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Some observations on the growth of a strain of Escherichia Coli in raw and in pasteurized milk Middlemass, James Douglas 1927

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SOME OBSERVATIONS ON THB GROWTH OF A STRAIN OF ESCHERICHIA COLI IN RAW AND IN PASTEURISED MILK. By James Douglas Middlemass, B.Sc. A Thesis submitted for the Degree of Master of Science in Agriculture done in the Department of Dairying. THE UNIVERSITY OF BRITISH COLUMBIA May 1927. TABLE 0? COITTSrTS Page Introduction. 1 The organisms of the coli-aerogenes group. 2 Media employed. 3 Methods 4 Tahles 1 and 2. 6 Interpretation of ta"bles. 10 Discussion of results. 12 Conclusions. 15 References. 16 Illustrations. 18 Appendix "A" Int roduct i on. 20 Media. 20 Methods. 20 Table 3. 23 Interpretation of tahle. 23 Discussion of results. 24 References. 26 Charts. Appendix "B" Introduction. 27 Media. 27 Methods. 27 Tahle 4. 29 Discussion of results. 30 References. 31 AcknowledgHe nt s. 32 SOME OBSERVATIONS ON THE GROWTH OP A STRAIN OP ESCHERICHIA COLI IN RAW AND IN PASTEURISED MILK. By J.D. Middlemass, B.Sc. INTRODUCTION. The Bacterium coli was first isolated in 1884 by Escherich (5) from the faeces of a cholera patient, subsequent work showing this organism to be a normal inhabitant of the intestiial canal of man and animals (9,11). The coli-aerogenes group, of which this organism of Escherich is typical, has since occupied a prominent position in the investigations of many bacteriologists, (9,11,12,16) who have demon-strated its widespread distribution. Two very important fields of in-vestigation are those of water supply and milk production, where the coli-aerogenes group has been shown to have outstanding significance (2,7,11). The occurrence of this group in water, milk and milk pro-ducts is extremely common, and the extent to which these organisms contaminate the water or milk supply has been the subject of much attention, following the realization of their pes sible origin. The possible ways of introducing organisms of the coli-aerogenes group into milk are almost innumerable, some workers (2) - 2 -concluding that even fresh milk, produced under the best conditions, always contains some organisms of this type, if a sufficiently large amount of milk is examined. On the other hand, the extent of the con-tamination can he deduced by considering more their absence from spe-cific dilutions, than their presence (13). The behaviour of these organisms, however, when once introduced^ would seem to merit further attention, the prime object of this study being an investigation into the behaviour of a strain of Escherichia coli in raw and pasteurised milk. THE ORGANISMS OP THE COLI-AEROGENES GROUP. The Standard Methods of water analysis (17) states -"It is recommended that the coli-aerogenes group be considered as including all gram negative non-spore forming bacilli, uto ich ferment lactose with gas formation, and grow aerobically on standard solid media." Recognition of the important difference between Bac-terium coli and Bacterium aerogenes was made first by Rqgers and Clark (12) in 1912. Their classification was based on the measurement of the gas evolved from the growth of the organisms in glucose broth. Bac-terium coli gave a constant ratio of one part hydrogen to 1 part car-bon dioxide, while Bacterium aerogenes gave a ratio of 1 part hydro-gen to 1.4 - 2 parts carbon dioxide. Subsequent work by Levine (10) agreed closely with this classification and emphasised the fact that the Methyl Red positive, Voges Proskauer negative, high gas ratio organism i.e., Bacteriwu aerogenes, is relatively rare in the faeces - 3 -of man and of animals, the supposition "being that it is a normal in-habitant of the soil. Present classification and nomenclature of the main groups, Escherichia and Aerogenes, is given "by Bergey (3). For presumptive, partially confirmed and completed "bacteriological tests for the coli-aerogenes group, the Standard Methods of water analysis (17) give detailed instruction "both as to the pre-paration of media and their routine use. Levine (9), covers a wider field, both in regard to modification of existing recipes, and use in routine practice. MEDIA EMPLOYES Peptone medium for Methyl Red determination (Standard Methods 17} was used for growing the inoculum of Escherichia coli. Afte,r various experiments, it was decided to use a bile salt agar for obtaining the plate count of Escherichia coli in the milk. As a known culture of Escherichia coli was used to contaminate the milk, it was unnecessary to differentiate "between the Escherichia and Aero-genes groups. Prescott and Winslow (11), refer to the advantages of a brom cresol purple agar for the detection of organisms of the coli-aerogenes group, hut omit to give the details of preparation. A modification of the ]actose bile salt agar as used by Savage (16) was therefore employed with brom cresol purple as indicator. Preparation of lactose bile salt agar. (16) 20 grams of dried agar were dissolved by boiling in 1 litre of distilled water, 20 gms. of Bacto peptone and 5 gms. of Bacto - 4 -Ox-gall were added with s t i r r i n g and the so lu t ion was then f i l t e r e d . 10 gms. of lactose and 2 ccs . of a 1.6$ a lcohol ic solution of brom cresol purple were added, the solut ion was tubed and then autoclaved for 15 minutes a t 15 l b s . the t o t a l period of subjection to heat being not more than 30 minutes. HffPHODB A 3 year old Jersey cow, 7 month's oalved, "Ubyssey Hogue»s Betty", from the University of British Columbia dairy herd was selected. Observing thorough cleanliness and with due precautions, half a pint of fore milk was withdrawn and discarded, the next pint being withdrawn direct into a sterile bottle. This milk was then imme-diately removed and 100 cc. portions were measured, with a sterile graduate, into four 250 cc. flrlenmeyer flasks. Flask 1 was then placed in a water bath and heated to 145° P. for 30 minutes with frequent agitation, the temperature being read from a thermometer immersed in a flask containing 100 ccs. of water and placed in the water bath close to the flask of milk. The milk flask was then removed and cooled in a water bath to the required inoculating temperature. Flask 2 was held as the raw sample at room temperature till the pasteurising of Flask 1 was complete, when it was adjusted to a the same inoculating temperature as Flask 1. Flask 3 was held on ice for 24 hours and then treated the same as Flask 1. - 5 -Flask 4 was held on ice for 24 hours and then treated the same as Flask 2 and used for comparative work with Flask 3. TJhen adjusted to the required temperature each pair of flasks was subjected to equal inoculations, subsequently numerically ascertained by the plate method, of 1 cc. of a specific dilution of a broth culture of Sscherlchia coll (isolated by Kelly,8). In all cases, the absence of other organi3m3 of the coli-aerogenes group from the original raw milk was proved by plating a measured quantity with the bile salt agar. At definite periods after inoculation, the plate count of the milk in each flask was ascertained by withdrawing 1 c c , diluting in 9 cc. water blanks, and plating the required dilution with the bile salt agar. The plates were incubated at 37° c. and were quite satis-factory for counting purposes when 24 hours old. The colonies were distinctly acid forming as evidenced by the change in colour of the indicator from purple to yellow, their appearance oeing clearly shown in Figures 1 and 2. Experimental plates of poor quality milk showed the easily distinguished growth of mould colonies on the madia, but all other types of growth, except those of the coli-aerogenes group, were inhibited. * - 6 -TABLE 1. THE COMPARATIVE RATE OP GROWTH OP ESCHERICHIA COLI IN PASTEURISED AIID RAW MILK HELD AT 37° C. Sxpt . MILK INOCULATED 'tfHEN FRESH I n o c . pe r ! Grow- j P l a t e Coxint c c . of t h JE.Coli per c c . of m i lk Hrs . ; m i l k . 63,300 P a s t . Raw MILK HELD 24 HRS.BEFORE INOCULATION Inoc .pe r j Grow-c c . of ! t h milk j Hrs. i i 2 4 6 8 10 12 620, 3 ,000, * * * * * Uncoi^ntab] 000 70,000 ooo: 1,800 10,400 114,000 :1 ,440,000 30,000,000 .e 56 ,000 3 6 9 24 Plate Count E.Coli per cc. of milk. Past. Raw 7,250,000 14,000 102,000,000 7,500 300,000,000 55,000 600,000,000 400,000,000 6,150 2 4 6 8 10 97,500 9,900,000 37,500,000 90,000,000 250,000,000 A D S . i n 1/1000 " 1/100* " 1/100 " 1/100 " i n 1/1000 15,000 2 140,000 2,450 4 3,240,000 430 6 65,600,000 240 8 270,500,000 65 10 345,000,000 Abs. in 1/100 * One plate (of 2 made) showed 1 colony only. - 7 -TABLE l.(,Con.) THE-COMPARATIVE RATE OP GROWTH OP ESCHERICHIA COLI IK PASTEURISED AID RAW MILK HELD AT 37° C. JBxpt. MILK INOCULATED WHEN PRESH MILK HELD 24 HRS.BEFORE INOCULATION Plate Count Inoc.per Grow-i Pla te Count Inoc.per Grow-cc. of .' t h E.Coli per cc . of. cc . of . th E.Coli per cc . of milk Hrs. milk. milk Hr3. milk. Past . Raw Past . Raw 3 18,900 2 4 6 8 10 30,500; 820,000 14,600,000 66,500,000 225,000,000 4050 1470 70 35 15 12,950 2 85,000 3,950 4 1,665,000 1,200 6 24,500,000 185 8 203,500,000 60 10 405,000,000 25 4 12,700 2 4 6 8 10 248,000 10,700,000 65,400,000 306,000,000 730,000,000 200 10 15 30 35 20,400 2 191,000 2,900 4 6,060,000 150 6 79,400,000 40 8 331,000,000 20 10 590,000,000 10 - 9 -TABLE 2. (Cant.) THB COMPARATIVE RATS OF GROTTH OF ESCHERICHIA COLI IN PASTEURISED AND RAW MILK HELD AT 17° C. MILK INOCULATED tfRKN FRESH MILK HSLD 24 HRS.BEFORE INOCULATION Inoc.per Growth Plate Count cc. of Hra. E.Coli per cc. milk milk. Inoc.perGrowth Plate Count of cc. of Hrs. E. Coli per cc. of milk milk. Past. Raw Past. Raw 18,900 2 4 6 8 10 21,000 21,000 20,500 17,000 22,500 19,000 17,500 18,500 19,000 17,000 12,950 2 4 6 8 10 13,550 11,550 12,000 23,950 52,000 12,500 15,500 14,000 10,200 9,250 12,700 2 4 6 8 10 16,800 17,900 42,000 112,000 380,000 14,400 13,500 5,150 800 1,000 20,400 2 4 6 8 10 16,900 18,100 22,800 38,500 112,000 21,000 18,800 19,200 17,100 17,500 - 10 -In all cases no organisms of the coli-aerogenes group were present in 1 cc. of the original raw milk with the exception of Experi-ment 2, Table 2, where the degree of absence was from 1/10 cc. INTERPRETATION OF TABLES If the bacteriological examination of milk for organisms of the coli-aerogenes group is to be of real value, it follows that the behaviour of this type in milk, both raw and pasteurised, merits attent-ion. By means of controlled experimental inoculation of a specific organism belonging to this group, into raw and pasteurised milk, under laboratory conditions, it.Is possible to study its behaviour when once introduced. The results obtained, though applicable only to specific cases, would seem to have a suggestive value in the wider sphere of commercial endeavour. The results in Table 1 may be summarised briefly as follows:-a)' A marked difference is noticeable in the rate of growth of Escherichia coli, when introduced into raw and pasteurised milk held at 37° C. b) A rapid and continuous growth of this organism in pas-teurised milk is seen in every case, whether the milk be fresh or held for 24 hours on ice. c) A definite reduction in count follows the inoculation*Sf this organism into raw milk in every case, whether the milk be fresh or held for 24 hours on ice. The time taken to reach a minimum count - 11 -would seem to vary with the rate of inoculation. The results in Table 2 may be summarised briefly as follows:-a) The difference in the rate of growth of Escherichia coli when introduced into raw and pasteurised milk held at 17° c. is not so marked as in Table 1. b) In the initial stages of growth, the plate counts in both the raw and pasteurised milk agree fairly closely. c) Later stages of growth show that Escherichia coli increases more rapidly in pasteurised than in raw milk, though the tendency is for the final count to approximate in both cases, as shown in Experiments I and 2. d) The marked reduction in count in the raw milk, apparent in Table 1, is not so evident and does not appear except to a limited extent in Experiment 4, where this reduction coincides with a noticeably rapid increase in the pasteurised milk. It will be noted that Experiments 3 and 4 in both tables provide a means of gauging the comparative effect of temperature on growth, as the initial inoculation in both pairs of experiments is the same. » - 12 -DISCUSSIOIT OF RESULTS Buchanan (4) divides the life cycle of a culture into 7 distinct phases, viz:-1. Initial stationary phase. 2. Lag phase or positive growth acceleration phase. 3. Logarithmic growth phase. 4. Negative growth acceleration phase. 5. Maximum stationary phase. 6. Accelerated death phase. 7. Logarithmic death phase. Prom the purely comparative point of view it is to be seen that these phases for the strain of Hscherichla coli used, differ mark-edly in raw and pasteurised milk. Alterations of the chemical constituents of various media was found by Salter (15) to have a distinct influence on the various phases of growth of Bacillus communis. The most important factor controlling the growth of organisms of the coli aerogenes group may be said to be temperature, the practical application being evident In the provision made for thor-ough cooling in commercial plants. In the estimation of contamination, by means of bacteriological analysis, the difficulty lie3 in the division of the original contamination from the subsequent increase as influenced by temperature. The investigations of Ayers and Clemmer (2) would see» to indicate that the temperature at which little cr no increase in numbers takes place is 10° C. The same workers made a careful study of - 13 -the limitations of the colon count as an index of cleanliness in milk pro-duction, the conclusions throughout being "based on growth in raw.milk. The inhibitive influence which raw milk exerts on germ content has long been noted. Stocking (18) bases this so-called germi-cidal action more on the inability of certain types of organisms to grow in milk, rather than on their actual suppression. Comparative work done by Allen (1) clearly indicates the different behaviour of the gas forming Bacillus aerogenes in raw and pasteurised milk. The method used was based on a direct measurement of the gas formed and demonstrated clearly that growth as measured by gas formation was more rapid in the pasteurised milk than in the raw when incubated at temperatures of 30-37° C. Working along somewhat similar lines but using plate count methods St. John and Pennington (14) al3 0 conclude that pasteuri-sation of milk affects the rate of growth of specific organisms. •Bacillus aerogenes was found to increase much more rapidly in pasteurised milk than in raw milk, when incubated at temperatures referred to as "room" and "ice-box". They conclude that the slower rate of increase in raw milk is traceable either to germicidal action or unnatural environ-ment. Stressing the importance of obtaining absolutely uncon-taminated milk from the cow, Evans and Cope (6) clearly demonstrate the bactericidal property of raw milk towards Bacillus coli communis. The milk from different cows was found to vary in this respect, and com-parative tests with pasteurised milk showed that the temperature of pasteurisation had a distinct bearing on the subsequent rate of increase - 14 -of the organisms in the milk so treated. They found that a decrease of 40% in 4 hours was obtained after inoculation of Bacillus coli communis in raw milk, but in these experiments the importance of the incubating temperature does not seem to be fully appreciated. These workers all show a reduction in the "colon count" of raw milk or at least a more rapid increase in the growth of-coli in aerogenes organisms pasteurised milk. Whatever the activating principle, to which this apparent decrease in raw milk can be attributed, there is no uniform opinion. Apart from the alterations of the growth phases as shown in the tabulated results, it is seen on examining Table 1, that a def-inite reduction occurred in the germ content below the number inoculated when the milk was incubated at 37° C. The maximum reduction occurred at a time influenced apparently by the age of the milk and the rate of in-oculation, though it is interesting to note that milk kept for 24 hours on ice shows a very marked germicidal action. On the other hand, as evidenced by the results in Table 2, when the temperature of incubation is lowered to 17" C, this reduct-so ion of germ content is not evident, the tables indicating that the growth phases are altered, but that there is no appreciable reduction in numbers of the strain of Escherichia coli inoculated, except in one case, possibly due to experimental error or faulty incubation. The indications are that at this temperature, the early 3tages show little change between the rate of growth of the organisms in raw milk and in milk pasteurised to 145° for 30 minutes, but that later stages show a very much more rapid increase in the latter. At the same - 15 -time, however, it is evident that the rate of increase in the raw milk must be maintained steadily as the very late stages show very high and some what similar counts in both milks. It follows that the arbitrary fixing of a "colon count" standard based on the presence, or even absence, of these organisms in milk is not justified unless a history of the milk is supplied. CONCLUSIONS The behaviour of a strain of Escherichia coli in raw milk derived from a specific cow in the Universityof British Columbia dairy herd and the same milk pasteurised to 145° for 30 minutes is markedly different. When this organism is introduced into raw milk derived from the above source, and incubated at 37° C. a definite reduction of germ content occurs, follov/ed by an increase. Under the same conditions, in the same milk pasteurised to 145° for 30 minutes, the organism maintains a rapid increase from the start. When this organism is introduced into raw milk derived from the above source and incubated at 17° C. the reduction in germ content is not apparent or only slightly so. The rate of increase, however, is slower than in the same milk pasteurised to 145° for 30 minutes under the same conditions. Raw milk has a definite power to inhibit the growth of Escherichia coli between the temperatures of 17° and 37° C. while the* pasteurising of milk to 145° F. for 30 minutes destroys all or part of this inhibiting factor. - 16 -RBFERSNOES (1) Allen, P.15T. 1917. Comparison of the rate of gas production "by-certain bacteria in raw and in pasteurised milk. Journal of In-fectious Diseases. Vol. 21. P. 219. Chicago, 111. (2) Avers and Clemmer. 1918. The significance of the colon count in raw milk. U.S. Dept. of Agric. Bull. Ho. 739. Washington, D.C. (3) Bergey. (SAB) 1923. Determinative Bacteriology. Williams and Wilkins. Baltimore. (4) Buchanan, R.E. 1918. Life phases in a bacterial culture. Journal of Infectious Diseases. Yol. 23. P. 109. Chicago, 111. (5) Sscherich, T. 1885. Die Darmbakterien des ITeugeborenen und Sauglings. Fortschritte der Medicin, III, 515 and 547. (6) Evans and Cope. 1908. Univ. Penn. Med. Bull. 21. No. 9. P. 264-274. (7) Harrison, Savage and Sadler, 1914. The milk supply of Montreal. Bulletin. Macdonald College. St. Anne de Bellevue, Quebec. (8) Kelly, CD. 1924. Further studies of the bacterial flora of the "Kingston" cheese. Journal of Dairy Science. Yol. VII. No. 6. Baltimore. (9) Levine, M. 1921. Bacteria fermenting lactose and their significance in water analysis. Iowa State College of Agric. and Mechanic Arts. Publication XX. 31. Bulletin 62. (10) Levine, Weldin and Johnson. 1917. The Voges Proskauer and correl-ated reactions of coli-like bacteria. Journal of Infectious Diseases. Yol. 21. P. 39. Chicago, 111. ! - 17 -(11} Prescott and Winslow. 1924. 31ements of water "bacteriology, 4th edition. Wiley and Sons. Hew York. (12) Sogers, Clark and Davis. 1914. The colon group of "bacteria. Journal of Infectious Diseases. Tol. 14. P. 411. Chicago, 111. (13) Sadler, 1. 1925. An index to the purity of market milk. Heai "before Western Canada Dairy Convention. Agric. Journal. Tol. 10. Ho. 2. Victoria, B.C. (14) St. Johrr>Pennington. 1907. The relative rate of growth of milk "bacteria in raw and pasteurised clean milk. Journal of Infectious Diseases. Vol. 4. P. 647. Chicago, 111. (15) Salter, R.C. 1919. Observations on the rate of growth of 3. coli. Journal of Infectious Diseases. Vol. P. 260. Chicago, 111. (16) Savage, W.G. 1912. Kilk and the public health. JTclIillan and Co. London. (17) Standard methods of water analysis. 1925. American Public Health Association. New York. (18) Stocking, W.A. 1905. The so-called germicidal property of milk. Bull. 37. Storrs Agric. 3xpt. Stn. Storrs, Conn. * - 18 -Hiotographs illustrating the appearance of colonies of Escherichia coli on the media referred to in the text;page 3. A. 1/1000 dilution. 4 hour3 after inoculation into raw milk at 17° C. B. " " 4 " " " " pasteurised milk at 1?°C. Both plates 26 hours old. » V - 19 -FIGURE Z. Photographs illustrating the appearance of colonies of Escherichia coli on the media referred to in the text,page 3. C. l/lOOO dilution. 10 hours after inoculation into raw milk at 17° C. B. " " 10 " " " " Pasteurised milk at 17°C. Both plates 20 hours old. - 20 -APPENDIX "A" SOME OBSERVATIONS ON THE GROOTH OP A STRAIN OP ESCHERICHIA OOLI IN THE MAKING OP CHEDMR CHEESE. INTRODUCTION In view of the fact that organisms of the coli-aerogenes group occur frequently in dairy products other than fluid milk, it was decided to carry the investigation a little further in its application to cheese making, the work being carried out along lines very similar to the previous investigation. Organisms of this group are often encountered in cheese i and have "been known and studied for a long time. (20). With modern developments in cheesemaking, however, such as pasteurisation of the milk supply, improvement in Btarters and the method cf their handling, it was thought advisable to investigate "briefly the behaviour of the same strain of Escherichia coli, together with other organisms of this in group which might be the raw milk, before, durirg and subsequent to the manufacture of cheddar cheese. MEDIA The same media, as previously described, was employed. METHODS One day 's "bulked milk of the Univers i ty of B r i t i s h Col-umbia dai ry herd was thoroughly mixed and divided in to two equal p a r t s , - 21 -one half "being heated to 143° F. for 30 minutes in a "Y/izard" 50 gallon pasteuriser, the other half remaining raw. Throughout the experiments every effort was made to obviate contamination. "Vats were thoroughly scalded, vat covers used and other factors considered which would tend to ensure a true comparison. The cheese made were of the Cheddar type and the process of manufacture was performed as given in the attached charts. The "colon count" of the raw milk was obtained by plating a specific dilution with the bile salt agar. It was realized that the colonies appearing, though belonging to the coli-aerogenes group, were not all those of Escherichia coli. In view of the fact that the pasteurising temperature used was well above the thermal death point of the organisms included in the coli-aerogenes group (19) and that the efficiency of the pasteur-iser had already been established, the pasteurised milk was considered as being free from organisms of this type. \7hen the two vats of raw and pasteurised milk had been adjusted to the same temperature, equal quantities of a specific dilution of a broth culture of Sscherichia coli (23) were inoculated into each. JLt the same time definite quantities of starter, composed of equal parts of cultures of "Ericsson"* and"D. 144"* organisms, free from organisms of the coli-aerogenes group were introduced into each vat. * Stock cultures of lactic acid organisms maintained for use as starters in the Dairy Department, University of British Columbia. - 22 -"Colon counts" were made from "both vats of:-(a) The milk just prior to rennetting. (b) The whey at running. (c) The cheese 22 hours after putting to press. The "colon counts" of both milk and whey were carried out as previously described in the first part of this study. The organisms in the cheese were ohtained in suspension dilutions using the method outlined by Kelly (23) these dilutions being plated as in the case of the milk and whey. • i - 23 -TABLE 3 . THS COMPARATIVE RATS 0? GR0T7TH OP ESCHERICHIA COLI IK RAW AMD PASTEURISED MILK BUSING TUB CHEESE MAKING PROCESS. Rate of Pasteurised Milk Raw Milk Inoc. of "Colon count" "Colon count" Starter E. Colli Milk when IShey at Cheese Initial Milk when Wxey at Cheese per cc. rennet running 22 fcrs. "Colon Rennet running 22 hrs. of added per cc. old Count" added per cc. old milk per cc. per gram per cc. per cc. per gram 1% 880 1305 1050 500 1200 4000 9000 26,000 2JisJS : 1740 2550 100* Absent in 580 2250 500* 3,100 1/200 * Lowest dilution made was 1/100 1 colony appearing on each plate from the pasteurised whey iand 5 colonies on the raw. INTERPRETATION OF TABLE 3. The cheesemaking process depends, among other factors, on the growth of various organisms. It follows that the control, during the process of any strain of organisms detrimental to the cheese, is of paramount importance. The methods of control are essentially dependent on the present state of knowledge concerning the various phases of the growth of such organisms. * The results of the table may be briefly summarised as follows:-(a) The milk received was contaminated with organisms of the coli-aerogenes group as evidenced by the initial "colon count". (b) The factor, previously mentioned, in the first part of this study, which inhibits the growth of Zscherichia coli in raw milk is not so apparent, as any reduction in germ content in it, occurs also in the cheese made from the pasteurised milk. (c) The increased amount of starter in the second experiment coincides with a markedly reduced "colon count" throughout the whole process of cheesemaking from "both pasteurised and raw milk. DISGUSSIQK OF RESULTS The reaching of definite conclusions based on the somewhat limited scope of the experiments outlined is undesirable, but the results obtained are of interest and suggest various lines of further investigation. The gas producing properties of organisms of the coli-aerogenes group have long been realized and their exclusion from the milk used in cheesemaking is an essential in the manufacture of good cheese. It follows that their control during the production of the milk and the manufacture of the cheese is of great importance. 7/ork by Hucker (22) points to the fact that the presence of colon and proteus types of organ-isms in the cheese examined is usually associated with a low grade of cheese. Once introduced into cheese, through the medium of contam-inated milk or faulty manufacturing methods, the growth of organisms of the coli-aerogenes group would seem to be affected by their total number as compared with the other bacterial flora. In addition , the ripening temperature has been shown by Harrison and Connell (20) to have a marked effect on the bacterial flora of cheddar cheese. Examination by Hastings, Evans and Hart (2l) of good quality cheddar cheese during - 25 -the ripening stages, lead them to conclude that the dominant group pre-sent in the early stages is the Bacterium lactis acidi group whose numbers fall off later with an accompanying development of the Bacillus "bulgaricus group. Pasteurisation of milk for cheesemaking on a commercial scale has many advantages according to Stevenson (25) who comments on the superior flavoured product obtained. Kurray (24) emphasizes the necessity of minimising post-pasteurisation contamination with gas forming organisms. The results in "both experiments point to the fact that raw milk as used for cheesemaking will "be infected with organisms of the coli-aerogenes type. An important controlling factor, apparently, makes its appearance in the amount of starter used. Other factors "being as equal as possible, it is seen that the increased amount of starter used from 1% to 2$0o coincides with a markedly reduced rate of increase and apparent actual suppression of organisms of the coli-aerogenes type, more especially in the pasteurised milk, where the inoculation of Escherichia coli appar-ently made little headway due to the addition of the starter. As previously stated, definite conclusions based on such shallow investigation cannot be drawn, but efficient control of the organisms of the coli aerogenes type in cheesemaking would seem to include-(a) Pasteurisation of the milk. (b) A heavy inoculation of starter, whether or not pasteuri-* sation is practiced. E X P E R I M E N T Date Made Series Number MILK a.m. • Past . 143° F, Mar. 2nd Haw do Past . 143° J\ Raw l i a r . 16th f t 67 68 69 70 2 do 16 do p.m. MILK Weight lbs. Fat % 1 190 do 190 15 ,. 172 do 172 STARTER Acid-ity % Ozt. Per 100 lbs. Amount Ozs. .8 . 1% .8 .76 Time Added S t a r t e r RECORD CHEDDAR CHEESE OF PROCESS OF MANUFACTUFE MILK AT T IME RENNET ADDED Rennet Test seoi. Acid-i ty % Rate of Rennet drams to lbs. Amount of Rennet drams Sr iccson 29 ozs. 10.0 j ; 144 29 ozs. 10.0 do do 2l% . 4 3/8} .76 2 i £ 4 3/81 ' i , Average • \ 10.20 \ 10.20 do do do do - , . 2 . 1-25 - .21 1-25 - . . 21 1-25 1 - . .21 , 1-25 7£ 7* 7 7 Time Temp. OF. 10.45 85° 10.45 85° • C U T TIME 11.25 11.25 10.55 85° 11.35 STARTED TO STIR Time 11.30 11.30 Acid-ity % .15 . 15! Time Started Acid-ity % CALD Time Finished Acid- Final Ity Temp. % o p . PITCHED Time 11.45 .15 12.25 11.45 11-40 .145 11.55 10.55 85° 11.35 11.40 , .155 11.55 .15E 12.25 • 14E 12.30 .15£ 12.30 Aoid-ity % .155 100° 1.10 .155 .16 100 ° 1-10 .165 .15 102 .155 102 0 1.10 .16 9 1.10 .165 • T A B L E 1 ! WHEY DRAWN C U T I N C U B E S . TURNED AND AND TURNED PILED Hours from Sett ing Time 3.10 3.10 2.45 2.45 1.55 Start Acid-ity % .16 1.55 -165 1.40 1.40 .165 .17 Finish Acid-ity % .185 .18 .2 .205 Time Acid-ity % Acld-Tlme ity % 1 2.15 2.15 2.5 .205 .205 .?7 TURNED AND PILED Time Acid-ity TURNED AND PILED Time I Acid-ity 2.45 2.45 ..23 ,3 .30 .23 3.30 2.30 .34. 3.0 2.5 .275 2.30 3ci 3 0 ' .28 .29 .40 4.15 4.15 3 30 .31 .405 O . «5U ' ' ' I JO IS c T3 C o O • - 27 -APPENDIX "B". SOME OBSERVATIONS ON THE GROWTH OF A STRAIN OP ESCHERICHIA CO LI IN THE MANUFACTURE OP BUTTER. INTRODUCTION. The growth of the same strain of Escherichia coli in the manufacture of butter was briefly investigated, as it was thought possible that such work would prove useful in supplementing that already-done on milk and cheese. The control of the organisms of the coli-aerogenes group in the manufacture of butter centres largely round the pasteurisation of the cream. At the temperature most generally used, (170° P. or higher for 10 minutes or longer) the destruction of organisms of this type maybe considered complete. (26). Their presence, however, in butter made from pasteurised cream has been clearly demonstrated (30) and, further, some defects in such butter can frequently be attributed to the action of these organisms.(30). Their presence therefore must be attributed to subsequent recontamination or faulty pasteurisation. * MEDIA The same media, as previously described, was employed. METHOIS The mixed morning and evening's milk of the University - 28 -of British Columbia dairy herd was separated, the cream obtained being halved. One portion was pasteurised to 170° P. for 10 minutes, by standing the can in a steam heated water bath and stirring the cream constantly. The other half remained raw. As the temperature used in pasteurising, was well above the thermal death point of the coli-aerogenes organisms, (26) their destruction was considered complete and the "colon count" of the raw cream was determined as previously described. Throughout the experiments every effort was made to obviate contamination. The churns used ( 8 gallon Eureka sanitary churns) and butter workers, were thoroughly scalded and other factors considered which would tend to ensure a true comparison throughout. The two lots of raw and pasteurised cream were cooled in a water bath to the same temperature and inoculated with equal quantities of a specific dilution of a glucose broth culture of the same strain of .Escherichia coli (28) as previously employed. The portions of cream were then held for a definite time at a fixed temperature in running water till ready for churning. "Colon counts" were then made:-(a) of the raw and pasteurised cream just prior to churning. (b) of the buttermilk when removed from churn. (c) of the finished butter at a definite period after making. Dilutions of the cream and buttermilk were made in the same way as described in the first part of this study. Dilutions of the butter were obtained by melting at 110° F. diluting in water blanks at the same temperature and proceeding as for the cream and buttermilk. - 29 -In all cases a churning temperature of 60° F. was used and two lots of washing water at 56° and 54° F. respectively. TABLB 4. THE COMPARATIVE RATS OF GROWTH OF ESCHERICHIA COLI IK RAW AND PASTEURISED CREAM XUBIITG THE BUTTER MAKING PROCESS. Pasteurised Raw Inoc. of Held "Colon Count"per cc. "Colon Count" per cc. % S. coli g per cc. Cream Butter- Butter Orig. Cream Butter- Butter ^ ° after milk Count after milk fr ro inoc.and inoc.and M ^ holding holding 1 9 18,800 2 hrs 15,200 20,900 305 150 15,300 14,500 50 at 48 hrs 48 hrs. 60° F. 2 9 18,800 24 hrs 18,500 82,000 150 16,500 * at ; 1/10 1/10 I 50° pi. 24 hrs 24 hrs 3.13 24,700 24 hrs 16,900 35,500 40 20,000 42,500 at 1/10 1/10 | 43° F. 96 hrs 96 hrs. 4i 10 41,700 24 hrs 27,250 54,900 25 27,250 . 41,500 Present \ j at ; 1/10 -in 1/10 43° F.i ' i20 hrs (1 plate ! of 2] I i i120 hrs * Plates spoiilt, » - 30 -DISCUSSION OF RESULTS In discussing "bacteriological examinations cf market butter made from pasteurised cream, the occurrence of organisms of the coli-aerogenes type is of definite significance and is a direct indication of recontamination as evidenced "by the work of Sadler and Yollum (30). Realization of this fact is an important aspect of control work. As the temperature of pasteurisation in all the experiments was well above the thermal death point of the organisms investigated, (26) the experimental inoculation is comparable to this recontamination. The undesirability of having this type of organism present in butter is well recognised and their presence is not indicative of high quality. Examination of the keeping quality of butter conducted by Rosenau, Frost and Bryant (29) points to the fact that Bacillus coli an tends to die out on storage, this fact being important consideration in conducting quantitative and qualitative determinations. Working along somewhat similar lines, Brown and Peiser (27) found that 30% of the organisms present in cream failed to grow after mechanical agitation in the churn and that washing and salting removed 50% from the unsalted butter. The reduction in germ content in both the raw and pasteur-ised cream after the inoculation and holding may be partly accounted for by the wide range of dilutions used, though inhibitive forces may also have been at work. In all cases, however, the most impressive feet is the tendency of the major portion of the organisms cf the coli-aerogenes - 31 -group to t»3 removed with the buttermilk. It would seem therefore that the complete removal of the buttermilk and thorough washing of the butter grains is an important step in control work. REFBRBHCBS 26. Ayers and Johnson. 1915. Ability of colon bacilli to survive pasteurisation. Journal of Agricultural Research, vol. III. Ho. 5. Department of Agriculture. Washington, D.C. 27. Brora, and Peiser. 1916. Keeping qualities of butter. Michigan State.Tech. Bull. 29. P.7-20. 28. Kelly, CD. 1924. Further studies of the bacterial flora of the "Kingston cheese". Journal of Dairy Science. Vol. VII. No. 6. Balti-more. 29. Hosenau, Frost and Bryant. 1914. A study of the market butter of Boston. Journal Med. Research 30. No. 1. P.69-85. 30. Sadler and Vollum. 1926. The relation of bacteria to the quality of graded butter. National Research Council. Report No. 16. Ottawa. - 32 -ACKN07/LEDGT.E1TTS I wish to extend my thanks to Professor U.S. Golding for his assistance and suggestions during the course of this study and to the University of British Columbia for laboratory and other facilities placed at my disposal. * 


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