ABSTRACT. even billions per gram or square centimeter, occur on or in poultry, fish, meats, vegetables,

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1 APPLIED MICROBIOLOGY, Jan., 1966 Vol. 14, No. 1 Copyright 1966 American Society for Microbiology Printed in U.S.A. Quantitative Ecology of Psychrophilic Microorganisms J. L STOKES AND MARY L. REDMOND Department of Bacteriology and Public Health, Washington State University, Pullman, Washington Received for publication 9 August 1965 ABSTRACT STOKES, J. L. (Washington State University, Pullman), AND MARY L. REDMOND. Quantitative ecology of psychrophilic microorganisms. Appl. Microbiol. 14: To obtain information on the importance of psychrophiles in nature, 95 samples of soil, water, mud, and various foods were quantitatively assayed for their content of psychrophilic bacteria and fungi and also for mesophilic and thermophilic bacteria and fungi. Thousands to millions of psychrophilic bacteria were present per gram of soil and represented 0.5 to 86% of the bacterial population. Also, about 25 % of the fungi in uncultivated soil were psychrophilic. In stream and river water, psychrophilic bacteria constituted to 47% of the bacterial population; in lake water, 41 to 76%; and in lake mud, 11 to 33%. Large numbers of psychrophilic bacteria were present in dairy products, meats, and other foods, and accounted for 35 to 93% of the bacterial population of meats. In contrast, thermophilic bacteria usually comprised 1% or less of the bacterial population in all of the materials examined. The data indicate that psychrophiles are both ubiquitous and numerous in nature, and probably play important roles in the cycles of matter. Downloaded from A large amount of information on the distribution of psychrophilic microorganisms has accumulated since they were first described by Forster in 1887 (8). The data clearly indicate that psychrophiles are ubiquitous. They occur in temperate as well as polar regions, on land and in water, on and in a large variety of plants and animals, and in foods. The group includes bacteria, yeasts and molds, and also morphological and taxonomic types similar to mesophiles. Thus, psychrophilic bacteria may be long or short rods, cocci or vibrios, gram-positive or gram-negative, sporeformers of nonsporeformers, and aerobic, facultative, or anaerobic. All of them have the common property of being able to grow at 0 C and lower temperatures, in contrast to the minimal growth temperature of about 10 C for mesophiles (7, 10, 15, 17). The significance of psychrophiles in nature is not fully clear, since data on the numbers of psychrophiles in natural habitats are limited. Most of the quantitative data are for foods, especially dairy products (3, 7, ). Many of these counts are difficult to interpret because incubation temperatures above 0 C were used (sometimes as high as 10 C, which would permit growth of mesophiles). But there is no doubt that high concentrations of psychrophilic bacteria, millions or even billions per gram or square centimeter, occur on or in poultry, fish, meats, vegetables, and dairy products, and also that hundreds or thousands of psychrophilic molds are present per gram of frozen pastries (11, 12). The present investigation was undertaken to determine the quantitative distribution of psychrophilic bacteria and fungi in 95 samples of soil, water, mud, and various foods as determined by counts at 0 C. Simultaneously, quantitative counts were made of the mesophilic and thermophilic bacteria and fungi present for comparative purposes. MATERIALS AND METHODS The soils included uncultivated, cultivated, and garden soil. The water samples were from streams, rivers, and lakes, and corresponding samples of lake bottom mud were taken at the same locations. The food samples included milk and other dairy products, chilled meats, frozen vegetables, and precooked frozen foods obtained from local stores. The dry weights of the soil and mud samples were determined by drying portions to constant weight at 105 C. The diluent for the microbial counts was % peptone. The dairy products were assayed by the procedures in Standard Methods for the Examination of 74 on March 26, 2019 by guest

2 VOL. 14, 1966 ECOLOGY OF PSYCHROPHILIC MICROORGANISMS 75 Dairy Products (2), and the other food samples by the procedures in Recommended Methods for the Microbiological Examinationi offoods (1). l counts were made on Trypticase Soy Agar by the spread plate technique. For psychrophiles, the plates were incubated for 14 days at 0 C; for mesophiles, 4 to 5 days at 20 C; and for thermophiles, 2 to 3 days at 55 C. Since the 20 C incubation for mesophiles would also permit growth of most psychrophiles, the true mesophile count was calculated by subtracting the psychrophile count at 0 C from the bacterial count at 20 C. Psychrophilic, mesophilic, and thermophilic counts of fungi were made in the same manner as for the bacteria except that acidified (ph 4) potato-glucose-agar was used. RESULTS Thermophilic fungi were not found in any of the materials examined. Such fungi, which have a maximal growth temperature of 50 to 60 C, exist, although they are not common (5). It may be that our incubation temperature of 55 C was somewhat high, since only a few thermophilic fungi can grow at 55 C. Soils. Representative data for psychrophilic, mesophilic, and thermophilic bacteria and also fungi (except thermophiles) are presented in Table 1. In the garden soils, mesophilic bacteria predominated, but psychrophilic bacteria averaged about 1 million per gram of soil and accounted for as much as 19% of the total bacterial population. They greatly exceeded the thermophiles, which represented % or less of the bacterial population. No psychrophilic fungi were present. The cultivated soils which were from wheat fields had fewer psychrophilic bacteria, as percentage of the total population, than did the garden soils, although one soil had 15% psychrophiles. Psychrophilic fungi were present, to the extent of 30% in one soil. The uncultivated soils differed markedly from the other two soil types. The total microbial population was smaller, as might perhaps be expected since such soils receive relatively little organic matter. But psychrophilic bacteria were usually predominant and represented as much as 86% of the bacterial population. As usual, relatively few thermophilic bacteria were present, less than 1 %. Considerable numbers of psychrophilic fungi were found, and they accounted for about 25% of the fungal population. In general, it appears that cultivation favors the growth of mesophilic bacteria, and this reduces the percentage of psychrophiles, although hundreds of thousands or millions of psychrophilic bacteria and hundreds or thousands of fungi were present per gram of soil (dry weight)-except for the absence of psychrophilic fungi in the garden soils. Forster (9) found 140,000 psychrophilic bacteria per gram of garden soil, and Lochhead (13) reported approximately 2,000,000 psychrophilic bacteria per gram of frozen field soil (compared with 32,000,000 mesophiles). Water and lake mud. The numbers of psychrophilic, mesophilic and thermophilic bacteria and fungi in different river and stream waters are shown in Table 2. Hundreds of psychrophilic bacteria per milliliter of river water were found, and these accounted for to 29% of the bacterial population. Mesophiles were predominant (70 to %), and thermophiles were relatively scarce (1 % or less). Psychrophilic fungi were absent. Stream water had a much higher population of psychrophilic bacteria, usually thousands TABLE 1. Psychrophilic, mesophilic, and thermophilic bacteria and fungi in soils Psychrophiles Mesophiles Thermophiles Psychrophiles Mesophiles No.* Per No. Per No. Per No. Per No. Per cent cent cent cent cent Garden soil la... lb... lc... Cultivated soil 2A... 2C... 2E... Uncultivated soil 3A... 3B... 3C... 1,000, ,000 1,700,000 23, , , ,000 0,000 3,, ,000,000 5,000,000 7,200,000 4,900,000 6,700,000 4,600, , ,000 1,000, ,000 15,000 10,000 79,000 30,000 61,000 4,000 6,000 7, < , 5, 4, ,000 32,000 29,000 1,600 35, ,000 12,000,000 12, * Per gram, dry weight.

3 76 STOKES AND REDMOND APPL. MICROBIOL. TABLE 2. Microbial populations of river and stream water Psychrophiles Mesophiles Thermophiles Psychrophiles Mesophiles No. per ml cpenrt No. per ml Per No. per Per No. per Per No. per cent ~~cent ml cent ml cent ml Per cent River water 7A... 7B... 7C... 7D... Stream water 4A... 4B... 4C... 4D... 1, 410, , , 3,300 TABLE ,000 3,300 3,600 3,800 24,000 3,600 4,300 3, Microbial populations of lake water and lake mud Psychrophiles Mesophiles Thermophiles Psychrophiles Mesophiles 8 No.^ Per No. Per No. Per No. Per No Per cent cent cent cent cent Lake water 5A < 10 SB C D Lake mud 6A... 28, , , < < 6B... 85, , , , C... 68, , , ,000 6D... 18,000 92, , * Per milliliter of water or per gram of mud (dry weight). per milliliter. In some samples, they were almost as numerous as mesophiles and represented more than 40% of the bacterial population. Thermophilic bacteria were present in less than 1% concentration. Psychrophilic fungi were found in only one of six samples of stream water. As shown in Table 3, lake water also contained hundreds of psychrophilic bacteria per milliliter. These organisms were predominant in several of the samples and represented about 70% of the bacterial population, compared with 1 % or less of thermophiles. No fungi of any type were found. The table contains the corresponding data for lake bottom mud. Mud 6A, for example, is a sample of the bottom material directly under lake water sample 5A. As would be expected, the bacterial counts of the mud are much higher than those of the overlying water. Thousands of psychrophiles were present per gram of mud (dry weight), comprising 11 to 33% of the bacterial population. Mesophilic bacteria predominated (65 to 88%), and thermophiles, although present in hundreds or thousands per gram, accounted for only about 1% of the population. Psychrophilic fungi were found in only one of the six samples of lake mud. In a bacterial analysis of 10 farm-pond waters, Malaney et al. (14) found, per milliliter, 3 to 2,300 psychrophiles, 30 to 29,000 mesophiles (standard plate count at 35 C), and 0 to 500 thermophiles. Unfortunately, the incubation temperature for the psychrophile plate counts fluctuated from 0 to 10 C and may have permitted growth of mesophilic bacteria. Foods. Numerical ranges of psychrophilic, mesophilic, and thermophilic bacteria in dairy products and frozen vegetables and fruits are given in Table 4. Hundreds of psychrophilic bac-

4 VOL. 14, 1966 ECOLOGY OF PSYCHROPHILIC MICROORGANISMS 77 TABLE 4. l populations of dairy products and frozen vegetables and fruits No. of samples a Psychrophiles Mesophiles Thermophiles Milk,raw ,0007,800 Milk, pasteurized Ice cream.5-15,000 3,400-13, Cream ,-,000 Butter ,500-11, ,000-1,,000 Cheeseb, ,300, ,000-24,000, Frozen vegetablesc ,000-10,000, Frozen fruitsd a Numbers of bacteria in the raw and pasteurized milk indicate the range per milliliter; for the other foods, the range per gram. I Cheddar, Swiss, American, cottage (two samples). c Corn, lima beans, string beans, broccoli, cauliflower, spinach. d Strawberries, raspberries. TABLE 5. l populations of meat, poultry, and fish per gram* Psychrophiles Mesophiles No. Per cent No. Per cent Hamburger... 54,000, ,000, Beef stew... 5,300, , Pork sausage. 13,000, ,000, Pork chops , ,000 7 Lamb chops , , Liver, beef... 8,000, ,000, Chicken, chilled t , , Fish sticks, frozen... 7, , * In all cases, the number of thermophiles was fewer than per gram. t Per square centimeter. teria were present in raw and pasteurized milk, and comprised 2 to 3% of the bacterial population in raw milk and 10 to 98% in pasteurized milk. Six of 12 samples of the latter, however, did not contain any psychrophiles. Thermophiles constituted usually 2% or less of the population in pasteurized milk. None of the milk samples contained psychrophilic fungi, and only two samples of pasteurized milk contained mesophilic fungi (two to six per milliliter). Some ice cream samples contained thousands of psychrophiles per gram but accounted for less than 10% of the population. Psychrophilic fungi were absent, and mesophilic fungi ranged from 0 to 50 per gram. No psychrophilic bacteria were detected in cream, but many were present in butter and some types of cheeses. Cottage cheese contained large numbers of psychrophilic bacteria and fungi. In general, numbers of psychrophiles in dairy products vary greatly depending on handling conditions, and our data are consistent with those of other investigators (3, 6, ). The frozen vegetables and fruits contained few or no psychrophilic bacteria and no psychrophilic fungi, although psychrophiles are known to occur frequently in such foods (7). Table 5 shows the numbers of psychrophilic, mesophilic, and thermophilic bacteria in meats, and also in one sample of chicken and one of fish. In general, the meats had large psychrophilic populations, containing hundreds of thousands or millions of bacteria per gram. Some of them had the highest psychrophile counts encountered in the present investigation, and the psychrophiles constituted 35 to 93 % of the bacterial population. Thermophiles could not be detected. Psychrophilic fungi were absent, although mesophilic forms were encountered, ranging from 5 to 10,000

5 78 STOKES AND REDMOND APPL. MICROBIOL. per gram. Chicken had hundreds of thousands and fish sticks several thousand psychrophilic bacteria per gram. DISCUSSION Considerably more quantitative data are needed before general conclusions can be drawn as to the importance of psychrophilic microorganisms in nature. But it is beginning to appear that psychrophiles are not only ubiquitous but also occur in sufficiently large numbers in natural habitats to warrant the conclusion that they are of considerable importance in the cycles of matter. In soil, psychrophilic bacteria range from thousands to millions per gram and may be the predominant bacterial flora in uncultivated soils. Psychrophiles also are major fractions of the bacterial population of stream and river water and may be predominant in lake water. Large populations of psychrophilic bacteria can be found in a great variety of foods, and here also they may greatly exceed the mesophiles. The situation is not as clear for psychrophilic fungi. They were not present in many of the materials examined, although appreciable numbers occurred in soil. A significant and perhaps surprising finding was the much larger populations of psychrophiles than of thermophiles in the materials examined. The latter rarely exceeded 1% of the bacterial population, compared with generally 10 to 90% for psychrophiles. This would seem to indicate that psychrophiles are of greater significance than thermophiles in nature, and may be related to the ability of psychrophiles to grow at the moderate as well as the low temperatures which most commonly occur in nature. Although no attempt was made to isolate psychrophiles from marine environments, psychrophilic bacteria have been isolated frequently from seawater and fish (4, 10). In view of the low temperature in the oceans, generally below 5 C, it seems likely that psychrophiles are the predominant bacterial flora, although systematic quantitative studies have not yet been made. ACKNOWLEDGMENT This investigation was supported by a research grant from the National Science Foundation. LITERATURE CITED 1. AMERICAN PUBLIC HEALTH AssocIATION Recommended methods for the microbiological examination of food. American Public Health Association, Inc., New York. 2. AMERICAN PUBLIC HEALTH ASsOCIATION Standard methods for the examination of dairy products, 11th ed. American Public Health Association, Inc., New York. 3. BAUMANN, D. P., AND G. W. REINBOLD The enumeration of psychrophilic microorganisms in dairy products. J. Milk Food Technol. 26: COLWELL, R. R., AND R. Y. MORITA Reisolation and emendation of description of Vibrio marinus (Russell) Ford. J. Bacteriol. 88: COONEY, D. G., AND R. EMERSON Thermophilic fungi. W. H. Freeman and Co., San Francisco. 6. DRUCE, R. G., AND S. B. THOMAs The microbiological examination of butter. J. Appl. Bacteriol. 22: ELLIOTT, R. P., AND H. D. MICHENER Factors affecting the growth of psychrophilic microorganisms in foods-a review. U.S. Dept. Agr. Tech. Bull FORSTER, J Ueber einige Eigenschaften leuchtender Bakterien. Centr. Bakteriol. Parasitenk. 2: FORSTER, J Ueber die Entwickelung von Bakterien bei niederen Temperaturen. Centr. Bakteriol. Parasitenk. 12: INGRAHAM, J. L., AND J. L. STOKES Psychrophilic bacteria. Bacteriol. Rev. 23: KUEHN, H. H., AND M. F. GUNDERSON Psychrophilic and mesophilic fungi in fruitfilled pasteries. Appl. Microbiol. 10: KUEHN, H. H., AND M. F. GUNDERSON Psychrophilic and mesophilic fungi in frozen food products. Appl. Microbiol. 11: LOCHHEAD, A. G The bacterial types occuffing in frozen soil. Soil Sci. 21: MALANEY, G. W., H. H. WEISER, R. 0. TURNER, AND M. VAN HORN Coliforms, enterococci, thermodurics, thermophiles and psychrophiles in untreated farm pond waters. Appl. Microbiol. 10: STOKES, J. L General biology and nomenclature of psychrophilic microorganisms, p In N. E. Gibbons [ed.], Recent progress in microbiology, VIII Intern. Congr. Microbiol., Montreal, Univ. Toronto Press, Toronto.. THOMAS, S. B., J. M. GRIFFITHS, AND J. B. FOULKES Psychrophilic bacteria in pasteurized milk. Dairy Eng. 77: WIrrER, L. D Psychrophilic bacteria-a review. J. Dairy Sci. 44: