r... _: 3...... ...2. 27:. . .. ..1.-....:.......E.....: 1 In; . w. x . . \I..-;l\\.. : . . ...: .... .11.... 7 ... .. a. . . .. . w . . 3.1. ...-..1 ' I 'r ‘- much—m ‘ ...-3w ‘xu —‘ m'ruons FOR ESTIMLTING THE PSYCMRUI’MILIC POPULATIUNS AND KEEPING QUALITY OF mm 137 Selwyn Arthur Broitman ABSTRACT OF THESIS Submitted to the School for Advanced Graduate Studies of :ichigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1956 Approvedm Q. Selvyn A. Broitman PART I PsychrOphilic organisms are best defined, from the practical aspect, as those organisms which are capable of relatively rapid growth at refrigeration temperatures (4.5 C). The mosticommonly encountered genera are Pseudomonas, Achromobacter, Flavobacterium, and Alcaliggngs. Members of the coliform group are encountered to a lesser degree. These organisms are responsible for deterioration of refrigerated dairy products and since they are destroyed by both h.T.S.T. and L.T.L.T. pasteurization, their presence in freshly pasteurized milk is indicative of post-pasteurization contamination. Many investigators have reported that the initial numbers of psychrophilic organisms in freshly pasteurized milk cannot be used as a "yardstick" to predict keeping quality of milk stored at refrigeration temperatures. That this is apparently true, is evidenced by the observations that certain species of organisms cause deteriorative changes in milk in a relatively short time vhile others ("inert" psychrophiles) reach extremely high popula-- tion levels over long periods of time without causing noticeable flavor defects. To date, the most satisfactory method for the enumeration of psychrOphilic organisms is a plate count,on tryptone—glucose- extract agar incubated at 4.5 C for 7 days. This procedure is Selm A. Broitman obviously too long for the routine enumeration of psychrOphilic organisms. Again, at the present time a rapid test for the predic- tion of keeping quality of milk at refrigerated temperatures is lacking. It was the aim of this thesis to: (1) develop a medium for the rapid enumeration of psychrOphilic organisms and (2) develop a method that would find application in the routine prediction of keeping quality of milk stored at refrigeration temperatures. A screening procedure using a solid medium was adopted to determine the most suitable peptone for the growth of representa- tives of the four psychrOphilic genera (Pseudomonas fluorescens, Flavobacterium rhenanus, Alcaligenes viscosus_and.Achromobacter gpperficiale). GrOwth determinations in liQnid media were carried out to determine the effects of varying concentrations of the peptone, nutritional additives, and hydrogen ion concentration. Since 20 C is the approximate Optimum temperature for growth of these organisms, it was selected as the incubation temperature. However, this temperature allows the growth of various mesOphilic organisms, which are part of the normal flora of milk. Consequent- ly, it was necessary to find an inhibitor which would prevent the growth of these mesophilic types, without interfering with the growth of psychrophilic organisms. Various dyes and surface active agents were tested and Naccgnol N.R.S.F. (an anionic wetting agent) in a concentration of 1:1000 was selected. Selwyn A. Broitman The Phytone-Nacconol medium was formulated as follows: Phytone 20.0 gm yeast extract 5.0 gm KH2P04 0.1 gm KEHPO4 5.0 gm Naccdnol N.R.S.F. 1.0 gm agar ‘ 15.0 gm distilled water 1,000 ml pH 7.5 (incubate plates at 20 C for 48 hours) This medium was tested against the procedure in Standard Methods for the Examination 2; Daigz Products, (10th Edition). A statistical analysis demonstrated that there was virtually little difference between the two techniques. PART II A keeping quality test is also presented whereby a 10 ml sample of pasteurized milk is aseptically pipetted in a tube con- taining 1 m1 of sterile Nacconol-tetrazolium test solution. The mixture is then incubated at 20 C for 12, 24, 86 and 48 hours. The appearance of a positive tube (rose red color) after 12 hours indicates milk will retain its acceptability under refrigera- tion for approximately 4.2 days; a positive tube after 24 hours indicates acceptability for 8.8 days; a positive tube after 36 hours or after 48 hours is indicative that the milk in question will remain acceptable for 12.6 and 15.6 days respectively. 4"“ ”UNI ..‘Efl/ The formula for Nacconol-tetrazolium test is as follows: 2, 3, 5 triphenyl tetrazolium chloride 0.1 gm 1.0 gm 5.0 gm 0.1 gm Naccdnol N.R.S.F. K2HP04 KH2P04 distilled water to make 100 ml 4. Seliyn A. Broitman MLTHODS FOR. ESTIMATING TH}; PSYCHHAOPHILIC POPULATIONS AND KEEWING QUALITY OF MILK By Selwyn Arthur Broitman A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1956 ACKNOWLLDGMENTS The author wishes to express his sincere appreciation to Dr. W. L. Mallmann for his patience, understanding and teachings and for the many helpful suggestions he offered throughout the course of this work. To the graduate students and members of this department, many heartfelt thanks for their valuable suggestions. Grateful acknowledgment is also expressed to Dr. G. M. Trout for his interest and help on the organoleptic tests. To My Wife VITA Selwyn Arthur Broitman candidate for the degree of Doctor of PhilosOphy Final examination: August 10, 1956 Dissertation: 'Hethods for Estimating the Psychrophilic POpulations and Keeping Quality of Milk Outline of Studies: Major Subject: Microbiology Minor Subjects: Mycology, Biochemistry Biographical Items: Born: August 30, 1931, Boston Massachusetts High School: Roxbury Memorial High, Boston, Massachusetts graduated June, 1948 Undergraduate Studies: University of Massachusetts, 1948 to 1952, 3.8., 1952 Graduate Studies: University of Massachusetts, 1952 to 1953, M.S., 1953, Major Professor: Dr. W. Litsky, Thesis Title: A Comparison of the Effects of Terramycin Hydrochloride and Amphoteric Terramycin on the Fecal Flora of Humans Michigan State University, 1953 to 1956, Ph.D., 1956, Major Professor: Dr. W. L. Mal lmann Affiliations: Society of American Bacteriologists Society of Applied Bacteriology (kngland) Society of the Sigma Xi ...- - “nan—...,“ rM—w‘w .7. .— TABLE OF CONTENTS Page mewcTIoN O I I O O O O I I O O I I O I O O O I 1 PART I A MEDIUM FOR THL RAPID ENUMLRATION 0F PSYCHROPHILIC ORGANISMS IN MILK Literature Review . . . . . . . . . . . . . . . 4 Experimental A. Selection of a peptone . . . . . . . . . . . 20 Experimental . . . . . . . . . . . . . . . . 2O Ike Bu]. ts O C I O I I O O O O O O O O I O O I 26 Discussion . . . . . . . . . . . . . . . . . 27 B. Effects of nutritional additives . . . . . . 29 merimental w o o e e o o o o o o w s s a c 29 Maults . . I . . . . . . . . . . I . . C . 31 Discussion . . . . . . . . . . . . . . . . . 58 C. Selection of an inhibitor . . . . . . . . . 61 Experimental . . . . . . . . . . . . . . . . 61 Results ...................62 Discussion . . . . . . . . . . . . . . . . . 62 D. Preliminary test of the Phytone—Nacconol Medium I I O O O O O O O O O O O O O O O 65 Experimental . . . . . . . . . . . . . . . . 65 Results . . . . . . . . . . { . . . . . . . 67 Discussion . . . . . . . . . . . . . . . . . 67 E. PhytOne-Nacconol agar vs. tryptone-glucose— extract agar . . . . . . . . . . . . . . . . 68 Experimental 0 o o o I o o o o o o o o o s a 68 Results . . . . . . . . . . . . . . . . . . 68 Discussion . . . . . . . . . . . . . . . . . 71 F. Use of tetrazolium salts as a coloring agent 73 Experimental 0 s o o o a o o o o o o o o o s 73 Results 0 o o I o o o o o o o o o o o o o a 73 Discussion . . . . . . . . . . . . . . . . . 73 SWY O I O O I C O O O I O O O C O O O C 7 5 PART II A TLST FOR DETERMINING THE KEEPING QUALITY OF MILK LiteratureReView...............77 TABLL‘ or CONleNTS (continued) Page '3: . prerimental.................78 \_ Results and Discussion . . . . . . . . . . . . 81 WY 0 O O O O O O O O O I O 0 O O O O O O 8 9 BIBLIOGWHY . O O O O O O O O O O 0 O O O O O O O 90 PART I A MLDIUM FOR Tm. RAPID INUMEIMTION OF PSYCHROI‘HILIC OHGANISMS IN MILK INTRODUCTION To the early settlers, the family cow served as the milk supply. Industrial develOpment, and increasing pOpulation necessitated the establishment of commercial milk supplies for the city dweller. From the original one or two cow Operation, there emerged the larger specialized dairy Operation. The dairy farmer was now producer, processor and distributor. Distribution at this time over established milk routes consisted of dispensing from earthen ware or copper transportation crooks via a pitcher to the consumer. This method presented a serious sanitation problem since it proved to be an excellent means of disease transmission. Through the joint cooperation of sanitarians, milk producers, processors and equipment.manufacturers a system was develOped where- by milk could be handled safely from farm to consumer. In recent years emphasis has been directed toward a,more efficient processing distributiontnd quality control. Recent technological advances in processing and distribution of milk products have materially reduced costs. Paralleling this more efficient utilization of man power, there has been a lengthen- ing in holding time of milk products prior to consumption. The advent of every other day deliveries at the consumer level, holding afternoon deliveries overnight for pasteurization the following day, the six day dairy Operation, necessitating holding milk an addition- al day, and bulk handling of milk from the producer to the dairy-- have all contributed in the lengthening of time that milk follows from "cow to consumer". Add these factors to the recent trends in the market milk industry of distribution over a wide area from a centralized pro- cessing plant and extensive interstate traffic in fresh pasteurized ‘milk1and the problem ofkeeping quality or storage life becomes of prime importance. Refrigeration has been used to prolong the shelf life of milk and other dairy products. In effect this serves to retard bacterial growth. However, certain microorganisms which may be present in dairy products as post—pasteurization contaminants are able to multiply with relative ease at refrigeration temperatures. In recent years, attention has been focused on these psychro- philic microorganisms since they are responsible for deteriorative changes in milk and milk products stored at low temperatures. It is generally agreed that these organisms are eliminated by both H.T.S.T. and L.T.L.T. pasteurization and that their presence in pasteurized milk is a more critical index of post-pasteurization contamination than the presence of coliform organisms. To date the selective culture of these organisms in freshly pasteurized milk requires an incubation temperature of 4.5 C for 7 days. This relatively long incubation temperature is impractical for the routine detection of these organisms as an indication of post-pasteurization contamination. "h' !' 10.44 -".. 1;. -..... . .' '7 1 INVESTIGATION SCOPL OF This study was undertaken in order to: (1) develop a plating xnedium.for the enumeration of psychrOphilic organisms in a shorter period of time than that currently used. A.medium which yields visible colonies rapidly, could effectivelybe used to detect posthasteurization contamination. . 1 I (2) DeveIOp awméthod baséd On the growth Of psychrOphilic organisms to predict the keeping quality of pasteurized milk at refrigeration temperatures. LITERATURL REVIEW Definition of psychrophilic microorganisms The general group of microorganisms which have received atten- tion as the causative agents of food and dairy product spoilage at refrigeration temperatures are most commonly referred to as psychrophiles. Psychrophile or "cold loving" has the connotation, as Zobell (1946) points out, that the Optimum conditions for growth are at "cold" temperatures. This does not hold true for most of the organisms which are generally considered as psychrOphiles by the food and dairy industry. Temperatures from 5 C to 10 C serve as Optimum for only a few of these. The majority, however, although capable of growth at refrigeration temperatures, appear to favor temperatures in the mesophilic range. Hence the term psychrophile, suggesting that Optimum conditions for growth are at "cold" tempera— tures, may be a misnomer. A controversy exists concerning Optimum conditions for these microorganisms. As Dorn and Rahn (1939)reported, there exist- four optimum temperatures for any organism on any medium; two are for metabolic activity and two are for growth. In each case, one opti- mum is for the rate of growth and the other for the amount of meta- bolic products or total crop yield formed on endpoint. If total crap is used as a criterion of growth, these bacteria are, as Greene and Jezeski (1954) found, truly psychrOphilic. Or if minimum generation time is taken as a standard, these organisms appear to favor temperatures within the range of mesophilic bacteria. To further complicate the question of definition is the dis— agreement on the most favorable temperature for "obligate" as com- pared to "facultative" psychrOphiles. These arguments are, of course, of academic interest, but from the standpoint of practicality, a psychrOphilic organism is one which is capable of relatively rapid growth at refrigeration temperatures. Psychrophilic microorganisms found in dairy products The psychrophilic group of organisms is- comprised largely of gram negative non—spore-forming rods. Most frequently encountered are members of the genera Pseudomonas, Alkali enes, Flavobacterium, and Achromobacter. Certain members of the colifonm group are encountered to a lesser degree. Van der Zant and Moore (1955) studied the growth character— istics and proteolytic activities of two psychrophilic cultures, Pseudomonas fluorescens and Pseudomonas putrifaciens. They recommended a plate incubation for three days at 25 C for the enumeration of bacteria growing in refrigerated milk and related products. Lawton and Nelson (1954) isolated 50 psychrophilic organisms from comercially pasteurized milk using an incubation temperature of 3 C for seven days. Eight organisms were selected as being typical of the entire group. Seven of these were members Of the genus Pseudomonas and one belonged to the genus Flavobacterium. EStudies on generation time at varying temperatures indicated that the temperature for Optimum growth appeared to be about 21 to 32 C. Flavobacterium a uatile, however, had its Optimum growth tempera- ture at 10 C and was thus considered to be an obligate psychrOphile. Elliker (1954) and Llliker, sun: and Parker (1951) isolated organisms causing defects of cottage cheese at low temperatures. One was identified as Pseudomonas viscosa and caused a brown slime film on cottage cheese. Accompanying this visual defect was a "putrid" flavor and "rotten" odor. Another organism was identified as Pseudomonas faggi and caused a white gelatinous film, "fruity" Odor and bitter taste. A third, Alcaligenes metalcali enes, caused a white gelatinous film on cottage cheese but little change in Odor or flavor. The source of these organisms was from soil and water. Davis and Babel (1954) isolated organisms from dairy water supplies which were capable of producing a slimy defect on cottage cheese stored at low temperatures. In addition to the organisms cited before, they found members of the genera Achromobacter, Aerobacter and Proteus. Pasteurization temperatures destroyed these organisms within three minutes. Greene and Jezeski (1951) and (1954) isolated organisms from deteriorated cottage cheese and creamery water supplies. They found the predominant causative organisws to be members of the genus Pseudomonas and Aerobacter aerogenes. Studies on the growth rates of these organisms at varying temperatures indicated that the lag phase at 0 C was 24 to 48 hours. As the temperature of incubation increased, the lag phase decreased while the growth rate was accelerated. ThOmas and Chandra Sekar (1946) indicted members of the genera Achromobacter, Alcaligenes, Flavobacterium, Pseudomonas and various micrococci for spoilage of raw and pasteurized milk at refrigera- tion temperatures. An investigation of "surface taint" of butter by Jezeski and Macy (1946) demonstrated that Pseudomonas was responsible for this defect in a majority of the cases. Alcaligenes and Flavobacterium, however, were found to be the causative agents in the remaining cases of "surface taint". Sherman Cameron and White (1941) noted that the bacteria responsible for the spoilage of milk held just above the freezing point were gram negative, non spore-formingxods; primarily of the Pseudomonas group. The low temperature spoilage of various cream samples were attributed to a Pseudomonas species by Anderson (1937). Long and Hammer (1941) described the isolation of Pseudomonas putrifaciens (formerly designated as Achromobacter putrifaciens) as the cause of a cheesy or putrid condition of salted butter. This organism was also isolated from raw milk, cream, soil and water. Stark and Shieb (1936) studied the lipolytic and caseolytic bacteria isolated from butter. They reported that a relatively large percent were identified as Pseudomonas aeru inosa, Alcaligenes bookeri and Alcaligenes faecalis. Of these, only Alcaligenes bookeri failed to grow at 5 C, but did show evidence of growth at 10 C. A smaller percentage were identified as .Achromobacter aromafaciens and Achromobacter lipolyticum. Both grew at 5 C. Anderson and Hardenbergh (1932) described an organism resembling Achromobacter as being responsible for lipolytic spoil- age of cream. It imparted a characteristic bitter flavor to the cream. The taste was a sensation similar to that of a sore throat. In later work by Anderson (1937), this gram negative, non-spore- forming rod was named Bacterium lipidis; but was later renamed Achromobacter lipidis N sp. by Allison, Anderson and Cole (1938). Rice (1936) isolated Alcaligenes viscosus as the causative organism of rOpy cream. More recently Gainor and wegmer (1954) isolated a psychrOphilic bacterium resembling Alcaligenes viscosus from a ropy sample of pasteurized milk. This organism produced ropiness in test milk samples incubated at 5 C and 25 C but not at 36 C. Boyd (1953) described Aerobacter aerogenes as one of the most common causes of flavor deterioration in milk stored at 4.5 C. This same organism was cited by Claydon (1943) as causing a medicinal flavor in market milk. This flavor became very intense after six days of refrigerated storage. Stark and Shieb (1936) identified 11 of 188 gram negative caseolytic rods from butter as members of the Escherichia- Aerobacter group. The growth of these organisms at 5 C varied from none to moderate. Hammer and Yale (1932) isolated 25 organisms from 17 samples of off-flavored butter. These were identified as members of the Escherichia-Aerobacter group . Olsen, Willoughby, ThOmas and Morris (1953) and Nelson and Baker (1954) showed that coliform bacteria vary from sample to sample in their ability to grow at refrigeration temperatures. Some milk samples demonstrated a marked increase in coliform count during storage at 5 C while others gave little if any increase. Primarily the gram negative non-spore-forming rods have appeared to be responsible in most cases of psychrophilic spoilage of dairy products. However, there have been occasional reports of organisms other than these that grow at refrigeration temperatures. Sherman and Stark (1931) reported that Streptococcus glycerinaceus and §t£gg. liguefaciens isolated from milk and §t£gp. Iaggglig from swiss cheese were capable of growth at 10 C. §t£_g. glycerinaceus grew at 5 C while §t£gp. lactis did not. They also reported that Lactobacillus, although capable of growth at 10 C did not grow at 5tC. Rogick and Burgwald (1950) stated that the psychrophilic flora in market milk was comprised of gram negative non-Spore-forming rods and cocci. 0n the other hand, Thomas 23 El (1949) studying milk bacteria which grow at refrigeration temperatures found the presence of micrococci to be comparatively rare. Thomas and Chandra Sekhar (1946) noted the occasional occurrance of various micrococci in a study of psychrophilic bacteria in raw and pasteurized milk. Again, the most frequently encountered psychrophilic organisms belonged to the genera Pseudomonas, Achromobacter, Flavobacterium —-———_r—:r-, at“: Alcaligenes. The coliform group appeared in somewhat fewer cases as the cause of dairy product spoilage at refrigeration temperatures. The primary sources of these organisms are from deteriorated dairy products, raw milk, soil and water. Shutt (1929), Provan (1941), Carley and Hammer (1942) and Wagenaar (1952) indicated that routine water analysis fails to detect psychrOphilic organisms. These organisms are frequently found in coliform free water supplies. While coliform determina- tions on water supplies may prove the water to be safe from a public health standpoint, no information relative to the psychrophilic papulation of that water would result. Furthermore a standard plate count on water supplies may lead to erroneous conclusions as many psychrOphiles do not grow at 35 C. Erdman and Thornton (1951) reported that not one of 722 psychrOphiles isolated grew at 35 C. Anderson and Hardenbergh (1932) showed that the Achromobacter and Alcaligenes groups did not grow at 35 C. Hiscox (1936) found 37 C incubation prevented the growth of certain Pseudomonas groups, while Greene and Jezeski (1954) observed that even 30 C was unfavorable for the growth of some Pseudomonas species. Jezeski and Macy (1946) stated that plate counts on creamery water supplies were higher at 20 C incubation than at 37 C. Thus, the "total plate count", in many instances does not give a complete picture of the total viable organisms present. Plate counts incubated at 22 C to supplement routine water analysis was suggested by Provan (1941). Doetch and Scott (1951) indicated that the determination of proteolytic and lipolytic organisms should be performed in parallel with routine water examinations. Elliker 339; (1951), Doetch and Scott (1951), Olsen e_t_g_l_l_ (1953) and Olsen, Parker and Mueller (1955) reported that proper sanitation and chlorination of suspect water supplies (2-3 ppm) were effective in minimizing the access of psychrOphilic organisms in dairy products. The influence of these organisms on the flavor of commercially pasteurized milk can generally be detected in 4 to 5 days. It is not uncommon, however, to find milk that is relatively free from flavor defects in 15 to 20 days storage. Flavor deterioration by these organisms is caused by the utilization of the various milk constituents and the accumulation of metabolic by—products in the fluid milk as growth proceeds. The most comonly found flavors are putrid, rancid, cheesy, fermented, fruity and sour. In many cases, there is a change in the physical state of the milk as a result of the growth of psychrOphilic organisms. Usually this occurs after flavor spoilage becomes apparent. Occasionally a thickening of the milk occurs, a condition often associated with ropy milk. Curdling and proteolytic changes may also be detected. It is not uncommon to find green or yellow discoloration at the surface of the milk resulting from psychro_ philic spoilage. These or similar defects caused by psychrOphilic organisms are also found in many other dairy products. Flavor defects in butter, cheeses, and other concentrated milk products are frequently caused by members of this group. Effect of pasteurization There has been some differences of opinion concerning the effect of pasteurization on psychrophiles. However, a review of the more recent literature reveals conclusively that psychrOphilic organisms in milk are destroyed by preper pasteurization. The relatively few organisms, which might remain after pasteurization, would probably not be a factor in flavor deterioration Over an extended period of refrigerated storage. Roadhouse and henderson (1941) state that certain psychro— philic organisms could withstand pasteurization at 143 F for 30 minutes. Kenedy and Weiser (1950), using laboratory pasteuriza- tion methods on pure cultures of psychrOphilic organisms, reported that 8 of 15 showed a 90 per cent reduction in numbers occurred during pasteurization. Five exhibited a 50-90 per cent reduction in numbers, while 2 organisms exhibited definite thermoduric charac- teristics. However, Erdman and Thornton (1951) observed that only 4 of 722 psychrophilic cultures survived laboratory pasteurization, Jezeski and Macy (1946) noted that only 6 out of 41 cultures of psychrOphilic bacteria could be recovered after pasteurization at 145 F for 30 minutes. Abel el Malek and Gibson (1952) isolated a heat tolerant psychrophile in 17 out of 28 samples of laboratory pasteuriZed milk. Alcaligenes tolerans N. sp. was the name given to this organism which withstood 63 C for 30 minutes. Kaufmann and Andrews (1954)’using 2 species of Pseudomonas conducted thermal death time studies and concluded that the organisms were killed by L.T.L.T. and H.T.S.T. pasteurization. The margin of safety was considerably less with the latter when skim milk was used as the heating medium. Sherman Cameron and White (1941) reported that good quality raw milk would keep for four weeks before spoilage was obvious; while good quality pasteurized milk would keep as long as eight weeks. Recontamination of the pasteurized milk with minimal quantities of raw milk significantly reduced the keeping quality. Presumably, this decrease in keeping quality was due to the presence of psychrOphilic organisms in the raw milk. Having gained access to the pasteurized product, these organisms prolifer- ated during refrigerated storage and thereby materially reduced its shelf life. They concluded that pasteurization effectively eliminated psychrophilic organisms. Watrous, Doan and Josephson (1952) supplemented laboratory pasteurization studies with commercial L.T.S.T. pasteurization investigations. They reported that psychrOphile counts of 75 samples immediately following laboratory pasteurization, and counts after 10 and 20 days of storage at 5 C, were less than one per milliliter. Psychrophilic bacteria were not detected in any sample from the holding vat when plated immediately or even after 5 C storage for 15 days. Similar results were reported by Rogick and Burgwald (1952). A comparison of the effects of commercial L.T.L.T. and H.T.S.T. pasteurization on the elimination of psychrophiles showed that both methods resulted in the destruction of these organisms. Initially psychrophilic organisms could not be detected in sample quantities as high as 4.1 ml. After seven days storage at 4 to 7 C, these samples did contain psychrOphilic organisms. Olsen, Willoughby, Thomas and Morris (1953) reported that samples rem0ved aseptically from H.T.S.T. pasteurizer did not contain any psychrophilic bacteria. The psychrophile counts did not reach a level of greater than 3 per ml. in seven days of storage at 4.5 C. Bottles of the finished product, although having relatively low psychrophilic counts initially, demonstrated a marked increase in numbers in five to seven days. This suggested the possibility that other heat tolerant mesOphilic organism might become adapted to, and multiply at, refrigeration temperatures; or that all the psychrophilic bacteria were not eliminated by pasteurization. Watrous, Doan and Josephson (1952) reported that there was little evidence to suggest that thermoduric organisms were readily adapted to psychrophilic conditions. These organisms did not show any evidence of growth in milk stored at 5 C. Studies on refrigerated storage of milk and cream by these investigators led them to the conclusion that organisms surviving pasteurization would not grow at refrigeration temperatures; and that the presence of psychrOphilic organisms in pasteurized milk was the real: Li result of post-pasteurization contamination. Coliform vs. psychrOphiles as indicators of post-pasteurization contamination. During the course of the early work on the coliform group, many investigators, Gage and Stoughton (1906), Ayers and Johnson (1951), Minkin and Gurgwald (1935) and Long, Hedrick and Hammer (1944) con— f cluded that certain strains of the coliform group exhibited varying ! degrees of heat tolerance. Many were not destroyed at pasteuriza— f r tion temperatures. t More recent investigations by Olsen, Macy and Halvorson (1952) L_ demonstrated that the incubation temperature at which the test organism was grown, markedly influenced its thermal death time at various temperatures. They reported that Escherichia 3211, after growth at 20 C, was destroyed by both h.T.S.T. and L.T.L.T. pasteurization. But after growth at 30 C to 37 C, its heat tolerance was increased to such a degree that the probability of its complete destruction, particularly by h.T.S.T. pasteurization, would be doubtful. These findings, along with those of Craige (1946), were instrumental in explaining the variation in survival studies of many of the earlier reports. Stark and Patterson (1936), Chilson, Yale, and Eglington (1936), Delay (1947), Prayer (1955) and others confirmed the ‘ eal‘lier work that the test for coliform organisms is of merit as an aid for the detection of post-pasteurization contamination. Buchbinder and Alff (1947) reported that there could not be any practical significance attached to the so-called resistant coli— forms in the routine performance of the coliform test of pasteurized milk. Dahlberg, Adams and held (1953) reported the absence of thermoduric coliform bacteria in raw milk. In 1937, Yale stated, with reference to the coliform test of pasteurized milk: "Bacteria forming red colonies upon these agar media, (violet-red-bile- agar or desoxycholate agar) regard- less of whether they ferment lactose with gas formation, are significant if destroyed by proper pasteuriza- tion, since their presence in fresh- ly pasteurized milk then indicates recontamination." If this statement were modified in lieu of current knowledge to read, - any organism which is killed by proper pasteurization is significant, for their presence in freshly pasteurized milk would indicate contamination after pasteurization — then there is just cause to consider the presence of psychrOphilic bacteria in freshly pasteurized milk as an indicator of post-pasteurization contamination. This group of organisms appears to be a much more logical indicator of post-pasteurization contamination, since it is possible to have a product contaminated after pasteurization by psychrophiles without coliform organisms. Under certain conditions, dairy water supplies, acceptable from a public health standpoint, may harbor psychrophilic organisms which could conceivably gain i access to pasteurized products. This was demonstrated by Shutt (1929), Provan (1941), Coreley and Hammer (1942) and Wagenaar (1952). Here there would not be any evidence of post- pasteurization contamination using the presence of the coliform group as an indicator. Whereas, this contamination might readily be detected if the presence of psychrOphiles were observed. In the current literature, Nelson and Baker (1954) stated that the coliform test should be retained as a quick index of contamination but negative results should be interpreted con— servatively, since the possibility exists that this test will not detect some important types of contamination. Watrous, Dean and Josephson (1952) reported that: "It seems obvious that determination of psychrophilic bacteria is a more critical index of post pasteurization contamination of milk than the coli- form determination." Many current reports have hinted at the possibility of using psychrophiles as an indicator of post-pasteurization contamination. These Opinions have been conservative owing to the length of time required to enumerate psychrophilic organisms at present. Again, there is the general reluctance to discard a test as well established as the coliform test for post-pasteurization con- tamination. Methods for estiaating psychrOphilic organisms Recently, Atherton 33 El (1954) described some attempts at possible methods to ascertain psychrophilic deterioration prior to the development of off-flavors. The resazurin reduction time of refrigerated bottled milk laroved to be so long at both 20 C and 37 C incubation, that it gave w ...-A only slight information concerning psychrOphilic activity. A test for the detection of phosphatase produced during the growth of these organisms proved to be of little value. As acid production is not a normal characteristic of psychrOphiles in milk at low temperatures, changes in pH were too slight to be of practical significance. The Storrs test, a measure of protein stability, appeared to be the most promising as a rapid method for the detection of psychrophilic activity. A 10 point drop in stability value below the normal for fresh milk was usually followed by off—flavor develOpment. This test, however, would be of limited application inasmuch as this dr0p in stability occurred too clase to the time of spoilage. The most frequently used method for the determination of psychrophilic populations in milk has been the agar plate method. Pennington (1908) incubated agar plates prepared from raw milk for 4 to 6 weeks at 0 C. Thomas and Chandra Sekhar (1946) reported that higher counts were obtained at the end of 21 days than at 7 to 14 days incubation at 3 C to 5 C. Kenedy and Weiser (1950) incubated plates at 10 C and suggested that psychrophiles appeared within 3 days, while adaptive mesOphiles required 8 to 5 days before colonies were visible. Burgwald and Josephson (1947) and Dahlberg 33 El (1953) employed a 10 day incubation period at 8 to 10 C. Watrous 31 El (1952) used a 10 day incubation time at 5 C. Boyd st 21 (1954) recommended that lasychrophiles be enumerated by incubation of plates at 5 C for at least 10 days but preferably 20 days. Standard Methods for the Analysis 2_r_ m Products, (mm 1953) states that psychrOphiles be enumerated by incubation of plates at 5 C for 7 days. Nelson and Baker (1954) suggested an incubation temperature of 25 C for 3 days or 21 C for 4 days on the basis that these times and temperatures detected samples giving high counts on plates incubated 10 days at 5 C in all cases. This last recommendation should be tempered with a word of caution. Although this method is the most rapid to date, it is subject to some degree of interpretation. PsychrOphilic counts (incubation of plates at 4.5 C for 7 days) on freshly pasteurized milk are generally very low. Higher counts are obtained by incubation at 20 C and 25 C since these temperatures would permit the growth of mesOphilic types as well as psychrOphiles. During the course of refrigerated storage, the 4.5 C counts increase. This increase in psychrOphilic organisms is reflected by the increase in counts at 21 C and 25 C but only partially reflected in the 35 C because only a few psychrOphilic types are capable of growth at this temperature. Usually after 5 to 6 days storage, the psychrophilic papulation greatly overshadows the non-psychrophilic p0pulation, hence the close agreement among the 4.5 C, 21 C and 25 C counts. A. SELECTION OF A PEPTONE EXPERDAINTAL The problem of develOping a selective plating medium must be considered along four lines of attack. Primarily, the medium should permit the growth of a given organism so as to obtain readily a visible colony in the shortest time possible. Secondly, it should allow the maximum recovery of viable organisms. Third, it should allow these organisms to exhibit a very short lag phase. Finally the inhibitory agent used must be capable of eliminating, as nearly as possible, all organisms exclusive of the desired group without exhibiting adverse effects upon the desired group. Representatives of four psychrOphilic genera commonly isolated from milk namely, Achromobacter superficiale, Alcaligenes viscosus, Egeudomonas flourescens and Flavobacterium rhenanus were used as test organisms. Colony size determination, total counts, and inhibition studies were carried out on solid media while the relatiVe effects of various nutritional additives were determined in liquid media. There is a large number of peptones on the market that vary in their nutritive values. They have not been tested in media for growing psychrophiles. In an attempt to determine the most suitable peptone for the growth of psychrophilic organisms, a screening procedure was undertaken. A peptone, incorporated into a. SOlid medium, which allowed the given organisms to exhibit "la-31mm colony size in the shortest time and also permit the maximum percentage recovery of the viable organisms would be the “Oat desirable. The following peptones were used in this study: peptmniized milk, trypticase, lactalysate, proteose peptone, milk protein hydrolysate, polypeptone, myosate, tryptose, thiotone, and phytone. To eliminate variables, the various media were made with two per cent of each peptone serving as a sole source of nutrition. Each medium was buffered with monobasic and dibasic potasium salts to a pH of 6.8 so that the total amount of both salts approximated 0.5 per cent. A 24 hour culture was transferred for each day for 4 days in brain heart infusion broth and diluted to approximately 50 to 100 organisms per ml. This dilution was then plated out in triplicate on the various media containing the peptones described above. The plates were incubated at 20 C and counted at 24, 36 and 48 hours. At each counting, the diameter of a representative number of colonies was measured. Since great variation occurred in the diameter of subsurface colonies owing to such factors as 02 tension and thickness of the medium, only the diameters of surface colonies were measured. Percentage recovery of viable organisms on each peptone was (letermined by comparison to a 48 hour plate count on tryptOn.- ggljicose—extract agar incubated at 20 C. This procedure was carried out five times on each peptone medium and with each of the four representative organisms. The results of these five trials were then averaged as shown in Tables 1 to 4 inclusive. TABLE 1 Average colony diameter and percentage recovery of Alcaligenes viscosus at 24, 36, and 48 hours at 20 C using various peptones incorporated into a solid medium Peptone Trypticase 0 heptonized Milk 0 Proteose Peptone 0 hilk Protein Hydrolysate o Lactalysate 0 Polypeptone 0 Tryptose 0.4 Thiotone 0.4 iyo sate 0 hytone 0.4 Average colony diameter in mm at ' ‘24 hrs 36 hrs 48 Eye] R Percentage recovery‘ based on 48 hr plate count of TGE agar 24 hrs 36 hr: 0.1 (0.1 0 0 l4 1 u t | i at 20 C ‘ s 48 hrs 0 21 3 3 41 86 7 38 14 79 41 69 79 103 28 69 86 121 90 121 aura-w. ... ..' TABLL 2 Average colony diameter and percentage recovery of Flavobacterium rhenonus at 24, 36, and 48 hours at 20 C using various peptones incorporated into a solid medium Average colony diameter in mm at Peptone count of TGE agar at 20 C 24 hrs 36 hrs 48 hrs 24 hrs 36 hrs 48 hrs :Trypticase ‘<0.1 0.1 0.1 0 2 11 'Peptonized Milk 0 0.4 0.9 0 56 105 ;Proteose Peptone 0 0.5 1.0 4 65 95 Milk Protein Hydrolysate (0.1 0.4 1.3 3 74 104 Lactalysate 0.1 1.3 2.1 42 105 119 Polypeptone 0.1 1.6 2.2 16 86 95 TryptOse 0.1 1.8 2.2 10 79 i 93 {Thiotone <0.1 1.3 2.3 21 88 91 Ellyosate 0.5 1.6 2.3 46 90 96 [fhytone 1.1 2.9 3.4 86 111 i 112 4 Percentage recovery based on 48 hr plate TABLE 3 Average colony diameter and percentage recovery of Achromobacter superficiale at 24, 36, and 48 hours at 20 C using various peptones incorporated into a solid medium fi—Average colony diameter in mm at Percentage recovery based on 48 hr plate I Peptone count of TGE agar E at 20 C 5 24 hrs 36 hrs 48 hr 24 hrs 36 hrs 48 hrs‘ Trypticase 0 0.6 1.4 ' 0 10 81 Peptonized Milk 0 0 1.5 0 0 133 Protease Peptone o 0.4 1.1 ' o 12 90 Milk Protein Ilydrolysate 0.3 0.6 '1’ 0.9 l 5 5 38 Lactalysate 0 0.8 1.6 0 105 112 P0 lypeptone 0.3 0.5 1.2 * 5 57 95 Tryptose 0.3 0.6 1.5 10 81 114 ThiOtOne 0 005 la]. 0 43 62 iyosote 0.5 0.9 1.6 10 48 86 hyt-one 0.5 ‘ 0.9 2.0 10 100 114 t l i .51]. TABLL 4 Average colony diameter and percentage recovery of Pseudomonas fluorescens at 36 and 48 hours at 20 C using various peptones incorporated into a solid medium Average colony diameter in mm at 1 l l ‘ I Percentage recovery based on 48 hr plate Peptone § count of TGE agar g] at 20 C . afighrs 48 hrs ‘1 .§§_EES 48 hrs Trypticase o (0.1 i 1 o 13 Peptonized Milk 1.0 2.8 i; 50 65 Proteose Peptone 0.5 0.9 2 L 51 89 Milk Protein Hydrolysate 1.0 1.4 64 91 Lactalysate 1.4 1.6 i 73 108 Polypeptoue 1.4 1.9 65 112 Tryptose 1.0 1.3 47 96 hiotone 1.4 1.9 61 105 yosate 1.1 1.6 82 99 lflhytone 1.8 2.3 99 127 fl i i | o RISULTS Examination of Table 1 demonstrates that the average colony diameter of Alcaligenes viscosus is not significantly different through the 36 hour measurement on the media containing tryptose, thiotone, and phytone. At 48 hours, however, the average colony diameter on the medium containing phytone is 0.3 mm greater than that on the tryptose and 0.5 mm greater than that on the thiotone. After 48 hours, only 69 per cent of the total count on tryptone—glucose-extract agar occurred on the medium containing thiotone, while both the tryptose and phytone containing media yielded counts after 48 hours of over 100 per cent. In Table 2 there was little difference between the effect of the peptones thiotone and myosate on colony size and percentage recovery of Flavobacterium rhenanus. The percentage recovery on the phytone medium, although over 100 per cent after 48 hours, is not significantly different from the other two peptones in question. However, a significant margin of difference may be noted with regards to colony diameters. The average colony diameter on the phytone medium was 0.6 n, 1.3 m and' 1.1 mm greater at 24, 36 and 48 hours respectively, than the average diameter of those colonies appearing on the myosate medium. In Table 3, the medium containing phytone yielded larger colonies of Achromobacter superficiale than did the myosate medium. The results indicate a difference of 0.4 mm in colony size as well as a 28 per cent difference in percentage recovery. The number of colonies of Achromobacter superficiale on lactalysate after 48 hours however, growth on M similar to that on the phytone medium. lactalysate mediumns not visible in 24 hours, while on the phytone medium there was a 10 per cent colony recovery in 24 hours. This may be due to either an extended stationary phase by the organism the lactalysate medium or a more rapid logarithmic phase on the In any event, the phytone medium was the most phytone medium. suitable peptone for the growth of Achromobacter superficiale. Pseudomonas fluorescens in Table 4 had a slightly larger colony on the medium containing peptonized milk, than on the medium containing phytone. The percentage recovery of colonies on the former is less than half of the latter. The phytone medium also gave a slight advantage for the growth of Pseudomonas fluorescens over the medium containing polypeptone. DISCUSSION In a general way there appears to be a similarity in the nutritional requirements of the four test organisms, as all appear to demonstrate larger colonies and higher percentage recovery on the phytone medium. Interesting to note is the observation that the peptones tryptose, myosate and phytone foster larger colonies of Alcaligenes viscosus, Flavobacterium rhenanus and Achromobacter augerficiale, than do the other peptones. Again this serves to demonstrate a closer similarity in growth requirements of these three organisms. More striking perhaps is the marked similarity of the sequence of poor to best peptones for the growth of LI 081 igenes viscosus and Flavobacterium rhenanus. The effects of phytone might be due to the presence of certain nutritional elements which promoted a more rapid growth. Possibly these nutrients were lacking or in lesser amounts in the other peptones, and thus account for the less luxuriant growth on the other peptones. The presence of toxic products in the other peptones could conceivably account for the differences in growth of the test organisms on the peptones tested. B. LFFECT 0F NUTRITIONAL ADDITIVES EXPLRIMINTAL Having selected phytone as the base medium peptone, the next step was to ascertain the optimum concentration of the peptone, the effect of additional metabolites and the effect of hydrogen ion con- centration. The method of Darby and liallmann (1939) of minimal inoculum was most suitable for these determinations. The effect of addition or omission of nutrients in a medium for the growth of an organism is most markedly demonstrated during its early phases of growth. Generally accepted is the idea that the lag phase is the most critical stage of growth. Huntington and Winslow (1937) showed that during this stage, the organisms exhibit all the characteristics of physiological youth. Generally, these young cells are more susceptible to adverse conditions. Thus, it is considered of prime importance to know if an organism can adjust to its new environment rapidly, undergo division, and survive. Accordingly, a diagnostic medium should not be evaluated in ‘terms of the final crop of organisms, but rather on the basis of idle behavior of these organisms during the lag and early lOgarith— nlic stages. Consequently, the following studies have been con— dIlcted utilizing minimum number of organisms which were stored at 4.5 C for 24 hours prior to use. The interpretation of results which follows are based on the behavior of the organisms during the lag and early logarithmic stages. Representatives of the four psychrOphilic genera, namely Pseudomonus fluorescens, Alcaligenes viscosus, Flavobacterium rhenanus, and Achromobacter superficiale were used as the test organisms. The procedure for determining the growth rates of these organisms was as follows: a 24 hour culture of each organism was transferred each day for 3 days in brain heart infusion broth. Incubation was at 20 C. Following the 24 hour incubation of the third transfer, each culture was placed in a refrigerator at 4.5 C for 24 hours. It was felt that this 24 hour refrigeration period would more closely approximate the actual conditions of psychrophilic organisms in refrigerated milk. After 24 hOur refrigerated storage, each culture was diluted so that between 20 aid 100 organisms per ml were present. These were seeded into flasks containing 100 ml of the respective media. Initial plate counts were made immediately after the original inoculation and every three hours thereafter up to and through the ninth hour. Another count was then made after 24 hours of incubation. These flasks were incubated at 20 C and subjected to a Inechanical shaking for two minutes prior to sampling. Plating was Inade with tryptose—glucose-extract agar and incubated for 72 hours Ext 20 C. Due to the relatively slight thermal resistance of these (Drganisms, the plating agar was always cooled to 48 C prior to P 1 ating. This procedure was repeated four times for each additive in the medium. Any great degree of variation in one or more of the trials in this series was deemed adequate grounds for repetition of the entire series. The results of the four trials were then averaged and the averages listed in Tables 5 to 24 inclusive. RLSULTS It may be noted in Table 5 that there was virtually no effect on the early stages of growth of Alcaligenes viscosus with increas- ing concentrations of phytone. This serves to illustrate that although a stimulatory effect was absent, the peptone was not toxic in concentration up to 3 per cent. Tables 6 and 7 clearly illustrates that lactose 0.5 per cent, dextrose 0.5 per cent and sodium chloride 0.5 per cent and 0.25 per cent did little to accelerate growth in the early stages of growth of Alcaligenes viscosus. The same was true of beef and yeast extract as seen in Table 8. Table 9 demonstrates that Alcaligenes viscosus was capable of growth over a wide pH range. However, this organism had an Optimum pH between 7.3 to 7.5. In these pH ranges, Alcaligenes viscosus had appeared in slightly greater numbers after 9 hours of growth than in the corresponding period at ph 6.5 to 6.8; and after 24 hours the laumber of cells in this pH range was approximately three times the IIumber of organisms growing in the range of pH 6.5 to 6.8. The effect of varying concentrations of phytone on the lag lDllase of Flavobacterium rhenanus is shown in Table 10. Again there 1'Ela apparently no stimulatory effect as the peptone concentration TABLE 5' Influence of phytone concentration on the early stages of growth of Alcaligenes viscosus gnedium in .-....._. ___.._.....__ T. O E U 1.5 0.275 0.275 27 75 456 2,530 Phy ton e K2HP04 KH2P04 Organisms per ml. ercent com osition 2.0 0.275 0.275 22 84 400 2,870 Phytone 2.5 K2HP04 0.275 KH2P04 0.275 33 89 456 3,000 Phytone K2HP04 KH2P04 3.0 0.275 0.275 28 81 430 3,360 TABLE 6 Influence of lactose and dextrose on the early stages of growth of Alcaligenes viscosus Hqu 5 Medium in Thytone 2.0 #2111304 0.275 K2HI’04 [CH2PO4 0. 275 Kli2ll’04 Lactose 0 22 3 84 6 400 9 2,870 0.275 0.275 0.5 22 95 553 3,660 Organisms per ml. ercent co 2 mposition ‘ Phytone 2.0 Phytone K2IIP04 KH2P04 Dextrose 2.0 0.275 0.275 0.5 26 100 513 3,430 TABLE 7 Influence of sodium chloride on the early stages of growth of Alcaligenes viscosus 2.0 HPO 0.275 2 4 1012204 0.275 0 22 3 84 6 400 9 2,870 “"0111 a“ Medium in Organisms per ml. 0.275 0.5 33 96 407 2,930 Phytone K2HP04 NaCl 2.0 0.275 0.275 0.25 28 95 540 3,200 'fi-r' WWW TABLE 8‘ Influence of beef and yeast extract on the early stages of growth of Alcaligenes viscosus {7 4], Organisms per ml. éHours: - Medium in percent compo 24 Phytone 2.0 Phytone 2.0 ‘K2u204 0.275 K2HP04 0.275 P0 0.275 KII2P04 0.275 2 4 Beef ex. 0.50 22 39 84 95 400 573 2,870 4,333 9,200,000 12,000,000 sition K2HP0 0 KH2P Phytone 2.0 0.275 0.275 4 4 Yeast ex. 0.50 35 100 613 2,950 7,800,000 II- Wham—w-“- 9". _.-..Al ”...—.— ---—-v TABLE 9 Influence of pH on the early stages of growth of Alcaligenes viscosus Hours 24 Phytone 2.0 Yeast ex. 0.5 K HPO FH2P04 pH 6.5 38 114 577 2,662 6,870,000 Organisms per ml. Phytone 2.0 Yeast ex. 0.5 K HPO 1012204 9“ 6‘8 35 100 613 2,950 7,800,000 Medium in percent composition_ Phytone 2.0 Yeast ex. 0.5 :3:::: pH 7.0 34 120 667 4,333 12,200,000 TABLE 9 (continued) Influence of pil on the early stages of growth of Alcaligenes viscosus Organisms per ml . Phytone 2.0 Yeast ex. 0.5 K HPO 2 4 pl! 8.0 10121’04 28 88 420 3,000 flour Medium in ercent com osition Phy tone 2 . 0 Phy ton e 2 . 0 Phy tone 2 . 0 Yeast ex. 0.5 Yeast ex. 0.5 Yeast ex. 0.5 K 2112904 K2HI’O K2HPO pH 7.3 pH 7.5 A pH 7.8 1(1121’04 10121’04 K112P04 0 29 40 29 3 141 136 111 6 696 637 640 9 5,700 5,067 2,900 24 21,400,000 20,000,000 11,300,000 11,000,000 TABLE 10 Influence of peptone concentration on the early stages 01' growth of Flavobacterium rhenanus Hours '1 l Hod ium in perce Organisms per ml . ! ghytone l .5 2HPO4 0 .275 114112204 0.275 73 183 983 4,830 l’nytone 2 . U haflP04 0.275 10121’04 0 . 275 72 177 827 5,600 1t “‘—“ ition Phytone 2.5 1(2111’04 0.275 lfll2PO4 0.275 74 150 765 4,600 Phy ton e 1(2111’04 [(1121’02 3.0 0.275 0.275 64 156 760 4,600 TABLE 11 Influence of lactose and dextrose on the early stages of growth or Flavobacterium rnenanus Hours Phytone 22304 mi2P04 2.0 0.275 0.275) 72 177 827 5,600 Phytone 112111’04 1012P04 Lactose ‘ Organisms per m1. Medium in Eercent comgosition 2.0 0.275 0.275 0.5 64 143 810 7,500 Phytone K2HPO4 KH2PO4 Dextrose 2.0 0.275 0.275 0.5 58 135 1,027 7,000 ii: ‘IPWiTT'QK-‘zr—fir“ . Influence of sodium chloride on the early TABLE 12 stages of growth of Flavobacterium rhenanus Organisms per ml. Hours Medium in percent composition Phytone 2.0 Phytone 2.0 Phytone 112111’04 0.275 K2HPO4 0.275 K2IIPO4 K112PO4 0.275 101le4 0.275 Kll2P04 N 1101 U o 5.. NaCl 0 72 65 3 177 120 6 827 703 9 5,600 6,000 2.0 0.275 0.275 0.25 51 120 703 6,000 HHWW TABLE 13 Influence of beef and yeast extract on tne early stages of growth of Flavobacterium rhenanus Hours Organisms per m . Medium in percent A ““"ition ”55"— 24 2 ‘Phytone 2.0 , . 7 2HPO4 O 2 5 KM P0 0.275 4 72 17‘ 827 5,600 25,000,000 Phytone 2.0 Phytone 2.0 K2HP04 0.275 K2flP04 0.275 KH2P04 0.275 KH2P04 0.275 Beef ext. 0.50 Yeast ext. 0.50 73 73 154 161 649 683 '4,200 5,833 45,000,000 69,000,000 '1? 3:19.32" 22‘. . “Devin—.1 TABLE 14 Influence of pH on the early stages of growth of Flavobacterium rhenanus II uvus q Phytone 2.0 Yeast ex. 0.5 K HPO p11 6.5 KH2P04 53 115 513 5,200 62,000,000 Phytone 2.0 Yeast ex. 0.5 K2HPO pH 6.8 KH2P04 73 167 683 5,833 69,000,000 Organisms per ml. ledium in percent composition Phytone 2.0 Yeast ex. 0.5 K HPO p“ 7.0 KH2PO4 47 116 533 5,233 67,000,000 Phytone 2.0 Yeast ex. 0.5 KZHPO 4 pH 7.2 EH2P04 56 127 773 7,433 70,000,000 TABLE 14 (continued) Influence of pH on the early stages of growth of Flavobacterium rhenanus Organisms per m1. ‘ Hours Medi Phytone 2.0 Phytone 2.0 Yeast ex. 0.5 Yeast ex. 0.5 K HPO K HPO4 pH 7.5 pH 7.8 KH2PO4 KH2PO4 O 66 75 3 176 201 6 893 963 9 11,134 7,233 24 81,000,000 69,000,000 um in percent composition _ Phytone 2.0 Yeast ex. 0.5 K HPO4 KH2PO4 pH 8.0 75 209 853 7,400 64,000,000 TABLE 15 Influence of peptone concentration on the early phases of growth of Achromobacter superficiale Organisms per ml. 1| Medium in ercent composition Phytone 1.5 Phytone 2.0 Phytone 2.5 Phytone 3.0 2HPO4 0.275 K211PO4 0.275 K2HPO4 0.275 K2IIPO4 0.275 KH2PO4 0.275 KH2PO4 0.275 KH2PO4 0.275 KH2P04 0.275 0 32 31 3O 32 3 76 86 79 85 6 580 573 570 493 9 2,300 3,970 2,500 2,530 Influence TABLE 16 of lactose and dextrose on the early stages of growth of Achromobacater superficiale Organisms per m1. in percent composition Hours Medium Phytone 2.0 Phytone K2hPO4 0.275 K2HI’O4 KB2PO4 0.275 Kll2PO4 Lactose O 31 3 86 6 573 9 3,970 2.0 0.275 0.275 0.5 29 86 647 3,433 Phytone K21m04 KH2P04 Dextrose 2.0 0.275 0.275 0.5 27 95 593 3,733 Influence of sodium chloride on the early TABLE 1? stages of growth of Achromobacter superficiale Bourq Phytone 2.0 K2HPO4 0.275 KH2PO4 0.275 0 31 3 86 6 573 9 3,970 Or anisms Phytone K2HPO4 KH2PO4 NaCl er ml. 2.0 0,215, 0.275 0.5 29 99 470 3,100 ’ Phytone K2HP04 KH2P04 NaCl Medium in percent composition 2.0 0.275 0.275 0.25 28 101 543 3,700 TABLE 18 Influence of beef and yeast extract on the early stages of growth of Achromobacter superficiale In“... Organi sms pe_r ml . 24 H Medium in percent concentration Phytone 2.0 Phytone 2.0 Phytone 2.0 K2HPO4 0.275 K211PO4 0.275 1(2111’04 0.27.5 Kll2PO4 0.275 l1id base medium described before. Preliminary studies indicated that the dyes were not suitable. 111 general, the concentrations of the various dyes necessary for ‘blle inhibition of the gram positive test organisms were also illhibitory to the psychrOphilic organisms. It was also found that sodium lauryl sulfate in concentrations greater thanlleOOO a'ystalized and became ineffective at 20 C. NELcconol N.R.S.F. was unaffected. This compound was found to be ‘ECIually as inhibitory as lauryl sulfate against gram positives in fiitndies by Mallmann and Darby (1941). Accordingly tests are re- POrted on this compound. To test the inhibitory action of Nacconol N.R.S.F. against * Product of Allied Chemical and Dye Corporation a representative gram positive flora of milk, representative Organisms were seeded into the base medium containing concentrations 01? Nacconol N.R.S.F. of l:100,000, l:50,000, l:10,000, 1:5,000 and 1 : 1000. The following organisms, representative of the gram positive flora of milk, were used: Streptococcus lactis, Streptococcus 1i. uefaciens, Streptococcus faecalis, Micrococcus _p. 102, Bacillus subtilis, Leuconostoc mesenteroides, Leuconostoc citroverum, Lactobacillus casei and Lactobacillus acidophilis. A 1 ml dilution, containing 50 to 100 organisms of a 24 hour culture which had been transfered for three days in brain heart infusion broth was used as the inoculum. Three separate trials, using triplicate plates were made with the base medium containing Various concentrations of the dyes and surface active agents. The plates were incubated at 20 C and observed after 48 and 72 hours, the counts were averaged and recorded in terms of percentage recovery as compared to the counts obtained on the base medium alone. This procedure was repeated using the four representative psychrOphilic organisms Alcaligenes viscosus, Flavobacterium x‘henanus, Achromobacter superficiale and Pseudomonas fluorescens. RESULTS AND DISCUSSION The results are shown in Table 25. Nacconol N.R.S.F. at a Concentration of l:10,000 in the base medium completely inhibited the gram positive test organism for 72 hours. On the other hand Alcaligenes viscosus, Achromobacter su erficiale, Flavobacterium afikoum o: * o o o o o o 0 Hana o o o o o o o Baum o o o o o o o soda" we we mv nv we no we Bonn” cog oou oou ooH cod con cod Becaua .3 .m: 2: 2: 2: 2: 2: 2: 2: o lMMNMHNMfiMMMIgonso Ebho>oa9meovwohoesbnsE mc~.mM\wsooou mwuwvnsm nsomOdmosv. awasoomu .A .A .A .A Iouowz .m .m .w essoo oesaatusos mp so some: namwsdwuo cansfib mo hho>oooa Home tom manusswao anew o>uaumoa Edam mo hao>oooa owaasooaoa one no saunas omen wagon a owns coasteauoosu .h. w m. z mosooooz mo uncapoaosoosou wsnhad> «o oeommfl mw mum<9 rhenanus and Pseudomonas fluorescens were uneffected at concentra- tions as great as 1:1,000. The investigatidn thus far suggested that the base medium containing l:l0,000 Nacconol N.R.S.F. might be used as a selective medium for psychrOphilic organisms in milk. Fr . ...... - A-;. .. D. PRELIMINARY TEST OF THL PHYTONLPNACCUNOL MEDIUM LXPLR ILflN TAL It was now necessary to determine if this concentration (1:10,000) of Nacconol N.R.S.F. would be adequate in preventing the growth of the gram positive flora of milk under practical conditions. Nacconol N.R.S.F. in final concentrations of l:l0,000, 125,000 and 1:1000 was added to the base medium. These media were then tested on 29 milk samples collected from the Lansing area. One ml quantities were plated out on three media and incubated at 20 C for three days. Colonies were picked at random and streaked on tryptone-glucose-extract agar slants and incubated at room temperature for 48 hours. The Cultures were then gram stained and transfered onto another tryptone—glucose—extract slant and incubated at 4.5 C for seven days in order to determine if the organisms isolated from the Phytone—Nacconol medium were psychrOphilic. These studies indicated that the media containing Nacconol N.R.S.F. in a concentration of l:l0,000 and 1:5,000 were not sufficiently selective. The relatively large number of gram positive isolates from these two media justified discarding these in favor of the medium containing a concentration of Nacconol n.R.b.F. of 1:1,000. This latter medium demonstrated marked selectivity as demonstrated in Table 26. .t“ " mm m' 1 Hr! v TABLE 26 Isolation of organisms from solid base medium with 131,000 concentration of Nacconol N. R. S. F. [Number Per Cent Organisms isolated 204 Gram negative rods 186 91 Other organisns" 18 9 Organisms showing growth at 4.5 C in 7 days 164 84 * Gram variable cocci Gram positive cocci Gram negative cocci RESULTS AND DISCUSSION Although a 1310,000 concentration of Nacconol N.R.S.F. was shown to be effectiVe against a gram positive flora in pure culture studies, it did not exhibit sufficient inhibitory properties against the gram positive flora in milk. Presumably this is due to the "tying up" of Nacconol N.R.S.F. with various constituents of milk or the presence of gram positive organisms in the milk which are resistant to the action of Nacconol N.R.S.F. In Table 26, it may be seen that of the 204 isolates from the base medium with a 1:1000 concentration of Nacconol N.R.S.F., 91 per cent were identified as gram negative rods while 84 per cent were capable of growth at 4.5 C. From the practical viewpoint, these results were considered to be adequate for a selective medium. Thus, the base medium contain- ing Nacconol N.R.S.F. in a. final concentration of 1:1000 was then employed in a field test. E. PHYTONE—NACCONOL AGAR VS. TRXPTONE—GLUCOSE—LXTRACT AGAR EXPLRIMLNTAL In order to test the effectiveness of the medium as compared to tdie method described in gagggggjilflgthods for the hgagination of lyiiqz_gnggggt§ for the enumeration of psychrophilic bacteria, the following investigation was carried out. Fbrty-five fresh pasteurized milk samples were obtained from clairies in the Lansing area. Replicate samples were plated accord- ing to Standard Methgdp on tryptone—glucose-extract agar and on Phytone-Nacconol agar of the following composition: Phytone 20.0 gms Yeast thract 5.0 gms K2HPO4 5.0 gms Iii-1214).4 0.1 gms Nacconol N.R.S.F. 1.0 gms Agar 15.0 gms. Distilled water 1000 ml Replicate tryptone-glucose~extract agar plates were incubated at 35 C for 2 days, 25 C for 3 days, 20 C for 4 days and 4.5 C for 7 days. Phytone-Nacconol agar plates were incubated at 20 C for 2 days. RESULTS A representative sample of the data is shown in Table 27. While this table does not include all the results obtained, those not included demonstrated the same trend. A count on tryptone—glucose—extract agar at 25 C for 3 days and 20 C for 4 days has been recently recommended for the estimation of Comparison of counts obtained on tryptose glucose extract agar incubated at 35 C for 2 days, 25 C for 3 days, 20 C for 4 days and 4.5 C for 7 days TABLE 27 Sample # 35 C 257C 20 C 4.5 C 1 2100 3800 3500 520 2 4500 11200 2000 4 3 15000 20500 7500 6 4 2800 3200 1700 1 5 20300 7200 1350 4 6 1300 3100 2500 210 7 13000 21100 14400 2 8 80 70 60 1 9 300 260 160 3 10 1800 1570 710 0 11 17100 20400 12000 12300 12 15100 16700 11000 2600 13 19800 22500 17200 6400 14 7300 7700 9100 4 15 5800 8900 6300 10 16 220 58 311~ 2 17 1700 1700 370 17 18 3400 6200 3800 1 19 6400 10400 6200 1 20 4900 8100 6300 90 21 2100 3200 1600 O TABLE 28 Statistical analysis of psychrOphile counts on tryptose glucose extract agar and Phytone-Nacconol agar Sample P-N 1‘0? Sample P—N fin ar ar ' fference # ar ar Difference 1 3 o 3 24 0 o o 2 42 40 2 25 70 30 40 3 50 40 10 26 24 15 9 4 21 6 15 27 0 o 0 5 1000 11200 -200 28 2 520 -518 6 60 53 7 29 3 4 -1 7 67 61 6 30 27 6 21 s 17 2 15 31 13 1 12 9 5 3 2 32 4 4 0 10 1500 1200 -300 33 120 210 —90 11 38 9 29 34 13 2 11 12 5600 6200 -600 35 0 1 -1 13 27 43 -16 36 5 3 -2 14 23 7 16 37 3 0 3 15 210 580 -370 38 30 4 26 16 210 21 189 39 31 10 21 17 570 620 -50 4o 9 2 7 18 26 130 —104 41 18 17 1 19 200 320 -120 42 0 1 -1 20 17 3 14 43 15 1 14 21 80 10 70 44 82 90 -8 22 7 o 7 45 6 0 __ __6_ ___ 23 40 40 o a = _1825 £d2= 985,755 t _ a :fld = 310.556 —0 s d ' n 45 t . _1.373 (gfgpsychrophilic organisms. These incubation times and temperatures yield reliable information on stored milk with high psychrOphilic counts. However, it is questionable if the same reliability exists fer the enumeration of psychrophilic organisms in freshly pasteur- ized milk. For this reason, the 25 C count for 3 days and the 20 C count for 4 days have been included in this study. It may be seen that counts at 25 C for 3 days and 20 C for ‘1 days on tryptone-glucese-extract agar on freshly pasteurized milk (are for the most part a reflection of the 35 C count. As such, these counts offer little information concerning the psychrophilic papulation. Table 28 represents a statistical analysis of the counts obtained at 20 C for 2 days on Phytone-Nacconol agar medium as compared to the counts obtained by incubation for 7 days on tryptone-glucose-extract agar at 4.5 C. DISCUSSION In the preparation of this table, the.t test as described 65 Stearman (1955) was used to test the differences in the two methods. The formula for the-t test is as follows: tad-ed g s d n — sum of the differences were d B “..-. om number of samples —1 32d 8 sum of {differences -d) number of sample -1 n - number of samples The hypothesis on this test is that there is no difference be- ween the two treatments; with this hypothesis pd=0. Alternatives to this hypothesis-'0 that the mean is greater than zero, in which case the counts obtained on the Phytone-Nacconol medium will be higher; or that the mean is less than zero, whereupon the counts on tryptone-glucose-agar will be higher. Using a 5 per cent level of significance and since there are 45 differences in the sample, there will be 45 -l = 44 degrees of freedom. From the 3 table, 3 with 5 per cent level of significance and 44 degrees of freedom is 2.016. Thus the critical region of the test will be values of i which are less than ~2.0l6 or greater than +2.016. The value of t as calculated by the equation is -l.373. This does not fall into the critical region of the test; therefore the mean plate counts of the two different methods are not significantly di fferent, statistically. F. THE USE OF TLTRAZOLIUM SALTS AS A COLORING AGENT FOR PSYCHROPHILLS ON PMYTONE—NACCONOL AGAR EXPERIMLNTAL During the course of this investigation, extreme care was exercised in counting colonies, particularly on those plates where 1 ml quantities of milk were used. The Opacity of the medium in these cases definitely impeded counting. In an attempt to alleviate tJiis situation, various dyes were screened as coloring agents for psychrophilic organisms. A reduction indicator, 2, 3, 5 triphenyl tetrazolium chloride (TTC) was selected. When .001 gm was incorporated into 100 ml of tryptone-glucose—extract agar and used to enumerate psychrophiles at 4.5 C for 7 days, it was found that almost without exception all psychrophiles reduced triphenyl tetrazolium chloride from colorless to red. This concentration of triphenyl tetrazolium chloride was ilicorporated into the Phytone—Nacconol medium. Forty-five milk samples were used to determine psychrOphilic numbers with this medium as compared to the Phytone—Nacconol medium without triphenyl tetrazol ium chloride . RESULTS AND DISCUSSION It was observed that there was little if any significant differences between the two media. Although no attempt was made to measure the size of the colonies on the two media, it appeared that the colonies on the medium containing the tetrazolium salts were larger. Presumably this was an Optical illusion resulting un—nuu—‘W from the vivid red colonies on a white background. There could be no dispute that the addition of the tetrazolium salts to Phytone—Nacconol agar definitely facilitated the counting of colonies; ww-s;au ”“- SUMMARY Of the peptones tested, phytone was found to be the most suit— able peptone for the growth of Alcaligenes viscosus, Flavobacterium rhenanus, Achromobacter superficiale and Pseudomonas fluorescens. A 2 per cent concentration of phytone was found to be Optimum, while concentrations up to 3 per cent did not demonstrate any marked toxicity for the growth of the test organisms. The addition of 0.5 per cent yeast extract to the medium was slightly stimulatory for the growth of Pseudomonas fluorescens. Marked differences were not apparent during the early stages of growth of these organisms grown on media adjusted from pH 6.5 to 8.0. A ph of 7.5, however, was found to yield a decidedly higher cdony coum;at 24 hours with all the organisms tested. Nacconol N.R.S.F. incorporated into the solid base medium in a final concentration of 131000 was found to be inhibitory to the gram positive flora of milk. There was no apparent effect on the representative psychrophilic organisms. To facilitate counting, 2, 3, 5 triphenyl tetrazolium chloride was added to the medium as a coloring agent. Almost without exception, psychrophilic organisms reduced TTC. A comparison of media with and without TTC showed little, if any, difference in the total numbers of colonies obtained. Phytone-Nacconol agar is suggested as a rapid selective method for the enumeration of psychrophilic organisms in freshly pasteurized milk. 2, 3, 5 triphenyl tetrazolium chloride, (1 cc of a sterile 1:100 solution per 100 ml of media) may be added to Phytone- Nacconol agar just prior to pouring plates. PsychrOphiles will reduce TTC. The red colored colonies thus formed facilitates counting. When tested, plate counts on this medium were similar statistically to those obtained by incubation at 4.5 C for 7 days on tryptose-glucose—extract agar. PART II A TI‘ST FOR DETEIMINING T111; iCLEPING QUALITY OF MILK LITERATURL REVILW The keeping quality of refrigerated pasteurized milk is largely determined by the number and activity of the psychrophilic popula— tion of the milk. In general, the number of psychrophiles in fresh- ly bottled milk is not considered a reliable index of the shelf life of the product,(Burgwald and Josephson 1947), Dahlberg 23 El (1953), Olsen 9193 (1953), Atherton 333; (1954). Wide variations occur in both the initial number of psychro- philes and the number at the time of spoilage. The organisms vary in their action on the various milk constituents as well as in the amount of by—products contributing to flavor defects. Occasionally, so-Called "inert" psychrOphiles reach high population levels with- out noticeable flavor defects. The psychrophilic population Of the milk prior to pasteurization reduces the shelf life of the pasteurized product. Weber (1956) reported that milk which had a high psychrophilic count before pasteurization would not keep as long as milk with relatively low counts prior to pasteurization. Added to these factors are inherent differences in growth rates at refrigeration temperatures of the various psychrOphiles. Day and Doan (1956) studied chemical tests for measuring flavor defects of milk. Changes in protein stability, acidity, and variations in nitrogen distribution of the non-casein fractions were too slight and inconsistent. Significant changes occurred too near the time of pronounced flavor defects to be of practical value. The methylene blue test has been used for many years as an ”mg a» indicator of bacterial activity and as such, gives a misleading indication of keeping quality. In some early studies, disco: gt_gl (1932) deviated from the usual standard of acid production as a criterion of keeping quality and used instead taste determination. They demonstrated a marked correlation between the reduction of methylene blue at 15.5 C and the keeping quality of milk at the same temperature. Almost without exception, as soon as the reduc— tion was complete, a flavor defect could be detected in a portion of milk reserved for tasting. This modification of the methylene blue test differed from others in the temperature of incubation. Thus, the test measured only the biochemical activity of organisms capable of relatively rapid growth at low temperature. The close agreement between reduction time and keeping quality provided a clue to the present investigation. A test which would indicate an extremely small pOpulation, i.e.- less than one organism per ml — as well as a relatively high p0p- ulation, and give an estimate of the activity of psychrOphilic organisms, might be used in predicting the keeping quality of milk. A test of such delicacy could be accomplished best by an enrich- ment technique using the natural substratum, the milk under test. EXEEBIIINIAL Earlier studies were conducted using 2, 3, 5 triphenyl tet- razolium chloride (TTC) as a coloring agent to facilitate counting of colonies where Opacity of the medium was a problem. Without exception, psychrOphiles growing on tryptose-glucose-extract agar, reduced TTC. A series of tests with pure cultures of psychrophiles comparing resazurin and TTC indicated that the latter was more sensitive and thus would be more suitable. 0n the basis of these studies, TTC in a concentration of l-l0,000 was selected as the reduction indicator for the demonstration of bacterial activity. To shorten the test time, the activity of the organisms had to be accelerated. As the minimum generation time of these organisms is at approximately 20 C, this temperature was selected as the incubation temperature for the test. An incubation temperature of 20 C is not selective for psychrOphiles and consequently the interfering organisms had to be inhibited. In the process of developing a selective plating medium for the enumeration of psychrOphiles, various surface active agents were tried. Nacconol N.R.S.F.* was shown by Mallmann and Darby (1941) to have similar inhibitory properties as sodium lauryl sulfate. Sodium lauryl sulfate, when tested, was found unsuitable for this plating medium because at the inhibitory concentration, it had a tendency to crystalize at 20 C. Consequently, Nacconol N.R.S.F. in a concentration of l-lOOO was selected. At this cencentration the surface active agent inhibited the gram positive organisms without exhibiting any adverse effect on the gram negatives. Because the gram negative pOpulations of milk is predominantly psychrophiles (Boyd, 1953), those not psychrophilic were minor and would likely not interfere seriously with the test. A buffer system was found necessary because some psychrophiles, * Product of Allied Dye and Chemical Company .grwnfnm ..I_:.:.=i_' ‘W ' v...“— being acid producers, lowered the ph of the milk and consequently interferred with the reduction of TTC. The addition of 0.5 per centK.2HP04 and 0.001 per cent KH2 milk from 7 to 7.5 depending upon the milk in question. P04would maintain the pH of the The indicator solution was prepared as follows: 2, 3, 5 Triphenyl tetrazolium chloride 0.1 gm. Nacconol N.R.S.F. 1.0 gm. KEHP04 5 gm. K112P04 0.1 gm. Distilled water to make 100.0 ml. Solution placed in dark bottle and autoclaved at 121 C for 15 minutes Sterile solution stored at room temperature In the performance of the test, 1 ml quantities of the Nacconol - TTC solution were pipetted into sterile tubes to which were added 10 ml samples of the milk in question. The tubes were shaken and incubated at 20 C and examined after 12, 24, 36 and 48 hours. The presence of a pale pink to rose red color was reported as positive at the particular time of reading. Occasionally a pink button was fonmed at the bottom of the tube. This was con- sidered negative in the absence of a pale pink color throughout the tube of milk. Twenty—three lots of fresh pasteurized milk were obtained from 12 dairies in the Lansing area. Three quarts of each lot were pooled in a large sterile vessel to eliminate possible variations in different quart bottles. This milk was then dispensed into 10 sterile half pint bottles which were chilled prior to filling. The bottles were capped aseptically and immediately stored at 4.5 C. Duplicate Nacconol - TTC tests were made on all samples _ w L._ “*3”. .3 1.1..-..'t--':-h, J} J' a531,.“ . V ' ~ " immediately following bottling. Replicate platings were made on tryptOse-glucose-extract milk agar. Sets of plates were incubated at 35 C for 2 days, 25 C for 3 days, 20 C for 4 days and 4.5 C for 7 days. Preliminary testing had demonstrated that most of the milks in the Lansing area were of relatively good quality. Therefore, in order to approximate conditions of poor quality, samples from each source were held at refrigeration temperatures for varying periods to allow an increase of psychrOphiles prior to testing. Milk samples were tested organoleptically on alternate days. Questionable samples were tested again the following day. An undisturbed bottle, representative of each lot, was used for each sampling. Bottles were coded to avoid bias. Prior to tasting, a portion was removed aseptically for measuring the number of psychrOphiles present. RLSULTS AND DISCUSSION In general, the initial psychrOphilic bacterial counts were not directly correlated with keeping qualities of the milk samples tested. These results are similar to those obtained by other investigators cited. There is, however, a correlation if broad ranges of psychrophiles are bracketed. Milks with initial psychrOphilic counts of less than 10 per ml exhibited a greater refrigerated storage life than milks with counts in the range of 10 to 10,000 per ml. These in turn showed a longer refrigerated shelf life than those which had counts ranging from 10,000 to 100,000,000 per ml. .j': i I l . w L . I “i3“fl!il ' , I. 'n . A — ‘ m a . A. -,v ‘ . p ‘1 . . .1 ‘ a g .4 - T k‘ I, 4. 101‘ T ‘ . ' V- J.'- .‘ L' ‘1 There is a relationship between the psychrOphilic counts of the milk at the time of spoilage, with few exceptions, and the presence of off-flavors. The counts at the time of the appearance of off- flavors ranged from 8 x 106 to l x 108 per ml. However, (Table 4) initial psychrOphile counts in milk approaching the above range did not necessarily have flavor defects. These high initial counts were a good indication that the samples refrigerated shelf life would be very short. Samples 31, 34, 36 and 39 (Table 3) had initial counts in the millions, yet they remained acceptable for 11, 10, 9 and 8 days respectively. These exceptions serve to point out the importance of the various types of organisms involved. These same samples demonstrate the importance of the difference in growth rates of various species. Assuming the generation time of a psychrOphilic pOpulation to range from 4 to 14 hours, it would take 20 to 70 hours for a pOpulation of 5 x 106 (acceptable flavor) to change to a pOpulations of 1.6 x 108 per ml (flavor already unacceptable). Within reasonable limits, the initial and final psychrOphilic counts, presented in Table 4, appear to fit into the above groupings. However, an examination of samples 31, 34, 36 and 39 illustrate some obvious discrepancies, because between 8 and 11 days were needed for various initial pOpulations of 2 x 106, 3 x 105, 6.9 x 106 and 2.7 x 106 to final pOpulations of 3.3 x 108, 6 7 1-2 3 10 , 1-3 I 10 and 3.3 x 108 respectively. These observa~ tions demonstrate the importance of generation time of a psychro- philic pOpulation. The results presented in Table l to 4 inclusive show the relationship between the positive Nacconol - TTC test and the keep- ing quality of various samples of milk. When a positive test was obtained in 48 hours, (Table l) the average keeping time of 5 samples was 15.6 days. The average keeping time of 14 samples showing a positive test in 36 hours (Table 2) was 12.6 days. hleven samples which gave a positive test in 24 hours (Table 3) had an average keeping—time of 8.8 days, while the 9 samples which exhibited a positive test in 24 hours (Table 4) had a refrigerated shelf life of 4.2 days. The data in Table 4 appear to confirm the work of Day and Doan (1956) who demonstrated reduction of neotetrazolium 3.75 days in advance of flavor spoilage. A direct relationship between reduction time and keeping quality is demonstrated in Figure 1 in which the average keeping time of the various milk samples was plotted against the period in which a positive test was obtained. 0f the 19 samples (Table 1 and 2) which have a prediéted keeping quality of 12.6 days or more, only 4 failed to meet this prediction, but even these had a refrigerated shelf life of 9 days. On the other hand, of the 20 samples (Tables 3 and 4) judged to haVe poor keeping quality, 19 exhibited a refrigerated shelf life of less than 12 days. On the basis of these results, milk samples which exhibited a positive test in 24 hours or less had poor keeping quality (a shelf life at 4.5 C of less than 12.6 days). A positive test in 12 hours would indicate very poor keeping quality. A negative test in 24 TABLE 1 Measurement of keeping quality by the Nacconol-TCC test of commercially bottled milk stored at 4.5 C Samples showing positive test in 48 hours Sample Total keeping Initial count Count at spoilage Off flavofi #' time in days 37 C 4.5 C 4.5 C 8 18 80 1 210,000,000 bitter 10 18 1,800 o 510,000,000 fermented 16 17 220 2 330,000,000 sour 17 10 1,700 17 420,000,000 fruity 18 15 3,400 1 690,000,000 sour ézszgge 15.60 TABLE 2 Measurement of keeping quality by the Nacconol-TCC test of commercially bottled milk stored at 4.5 C Samples showing positive test in 36 hours Sample Total keepingl Initial count Count at spoilage Off flavor # time in days 37 C 4.5 C . 1 9 2,100 520 890,000,000 sour 2 13 4,500 4 80,000,000 bitter 3 19 15,000 6 12,000,000 fruity 4 18 2,800 1 7,000,000 fermented 5 13 20,000 4 260,000,000 bitter 7 13 13,000 2 180,000,000 fruity 9 9 300 3 270,000,000 rancid 12 10 15,100 11,000 460,000,000 rancid 15 12 5,800 10 100,000,000 bitter 19 13 6,400 1 250,000,000 bitter 20 9 4,900 90 210,000,000 bitter 21 15 2,100 0 220,000,000 fermented 23 10 1,000 1,000 210,000,000 bitter 26 13 25,000 8,000 12,000,000 fermented Average 12.57 TABLE 3 Measurement of keeping quality by the Nacconol—TCC test of commercially bottled milk stored at 4.5 C Samples showing positive test in 24 hours 7 Total 4W keeping count at Sample time in . Initial count _ spoilage Off flavor # days 37 C 4.5 C 4.5 C 6 10 1,300 210 340,000,000 sour 11 9 17,100 12,000 1,000,000,000 bitter 13 10 19,800 6,400 1,100,000,000 bitter 25 7 23,000 510,000 80,000,000 bitter 29 12 1,800 47,000 210,000,000 fermented 31 11 2,400,000 20,000,000 330,000,000 sour 33 4 330,000 3,500,000 80,000,000 bitter 34 10 100,000 3,000,000 12,000,000 fermented 36 9 69,000,000 180,000,000 fruity 39 8 6,900,000 27,000,000 330,000,000 sour 40 7 100,000 100,000 7,000,000 fermented Average 8.82 awn-"M“- .11-..-qu TABLE 4 Measurement of keeping quality by the Nacconol-TTC test of commercially bottled milk stored at 4.5 C Samples showing positive test in 12 hours Total keeping .L_ count at Sagple t1::I;n agnétial ggfig§_6_+ spZigage Off flavor 22 3 1,200,000 27,000,000 250,000,000 bitter 24 3 3,800,000 30,000,000 890,000,000 sour 30 3 540,000 24,800,000 1,000,000,000 bitter 35 2 14,000,000 190,000,000 340,000,000 sour 14 4 100,000 33,000,000 260,000,000 bitter 0 37 7 14,000,000 25,000,000 512,000,000 fermented 38 5 3,200,000 80,000,000 690,000,000 sour 41 6 164,000,000 190,000,000 210,000,000 fermented 43 5 40,000,000 106,000,000 210,000,000 bitter Average 4.22 '11.”- FOZCOO>Z H