C. developed typical lesions of the disease. Similar results had previously been reported by such investigators as Carpenter, Curren, Fuddleson (l7), and others, not only in connection with the tubercle bacillus but various other pathogenic organisms as well. Resistance of Certain Types 2: microorganisms [to the Pasteurization Process Since one phase of the’present work will deal to some extent with thermophilic and thermoduric microorganisms, a brief review of some of the work of the leading investiga- tors in this field might prove of interest. It must be said, however, that the work here reviewed has been.done mainly on the holding methods of pasteurization. Practi- cally no work of this nature has thus far been done in con- nection with the electropure process. Robertson, Yale, and Rreed (9), 1926, found that of 140 cultures of spore-fonning non-thermophilic bacteria isolated from either freshly pasteurized milk or pasteurizing eeuip- ment, the greater majority proved to be such organisms as 11 Bacillus mesentericus, Bacillus Subtilis, Racillus vulgatus, etc. These workers found the above species to abound in the dried material cooked onto the pasteurizing eouipment. 'Hucker (10), 1928, in his work on cocci which resist pas- teurization temperatures, found that the holding temperatures previous to pasteurization did not appear to effect the numbers of organisms appearing on plates incubated at 45° 0. made from pasteurized milk. He found, however, that strep- tococcus thermOphilis appeared to be more prevalent in samples of milk which were held at 300 C. or higher for four hours or more previous to pasteurization. Fay (11), 1927, states that when milk is judged on a basis of its bacterial count, the improvement in its sanitary oualities through pasteurization may not be recognized, if various thermoduric organisms (pin-point colonies) constitute a major part of the flora. The omnipresence of thermo-resistant and thermo- philic bacteria in pasteurized milk is usually considered to be of little significance, since they seldom find conditions favorable for their rapid multiplication. Fay (ll) further states that the most resistant cultures isolated during his investigations were killed by heating in the steamer for one minute, or by treating for an enual time with a commercial hypochlorite disinfectant. If the organisms are resistant to pasteuriZation, then subsenuent destruction in milk, particularly by the holding processes of pasteurization, is ‘ hopeless, and the solution of the so-called pin-point 12 colony problem must be through preventive measures. North (12) in his investigations of the resistance of Hycobacterium tuberculosis strains found that there is no material dif- ference in the resistance to heat between bovine and human strains of tubercle bacillus and between different strains of either species, or between the tubercle bacilli of naturally infected milk, tissue, lesions, and pure cultures. Eckford (13), 1927, states that thermo-tolerant or-v ganisms nmy cause annoyance to pasteurizing plants by multi- plying during the process and giving the pasteurized milk a high bacterial count. They may cause pin-point colonies at 37° C. only two groups of these organisms isolated by this investigator ferment lactose. He found thermOphiles to sur- vive not only pasteurization but 100° to 120° C. for fifteen minutes in the De Khotinsky Water bath. Breed (14), 1928, in his studies on bacteria resisting pasteurizing tempera- tures concludes that all the procedures that devalop true thermophilic bacteria in pasteurizing and milk powder plants seem to be properly classed as faulty or undesirable rather than as unsanitary plant practices. The original presence of thermophilic bacteria in raw milk, on the other hand, seems to be due to the inoculation of the milk with dirt, dust, etc. Breed (14) further showed that the real develop- ment of the truly thermophilic organisms, when they occur in pasteurized milk, takes place during the process of pasteuri- zation. Such faulty procedures as allowing foauxto remain 15 in the Vats, holding of milk for pasteurization, dead ends, rough surfaces, etc., all offer places for thermophilic bacteria to grow and should be eliminated as far as possible. Breed and Brickett (15), 1929, conclude that it does not seem probable that especially favorable conditions for growth of milk thermophiles occur‘normally before the milk reaches the pasteurizing plant. The significance of the presence of occasional large numbers of these organisms in milk is largely dependant upon their origin, upon the con- ditions under which they grow in the milk, and upon the effect of their activity in the milk. Prolonged holding at high temperatures favors the development and growth of thermOphiles. host thermophiles found in pasteurized milk are non-pathogenic, but many of these types are linuefiers and alkali producers and are objectionable for these reasons. 14 IV. EXPERIMENTAL A. To attempt to demonstrate the efficiency of the electro- pure treatment of market milk with special reference to the occurrence of members of the colon-eerogenee group in the treated product. A series of preliminary tests were conducted with a laboratory model of the electropure machine on various lots of good, average, and poor market milk in order to determine the percentage reduction in bacterial numbers at various temperatures, and also to ascertain, if possible, the o occurrence, if any, of Escherichia _o_o_l_i and Aerobscter m- m, in the milk after treatment. The earnplee for these tests were obtained from the re- ceiving vat of the college creamery. The milk of various patrone was selected at random, and at least two litere were collected from each lot in a flank of suitable size. The sample- wcre immediately taken to the laboratory and a stan- dard plate count was made before subjecting them to the electropure treatment. The samples were run through the machine at such a. rate that a constant temperature ‘of 71° C. was maintained, cooled imadiately to 25° 0. (tap water temperature), and again plated. Dunhan fermentation tubes containing lactose broth were inoculated with the milk (0.1 cc... 0.5 00., and 1 co. respectively ) both before and after treatment, and smears were made on pletee containing Salle'e medium from those tubes showing gel. 15 All plate counts were made on.standard nutrient agar adjusted to pH 6.8. Dilutions of 1-100, 1-1000, 1-10,000, and 1-100,000 were made on.the raw milk, and 1-10, 1-100, and 1-1000 were made on the treated milk. In general the methods as set forth by the Anerican Public Health Associa- tion were used for all plate counts. The medium (Salle's) used for’demonstrating the presence of‘and differentiating the colon-aerogenes group was made up as follows: Peptone - - ----- - ----- 5 gm. KzfiPO‘ - ------------ 5 gm. K32P04 - ------------ 1 gm. Powderedagar ---------ZOgms. Distilled water ------- 1000 cc. When made up, the agar was sterilized'in 100 cc. por- tions, and immedhately before using, the following ingre- dients were added to each 100 cc.: Erythroein (2% aqueous) - - - - - 2 cc. Methylene blue (1% aqueous) - - - 1 cc. Bromcresol purple (1% aqueous) - 2 co. Lactose - ---------- - 1.5 gm. This medium gave excellent colon-aerogenes differen- tiation in all cases tried. The inoculated plates were incubated at 37° 0. for twenty-four hours. In order to obtain a fair representative sample of milk, and also in order to secure proper adjustment of tem- perature and milk flow in the experimental machine it was found necessary to use at least two liters of milk for each I' 16 trial e The results of the trials at the various temperatures, the occurrence of the colon-aerosenes group before and after treatment, and the percentage reduction in total numbers are given in the tabulations Nos. I and II. The accompanying photograph shows the complete apparatus used in this work. The Electropure Machine with which all tests presented he rein were conducted LahoraLt'o—rymodel‘in“operation-showing a sample of milk inoculated with a pure culture being passed through. 1’7 18 No. 1.. "Electro-purification" of Milk Raw ‘ v ‘r_ Lactose 10 Q , Sam 18 I Plate I I Lalle E medium p , , fermented I NC. ' count 1 001 I 0.5 ' 1.0 V Ee 0011 I Ae aerogenee I V CC. I OOe ' 000 ' ' v I I rq I I 1 I 110,000 I + I + I + I +++ I ++ 2 I 185,000 I - I +' I + I +++ I - 3 I 125,000 I + I + I + I +++ I + 4 I 550,000 I + I + I + I +++ I + 5 ' 65,000 I + ' + I + I +++ , + 6 I 60,000 I + I - I + I +++ I + 7 I 108,000 I - I + I + I +++ I + 8 I 295,000 I + I + I + I +++ I .+ 9 I 210,000 I - I - I + I ++ I + 10 I 16,000 I - I - I - I - I - 11 I 145,000 I + I + I + I +++ I + 12 I 150,000 I + I + I + I +++ I + 13 I 155,000 I - I + I - I ++ I + 14 I 233,000 I + I + I + I +++ I ++ 15 I 175,000 I - I + I + I +++ I + + - Few colonies ++ 8 Medium growth +++ - Heavy growth No growth 19 "Electro-purification" of milk 2. HO . Treated m u .1 e .0 A 8 e 1 1 a O S E .0 O 1 d 68 I S+u non no t e ..m Au 8“; The I :1 1 O .U ..P 0 unu do 8;; ??t “Wt am PO ..e a.Uno rD mp % e mmue e r mftné . m.e . 3110 «DnrN . aerogenes I 0011 ' 00. I cc. ' 00. , 22,000 . 80.0 65 87.8 7 , 22,580 . 68 , 12,000 , 90.5 70 , 35,000 , 90.0 70 92.3 4,000 , 71 ' , 98.0 1,200 72 98.6 , 4,000 , 74 , 3,560 , 90.4 74 . 97.8 350 ' 99.6 610 , 99.4 750 75 13 . 76 15 . + 3 Growmi N0 growth 20 B. To test the resistance of a series of the various milk borne disease producing bacteria, and to find, if possible the safety range for these organisms in the electropure process of milk "purification". The f0110Wing tests were conducted in order to point out the efficiency of the electropure process in destroying various milk borne disease producing bacteria, and to deter- mine,if possible, the extent of the Safety range for each individual organism, A two liter Sample of fresh raw whole milk Was rendered sterile by autoclaving for twenty minutes at eight to ten pounds pressure. A plating was made to check its sterility. A twenty-four hour agar culture (in the ease of those or- ganisms capable of growing on agar medium) was suspended in a physiological saline solution and thoroughly shaken and filtered through cotton to remove all bacterial clumps. The sterile milk was then heavily inoculated with this suspension, from 0.5 to 1 cc. of inoculum being used in each case. This inoculation usually gave a preliminary count of from 100,000 to 1,000,000 or more organisms per 00. of milk. After thor- oughly shaking the inoculated sample in order to insure even distribution of the organisms, a plate count was made to determine the extent of the initial inoculation. The milk was then subjected to the electronure process at temperatures ranging from 65° C. to 75° 0. Small samples (about 50 00. per sample) were collected aseptically at the various tem- 21 peratures in small sterile flasks and cooled immediately to tap water temperature. The samples were plated as soon as possible after the last one was collected. The above pro- cess was repeated for each organism used in these tests. The milk was admitted to the machine in a specially improvised flask arrangement which permitted of easy cleaning and thorough sterilization. The temperatures were easily attained and held constant by adjusting the rate of flow of the milk into the machine. The media used in these tests varied with the type of organism being tested. Whenever possible "Difco" dehydrated media were used. In the case of the haemolytic streptococci the blood agar used was prepared by adding 5 cc. of sterile rabbit blood obtained by heart punctures to each 100 cc. of dextrose agar containing 2.5 per cent agar. The presence of the haemolytic streptococci in the milk before and after treatment was demonstrated by making smears on blood agar plates of the sediment obtained by thoroughly centrifuging 10 to 12 00. lots of the milk. The organisms used in these tests are listed in table III, and the results of the various tests are shown in table IV. 22 No. III. Cultures Used in Experiment No. I Organism 1 T_ Source 1 IV Eberthella typhi :_»(Rawlings) state Lab. 2 _:> Salmonella paratyphi i H. S. C. Lab. 3 j—salmonella schottmfillerii(Schotmuller) M.S.C. Lab. 4 I Staphlococcus aureus i Souibb and Son 5 ‘WTStreptococcus scarlatinaeistock # 1171 (State Lab.) 6 I Escherichia coli 3* Yale S. of M. 7 I Aerobacter aerogenes E Yale S. of M. 8 ‘T Eberthella dysenteriae i (Flexner) State Lab. 9 : Eberthslla dysenteriae : (shiga state Lab. 10 I Alcaligines melitensie Ai M. S. C. Lab. 11 Ti Alcaligenes abortue E Bovine (M. S. C. Lab.) Ho . IV ' Temperature Range I I cufifiure' Medium used ' Initial ' Degrees c. o I I. I inoculation‘335—375—3g5—7I3—73u-750 I I I I I I I I 1 ' Standard agar '17,000,000 ' + ' + ' + "- ' - ' - I I I I I I I I 2 ' Standard agar ' 1,250,000 ' + ' + ' + ' - ' - ' I I I I I I I I 3 ' standard agar ' 8,300,000 ' + ' + ' + ' - ' - ' I I I I I I I I 4, : Standard agar : 1,780,000 : + : + I + : - z - I I I I I I I I I 5 ' BlOOd agar ' heavy ' + ' + ' + ' ~ ' - ' 6 ' Standard agar '21,000,00C ' + ' +A' - ' - ' - ' w I I I I I I I I 7 ' standard agar '14,800,000 ' + ' + ' + ' - ' - ' I I I I I I I I 8 ' Standard agar '22,600,cco ' + ' + ' - ' - ' - ' I I I I I I I I 9 ' Standard agar '19,1oo,000 ' + ' + ' - ' - ' - ' I I I I I I I I 10 'Liver Infu. agar' 455,000 ' + ' + ' + ' - ' - ' I I I I I I I I I _ I I I I _ I _ I _ 1 ll 'Liver Infu. agar' 6,500,000 ' + ' + ' ' ' ' + ' Growth - = No growth 24 C. To study to a limited extent the effect of the electro- pure process upon thermoduric organisms, and to compare their resistance to this process with that of the holding me thod. The cultures used in this experiment were isolated from several samples of milk pasteurized by the holding method at the Michigan State College creamery. Fractically all of the cultures appeared as pin-point colonies on standard agar plates after forty-eight hour incubation period at 37° C. The holding method was simulated by placing 10 cc. lots of skimmed milk in test tubes and autoclaving these at ten pounds pressure for twenty minutes. Platings were made to check the sterility of the milk. Skimmed milk was used to avoid any interference from the fat layer in the test tubes. Each tube Was then inoculated with a 0.1 cc. broth culture (24 hour culture) of the particular organism to be tested. Dilutions of 1-100, 1-1000, and l-10,000 were made, and 1 cc. from each of these was plated on plain nutrient agar cleared with egg albumin and adjusted to pH 6.9. In this manner initial counts were taken for each organism be- fore pasteurization. The tubes were then placed in an auto- matically controlled water bath at 62.70 C. and held at that temperature for thirty minutes (five minutes being allowed for the milk to reach the desired temperature). At the.cnd of thirty minutes the tubes were immediately transferred to an ice water bath and cooled to 100 0. Each tube was then 25 plated, (dilutions of 1-10, 1-100, 1-1000, and 1-10,000 being used), and the percentage reduction before and after treatment, if any, was determined. The above process was repeated in order to check the results of the first trials, and these final results were found to check with the first within reasonable experimental error. Each culture used in the above tests was individually subjected to the electropure process according to the following procedure. A two liter sample of skimmed milk previously rendered sterile by autoclaving at ten pounds pressure for twenty minutes was heavily inoculated with a saline suspension of’a pure culture of the organism to be tested. The suspension was obtained by washing a twenty- four hour agar slant culture into a tube of sterile salt solution and thoroughly shaking and filtering to remove any bacterial clumps. All milk samples used were carefully checked for sterility before making inoculations, and all results for that particular trial were immediately discarded when control plates showed any signs of growth. The flasks of milk were inoculated with such ouantities of suspension that 1 cc. of the milk gave a plate count of 50,000 or over in dilutions of 1-1000 to 1-100,0CO before treatment. After thoroughly shaking to insure even distribution of organisms, the inoculated milk was plated according to standard methods of milk analysis in order to obtain accurate initial counts. The milk Was then subjected to the electropure treatment at 26 a temperature of 71° C. A sample was drawn in a sterile flask (about 75 to 100 00.) after the rate of flow and tem- perature had been properly adjusted and was remaining con- stant. Immediately upon drawing the sample it was cooled to the temperature of tap water and plated as above. The organisms used in these tests are listed in table V and partly described in table VI. The results of the tests are shown in tables VII and VIII. Ho. V Organisms Used in Experiments culture I Source 1P : Pasteurized milk 2? I " " 3? I " " 413 I " " 5? I " " 61’ I " " 71’ I " " 8P : n n 9:? I " " 10;? I " " 1113 I " " 1213 I " " 1:513 I " " 1439 I " " 15:» I " " 28 I . + . I . I . I . I . I . I . + . + . GSHHw .dwod . + . I . I . don Phofim. mama . . . h . . . . . . . . . . . I . + . I . I . + . + . + . I _ + . + . doapofidmm . I . I . I . don .wfloa. mug” . . . b . . . . . . . . . . . I . + . + . I . + . + . + . I . .+ . + . .poaoom .mmoo . I . + a. + . muooooofimmnpm. mma . a . a . . _ . . . . . . . . + p + u I p I u I n I p I p I a + — + — Oopgmm omfiuoo — I. - II a + - don figmga— mNH _ u . . . . p . . _ _ . . . — I . I . I . I . I. . +. I . + . + . +. .IWQOO £5.54 . I . + . + . 60H mung. MHH . . . all . . . . . .. . . . . . I . I . I . I . I . I . + . + . + . + . oodcmm .dfiod . I . I . + . mEHOH mdooov. QOH . . . t . . . . . . . . . _ . I . + . I . I . I . I . I . + . + . + . 39mm .maoo . + . I . + . mucoooonowé Mm . . . r . .. . . . . . . _ . . I . + . I .. + . + . + . I . I . + . + . noggemm . I . I _ I . con anonm. mm _ . p p . _ . _ . . . . . . . I . I . I . I . I . I . I . + . I. . + . omndzo oz . +++ . I . + . muoooooamcgmpm. m: . . . L . . . . . . . . . . . J I . + . I . + . I . + . + . + . + . + . omwoo 79:39 . . I . I . wfiflaaownovgmhwm. Mo - p u . _ . _ _ u . . _ p . . iii. I u I u I . I . I a I . I . I . + . I . omflflgo OZ. — +++ . I . I . dOaH HQOHQHM. mm . p p P . . . . . . _ _ . . . I p I p I u I _ .9 . I p + p I . I . I . oOanmnH omdwOO . I — I — I . 60H PHOSW. m¢ . . _ P . . . . . . . . . . _ I — I. _ I u I . I . I . I _ I . + . + . QmSEO Oz . I a I — I . don PHOSm. Wm” . u . . . . . . . _ . u _ . . I . I . I . I . + . I . I . + . + . + . noapoaeom . I . I . + . mdoooahnmwpm. mm F p L . . . . . . _ . _ . . . I - I u I p I p I u I — I — + — I p + - CHOaW - I . I p + — gOOOHNnCrngQ g r _ p . . . . . _ . . _ . . . U . d _ G . 4 . O . 4 . a . aw . U . 4w . . . . . . . _ 2. . omen» . . . H8303”. . . . mmonosm. @3333. mmoflatr amoeba. INon _ Mafia mdaflh . .3035? I283. Sago. hmoaogmnoa . oz _ L L 'F . . mmwfihpfifi. and 0 . _ DHSPHH: noggnmanow nwmwm . . .93 o. h H> .oa 29 «.0 0H0. mmmooam onsgonpooax A.naa om I .oos.mo. mmooohm mswcaom {0.05 ” -000.HH H 000.0e H” 0.0e H 000.e0 H 000.¢ma H mmfi 0.00 ” 000.00H ” 000.0b0 ”n 0.00 H 000.0me H 000.000 ” mea 0.00 ” 000.000.m ” 000.000.¢ H” 0.0a u 000.000.H H 000.000.m H 000 0.00 ” 000.00e ” 000.000.0 H” «.00 H 000.0em ” 000.0mm.¢ ” mmH 0.00 H 000.0em ” 000.000 ”M p.00 H 000.¢0 H 000.00H ” mad 0.¢ ” 000.000 ” 000.0s0 u” s.0 H 000.000 H 000.a00 H smoa 0.H0 ” 000.000 ” 000.00e.0 ”n 0.He ” 000.000.H H 000.0mm.m ” mm 0.00 H 300.000 ” 000.00H.0 H” 0.0e ” 000.000.H H 000.000.m ” mm 0.05 ” 000.000.0 ” 000.0em.mm u” 0.HH H 000.sa0.m H 000.00m.m H ma 0.e0 H 000.000 H 000.00s.n n” 0.ee ” 000.0H0 M 000.000.H ” m0 oqem H 00¢.0 M 000.00 n” 0.H0 H 00¢.0m ” 000.0em ” mm 0.00 H 000.00 ” 000.000 H” 0.00 ” 000.e> n 000.0NH ” me 0.00 M 000.00 ” 000.H0 ”n a.0~ H 000.000 ”I 000.00b H mm p.00 H 000.000 H 000.000.H n” .H.0m H 000.e00 H 000.000 ” mm H.0H ” 000.0s0 H 000.0HN.H n” m.b H 000.He0 .4 000.000 ” ma qoflwseom ” 0500 H32 ” 056.0 33:; ”wnoEmSmm H 0550 350 .q £800 333 H 93:50, a p . . - W HH> .00 W 0.00 ” 000.000 ” 000.000.0 .HH 0.00 ” 000.000 ” 000.000.0 ” 000 0.00 ” 000.000 ” 000.000 H” 0.00 ” 000.000 ” 000.000.0 ” 000 0.00 H 000.000 H 000.000.0 H” 0.00 H 000.000 ” 000.000 H 000 0.00 M 000.00 H 000.000 H” 0.00 M 000.00 H 000.000 ” 000 0.00 ” 000.000.0 H 000.000.00 H” 0.00 H 000.000 H 000.000 ” 000 0.0 H 000.000.0 H 000.000.0 H” 0 H 000.000.0 H 000.000 H 000 0.00 H 000.000 ” 000.000.00 ”n 0.00 ” 000.00 H 000.00 n 00 0.00 ” 000.000.0 ” 000.000.00 n” 0.00 H 000.000 H 000.000 H 00 LL0.00 ” 000.000.0 H 000.000.00 ”H 0.0 ” 000.000 H 000.000 H 00 0.00 H 000.00 ” 000.000 ”H 0.00 ” 000.000 H 000.000 H 00 0.00 0 000.00 ” 000.000 ”n 0.00 n 000 H 000.0 0 00 0.00 ” 000.00 ” 000.000 ”H 0.00 ” 000.00 M 000.00 M 00 0.00 0 000.000 ” 000.000 n” 0.0 ” 000.000.0 ” 000.000.0 H 00 0.00 ” 000.000 ” 000.000 ”H 0.00 ” 000.000.0 ” 000.000.0 H 00 0.00 H 000.000 ” 000.000 H” 0.0 ” 000.000 H 000.000 U 00 ugwgoom H 0000000 00000.0 N 000000 00090000 n” dogmmémm ” 0.5000 00000.0 N 05000 00.30000 N 0.005005 .. . .0 - HHH> .om 51 D; To compare the resistance of sore milk thermOphilic organisms to the holding process of pasteurization with that of the electropure process. The organisms used in these tests were obtained from the Tanner collection at the University of Illinois. The methods used were similar in most respects to those used in the preceding experiments. Agar slant cultures grown at 45° C. were suspended in physiological saline solutions,and thoroughly shaken and filtered to remove clumps. Flasks containing two liters each of sterile skfinmed milk were heavily inoculated with each culture respectively. Pach flask of inoculated milk was then subjected to the holding process of pasteurization at 62.70 0. for thirty minutes, and similarly inoculated flasks were then subjected to electropure process at 71° C. In.the case of the holding Operation, 10 cc. portions of sterile skimmed milk in test tubes were inoculated with about O.l cc. of the suspensions of the respective organisms, thoroughly shaken to insure even distribution of the organisms, exposed to a temperature of 62.70 C. for thirty minutes in.an automatic water bath, and cooled immediately thereafter to 25° 0. In the case of the electropure process a 50 to 75 cc. sample was drawn directly from the delivery spout of the machine a minute or so after the temperature had been adjusted to the desired point. The sample was immediately cooled to 25° C. and plated within a few minutes after drawing. The time of’exposure of 52 the milk to the electric current in the nachine was approxi- mately 14 seconds. Standard plate counts on "Difco" nutrient agar were made of each sample immediately before and after each treatment using in each Case dilutions of 1-100, 1-1000, l-l0,000, and l-lO0,000. Duplicate plates were poured of each dilution, and all counts were made with a counting plate when necessary and a counting lens. All plates were incu- bated at 45° 0. and counted at twenty-four and forty-eight hours. In order to insure consistent results it was found very necessary to use cultures of exactly the same age for each trial, as the older the culture the more heat resistant it was found to be. Therefore, in order to standardize conditions as much as possible, cultures which were exactly twenty-four hours old were used throughout. A series of seventy-two hour old cultures, however, were also used for comparison. The results of these tests (Plate counts and percent reduction in each case) are shown in tables X and XI. The cultures used in these tests are listed in table IX. No. II! Organisms Used in Experiments Culture I Source 27 i U. of 111. (nilk) 51 i U. of 111. (Milk) 41 I U. of 111. (Milk) 49 i U. of I11. (Milk) 50 i U. of 111. (Milk) 56 I U. of 111. (Milk) 66 i U. of I11. (Milk) 75 i U. of 111. (Milk) 155 I U. of Ill. (Feces) 142 I U. of 111. (Feces) 54 2 lab Ln momO . OON0mH 0 u 0 Om 0 . OOO.OON0§ —. momm 0 300. 0.00 H 00 H 000.000 n” . . 000 00 ” 000.000 . . 000.0 . . .. 0 00 . 00 . . . 000 0 OOO mom 0 0 n OEH . OOH 0 . 0 mm 0 0 OOO 0mm 0 p O a a p a 0mm. - - GNH c p 003 OFH - - ¢ 0 0 mm . 0 0 OO . 0 COO 0mm 0 . ONm H 0 000.0 . .0 H . O 0 0 Mb moam u - mm H u p - COO. - . 000.0 . 000. .. 000 n 0 . 000 . 00 OCH 0 0 0mm 0- CO 0 - OOOooom 0 n o - OOOQNQ lul- H u o u n on @000 0 u b a. — OOO. - . OOO.mH . 0 .- OOH 0 O . mHm 0 0* 0 0 OOO Omm .0 0 OO 0 N0 0 0 mo¢® - a O NWM — . 00 Hm" - 9 p a OHNO - n on 0 0 000 0mm .0 0 ON O 0 OOH 0 O 0 .- mm 0 0 0 OO &0& . Hfl gowpvfidm 0 Oflmgmm 0 000 m 0 0 0 m — HIM — — 00 u - OOO HHm — . p 000 00:00 . 0:00 .. 0 . 0 . . 00 . o HwapanH .. noapoddmm . . ooo.SN . 0m - p 0\ PHHHHOO H u - 0 mafia . . . 0.0800 000 000 . a n H - oz 0 0 A 0 000. 0000000 00000000000 AOHHHE O) I- O K O OboNOv mmmOOHm mflddHOm 00000000 050m 00 w . oz 35 .V. 5 0.00 M 000.00 ” 000.000 n” 0.00 H 000.00 H 000.00 H 000 0.00 H 000.000 H 000.000 n” 0.00 H 000.000 w, 000.000 ” 000 0.00 H 000.000 H 000.000 n” 0.00 ” 000.000 H 000.000 H 00 0.00 H 000.0 M 000.00 ”H 0.00 H 000.0 M 000.00 M 00 0.00 H 000.000 H 000.000 n” 0.00 H 000.000.0 n 000.000.0 H 00 0.00 M 000.00 M 000.00 H” 0.00 ” 000.000 H 000.000.0 ” 00 0.00 H 000.000 H 000.000 ”H 0.00 H 000.000.0 H 000.000.0 H 00 0.00 H 000.000 H 000.000.0 H” 0.00 H 000.000 ” 000.000 H 00 0.00 H 000.00 M 000.000 ”H 0.00 ” 000.000 H 000.000 H 00 0.00 M 000.00 H 000.00 n” 0.00 M 000.00 M 000.000 . 00 0000M0000 N 00000 00000 M 00000 0000000 ”n 0000W0000 N 00000 00000 . 00000 0000000 .00 .. .. 0.0 0000 0000000 000900000Hm 0.008 on I .o 00.N0O 0000000 000000m 00000000 0000 m0 HM .00. E. To determine the effect of the electropure process upon some spore-forming orgarusss commonly or occasionally found in milk. The organisms used in these tests were all obtained from the stock cultures of the lichigan state College Bacteriolo- gical laboratories. The general techninue ard methods em- ployed were essentially the sane as those outlined in the previous experinerms. The cultuzes were grown for four to six weeks on agar'slants at room temperature. Before using each culture was examined microscopically to determine tie approximate extent of sporulation. All cultures showed from 80-90 % spores when used. The cultures which were too¢iry to be put in suspension by the usual method were ground in a sterile mortar with sterile sand, then washed with salt solution and filtered through sterile filter paper. Flasks of sterile skimmed milk were heavily inoculated with these suspensions. The remainder of the tests were carried as previously described. The spore-formers used in the above tests comprise 'culture members 153, 163, 173, 183, and 193 and are described in table XII. . The results of these tests, including plate counts and percent reduction before and after each method of pasteurization, are shown in detail in table XIII. No. XII spore-formers Used in Experiments : Culture : Source : : 15s : Bacillus anthracis : : 16s I Bacillus megathorlumm.s.c.) I T Y A7 I 173 I Bacillus subtilis (10.5.0.) I : lss : Bacillus mycoides (M.S.C.) I Tfi r ‘4‘ I ' 198 :Bacillus mesentericus (M.S.C.)u 38 ..o oapv mmmoonm ondmonaomam ..¢Ha om - .o op.mo. mmmoonm mqfldflom o.¢m H ooo.oam H cooloom.m “H m.o H ooo.oofl.m ” ooo.ooH.m H mmH $.00 “ ooo.m H coo.oom.¢ ”n o.m ” ooo.oo¢.a ” ooo.30w.a H mmH o.ma H ooo.ma ” ooo.o¢H.H ”n m.o ” 000.com.a ” 000.com.H H mpa o.o® “ coo.Opm H ooo.oom.¢fl ”H o ” ooo.om>.n ” ooo.om§.n ” mmfl p.00 ” ooh.¢ H ooo.omm.a n” . n.o ” coo.¢¢m H ooo.>¢m H mma noapwwummh pqsoo Hmnflm H pusoo HprHnH n” moapmwcmm H assoc Hanflmn ansoo HprHqH H .02 A.o oauv mmooonm madmonpooam H” ..nws on I .o or.mo. mmmoonm mnwcaom H Hmaup cqoomm L. o.mm p ooo.omH H ooo.oom.b n” b.H H ooo.o¢H.H ” ooo.moH.H H mma m.mm H ooo.m H coo.ooo.n n” o H ooo.mmo ” ooo.mbo ” -mma o.mm .H 000.com H ooo.ooa.m ”n o H ooo.omm ” ooo.om¢ ” mma m.ab H ooo.o¢p H ooo.ooo.m ”n o H coo.mam.¢ H ooo.oam.¢ H moa m.mm H oom.¢ H ooo.oo¢.m n” §.m ” coo.OHH.m H ooo.oba.m . mad QOHdecmm ” undoo Hmmwm ” pmdoo HwfipanH H“ noprWCmm H pnsoo Hmcam H unaoo HprHnH .oz .. _. Hwfige pmpfim HHHM . oz 59 V. SUMMARY AND ODllCLUSION S l. A series of samples of raw market milk taken at ran- dom from'various patrons of the Michigan State College creamery were subjected to the electropure treatment at temperatures ranging from 65° C. to 760 C. The results of plate counts made before and after treatment, and the occurrence of mem- bers of the colon-aerogenes group were tabulated. The con- dition of the milk after being subjected to the various temperatures as to flavor and cream line were noted. From the results obtained in these tests it would seem that temperatures ranging from 71° - 75° C. are the most efficient. The percent reduction in total count at these temperatures ranged from 92.5 to 98.2. Though the percent reduction is greatly increased at 74° C. and 75° C., a marked effect on flavor and cream line becomes apparent at temperatures above 75° C. Some of the colon-aerogenes group, apparently, occasionally escaped destruction for some reason or other at temperatures up to 72° C. The extent of the reduction will naturally depend upon the kinds of organisms present and very probably upon the age of the organisms themselves. That the age of non-spore-forming organisms in milk affects their heat resistance has been clearly demonstrated by Robertson (16) and to a limited extent by the author in some of the work presented in this thesis. 2. A series of pathogenic organisms commonly or occasion- ally found in milk was subjected to the electropure process in 40 pure culture. 'Temperatures ranging from 65° C. to 75° C. were used on each organism tried. The object was to test their individual resistance to the process, and also to determine, if possible, the safety range in temperature for these various organisms. The results obtained in these trials tend to indiCate that though the safety range is very narrow, it is clear-cut and apparently very constant, that is, most pathogens escaped total destruction at 69° C. but were all destroyed at 71° C. From this narrow safety range in temperature it natu- rally follows that the success of the electropure process will depend in the last analysis upon an efficient and ab- solutely dependable mechanical device which will control the temperature to within at least three degrees. 3. A number of organisms which seemed to persistently resist the holding process of pasteurization were isolated and studied in pure culture. fach of these organisms were subjected to the holding method of pasteurization and also to the electropure process. At least two trials (and in some instances four and five) were made for each culture. -The percent reduction in numbers before and after treatment for each method of pasteurization was carefully noted. Al- though as a whole the organisms studied were very resistant to both the holding and the electropure processes, the difference in percent reduction between the two methods is markedly in favor of the electropure process, there being only two instances out of the thirty trials recorded where 41 the reduction was greater by the holding process. These results are certainly worthy of note, especially when the brief period of tine at which the milk was exposed to the desired temperature (usually less than ten seconds) is taken into consideration, and this would undoubtedly lead one to believe that some factor or factors other than heat are most probably responsible for the destruction of bac- terial cells. Whatever these factors may be, however, still remains to be determined. Various suggestions have been offered in explanation for the phenomenon manifested in the chamber of the electropure machine. Some of these, such as the possible production of ozone, electrolysis, production of ultra-violet or other rays, etc., are merely speculative, however, and the field still lies open for experimermation and further investigation. 4. A number of thermOphilic organisms were subjected both to the electropure process and holding method of pasteuriza- tion, and their individual resistance to each process was compared. The results of these tests do not appear nearly as favorable with.resrect to the electropure process as some of the results previously obtained when other types of or- ganisms were used. Though the cultures here employed grew well at 45° C. to 50° 0., they proved to be not very resis- tant to pasteurizing temperatures. In this series of cul- tures No. 155, an organism of fecal origin, was the only one which showed any great degree of heat resistance. This organism was ecually resistant to both processes. 42 From the data collected in these tests it would seem reasonable to state that the electropure Immcess, although as fully efficient as the holding method in.redueing the numbers of microbes present, shows no particular'adventage over the holding method in this respect. In view of the present conception, however, that the role played by there mophiles in milk is of no great significance, the electro- pure process could not very'well be severely criticized from this point of view. here again we have evidence of an increase in heat tolerance of an organism with an increase in age of the culture. Table X shows that the cultures were markedly more heat resistant at 72 hours than they had been at 24 hours. The percent reduction, however, in the 24 hour’and 72 hour cultures was, in general, proportionately the same for each method of pasteurization. 6. A series of tests were conducted with some common spore-forming organisne in.order to compare their relative resistance to the electropure and holding methods of pes- teurization. The cultures were from six to eight weeks old when used, and microscopic examinations showed them to con- tain from 80 to 90 percent spores. The results obtained in these tests, as shown in table XI, are nerkedly in favor of the electropure process. Earticular attention is drawn to those results obtained with Bacillus anthracis (No. 165) which is known to be an extremely resistant organism. The percent 45 reduction in the case of this organism ranged from 0.5 to 2.7 in the holding method, and from 99.5 to 99.7 in the electropure process. The results of the tests with 33311135, subtilis, Bacillus mycoides, sacillus mesentericus, and Bacillus megatggrigmare enually striking. From the figures shown in table XI, the electropure process is undoubtedly effective in the destruction of spores. Indications of this fact were found in the early part of this work when raw milk samples were being subjected to the treatment. At this time the organisms escaping destruction were isolated and examined microscopically. N0 spore-formers were found during the course of these examinations. The results of this experiment indicate beyond further doubt that there is something else besides the heat factor which causes the destruction of bacterial cellsin the electropure process. There seems to be a strong possibility, however, that, in the case of spores, the destruction might be brought about indirectly by the effect of the electric current. It is a Known fact that the more concentrated an electrolytic solution becomes, the less resistance it offers to an alternating current, and the greater the amount of heat produced in consequence. The cytoplasm in the sporu- lated cells becomes more concentrated due to loss ofwaater, and consequently the electrolytic substances in solution within the cells offer less resistance to the electric current than does the surrounding medium (milk). As a 44 result, an instantaneous and marked increase in temperature within.the cells themselves is affected.- The heat thus created is probably sufficiently intense to cause the de- struction of the spores. The idea brought out in the above statement has been demonstrated in the laboratory by immersing an artificial cell (consisting of a parchment sack filled with a concen- trated Neel solution) between two carbon electrodes in a vessel containing a salt solution of lesser concentration. A sensitive thermometer was suspended in each solution respectively, find, when the current was applied (110 v.AC), the rise in temperature in the parchment sack was shown to be much more rapid than the rise in temperature of the surrounding medium. The medium in the parchment sack con- ' tained the greater amount of free ions.and, therefore, offered the least resistance to the current, and as a result more current flowed through the cell, and consecuently more heat was generated. Since the results of this experiment show so definitely that the "purification" of milk by the electropure process is brought about by the aid of another factor besides heat, then, the process, by definition, cannot properly be called a pasteuriaation process. whatever the factor or factors might be, whether their effect is direct or indirect, the process is superior to pasteurization and should not be classed as, regarded as, or confused with "Pasteurization". .,3. 4. 5. 8. 45 BIBLIOGRAPHY stone, Q. E.'- Influence of Electricity on Kicroorganisms. Bot. Gaz., XLVIII, No. 5, 1909, pp. 359-579. Beattie, J. M. - Electrical Treatment of xilh for Infant Feeding. The British Journal of state Hedicine, 1914. Anderson, A. K. and Finkelstein, R. - Electro-pure ?ro- cess of Treating Hilk. Journal of Dairy Science, Vol. II, 1919. Christian, K. - Disinfection and Disinfectants. pp. 43-44. (Electric Currents and Mechanical Influences). Brannon, J. H. and Prucha, M. J. - The Effect of the Pasteurization Temperature on Individual Germs Found in Milk. Jour. of Dairy Science, Vol. X, pp. 263-268. Ayers, H. 8., Johnson, N. T. - A Study of Bacteria which Survive Pasteurization. U. S. D. A. Bulletin 161, 1913. .A. Tanner, P. W. and Dubois, G. C. - Some notes on the Effect of Heat on xembers of the Colon-Typhoid Group in milk. Journal of hairy Science, Vol. 8, pp. 47-54. Carpenter, C. M. - The Lffect of Heat Troduced by an Alternating Current on Tubercle sacilli in iilk. Journal of Bact., Vol. 17, No. 1, pp. 38, 1929. Robertson, A. H., Yale, H. T., Breed, R. S. - Non- thermophilic, Spore-forming 3acteria Associated with Pasteurizing Equipment. H. Y. Agr. Exp. Sta. Bulletin 119, 1926. 10. 11. 12. 15. 14. 15. 16. 17. 46 Hucker, G. J. - A Study of the Cocci Resisting Pasteuri- zation Temperatures. N. Y. gori. Exp. Sta. Bulletin 134, 1928. Pay, A. C. - Thermo-tolerant Organisms as a Cause of So-called Bin-point Colonies. Jour. of Bact., Vol. 15, pp. 547-377, 1927. North, C. E. and Bark, Y. H. - standards for Milk Pasteuri- zation. American Journal of Hyg., Vol. 7, pp. 147. Echford, K. C. - Thermophilic Bacteria in Milk. Amer. Jour. of Hyg., Vol. 7, pp. 201-220, 1927. Breed, R. S. - Heat-resistant and Heat-loving Bacteria in their Relation to the Pasteurization of Vilk. H. Y. Agri. Exp. Sta. Bulletin No. 559, 1928. Breed, R. S. and Prickett, B. S. - The Significance of Thermophilic Spore-forming Bacteria in Pasteurized Yilk. Jour. of Bact., Vol. 17, pp. 37, 1929. Robertson, A. H. - Effect of Age of Culture on the Heat Resistance of Non-spore-forming Bacteria. Vermont Agric. College Bu11., No. 275, (1927). 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