THE VIABILITY OF MICROORGANISMS ISOLATED FROM FRUITS AND VEGETABLES WHEN FROZEN IN DIFFERENT MENSTRUA BY ALEXANDER HUNTER JONES A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology and public Health 1950 ProQuest Number: 10008698 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008698 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ACKNOWLEDGMENTS The writer wishes to express appreciation to Dr. A. G. Lochhead, Dominion Agricultural Bacteriologist and Dr. C. K. Johns, Senior Bacteriologist, Division of Bacteriology and Dairy Research, Science Service, Department of Agriculture, Government of Canada for their interest and criticism at various times in the course of this study. The writer is also deeply grateful to Dr. F. W. Fabian, Professor of Bacteriology and Public Health for his many helpful suggestions in this study. Thanks are also due to the Division of Horticulture, Central Experimental Farm, for the use of their freezer facilities and for the technical advice by personnel of that Division during the course of this investigation. In the analytical portion of these studies thanks are also due to Mr. W. E. Ferguson, Technical Officer, Division of Bacteriology and Dairy Research, for his assistance. 244589 TABLE OF CONTENTS Page INTRODUCTION ...................................... 1 REVIEW OF THE LITERATURE Frozen Fruits and Vegetables ........... 4 Pure Culture Studies ............................. 9 Discussion of the Literature Review ............. 19 INVESTIGATIONAL FRUIT AND VEGETABLE PRODUCTS Procedure for analysis of fresh and frozen products ....... 21 Results of analyses of products ................ 23 Slow freezing vs. quick freezing on numbers of microorganisms in frozen fruits and vegetables .. 33 PURE CULTURE STUDIES Methods ......................................... 37 Preparation of cultures for freezing ........... 39 Effect of freezing on bacterial cells .......... 45 Effect of freezing different species of bacteria in water at 0°F. (-17.8°C.) ..................... 47 Effect of freezing three cultures of Escherichia coli ............................................ 57 Effect of freezing bacteria in different substrates ................... 59 Discussion of fluctuations in counts ........... 82 Comparison of survival of spores and vegetative stages of bacteria subjected to freezing ....... 85 Survival of bacteria frozen in different phases of growth at 0°F ..................... 89 Table of Contents (continued) Page Survival of microorganisms frozen continuously and intermittently in water at different temperatures .................................... 93 Tbe effect of antibiotics on bacteria in vegetable products .............................. 105 DISCUSSION ......................................... 109 S U M M A R Y ........................................... 115 LITERATURE CITED .................................. 119 INTRODUCTION The effect of low temperatures on microorganisms in fruits and vegetables has been a subject of study by many investigators. It is now well known that the microbial content of these products undergoes con­ siderable change in the process of freezing and subse­ quent freezing storage, and it is generally agreed that freezing temperatures result in a reduction of the initial microbial load. The rate of reduction varies tremendously, both in similar and different products, and is dependent upon a variety of conditions associated with the freezing process. The present study is concerned with a number of phases of the problem, to determine the extent which the various factors contribute either to the protection or the destruction of microorganisms during freezing. The initial microbial load on fruits and vegetables is comprised of many species of bacteria, yeasts and moulds. Primarily, these organisms are soil types and have little, if any, public health significance. However, if the products are held, either before or after freezing, under conditions favourable for the development of microorganisms, undesirable changes take place in the product and in a comparatively short - 2 - period of time tiie product lias spoiled* The rate of spoilage is dependent to a large extent on the numbers and types of microorganisms present. Thus, any measures taken with the initial product to reduce the microbial load will control, to some extent, the rate of spoilage following defrosting of the product. Such practices as washing and blanching, particularly the latter, have a very great influence on the numbers and types of microorganisms present on the product going into the freezer. Properly conducted, the blanching process should reduce the microbial load in excess of 99 per cent. However, even although the blanching is adequate, faulty practices in many commercial plants are respons­ ible for recontamination of products following the blanch process. Recontamination occurs largely from conveyances of the product, such as flumes or belts, or by the handling of the products by workers during inspection or packaging. Pure culture studies of microorganisms which have been made by many workers have, unfortunately, been limited to comparatively few species of bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhosa and a few other well known organisms. While such studies are of interest from an academic viewpoint the writer has found that results of these studies have little, if any, bearing on the effect of freezing - 3 - on the heterogeneous microbial flora found on fruits and vegetables. The present investigation is a continuation of previous studies conducted by the writer on the microbiology of frozen fruits and vegetables with particular emphasis on pure culture studies of organisms found on fruits and vegetables - 4 - REVIEW OF THE LITERATURE FROZEN FRUITS AND VEGETABLES Previous to 1930 there were few investigations conducted on the microbiology of frozen fruits and vegetables* The earliest reference to studies on these products which provides any authentic informat­ ion is a paper by Prescott, Bates and Highlands (24). Among other products they examined samples of commercially frozen strawberries, raspberries, orange juice and spinach. These authors found a distinct reduction in the initial microbial load when the products were subjected to freezing temperatures. They also made the observation that higher storage temperatures e.g. - 10°C. (14°F.) were more destruct­ ive to microorganisms than lower temperatures. Smart (30) (31) (32) examined a number of fruits and vegetable products before and after freez­ ing. She found considerable variation in the microbial content of different lots of the same product. Scalding, freezing and storage for periods of 5 to 7 months reduced the average microbial content 94.6 to 99.8 per cent. Even with these striking reductions some frozen products were found to contain up to 1,000,000 microorganisms per gram. - 5 - In a later publication (33) this same author reported that storage of blueberries for 9 months at 0°F. resulted in the reduction in microbial content of 59.7 per cent., while a higher storage temperature 6.67°C. (20°F.) for the same period resulted in 99.9 per cent reduction. This agrees with the earlier findings of Prescott, Bates and Highlands (24). McFarlane (20) studied the distribution and survival of microorganisms in frozen sugar-packed raspberries and frozen brine-packed peas. Among other results McFarlane noted greater decreases with raspberries at higher storage temperatures while the reverse was noted with peas. With raspberries he found the microbial content at -20°C. (-4°F.) greater in sections from the lower half of the container and this corresponded with a higher soluble solids content in this area. Similarly, the microbial content of frozen peas stored at -20°C. (-4°F.) appeared to be correlated with the soluble solids content with the highest count being obtained from the central, coneshaped area and in the surface. Pederson, (23) in a study on plate counts on stored, quick frozen vegetables, in some instances, found increased counts as the period of freezing - progressed. 6 - Microscopic examination indicated that the increased counts were due to the breaking up of bacterial clumps during freezing, a point which has been suggested by other workers (19) (36)• Pederson makes no mention of the method used for the preparation of products prior to analysis. Recent studies made by the writer (12) have indicated that if the Waring Blendor is used for comminution of products the grinding action of this machine may result in increased counts due possibly to the disintegration of bacterial clumps. Van Eseltine et al. (36) also noted increased counts resulting from the use of the Blendor for preparing products for analysis. These same authors found that freezing products in liquid air and in an air blast at -60°F. resulted in such quick freezing that the bacterial content of the foods was essentially unchanged. Luyet and Gehenio (17) also noted this fact and stated that during such rapid freezing the water in the cell is not changed to ice crystals but to a glass-like amorphous mass which they term the “vitreous state11, and this results in less injury than freezing at slower rates. Van Eseltine et al., summarizing their results, state that freezing at slower rates allowed multiplication of bacteria before the freezing temperature was reached. This r e ­ statement is not substantiated by data presented in graphs nor by other material in the text since with peas they show a marked increase in count during the early hours of freezing storage followed by a levell­ ing off and finally, after 30 hours, a decided decrease. However, with corn the trend was entirely different with a decided decrease during the early hours of freezing followed by a levelling off and finally a more gradual decrease. These findings are somewhat unusual since corn, which is relatively high in starch, might be expected to provide greater protection to microorganisms against freezing and also, in the event of multiplication of microorganisms before the product is actually frozen, corn might provide a greater amount of food material for microbial growth. Previous studies by the writer (15) on frozen vegetables and fruits showed that the microbial load of freshly packed product may be subject to wide variations which tend to become obliterated during freezing storage at -17.8°C. (0°F.). With the vegetables studied there was a pronounced decrease in numbers of microorganisms during the first weeks of storage after which they declined slowly or remained stationary. With fruits the decrease appeared to be more gradual. Bacteria were the predominant organisms in frozen vegetables while yeasts and moulds were more numerous in the acid - fruits. 8 - Even after 9 months storage at -17*8°C. (0°F.) frozen vegetables and fruits contained an appreciable number of microorganisms. Micrococci and species of Flavobacterium were found to be relatively more resistant to freezing than other types of bacteria encountered in frozen vegetables (16 ) (10). More recently a study by Burton (3) of this Division showed that the coliform test was more efficient for detecting contamination in foods prior to freezing and storage while the fecal streptococci are the superior indicator in frozen foods, as the coliform organisms seem less able to survive the storage temperatures. The fact that the microbial content of fresh fruits and vegetables harvested from the same area of particular fields showed wide variations from (day to day led to a study (11) to determine reasons for these variations. It was found that the variations in the microbial content were largely due to weather factors, definite correlation being obtained with bacteria counts and the temperature and humidity prevailing and preceding N the time of harvesting. Products harvested during a - 9 - period of relatively high temperatures and high humidity generally showed a high microbial content. Material from low growing plants generally carried a heavier microbial load than that further removed from the soil. Pure Culture Studies The literature on the effect of subjecting cultures of microorganisms to freezing temperatures is very extensive. Since our main interest is concerned with the range of commercial freezing temperatures the review of the literature, with some exceptions, will cover studies conducted within the temperature range of -28.89°C. (-20°F.) and 0°C. (32°F,). Wallace and Tanner (38 , 39) have presented an excellent historical review dealing with early work on the freezing of pure cultures of micro­ organisms, Unfortunately, as these authors point out, there was considerable contradiction from one atithor's work to another and considerable confusion exists even at the present time. As a result of some of the earliest studies recorded it was generally believed that bacteria were quite resistant to freezing. About 1900 another group - 10 - of studies indicated that bacteria were easily destroyed by low temperatures* As these authors point out the trend of thought resulting from more recent studies has changed again and it is now generally accepted that bacteria are resistant to low temperatures* Probably mention should be made of the work of Keith in 1913 (13) since it was concerned with the freezing of Escherichia coli. an organism studied extensively in the present investigation* Keith froze this test organism solidly in tap water at -20°C. (-4°F.) and found that only a fraction of one per cent of the original number remained alive at the end of 5 days. This finding confirmed that of Sedgewick and Winslow (27). when the same test However, organism was placed in tap water and cooled as “water ice or sherbetM (not solid) and held in this condition at -20°C. (-4°F.) a large percentage remained alive for many months. latter test is far from clear. This The writer is at loss to understand how it would be possible to hold this test material, “water ice or sherbet*1 at -20°C. (-4°F.) without it being frozen solidly. If it is assumed that the temperature is incorrectly stated or that some other factor was introduced to maintain - l i ­ the test material in a semi-frozen condition then the results are at variance with an earlier study by Prudden (25) who obtained the opposite results. The most authentic work published in the early years is that reported in a paper by Hilliard, Torossian and Stone (7). They found that suspensions of Escherichia coli frozen in tap water showed a reduction in total number of cells of 93 per cent within 3 hours. A spore-forming bacterium, Bacillus subtilis, in the vegetative stage, showed a variable resistance to freezing while the spores were decided­ ly more resistant to freezing. They also found that intermittent freezing was only slightly more injurious to bacterial cells than suspensions held continuously in the frozen state. In a later work Hilliard and Davis (8) modified earlier conclusions and stated that in the later studies they found intermittent freezing of bacterial cells much more injurious than continuous freezing. In this later paper they also found that the degree of cold was insignificant insofar as reduction in numbers of microorganisms was concerned. This finding was directly opposed to the e arlier report by the same senior author. Like other workers Hilliard and Davis found that freezing bacteria in menstrua other than tap water, such as milk or cream, resulted - 12 - in appreciably less destruction to bacteria* indicating that the organisms were afforded pro­ tection by these foods. Berry (2) studying the destruction and survival of microorganisms in frozen pack foods, presented data indicating that lactobacilli and to some extent coliform organisms in peas remained viable for at least 2 years at -10°C. (14°F.). Data are presented on the destructive effect of ice formation on cells of Saccharomyees sp. in wort. The number of viable cells in liquid wort was greater over a period of 6 days at -10°C. than in frozen wort at the same temperature. A table presented in this paper indicates that viable cells in the frozen wort had practically disappeared. Wallace and Tanner (40,41,42) studied pure cultures of microorganisms isolated from frozen fruits and vegetables. In all instances they found a rapid drop in numbers during the first months of freezing. After the eighth month the numbers dropped slowly. They also noted that spore formers were the most resistant to freezing and that yeasts and moulds were generally more resistant than non­ spore forming bacteria. In fruit juice and broth with a decidedly acid reaction, the organisms died - out quite rapidly. 13 - These authors, in contrast to work by other authors, present data showing that alternate freezing and thawing probably is no more destructive to microorganisms than continuous freezing, in fact, the data given in tables indicate that it is even less so. They suggest that in view of the fact that this finding conflicts with those of other workers, previous studies had been based on tests in which alternate freezing and thawing were done repeatedly at intervals of just a few hours; their studies consisted of thawing the menstrua at daily intervals and immediately refreezing. They also found that the degree of cold had very little effect on microorganisms although in some instances the lower temperature did not seem as lethal as did -16°C. (-2°F.). One of the most interesting studies in recent years was conducted by the late R. B. Haines (5) who studied the effect of freezing pure cultures of bacteria. Cultures of Pseudomonas aeruginosa. Escherichia coli and Staphylococcus aureus in aqueous suspensions were used in this study. Using temperatures ranging from -20°C. (-4°F.) to -1°C. (30.2°F.) Haines, like other workers, found the higher storage temper­ atures more destructive to bacterial cells. Of the 3 organisms studied P. aeruginosa was the most sensitive - 14 to freezing with Staphylococcus aureus the most resistant. the two. Escherichia coli was intermediate between Spores of Bacillus mesentericus were resistant to all temperatures used showing no reduction over a period of 150 days storage. Haines suggests that two factors are responsible for the death of bacterial cells on freezing; one unknown, but apparently not mechanical, and the other, some change leading to flocculation of the cellular proteins; or else there is in reality one process, leading to coagulation, but with a time lag, so that coagulation is not found on immediate freezing and thawing. Smart and Brunstetter (29), in a study of the types of organisms found on fresh and frozen spinach and kale, state that the predominating types of bacteria on these two vegetables are Achromobacter. Bacillus. Flavobacterium and Pseudomonas species. According to data presented, the microbial flora members of the genus Achromobacter had disappeared during the process of freezing. However, in a later paper by Smart (3 $) Achromobacter types were prominent in frozen peas, beans and corn. No mention is made of the proportion of types in either the fresh or frozen products. Hegarty and Weeks (6) conducted an interesting - 15 - study on the sensitivity of Escherichia coli to cold shock. They confirmed earlier findings of Sherman and Albus (28) that young cells of E. coli are susceptible to an Initial cold shock of 0°C. (32°F.). This sensitivity to cold shock extends throughout the entire logarithmic phase of growth. Mature cells were not affected by either an initial cold shock or prolonged holding at 0°C. McFarlane (21) studied the effect of freezing Saccharomyces spp. of yeasts and Escherichia coli in distilled water and in concentrations of sucrose solutions varying from 1 to 50 per cent and adjusted to different pH values ranging from pH 3.6 to 6.5* He found that when the hydrogen-ion concentration was the only variable, a reaction of pH 6.5 was more favorable than one of pH 5 for hastening the destruction of yeast cells in some of the sucrose media. With Escherichia coli destruction was greatest in those samples which possessed the greater hydrogen-ion concentration. When temperature was the only variable greater kills tended to occur after sevdral weeks storage, at -10°C. (14°F.) than at -20°C. (-4°F.). McFarlane and Goresline (22) studying microbial destruction in water and sugar syrup stored at -17.8°C. - 16 - (0°F.) over periods of from 1 to 60 weeks, found that greater destruction of microbial cells occurred in water than in any of the syrups used. Escherichia coli. Saccharomvces cerevisae. Saccharomyces elliusoideus Hansen. Schizosaccharomyces octosporus. a strain of brewer's yeast and an unidentified yeast exhibited greater cold resistance in sucrose than in invert syrup. A Torula s p .. Aspergillus nidulans. Zygosaccharomyces pastori and Zygosaccharomyces .iaponicus exhibited greater cold resistance in invert than in sucrose syrup. Kiser (14) studied the rate of destruction of an Achromobacter sp. by freezing at -28°C. (-l8.4°F.). He found a slight increase in count up to a period of 10 hours following which there was a steady decrease up to a period of 300 hours. After this time there was a distinct decrease in the rate of death of bacteria. Weiser and Osterud (44) in a recent publication on the death of bacteria at low temperatures, provide some useful information on the mortality of Escherichia coli exposed to freezing temperatures. They report that the mortality due to immediate death by freezing is marked but does not vary with the intensity of the freezing temperature. Data are presented which indicate that immediate death occurs at a brief stage in the freezing process during which extracellular ice formation is - being completed. 17 They report that the rate of storage death at the higher freezing temperatures is very rapid and is much greater at temperatures above -30°G. (-22°F.) than at lower temperatures. Repeated freezing was more lethal than a single freezing. They also found that freezing is much more lethal than supercooling and that repeated fluctuations of temperature of frozen suspensions do not exert a lethal action additional to that of the storage. Gunderson and Rose (4) studying the survival of bacteria in pre-cooked fresh-frozen chow mein, found that pathogenic enteric bacilli are killed rapidly during the first 5 days at -25*5°C. (-14°F.). After that the r ate of death drops off until a more or less resistant population remains. The normal saprophytic flora appears to be killed less rapidly. In either instance the numbers remaining after any given storage time depend primarily upon the initial contamination. When this is high even storage for as long'as nine months does not assure a low count. Studies on food poisoning bacteria, in particular Clostridium botulinum, indicate that spores of this organism survive freezing for long periods. Wallace and Park (37)> Straka and James (34, 35), James (9) showed that spores and toxin of this organism - 18 - survived freezing for as long as 14 months. McCloskey and Christopher (18), in a study of some pathogenic bacteria in cold-pack strawberrries, found that in sliced, sweetened strawberries held at -18°C. (-4°F.) Salmonella typhosa could be recovered after 6 months; Staphylococcus aureus after 5 months; Salmonells typhimurium and Salmonells schottmuelleri after one month. Salmonella paratyphi could not be recovered at any time from the frozen berries. - 19 - DISCUSSION OF THE LITERATURE REVIEW Many of the studies reported in the literature have added a great deal to our knowledge of the effect of freezing on microbial cells. There appears to be agreement that freezing generally effects a reduction in numbers either during the initial freezing process or during subsequent freezing storage. However, results of studies on the rate of reduction of microbial cells are extremely variable. If we compare the results with one test organism, Escherichia coli. an organism which has been used extensively by numerous workers, it will serve to illustrate this point. Wallace and Tanner (43) report freezing cells of E. coli in water at -16.11°C. (3°F.) for periods of 1, 5, 6, 9 and 12 weeks and showed reductions in numbers of 20, 50, 60, 80 and 94 per cent respectively. Weiser and Osterud (44), using this test organism in a 0.5 per cent peptone at -15°C. (5°F.), showed a 78 per cent reduction in a period of 3 to 10 minutes. Freezing for 2 minutes at slO°C. (14°F.) effected a reduction of 54 per cent. McFarlane (21) froze the same test organism at -20°C. (-4°F.) and showed a reduction of 99 per cent after 1 week storage. Hilliard, Torrossian and Stone (7) froze this organism in water at -15°C. (5°F.) and effected a reduction of 99.9 per cent in 3 hours. Haines (5) - 20 - studied the effect of freezing E. coli in aqueous suspensions and found that after 150 days at -20°C. (-4°F.) a very slight reduction in numbers occurred and at -10°C. (14°F.), a temperature comparable to that used by Weiser and Osterud, a reduction slightly over 50 per cent in the same period of time. The time required to effect a reduction of approximately 99 per cent was between 50 and 140 days. Contradictions on the effect of freezing micro­ organisms in fruits and vegetables are also numerous. Berry (1) states that after one year's storage the microorganisms in frozen foods were so changed that they could not cause spoilage. This statement has not been supported by any other worker. A year after the first paper the same author writes about spoilage of frozen pack peas (2). These discrepancies do not reflect in any way on the studies conducted by the various workers but rather point to the extreme variability encountered with microorganisms subjected to the freezing process, and also to the fact that, in may instances, experiments were conducted by the various authors under entirely different sets of conditions. The variability of microorganisms will be discussed at greater length in a later portion of the present work. - 21 - INVESTIGATIONAL FRUIT AND VEGETABLE PRODUCTS The studies reported in this section were conducted over a period of years and are included to demonstrate the degree of variation in the microbial content in similar and different frozen vegetables which had been initially harvested from the same general area of the Central Experimental Farm, Ottawa. All products were packed by the Division of Horticulture and were given essentially the same treatment each year. Vegetable products were blanched, usually until a catalase negative reaction was obtained, and were packed in pint-size waxed cartons in either a 2 or 3 per cent brine, depending on the product. Procedure of analysis for fresh and frozen products: The procedure for the analysis of fresh and frozen products follows exactly the same pattern. The frozen product is removed from the freezer, placed in an incubator at 37°C. (98.6°F.) and defrosted to a point where the ice could be separated from the product. One hundred grams of the vegetable are weighed into sterile pint-size sealers and 50 grams of ice or brine solution added. The product is then macerated, using either a flamed, 6-prong cutting knife or a flamed pestle or both, depending on the nature of the product. To this - 22 - ground-up mixture is added 100 grams sterile water. This provided the first dilution with a ratio of 1 part product to 2.5 parts water. The whole was then shaken for 5 minutes using a reciprocating type mechanical shaker. Further dilutions were prepared in the usual manner by pipetting 1 millilitre of the first dilution into a 99 millilitre sterile water blank. A number of tests were made to determine different groups of bacteria but for the studies reported in this section only the method used for determining total numbers of mesophilic bacteria will be given. Duplicate plates were prepared from each dilution. The plating medium was tryptone glucose extract agar and plates were incubated at room temperature, 20 to 25°C., for a period of 4 days. Isolation of colonies for the purpose of determining the proportion of types consisted of picking 50 colonies from plates of suitable dilution. Where there were more than 50 colonies on a plate the plate was divided into sections and 50 colonies picked from one section. Colonies picked were transferred to tryptone glucose extract agar slopes and incubated for a period of 4 days at 20 to 25°C. Classification into genera was made by macroscopical, - 23 - microscopical, and, when required, by biochemical tests. RESULTS Data are presented in Table 1 and in Figs. 1, 2, 3 on plate counts obtained with 4 individual samples of asparagus, 7 samples of peas and 5 samples of corn. All of these products were prepared and frozen during the same or in different years from 1935 to 1949. From these data it is evident that there are wide differences in the initial microbial content of the different products. Perhaps more striking are the wide variations in the rate of survival of microorganisms on these products at the different periods of analysis over a period of 30 weeks. In Fig. 1, showing the results of analyses of four samples of asparagus, one sample (No. 4) shows almost complete destruction of microorganisms at the six-week period of analysis while three other samples in the same period show survivals varying from 33 to 84 percent. One other point of interest from the data presented in this graph is the fluctuation in the microbial content with two of the samples (No's 2 and 3) following the initial freezing process. Since these fluctuations have been noted with some samples of all products it might be well to discuss them at this point. 24 - 100*- 90. SAMPLE NO. 1 80 . 70- PERCENT SURVIVAL OF BACTERIA 60 50. 40 30 20 10 K K— f 8 “T 12 18 24 PERIOD OF FREEZING STORAGE FIG. f . SURVIVAL OF BACTERIA IN ASPARAGUS FROZEN AND STORED AT 0* F. I8" 30 weeks - 25 100 SAMPLE NO . 5 A ii ii 6 □ n ii 7 0 it it 8 X it ii 9 ▼ ii ii 10 • ii n 11 70 PERCENT SURVIVAL OF BACTERIA 60 20 12 =f= 18 24 PERIOD OF FREEZING STORAGE FIG.2 . SURVIVAL OF BACTERIA IN PEAS FROZEN AND STORED AT 0a F. ---30 weeks “ 26 00 SAMPLE HO. 12 90 A " " 13 ii n j_ 4 □ O " " 15 X 80 70 60 v 50 40 30 20 10 xa 0 1 --------------------- 1---------------------1---------------------1--------------------- 1------- 6 12 18 24 PERIOD OF FREEZING STORAGE 3• SURVIVAL OF BACTERIA IN CORN FROZEN AND STORED AT 0° F. 30 weeks - 27 Table No.I Survival of Bacteria In Frozen Vegetables Bacteria Count per Gram and Percentage Survival Period of Freezing 6 weeks 12 weeks 18 seeks Product No. Year Fresh Count % % survlval Count vlval 24 seeks % 30 weeks % survlval ount Vlval % survlval Asparagus n i 1935 4,500 1,535 34 2,3 0 0 51 1,1 0 0 24 2,1 0 0 47 860 19 2 1935 18,0 00 9,0 0 0 50 7,200 40 6,800 62 8,6 0 0 42 2,400 13 ii 3 1936 38,000 32,000 84 26,000 68 19,000 50 12,000 32 8 ,6 0 0 23 n 4 1940 5,5oo 40 0.7 30 0.5 30 0.5 20 0.4 20 0.4 5 1935 15,ooo 750 5 1,750 12 900 6 1,550 10 950 6 1935 62,0 00 24,000 39 36,000 58 42,000 68 8 ,0 0 0 13 18,000 29 50 Peas H 6 » 7 1936 3,8 0 0 4,600 100 3,200 84 1,600 42 2,400 63 1,9 0 0 it e 1938 8,7 0 0 4,300 49 6,400 74 2,000 23 2 ,3 0 0 26 1,600 18 ii 9 1940 12,000 20 0.2 20 0.2 20 0.2 20 0.2 10 0.1 it 10 1949 34,000 28,000 82 34,000 100 16,000 47 12,000 35 8,000 24 n ii 1949 80,000 4,000 5 4,800 6 5,200 7 3 ,6 0 0 5 2,400 3 12 1935 55,000 36,0 00 65 14,000 25 ■I 13 1935 156,000 79,000 n 14 1935 78,000 64,000 15 1949 172,000 16,000 9 12,000 7 8,000 5 9,600 6 8,200 5 16 1949 96,000 63,000 66 46,000 48 54,000 56 25,0 00 26 16,000 17 Corn a H - - - 51 - - - 6,5 0 0 4 82 - - - 14,000 18 - 28 Pederson (23) noted these increased counts following the initial freezing process and attributed them to the breaking up of bacterial clumps during freezing. He based his findings on microscopic examinations of the organisms in the products before and after freezing. However, in the opinion of the writer, while these findings might be partly true, they fail to explain how these fluctuations persist beyond the initial freezing process when the breaking up of bacterial clumps might be expected. demonstrates this point. Sample No. 2, Fig. 1, After 6 weeks1 storage at 0°F. the microbial content of this product showed a survival of 50 per cent of the initial count. After 12 weeks there was a further reduction in count and the survival was approximately 40 per cent. At the 18-week period of analysis the count suddenly jumps and 62 per cent of the organisms are found to have survived the freezing process. From this period on there is a steady decrease in the microbial content through to the 30 week period. These fluctuations are more likely due to the uneven distribution of microorganisms on the product or to some properties peculiar to the particular types of organisms present. This latter statement will be discussed at greater length in connection with the pure culture studies which follow. With the seven samples of peas the trend of counts - 29 - was similar to those of the asparagus (Fig. 2). Three samples showed a rapid reduction in counts during the first 6 weeks of freezing storage followed by a more or less stationary count which persisted throughout the entire storage period. Four other samples of peas showed survivals varying from 100 per cent to 39 per cent during the first 6 weeks of storage. These same four samples show considerable fluctuation in counts following the six-week analysis. After 30 weeks storage the seven samples of peas showed survivals of bacteria varying from 1 to 51 per cent. While the results are not as complete as for asparagus and peas, five samples of corn, show the same degree of variation in the rate of survival of bacteria at the 6-week period of analysis (Fig. 3)After 30 weeks' storage survivals varied from 4 to 27 per cent. Results of a study of the types of bacteria from 6 samples, both from the fresh products and from those frozen for 30 weeks, are given in Table 2. Sample No. 3, which showed a steady decline in numbers of bacteria from 84 per cent survival after 6 weeks' storage at 0°F. to 23 per cent after 30 weeks, showed a predominance of micrococci (60 per cent) with Flavobacterium. - 30 - Table No. 2* Survival of Types of Bacteria In Frozen Vegetables Classification — Lcatlon . # Product bacillus Bacillus on Colonies coccus FlavoChromoAchromo- Escher­ Aerobacter Proteus Examined ichia Transfer Serratla bacterlum bacterlum bacter 3 Asparagus (Fresh) 100 19 3 Asparagus (Frozen)## 100 60 - 15 ♦ Asparagus (Fresh) 100 11 . 14 4 Asparagus (Frozen)## 8 12 - Peas (Fresh) 50 17 1 16 Peas (Frozen)## 50 44 - 22 100 4 - 7 - 7 7 9 Peas (Fresh) Peas (Frozen)## Percentage of Types 14 4 - 9 1 - 8 . 3 2 4 a. 12 • 26 . • 6 25 16 • 12 2 1 3 1 . . 30 _ . 1 74 2 1 - 5 4 2 25 - 25 - - - - - . 4 . 46 15 16 . 2 4 4 - . . 4 • 6 6 . 8 1 _ 23 12 16 - 10 3 2 . 53 5 . 50 2 . 11 4 ?0 Corn (Fresh) 100 6 Corn (Frozen)## 100 58 • 24 - 8 16 Corn (Fresh) 100 14 . 31 - 28 12 16 Corn (Frozen)## 100 32 _ 46 3 1 9 15 15 1 # Taken from Table 1, ## 30 weeks plus or minus 6 weeks. 1 12 6 - 31 - Achromobacter and lactobaeilli following with 15 j 10 and 9 per cent respectively. Escherichia. Aerobacter, and Bacillus were present in small numbers. Sample No. 7» which showed no reduction in numbers after 6 weeks' storage and 50 per cent after 30 weeks, showed a large proportion of micrococci (44 per cent) with lactobaeilli and Flavobacterium following with 30 and 22 per cent respectively. Sample No. 15, which showed a very rapid decline in numbers after 6 weeks of freezing storage, showed 58 per cent of the colonies isolated from the frozen product to be micrococci, 24 per cent were Flavobacterium. 8 per cent were Achromobacter and 4 per cent, were lactobaeilli. Sample No. 16, which showed a survival of 66 per cent in numbers after 6 weeks and 17 per cent after 30 weeks, showed 46 per cent of the colonies in the frozen product to be Flavobacterium and 32 per cent micrococci. Twelve per cent wer lactobaeilli with Achromobacter and Escherichia forming a small part of the microflora with 3 and 1 per cent respectively. Too few colonies were examined from the frozen samples of Nos. 4 and 9 to permit making comparisons with other sapples. - 32 - While it is impossible to state definitely from the data presented that the types of bacteria influence the survival of numbers of bacteria in frozen products, there is some indication that the types present play an important role. Of particular interest is the fact that the number of types in the frozen product is considerably reduced when compared with those present in the fresh product. From the data presented in Table 2 it will also be noted that in the frozen product of all samples studied bacteria belonging to the genera Micrococcus and Flavobacterium formed a higher percentage of the total microflora than did the corresponding fresh sample. 33 SLOW FREEZING VS. QUICK FREEZING ON NUMBERS OF MICROORGANISMS IN FROZEN PACK FRUITS AND VEGETABLES Until a comparatively few years ago the usual method of freezing consisted of placing products to be frozen in a refrigerated room maintained at temperatures varying from -12.22°C. (10°F.) to -28.89°C (-20°F.). Although the air within such a room will circulate to some extent by convection, no provision was made for forced air circulation. The relatively still air is a poor conductor of heat and products are frozen at a comparatively slow rate, many hours or days being required before the products are completely solidified. This method is commonly known as the slow or 11static" method of freezing. The term "quick freezing" refers to any method which incorporates some feature which speeds up the rate of freezing such as "contact" or "air blast" methods* Few comparisons have been made of the effect of slow and quick freezing on microorganisms in frozen pack fruits and vegetables. Van Eseltine et al. (36), studying the effect of freezing on the bacterial content of frozen vegetables, subjected peas and corn to temperatures of liquid air, -5l»l°C. (-60°F.), and -17.8°C. (0°F.). At the latter temperature products were frozen in "still air" both exposed freely to the atmosphere and in insulated boxes. These authors - 34 - state that freezing in liquid air and in an air blast at -60°F. resulted in such quick freezing that the bacterial content of the foods was essentially unchanged. However, this statement is not substantiated by the data presented since pronounced increases in counts are recorded in the -5l.l°C. series. It is further stated that freezing at slower rates allowed multiplication of bacteria before the freezing temperature was reached. The Data presented show no greater increase after 1 and 2 hours' freezing at -17.8°C. than after 2 hours at -5l.l°C. Substantial increases in counts are recorded in the series frozen in insulated boxes at -17.8°C. but this might be expected under such unusual circumstances since the rate of freezing is retarded considerably as these authors have demonstrated by the use of thermocouples. Experimental The present study was made with three products, asparagus, peas and strawberries. packed dry in rectangular cartons. The products were Some were frozen by the "static" method at both -28.89°C. (-20°F.) and -17.8°C. (0°F.), others by placing the cartons of the product between refrigerated coils at -20°F. Some products frozen by this contact method were also stored at 0°F. Initial analyses were made of the fresh products and again after one and six months' freezing storage. - 35 - The results of these studies shown in Table 3 indicate a higher percentage survival of bacteria in vegetable products frozen contact at -2Q°F. and stored at the same temperature when compared with the static frozen products stored at either -20°F. or 0°F. The only exception to this was with the psychrophilic bacteria in peas which showed about the same percentage survival with both methods of freezing. Vegetable products frozen by contact (-20°F.) and stored at 0°F. compared with those frozen and stored at 0°F. showed variable results. With strawberries the rate of freezing or temperature of freezing storage appeared to have little, if any, influence on the percentage survival of microorganisms. A summary of the data presented in this table of both vegetable products shows an appreciably higher percentage survival with the contact method of freezing. - 36 - 03 fa P •G 2 0 O o CO • o CM • CN rH CM • OO CM • Vs rl Us • m rH Cv. • CM • O rH 00 • CM rH cry rH • m CM • O • On nO o o (V U .q oP P £ 0 P U fa £ cfl f CO o ofa £ o (0 rH « O 0 CM CM ♦ rH P C n- 00 NO CM ON \£> CM On vO NT C n. • ON p p id p o V\ p rH • Vs H OD ro rn CO • CM CM 'T • Vs m oo » P m 03 0) P t. 03 P G InO CO P > fa F iP O £ O G 4 3 N O P co 0 fa ir\ • CM • O C M r l CO ♦ CnVs Cv • NO P oo 00 • m • C M Vs Vs • v \ p so O n O

fa O P O S > o J H v£> g <0 ttl u <«H o w o> fsa p £ o h0 G P (4 0) 0) F h fa O * V\ CM P CO TJ o o F* o CO • o CM a> f ri£ o P £ a> 0 t* o P £ V co o 0 fa ir \ CM n£> CO o CM P I cd P £ O u cd 03 X • .1O^ O f a Fi P CO CO fa •p fa £ o o 0s 1VO v\ oo Cn. o Vs Vs *fa O£ On • P o CO s NO NO • o Vs c0 o P • C T J P o ro O o 00 » cO C n• P CM o £ CM C M Fh p o fa Vs • cO NO • 00 O P 3 O* 03 > * o i-4 +-» ^ f0a } £^ e 0) p 0) co F h 00.(4bO fa o o Vs OJ Vs Vs o CM «s p NO CM V\ p CM P CO p o O o 00 O O V* Vs p tN CO CO 4h O • P P O C O o co fa fa p o C O 0) p o C O fa fa fCaO ♦ • oo oo o Nt CM C O co P f-. 0) p p L, a> P o o ai aj fa fa 0 3 a> 0H p p o p T3 O P-. a, P bO co cd f a (ft co 0) fa u t* 0) 'S aj U 0) q P bo a> PTJ 03 fa Fh £ CO C O C OC OQ > fa fa Fh tft 6,600 48 100 820 41 6,100 100 t7 78 1,400 4i 116 1,100 5 37 32 1,100 1 36 6 60 720 18 480 46 10,000 12 * 37 6,100 1 38 24 54 28 10 30 4 1.4 6.9 11 90 12,000 100 13,000 100 12,000 100 100 130 72 150 83 155 86 4 2 1 1 44 2 18.6 40 42 48 62 45 82 II 5 42 50 37 47 Old Cultures 58 62 63 44 69 72 73 Vi Flavobacterluu * Micrococcus R Achronobacter R Chromobacterium 70 Serratla 1,120 Sarcina Bacillus 11 90 0 70 1 70 7,0 0 0 4,000 260 120 1,2 00 80 70 0 1,1 770 S5 120 750 .70 1,000 Average 37 64 110 35 58 32 90 33 57 46 4,400 140 63 54 68 11 984 82 100 36 67 58 68 6 830 110 720 53 960 9 74 91 72 64 7 V 59 62 49 3,000 130 43 50 768 750 64 86 516 670 43 77 .01 1 ?? OO 38 .01 770 57 160 52 71 42 55 3,700 140 53 3,000 54 170 43 65 3,500 130 50 50 3,400 160 1,100 800 88 92 960 600 80 69 1,100 400 92 46 .035 770 1 69 77 .01 910 13° 1 81 100 68 .01 840 88 850 1 75 Z3 85 54 §3 53 10 56 87 30 62 100 530 62 15 30 160 860 18 28 33 3,800 4.8 46 27 14 65 13° 80 900 75 870 100 68 73 27 71 47 30 93 460 46 62 680 59 52 660 79 380 50 1 77 73 7 16 - 91 - Table No. 17 Survival of Bacteria Frozen in Different Phases of Growth at 0°F, Series "B". Count per ml. Substrate in Thousands and Percentage Survival 1ft Period of Freezing 4 hours 8 hours 24 hours 48 hours 72 hours 96 hours 192 hours 1 month Generic 1 * % f % < * 4 Cult. Class- Initial sursur­ sur­ sur­ sur­ sur­ sur­ sur­ No. lficatlon Count Count vlval Count vival Count vival vival Count vival Count vival Count vival Count vival Avers Vnunv Cultures • 125 126 127 128 129 130 131 132 Achromobacter 10,400 5,5oo 48 8,000 5,000 53 6,500 63 11,300 100 77 6,200 60 6,100 59 5,400 52 Flavobacterlum 6,300 6,800 100 6,100 97 3,000 48 5,600 5,700 2° 4,900 78 5,000 P 5,500 ?7 89 ii 7,500 8,000 100 10,000 100 6,100 81 7,300 97 6,200 63 7,000 93 6,000 80 4,900 65 Achromobacter 14,600 4,700 32 5,100 35 7,900 54 7,300 50 4,400 30 6,500 45 4,200 29 5,600 38 Micro39,000 36,900 90 31,000 78 10,000 26 33,500 86 38,000 95 35,000 coccus 90 38,000 97 32,000 81 Achromo11,000 4,800 44 4,800 44 6,000 55 4,600 42 4,000 36 6,200 56 4,900 4,200 bacter 45 ft 2,400 65 2,200 59 2,100 62 2,100 57 1,900 51 2,100 3,700 2,300 57 2,300 62 57 Klcro1,800 4,000 100 3,700 100 2,500 100 2,700 100 4,300 100 6,000 100 4,200 100 3,200 100 coccus Average 72 73 72 72 65 73 68 90 1,760 100 2,150 610 91 95 64 84 87 39 80 45 59 100 65 70 1,420 83 93 2,200 570 92 94 88 Old Cultures 125 126 127 128 129 130 131 132 Achromo1,720 bacter Flavobacterium 2,360 H 640 A&iromo170 bacter Micro760 coccus Achromo* 246 bacter M 670 Micro3,900 coccus Average 100 1,910 100 1,550 2,100 560 2,400 100 650 100 1,340 78 1,640 95 1,930 100 1,900 2,230 480 94 75 2,380 100 2,200 410 64 600 94 94 2,370 100 650 100 50 29 54 32 40 24 50 29 95 56 54 32 25 15 33 520 68 400 53 460 61 650 86 810 100 780 100 690 91 580 76 79 203 86 99 158 600 64 94 175 71 690 100 194 710 79 100 70 I71 840 100 225 700 91 100 191 78 720 100 201 650 82 660 97 78 99 3,800 97 2,400 63 64 2,400 62 2,670 68 69 75 79 78 71 2,500 76 82 75 1,850 88 87 44 47 80 2,930 75 89 2,930 75 83 89 - 92 - 40 PERCENT SURVIVAL OF BACTERIA 100 20 YOUNG CULTURES ( SERIES A ) OLD CULTURES SERIES A ) YOUNG CULTURES ( SERIES B ) 10 OLD CULTURES 24 48 SERIES B ) 1.68 hours 1 month PERIOD OF FREEZING STORAGE FIG.I&. SURVIVAL OF BACTERIA FROZEN IN DIFFERENT PHASES OF GROWTH AT 0°F= SERIES A AND B. - 93 some individual cultures did show less resistance to freezing in the logarithmic stage there were just as many that showed less or equal resistance to freezing in the old cultures. Of some interest are the results with members of the genus Achromobacter. Of the six cultures studied in the two series, five showed less resistance to freezing in the logarithmic phase of growth. None of the other genera represented showed any definite trend, some showing greater resistance to freezing in the logarithmic phase, others the opposite. Attention should be drawn to the fact that the temperature of freezing used by Hegarty and Weeks in their studies was considerably higher than that used in this study. They were using a temperature of 0°C. (32°F.) compared with -17.8°C. (0°F.) in the present study. Also their results were based on findings with only one organism, Escherichia coli. From the present studies it may be concluded that there are no important differences in the rate cf survival between cultures of bacteria in the logarithmic phase of growth and of old cultures frozen at -17.8°C. (0°F.). SURVIVAL OF MICROORGANISMS FROZEN CONTINUOUSLY AND INTERMITTENTLY IN WATER AT DIFFERENT TEMPERATURES Previous studies by other workers have shown, as - 94 indicated in the review of the literature, that intermittent freezing is more destructive to bacteria than continuous freezing. A study on this phase was conducted in the present investigation on four species of bacteria and three species of moulds isolated from fresh vegetable products. The three species of moulds used formed definite colonies on agar plates. Any spreading type was rejected since it would be difficult with the usual bacteriological technique to determine whether or not they were affected by freezing. All cultures were frozen in water at 4 different temperatures, -28.89°C. (-20°F.), -17.8°C. (0°F.) -9.44°C. (15°F.) and 0°C. (32°F.). For each organism studied one suspension was used throughout for the intermittent freezing. This tube was removed at each period of analysis and after holding for 2 hours at room temperature following defrosting was returned to the freezer. For the continuous freezing the procedure was the same as for previous studies and separate suspensions were prepared simultaneously and frozen. One tube of suspension was removed from the freezer at each period of analysis and when the plating was complete was discarded. Analyses were made of each test organism initially and after 1, 2, 4, 7> 8, 9 and 10 days for bacteria and at intervals of 5 and 24 hours, 1, 2, 4, 5 and 6 weeks for moulds. - 95 The results for bacteria shown ±1 Table 18 and Figs. 19 to 22 give some interesting findings. With all four cultures studied the continuously frozen series showed considerable fluctuation from one period of analysis to another at all four temperatures. The series frozen intermittently showed no fluctuation and indicated pronounced reductions as compared to the continuously frozen series. It is of interest to note the results with two of the organisms held at 32°F. (culture No. 122 - Lactobacillus culture No, 123 - Micrococcus sp.). s p . and From the data presented it would appear that at 32°F. these two organisms showed a greater resistance to freezing than at any of the other temperatures used. However, it should be pointed out that at 32°F. the suspensions were not frozen and subsequent tests with these two organisms showed that growth occurred, particularly during the early hours of storage. There is little doubt from the results that, as a rule, intermittent freezing is more injurious to bacterial cells than continuous freezing at any of the four temperatures used. Studies with the three cultures of moulds showed some interesting facts. 23 to 25). (Table 19 and Figs. The most noticeable feature is the pronounced resistance of moulds to both intermittent and continuous freezing as compared with the bacteria. 96 - Table ffo./§ Survival of Bacteria Frozen Continuously and Intermittently in Vater at Different Temperatures Count per ml. Substrate In Thousands and Percentage Survival Org« Generic Freezing Mo. Classification Treatment 121 Flavobacterlum Continuous l day 2 days * days^ 7 daya 8 day* 10 day* 9 taji * aurFreezing Initial sursursur­ iur* aursur­ Temp* Count Count vlval Count vlval Count vlval Count rival Count vival Count vival Count vival * -20?F. 6,800 qTr. 1 5&. 32°F. Intermittent -20°F. 5,600 5,400 4,000 4,900 82 80 59 72 21,500 6,200 12,800 7,600 19,700 29 60 35 92 32°F. -20°F. 0°F. 15°T. 32°F. Intermittent -20°F. 0°F. Continuous 7,600 5,400 5.900 3,800 6.900 i fc. 32°F. 6,700 4.000 1.400 7i 78 50 91 11,200 14.700 12,800 24,400 24,300 3,800 1,800 21 210 2 2.5 274 50 52 16,900 68 11,700 60 11,600 22,400 100 18,600 560 54 3 4,200 20 490 24 2 520 18.700 87 18,000 6.000 6.400 4,600 8,300 & 61 100 4,100 54 300 28,000 o°f . I 3 84 6.900 36,200 27,300 S: Intermittent -20°F. 66 4.300 5.700 3,000 }S: 124 Flavobacterlum Continuous 4.500 90 99 59 3.400 15>. 32£* Intermittent -20“F. 0°F. 123 HIcfococcus 82 72 156 58: 122 Lactobacillus Continuous 5,600 6,100 4.900 75 27,100 77 24.700 67 19,200 67 31,100 17,200 14.900 5,900 20.900 39 4 91 5.300 4.700 3,900 6,600 920 580 s <1 4 ?4 si s & 12 8 24,500 22,400 68 62 49 25,100 7,000 3.500 2,000 6,000 4.400 3.400 3),500 380 22 <1 16 58 26 17 7,000 & 17,700 48 41 56 92 .13 3.1 88 5,200 65 50 t;2oo 111 <1 <1 <1 10,400 14,800 5,500 19,300 48 11 <1 <1 <1 94 4.2 12,200 7.3 .11 1 2 <1 <1 <1 31 2 .8 .1 .1 <1 <1 <1 <1 62 26 90 4.9 20 1 57 13,400 <1 <1 <1 62 2.4 8.3 .4 9,800 <1 <1 <1 17 4 .13 6,000 33 3 .01 .12 <1 2.3 <1 .5 .01 <1 79 5,900 <1 <1 <1 <1 15,500 1 .2 4.8 .4 46 6,600 <1 <1 <1 78 5.400 5.400 5.400 7,100 .23 .02 .005 5,000 1 4.400 7,900 100 1 93 52 <1 .9 <1 7,200 95 23,300 64 24,800 68 21,000 58 930 3 2,700 7 420 1 .8 <1 76 <1 .015 4* <1 <1 <1 <1 12,200 12,800 8 600 5,700 6,600 3 *8?5 1 .6 .07 13 <1 .1 <1 .01 <11 .01 <1 <1 31 71 n 71 n <1 2 26,000 20,000 I 22,300 62 14,400 40 .005 <1 .005 <1 .025 <1 .01 <1 - 97 - 40 PERCENT SURVIVAL OF BACTERIA 100 INTERMITTENT 20 10 O*' 1 2 4 7 8 9 10 days PERIOD OF FREEZING STORAGE FIG. 19. SURVIVAL OF MESOPHILIC BACTERIA ( CULTURE NO. 121 - FLAVOBACTERIUM SP. ) FROZEN CONTINUOUSLY AND INTERMITTENTLY IN WATER AT DIFFERENT TEMPERATURES. - 98 - 40 PERCENT SURVIVAL OF BACTERIA 100 •X CONTINUOUS 20 INTERMITTENT — — -20 10 10 days PERIOD OF FREEZING STORAGE F I G .20. SURVIVAL OF MESOPHILIC BACTERIA ( CULTURE NO. 122 - LACTOBACILLUS SP. ) FROZEN CONTINUOUSLY AND INTERMITTENTLY IN WATER AT DIFFERENT TEMPERATURES. - 99 100 a 40 PERCENT SURVIVAL OF BACTERIA •x CONTINUOUS INTERMITTENT - 20 20 10 10 days PERIOD OF FREEZING STORAGE FIG.il . SURVIVAL OF MESOPHILIC BACTERIA ( CULTURE NO. 123 - MICROCOCCUS SP. ) FROZEN CONTINUOUSLY AND INTERMITTENTLY IN WATER AT DIFFERENT TEMPERATURES. - 100 - IOC CONTINUOUS 90 --- INTERMITTENT — - 20 F. OF. 80 A □ 15 F. O 32 F. X 70 60 50 40 30 20 \ 10 0 T T 2 4 10 days PERIOD OF FREEZING STORAGE .2,2 SURVIVAL OF MESOPHILIC BACTERIA ( CULTURE NO. 124 - FLAVOBACTERIUM SP.) FROZEN CONTINUOUSLY AND INTERMITTENTLY IN WATER AT DIFFERENT TEMPERATURES - 101 - Table No. f$ Survival of Moulds Frozen Continuously and Intermittently in Water at Different Temperatures Count per ml* Substrate in Thousands and Percentage Survival 5 hours Org. Generic Freezing No. Classification Treatment 1 Penlcilllua Continuous Intermittent Aspergillus Continuous Intermittent 4 Penlcillium Continuous Intermittent Period of Pressing 1 week Z weeks 4 weeks 5 weeks 6 weeks Freezing Initial sursursursursursursurTemp. Count Count vlval Count vlval Count vlval Count vlval Count vlval Count vlval Count viva -20°F. 0°F. 15°P. 32°F. 63 76 64 69 54 100 100 100 86 -20 °?. 0°F. l52 * 32°F. 3 24 hours -20°F. 0°F. 15?F. 32T1, 39 59 42 58 46 100 100 100 100 -20 °F. 0 °F. 15^. 32 °F. -20°F. <£*• 15°F. 32°F. -20°F. 0°F. 1$°F. 32°F. 65 64 P 80 13 86 12 84 16 100 100 56 60 89 62 P 84 55 50 69 98 79 100 69 58 48 98 79 40 43 25 52 86 53 74 100 92 79 71 50 39 27 72 100 100 100 59 52 41 ?4 100 66 100 64 44 40 83 37 32 17 54 59 51 27 86 9.2 24 92 76 83 65 59 *6 43 50 94 76 69 79 70 51 44 78 100 100 58 50 45 91 36 38 37 46 40 54 100 100 100 100 41 66 55 40 51 100 100 100 100 26 92 97 95 67 29 43 74 45 40 39 42 100 100 100 100 40 34 18 43 100 33 39 39 32 100 82 14 13 35 28 62 69 38 64 56 67 90 27 15 25 53 22 100 100 36 33 29 51 9.8 7.8 7.6 25 25 20 19 64 11 12 P 80 13 ll 69 69 55 8 8.9 100 54 65 52 90 61 9.5 7.7 13 8 .6 15 9.7 8.5 7-3 13 58 57 10 10 8 .1 66 11 4>? 4.6 2*6 8 .1 33 31 24 55 4.6 1.5 4.3 26 15 100 100 100 100 8.4 15 § -4 8.3 9.3 15 81 70 85 62 100 15 64 56 63 7.8 5 6.9 100 12 100 72 100 88 73 73 100 53 34 t7 80 28 62 72 28 18 20 100 62 46 51 50 90 16 31 11 20 3.5 3.9 10 1 .2 29 1.7 24 2i 8 12 15 21 24 33 15 37 100 87 4 7.4 9.1 59 5° 62 1.9 1.9 .5 •5 13 13 3 3 " 1 0 2 «» 100 a, «A 90 80 •a 70 6o 50 40 \ •a 30 CONTINUOUS INTERMITTENT 20 - 20 •o 10 0 i 5 i 24 hours i 1 i 2 i 3 r 4 i 5 i 6 wks. PERIOD OF FREEZING STORAGE Z3. SURVIVAL OF MOULDS ( PENECILLIUM SP. - ORG. NO. 1 ) FROZEN CONTINUOUSLY AND INTERMITTENTLY IN WATER AT DIFFERENT TEMPERATURES. - 103 100 •a/ x«< / / A*' x*A — \— •' 40 PERCENT SURVIVAL OF MOULDS •X CONTINUOUS INTERMITTENT----- \ \ ** -20 20 10 6 wks. 24 hours PERIOD OF FREEZING STORAGE FIG.2 if. SURVIVAL OF MOULDS ( ASPERGILLUS SP. - ORG. NO.3 ) FROZEN CONTINUOUSLY AND INTERMITTENTLY IN WATER AT DIFFERENT TEMPERATURES - 104 100 4-0 PERCENT SURVIVAL OF MOULDS •x CONTINUOUS INTERMITTENT 20 / \ 20 10 ’• o x 24 hours 1 2 4 6 wks PERIOD OF FREEZING STORAGE FIG.Z5. SURVIVAL OF MOULDS ( PENECILLIUM SP. - ORG. HO, 4 ) FROZEN CONTINUOUSLY AND INTERMITTENTLY IN WATER AT DIFFERENT TEMPERATURES - 105 Tiiis was particularly evident witli tiie continuously frozen series during tiie early period of freezing storage and to some extent throughout the entire period of storage* With the intermittent series there was little doubt that moulds frozen intermittently were considerably more resistant than bacteria similarly treated* From these results it may be concluded that intermittent freezing is more destructive than continuous freezing, both to bacteria and moulds. The data indicate that intermittent freezing is more destructive to bacteria than it is to moulds* Temperature generally is not an important factor in the range of -20°F. to 15°F. At 32°F. some of the test organisms showed growth* THE EFFECT OF ANTIBIOTICS ON BACTERIA IN VEGETABLE PRODUCTS Since the problem of natural antibiotics in vegetable products is so extensive in its scope no attempt was made in the present study to make a thorough investigation of the proportion of organisms producing substances which control the growth of other organisms. The only purpose in including the work here reported is to point out just one more complexity associated with studies on - 106 the microbiology of frozen fruits and vegetables. The appe arance of an antibiotic in the present st^dy was accidental and came about when pure cultures of Serratia marcescens. Sarcina lutea, Staphylococcus aureus and Chromobacterium viola ceum were inoculated into fresh and blanched peas. The purpose of the study was to determine the rate of growth of these test organisms in fresh and blanched peas both before and after freezing. The peas used for this study were not sterile, the rate of growth of the test organisms being determined by plate counts of only those colonies of organisms corresponding to the original test organism. The results presented in Table 20 are for the fresh, unfrozen series. The results for the frozen series were quite similar to those of the fresh product with the exception of results with one test organism, Chromobacterium violaceum. Apparently this organism is very sensitive to freezing since in all tests it completely disappeared after 24 hours at 0°F. With Serratia marcescens in the fresh product it is of interest to note the more rapid rate of growth in the blanched product as compared with the counts in the unblanched product. This is likely due to the release of soluble solids in the blanch process which provides additional nutrients for the growth of this organism. Of particular interest were the results - 107 with Sarcina lutea and Staphylococcus aureus in both blanched and unblanched psas. Held 24 hours at room temperature both of these test organisms had disappeared. Since the pH of the peas had dropped from a normal of 6.2 to 5*0 it was first thought that the increased acidity was responsible for the disappearance of the test organisms. However, the fact that Serratia marcescens showed only a slight decrease in numbers in the unblanched product and a normal rate of growth in the blanched product suggested that in addition to the acid an antibiotic might be the factor responsible for the peculiar results. Further studies in which the pH was held at 6.2 using a titrimeter indicated definitely the presence of an antibiotic which as subsequent plate tests showed was produced by a spore former and was active against Sarcina lutea and Staphylococcus aureus but not against Serratia marcescens or the Chromobacterium violaceum. The increased acidity was probably responsible for the disappearance of Chromobacterium violaceum in the unblanched product. Why it also disappeared in the blanched product in the Series k is not known unless an acid-producing type organism survived the blanch process and produced sufficient acid to inhibit this organism but not the others. Chromobacterium violaceum has been noted in other studies to be very sensitive to various agents. - 108 - Table Mo. 2 0 Rate of Growth of Pure Cultures of Bacteria In Unblanched and Blanched Peas Series A - pH Unaltered - Dropped from Normal pH 6.2 to 5.0 Count per Gram Product Serratia Marcescens Sarcina Lutea Staphylococcus Aureus Chromobacterium Violaceum Unblanched Initial 2 hours 4 hours 8 hours 12 hours 24- hours 12,950,000 14.700.000 1 0 .500.00 0 5.600.000 9.450.000 3.500.000 875,00 0 1,200,000 700,000 6 ,300,000 700,00 0 900.000 6 0 0.00 0 0 0 0 3,500 0 0 9.450.000 7 .700.000 3,500 0 0 Blanched Initial 2 hours 4 hours 8 hours 12 hours 24 hours 12,300,000 22,000,000 6 5 ,00 0 ,0 0 0 149.000.000 3 5 0 .00 0.00 0 2,100,000,000 263.000 455.000 1.400.000 1.400.000 350.000 <3,500 Series B .- pH Adjusted Serratia Marcescens 3 .600.00 0 1 .800.00 0 760,00 0 6,300,000 1 4 ,350,000 1,800,000 35,000 0 0 350,00 0 0 0 - Held at Constant pH 6.2 Count per Gram Product Sarcina lutea Staphylococcus Aureus Chromobacterium Violaceum Unblanched Initial 2 hours 4 hours 8 hours 12 hours 24 hours 22,000,000 36,000,000 8 2 ,0 0 0 ,0 0 0 96,000,000 1 ,9 0 0 ,0 0 0 ,0 0 0 3,600,000,000 6,000,000 7,200,000 4,800,000 960,000 350,000 <3,500 14,000,000 12,600,000 9.400.000 6.200.000 3 ,500,000 <3,500 600,00 0 840,000 1,200,000 3 .100.00 0 4.800.000 12,000,000 - 109 DISCUSSION A study of microorganisms associated with frozen fruits and vegetables presents many difficult­ ies, as may be gathered from the work here reported. There are many variables to be considered which may have a definite influence on the rate of survival of microorganisms during freezing and subsequent freezing storage. Probably the most important factor involved is the type of organism present on the products. Results of the present study with almost 100 different species, isolated from fruit and vegetable products, clearly indicates that some species of bacteria show enormous reductions in numbers when stored at 0°F. for a period of 6 weeks while others show considerable resistance to freezing with a lesser reduction in numbers during the same period of freezing storage and under identical conditions. There are also tremendous differences in the rate of destruction of different species since many showed no reduction during the freezing process and early freezing storage; others showed a marked susceptibility to freezing with almost complete destruction during the first 4 hours of freezing. Differences were also noted within the species when cultures of Escherichia coli were studied. An important point is that all organisms studied in - 110 this work did show reductions in numbers after 6 weeks freezing storage. The substrate in which the organism is suspended for freezing has been considered an important factor by many workers. However, the results presented in this study, while indicating a definite protection to some organisms against freezing in substrates such as a vegetable extract and 40 per cent sucrose, show that other organisms are not appreciably protected by these substrates. The results suggest that the character of the substrate may not be nearly as important as was previously thought, since the results presented in a graph (Fig. 16) of the average of all organisms studied in the different substrates indicate that the difference between the percentage survival in vegetable extract and in water was 17 per cent. It is doubtful if this figure is significant since, if different cultures had been used, such as spore formers, then the difference in the rate of survival after 6 weeks freezing storage would be negligible. The temperature of freezing has been thought by many to influence the effect on microorganisms. However, in this study, within the range of commercial freezing temperatures, -20°F. to 15°F., temperature did not appear to be an important factor, although some - Ill individual cultures do show some difference in rate of survival at the different temperatures used. In these instances -20°F. appears to offer greater protection. Again the importance of this factor varies with the species of bacteria. Definite evidence is presented in this and other studies on the effect of intermittent freezing as compared with continuous freezing. There is little doubt that intermittent freezing is more destructive to microorganisms than continuous freezing. From the point of view of evaluating the quality of frozen fruits and vegetables on the basis of the microbial content this is an unfortunate finding since a product, although showing a marked deterioration in quality due to defrosting and refreezing, will also show a relatively low microbial content. This means that in any analysis of frozen fruits and vegetables, particularly when the counts are low, the complete freezing history of the product must be available to the analyst. The results also suggest that the microbiological findings alone cannot be used in assessing the quality of frozen fruits and vegetables. From the results presented it is obvious that it would be impossible to predict the effect of freezing on the microbial load of a fresh fruit or vegetable going into the freezer. However, attention should be - 112 directed to newer methods in the commercial processing of frozen vegetables. Pre-processing procedures, in many plants, now employ a longer blanch to a peroxidase trace or negative reaction rather than the older procedure of using tests for the enzyme catalase which is inactivated at lower temperatures than peroxidase. The longer blanch results in a marked diminution in the initial microbial load and, perhaps of greater significance, a reduction in the number of types of microorganisms. This longer blanch should eliminate most bacteria other than spore formers and these, as indicated in the present study, show less resistance to freezing in the vegetative stage than many other types of bacteria. Thus, by reducing the opportunity for recontamination following the blanch process, and with continuous line operation, it is possible to produce frozen vegetables with a relatively low microbial content. With fruits which are not subjected to blanching, the picture is entirely different. Up to the present no method has been devised to control the numbers of microorganisms on products going into the freezer. However, natural protection due to the lower pH of fruits will limit the types and growth of some of the microorganisms present on the product. While no reference has been made in this paper - 113 to the various theories which, have been advanced concerning the death of microbial cells by freezing it might be well at this point to discuss some of these theories and present the views of the writer. Luyet and Gehenio (17) have dealt fully with these theories in their writings so no useful purpose would be served by going into detail in the present work. They list the theories which a number of workers have attributed the mechanism of death by freezing to as follows: 1. A withdrawal of energy 2. The attainment of a minimal temperature 3. Mechanical injury 4. Too rapid thawing 5. Dehydration 6. Various physiological, physical and chemical changes Each one of these theories has some point in its favour but unfortunately many of the theories are based on observations with higher plants and animals and attempts have been made to apply these observations to microbial cells. Many workers have subscribed to the theory of death of cells by mechanical injury. It was suggested that the production of ice crystals during the freezing process resulted in disintegration of microbial cells. However, Haines (5) was unable to find any change in - 114 the microbial cells due to freezing. This finding was substantiated in the present study. Cells which survived freezing showed no apparent change in the metabolic processes of the organisms studied. Rahn (26) states that "freezing involves several causes of death, and the most common cause, injury by ice crystals, is quantitatively unpredict­ able. Thus, no order of death can be expected and no order has been observed1'. Rahn does not present any data to substantiate his statements. Based on observations in the present investiga­ tion it is the opinion of the writer that none of the first five theories listed are applicable to microbial cells since none account for the fact that some cells in an actively growing culture of bacteria are killed during the process of freezing while others apparently suffer no cnange. The writer suggests that "weak cells" in a bacterial suspension are the ones that are killed while the stronger cells survive. The reason for some cells being weaker than others in the same suspension is not known but it could be associated with some of the factors mentioned by Luyet and Gehenio (physiological, physical and chemical changes). - 115 SUMMARY Data are presented on the effect of freezing vegetables harvested, from the same general area at various times showing wide differences in the rate of survival of bacteria. These data also indicate that freezing of vegetables effects a reduction in the number of types of bacteria present on the initial product. In the products studied, members of the genera,.Micrococcus and Flavobacterium were prominent in the frozen products. Studies on the effect of rate of freezing on microorganisms in fruits and vegetables generally showed a higher percentage survival in vegetable products frozen by the contact method at -20°F. and stored at -20°F. as compared with products frozen by a static method and stored at either -20°F. or 0°F. With fruits, neither the temperature nor the rate of freezing had any important influence on the survival of microorganisms. A study of pure cultures of five bacteria and one yeast substantiated earlier findings of Haines that microbial cells show no apparent change in appear­ ance due to freezing. Further studies showed that the metabolic processes of cells which survived freezing were not affected since the rate of growth - 116 and the ability to ferment a sugar broth in a given time were similar with both "fresh" and frozen cells. The effect of freezing aqueous suspensions of 80 pure cultures of psychrophilic, mesophilic and thermophilic bacteria indicated that the latter group of bacteria "were more susceptible to freezing with the psychrophiles and mesophiles showing greater resistance, both in the initial freezing process and througout the period of freezing storage. Considerable fluctuations in counts in individual cultures were noted from one period of storage to another and these have been attributed to uneven distribution of the cells of organisms in the suspensions. These fluctuations were more prominent with some species of bacteria than with others. Considerable differences in the irate of survival were also noted with different species of bacteria and, with one organism studied, within the species. Bacteria belonging to the genera Serratia. Sarcina. Micrococcus and Flavobacterium were more resistant to freezing than any of the other types studied. Freezing seven pure cultures of bacteria in suspensions of water, 3 per cent salt, a vegetable - 117 extract, and concentrations of sucrose varying from 5 to 40 per cent showed that the vegetable extract and 40 per cent sucrose generally provided the greatest protection to the organisms against freezing, while water, 3 per cent salt and 5 per cent sucrose showed the least protection* Sppres and vegetative forms of members of the genus Bacillus* subjected to freezing in aqueous suspensions, indicated that the spore stage of organisms is more resitant to freezing than the vegetative stage. Thermophilic spores showed less resistance to freezing than mesophilic spores. Bacteria frozen at 0°F. in aqueous suspensions, in both the logarithmic phase of growth and in older cultures, showed that, although members of the genus Achromobacter were less resistant to freezing in the logarithmic phase, the over-all picture suggested that there was no important difference in the rate of survival between cultures in the logarithmic phase of growth and in older cultures. Cultures of both bacteria and moulds frozen continuously and intermittently in aqueous suspensions at -20°F., 0°F., 15°F. and 32°F. showed that intermittent freezing was much more destructive to both bacteria and moulds. Moulds were generally more - 118 resistant to freezing than bacteria both in the continuously and intermittently frozen suspensions. Temperature did not appear to be an important factor although there was a slight indication that -20°F. was less destructive to microorganisms in the continuously frozen series. However, with intermittent freezing temperature was definitely unimportant. In a study in which pure cultures of chromogenic bacteria were inoculated into fresh and blanched peas it was found that with those organisms not inhibited by a natural antibiotic which was presnt, the rate of growth was greater in the blanched product, due possibly to the release of soluble solids in the blanch process. The growth of two organisms, Sarcina lutea and Staphylococcus sp., was inhibited by the antibiotic. - 119 LITERATURE CITED 1. Berry, J.A. Microbiology of the frozen pack. The Canning Age, 13, 251. Western Canner and Packer, 25, Feb. 1932. 2. Berry, J.A. Destruction and survival of microorganisms in frozen pack foods. J. Bact., 26 (5), 459-470. 1933. 3. Burton, M.O. Comparisons of coliform and enterococcus organisms as indices of pollution in frozen foods. Food Res., 14 (5), 434-438. 1949. 4. Gunderson, M.F., and Rose, K.D. Survival of bacteria in a precooked, fresh-frozen food. Food Res., 13 (3), 254-263. 1948. 5. Haines, R.B. The effect of freezing on bacteria. Proc. Royal Soc. of London. B, 124, 451-463. 1938. 6. Hegarty, C.P., and Weeks, O.B. Sensitivity of Escherichia coli to cold-shock during the logarithmic growth phase. J. Bact., 39 (5), 475-483. 1940. 7. Hilliard, C.M., Torrossian, C., and Stone, R. Notes on the factors involved in the germicidal effect of freezing and low temperatures. Science, 42, Nov. 26. 1915. 8. Hilliard, C.M., and Davis, Mildred A. The germicidal action of freezing temperatures upon bacteria. J. Bact., 3, 423-431. 1918. 9. James, L.H. The microbiology of frozen foods. The Fruit Products Journal, 12 (4), 110-113. 1932. 10. Jones, A.H., and Lochhead, A.G. A study of micrococci surviving in frozen pack vegetables and their enterotoxic properties. Food Res., 4 (2), 203-216. 1939. 11. Jones, A.H. Factors affecting the microbial load of vegetables and fruits for frozen pack. Food in Canada, 2 (2), 13-17. 1942. - 120 12, Jones, A.H., and Ferguson, W.S. A study of Methods of preparing food products for microbiological analyses. Food Technology. In press. 13. Keith, S.C. Jr. Factors influencing the survival of bacteria at temperatures in the vicinity of the freezing point of water. Science 37, 877-879. 1913. 14. Kiser, J.S. A quantitative study of rate of destruction of an Achromobacter S p . by freezing. Food Res., 8 (4), 323-326. 1943. 15. Lochhead, A.G., and Jones, A.H. Studies of numbers and types of microorganisms in frozen vegetables and fruits. Food Res., 1 (1), 29-39. 1936. 16. Lochhead, A.G., and Jones, A.H. Types of bacteria surviving in frozen-pack vegetables. Food Res., 3 (3), 299-306. 1938. 17. 18. 19. 20 . Luyet, B.I., and Gehenio, P.M. Life and death at low temperatures. Biodynamica, Normandy, Missouri. 1940. McCloskey, C.S., and Christopher, W.N. Some factors influencing the survival of pathogenic bacteria in cold-pack strawberries. Food Res,, 6 (8), 327-333. 1941. McFarlane, V.H. Behaviour of microorganisms at sub-freezing temperatures. I. Freezing redistribution studies. Food Res., 5 (1)j 43-57. 1940, McFarlane, V.H. Behaviour of microorganisms at sub-freezing temperatures. II. Distribution and survival of microorganisms in frozen cider, frozen syrup-packed raspberries and frozen brine-packed peas. Food Res., 5 (1)? 59-68. 1940. 21 . McFarlane, V.H. Behaviour of microorganisms at sub-freezing temperatures. III. Influence of sucrose and hydrogen-ion concentrations. Food Res., 6 (5)5 481-492. 1941. 22. McFarlane, V.H., and Goresline, H.E. Microbial destruction in buffered water and in buffered sugar syrups stored at -17.8 C (OF.). Food Res., 8 (1), 67-77. 1943. - 121 23. Pederson, C.S. Significance of bacteria in frozen vegetables. Food Res., 12 (6), 429-438. 1947 24. Prescott, S.C., Bates P.K., and Highlands, M.B. Numbers of bacteria in frozen food stored at several temperatures. Am. J. Pub. Health, 22 , 257-262. 1932. 2*. Prudden, T.M. On bacteria in ice and their relations to the ice supply of New York City. Med. Rec., 31 , 341, 31 , 369. 1887. 26 . Rahn, Otto. Physical methods of sterilization of microorganisms. IV. Death by low temperatures. Bacteriological Reviews, 9 (1), 21-33- 1945. 27. Sedgwick, W.T., and Winslow, C.E.A. Experimentelle und statistische studien uker den einfluss der kalte auf den typhusbacillus und seine verteilung. Cent. Bakt., 27, 684. 1900. 28 . Sherman, J.M., and Albus, W.R. in bacteria. Physiological youth J. Bact., 8, 127-139- 1923. 29. Smart, Helen F., and Brunsteller, B.C. Spinach and kale in frozen pack. I. Scalding tests II. Microbiological Studies. Food Res., 2 (2), 151-163. 1937- 30. Smart, Helen F. Microbiological studies on cultivated blueberries* in frozen pack. Food Res., 2 (5), 429-434. 1937. 31. Smart, Helen F. Types and survival of some microorganisms in frozen-pack peas, beans, and sweet corn grown in the east. Food Res., 2 (6), 515-528. 1937. 32. Smart, Helen F. Microbiological studies on commercial packs of frozen fruits and vegetables. Food Res., 4 (3 ), 293-298. 1939. 33. Smart, Helen F. Further studies on behaviour of microorganisms in frozen cultivated blueberries. Food res., 4 (3), 287-292. 1939- 34. Straka, R.P., and James, L.H. A health aspect of frozen vegetables. Am. J. Pub. Health, 22 (5), 473-492. 1932. - 122 35. Straka, R.P., and James, L.H. Frozen vegetables. Am. J. Pub. Health, 23 (7), 700-703. 1933. 36. Van Eseltine, Wm. P., Nellis, Lois F., Lee, F.A., and Hucker, G.J. Effect of rate of freezing on bacterial content of frozen vegetables. Food Res., 13 (4), 271-280. 1948. 37. Wallace, G.I., and Park, S.E. Microbiology of frozen foods. V. The behaviour of Clostriduim botulinum in frozen fruits and in vegetables. Jour. Inf. Dis., 52, 150-156. 1933. 38. Wallace, G.I., and Tanner, F.W. Microbiology of frozen foods. I. Historical review and summary of results. The Fruit Products Journal, 13 (2), 52-54:56. 1933. 39. Wallace, G.I., and Tanner, F.W. Microbiology of frozen foods. I Historical review and summary of results. The Fruit Products Journal, 13 (4), 109-113. 1933. 40. Wallace, G.I., and Tanner, F.W. Microbiology of frozen foods II. Studies on frozen fruits and vegetables. The Fruit Products Journal, 13 (9)5 274-277. 1934. 41. Wallace, G.I., and Tanner, F.W. Microbiology of frozen foods. II. Studies on frozen fruits and vegetables. The Fruit Products Journal, 13 (12), 366-369:377. 1934. 42. Wallace, G.I., and Tanner, F.W. Microbiology of frozen foods. II. Studies on frozen fruits and vegetables. The Fruit Products Journal, 14 (5), 145-147:151. 1935. 43. Wallace, G.I., and Tanner, F.W. Microbiology of frozen foods. III. Longevity of pure cultures of microorganisms frozen in various menstra. The Fruit Products Journal, 14 (8 ), 235-237* 1935. 44. Weiser, R.S., and Osterud, Clarice M. Studies on the death of bacteria at low temperatures. I. The influence of the intensity of the freezing temperature, repeated fluctuations of temperature, and the period of exposure to freezing temperatures on the mortality of Escherichia coli. J. Bact., 50 (4), 413-439. 1945.