METABOLIC INJURY TO BACTERIA AT LOW TEMPERATURES

METABOLIC INJURY TO BACTERIA AT LOW TEMPERATURES ROBERT P. STRAKA AND J. L. STOKES Western Regional Research Laboratory,' Albany, California Received for publication January 19, 1959 The death of bacteria may be rapid during freezing or slow during extended storage at low temperatures (Weiser and Osterud, 1945). In the latter case, especially, one may expect to find that the first stages involve nonlethal physical or metabolic injury to the bacteria and that as injury becomes progressively more extensive or critical, it eventually leads to the death of the cell. Destruction of bacteria by low temperatures has been investigated extensively but the intermediate stages of injury have received little attention. The present paper deals with the extent and nature of nonlethal injury to bacteria by storage at subfreezing temperatures. METHODS AND MATERIALS Determination of injury and death. The criterion of injury was metabolic injury as evidenced by a change in nutritional requirements. Strains of bacteria were chosen which grew equally well initially on a minimal agar medium composed of inorganic salts, citrate, and glucose and on a rich, complex medium, trypticase soy agar. The bacterial strains, when plated, gave rise to the same number of colonies on both media although the colonies were smaller on the minimal medium. It was thought that metabolic injury due to low temperatures, which impaired the ability of the bacteria to synthesize essential cellular components, might prevent the cells from growing on the minimal medium but not on the complex nitrogenous medium rich in preformed amino acids, peptides, vitamins, purines, and pyrimidines and other essential cellular materials. This was found to be true. Although the bacterial counts decreased on both types of media as injury and death occurred during storage at low temperatures, the decrease was invariably greater on the minimal agar. We have defined the various states of the bac- 1 A laboratory of the Western Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. terial cells after exposure to low temperatures in the following, necessarily arbitrary manner: (1) Injured cells:-those which grew on trypticase soy agar but not on minimal agar after exposure. They were determined quantitatively by the difference in plate counts on the two media. (2) Dead cells:-those which failed to grow on trypticase soy agar after exposure. The difference between the initial trypticase soy agar count and that after exposure indicated the number of cells killed. (3) Unharmed cells:-those which grew on minimal agar after exposure to low temperatures. Cultures and cell suspensions. Strains of Escherichia coli, Pseudomonas fluorescens, Pseudomonas geniculata, and Pseudomonas ovalis were used. E. coli was grown at 37 C and the species of Pseudomonas at 30 C. Cell suspensions were prepared from 1 day old trypticase soy agar slant cultures. The Pseudomonas species were initially suspended in 0.1 per cent peptone to eliminate destruction during dilution (Straka and Stokes, 1957) and E. coli was diluted in 0.0003 M phosphate buffer, ph 7.2 (Butterfield, 1932). The suspensions were adjusted to give a reading of 50 on the Klett Summerson photometer (red filter) and these contained approximately 2 X 108 cells per ml. These master suspensions were then diluted 100-fold with the particular suspending fluid desired for freezing and storage of the cells at low temperatures. This was usually 0.5 per cent beef extract. Thus at the time of freezing the cell concentration was approximately 2 X 101 cells per ml. The exact number of cells was determined by plating aliquots of each suspension on trypticase soy agar and minimal medium. Ten-ml portions of each cell suspension were pipetted into a series of 15 by 150 mm screw cap test tubes. The suspensions were frozen rapidly by immersing the tubes in dry ice (-78 C) and were stored in refrigerators at -7 C, -18 C, and -29 C. At intervals of one to several days duplicate tubes of each treatment were removed, thawed 181

182 STRAKA AND STOKES [VOL. 78 under cold tap water, and plated on the minimal and trypticase soy agar to determine the condition of the bacterial population. The minimal agar, ph 7, contained K2HPO4, 7.0 g; KH2PO4, 3.0 g; sodium citrate *2H20, 0.1 g; MgSO4 7H20, 0.1 g; (NH4)2SO4, 1.0 g; glucose (autoclaved separately), 2.0 g; and agar, 15 g, dissolved in 1 L of distilled water. As described previously, the pseudomonads were diluted for plating in 0.1 per cent peptone and E. coli in phosphate buffer. The duplicate samples were plated in duplicate or triplicate and the counts were averaged. The pseudomonad colonies were counted after 3 to 5 days of incubation and the coli plates after 2 to 3 days of incubation. The data in the tables are representative and are recorded as percentages of the initial cell counts made just prior to the freezing of the suspensions. Numerous experiments indicated that the freezing process itself destroyed or injured usually less than 10 per cent of the bacterial population. Therefore the changes observed in the state of the cells were due primarily to the effects of storage at the low temperatures and not to the freezing process. Additional details of technique will be presented later in the text as needed. RESULTS Extent of injury. The fate of bacterial cells suspended in 0.5 per cent beef extract, frozen, and then stored at -18 C is shown in table 1. After 1 to 2 weeks, approximately 25 per cent of the cells of all of the Pseudomonas species and E. coli exhibited metabolic or nutritional injury in that they were no longer able to grow on the minimal agar although they could still develop on trypticase soy agar. There was little variation in the TABLE 1 Low temperature injury to bacteria Species Days Held Days Held at -18 C Population* Injured Killed hauned Pseudomonas fluorescens.. 7 25 56 19 Pseudomonas ovalis. 5 27 38 35 Pseudomonas geniculata... 6 17 61 22 Escherichia coli 13 23 45 32 * Frozen in 0.5 per cent beef extract. TABLE 2 Influence of time on cold injury of Pseudomonas ovalis Days of Storage Population* 1 15 22 63 5 27 38 35 11 24 44 32 19 18 53 29 * Frozen in 0.5 per cent beef extract and stored at -18 C. extent of injury among the different species. An even larger percentage of the cells, 40 to 60 per cent, were killed in the same period of time in the sense that they could no longer grow even on trypticase soy agar. It is reasonable to assume that prior to death these cells also exhibited nonlethal nutritional injury and that death was the end result of continued injury. Only 20 to 35 per cent of the initial populations remained unharmed under the above conditions. Storage period. It is to be expected that injury and death are interrelated parts of a continuous process and the data in table 2 appear to bear this out. The data were derived from a study of the effects of time on injury and death of P. ovalis by cold. Appreciable injury and death occurred within 1 day at -18 C. On continued storage, injury increased from 15 per cent to a maximum of 27 per cent after 5 days but subsequently decreased to 18 per cent after 19 days. In contrast, death continuously increased during the entire storage period and rose from 22 per cent after 1 day to 53 per cent after 19 days. As injury leads to death there are fewer cells left to be injured and therefore the percentage of injured cells decreases. These considerations limit the significance of the percentage figures for injury. A better measure of total injury is the sum of the injured and dead cells since the latter were injured cells at one stage. In this sense the per cent of cells in the original population which are injured never decreases but rather increases progressively with time. This is evident if the percentages of injured and dead cells at each interval are added and also from the continuous decrease in percentage of unharmed cells. Injury and death occur more rapidly during the early periods of storage than later and this is in

1959] INJURY OF BACTERIA AT LOW TEMPERATURES 183 agreement with the results of other investigations on the death of bacteria at low temperatures (Haines, 1938; Gunderson and Rose, 1948). Suspension fluids. The nature of the suspending fluid in which the bacteria are frozen and maintained has a very marked effect on the extent of injury and death as shown by the data on E. coli in table 3. The various fluids are arranged in order of increasing protective effect. Death, and therefore injury, is so extensive in 1 per cent peptone that virtually no cells are alive after 13 days at -18 C. At the other extreme, 10 per cent skim milk fully protected the cells and virtually all were alive and uninjured after 13 days. Beef and yeast extracts were intermediate and permitted some injury and some death. These differences cannot be due entirely to the variations in substrate concentrations since markedly different results were obtained with peptone and yeast extract although both were used in a 1 per cent concentration. The nature of the substrate in the suspension fluid therefore must be of importance. Temperature. The data in table 4 show that as the temperature of storage is lowered from -7 C to -29 C, the percentage of injured cells of P. fluorescens increases whereas that of killed cells decreases. This inverse relationship is due, again, to the fact that extensive death leaves fewer cells in the intermediate injured state and, conversely, as death is retarded, more of the cells are maintained in the nonlethal, injured state. The retardation of death as temperatures are lowered below the freezing point has been observed frequently and our results confirm this phenomenon. But more significantly, our data indicate that a large number of the cells which do not die at the lower temperatures may exhibit injury. TABLE 3 Effect of the suspension fluid on Escherichia coli cold injury of Suspension Fluids Population* Peptone, 1%... <1 99 <1 Beef extract, 0.5%... 23 45 32 Yeast extract, 1%... 14 37 49 Skim milk, 10%... 0 7 93 * Stored at -18 C for 13 days. TABLE 4 Cold injury of Pseudomonas fluorescens as a function of temperature Temperature Tempeature Days in Population* Storage- -7 C 1 25 51 24 7 17 82 1 15 1.9 98 0.1-18 C 1 41 51 8 7 34 63 3 15 12.8 87 0.2-29 C 1 35 48 17 7 39 54 7 15 36 59 5 * Frozen in 0.5 per cent beef extract. Cell concentration and ph. The extent of injury and death, with P. fluorescens, is independent of cell concentration within the range of 100,000 to 8,000,000 cells per ml. The cells were suspended in 0.5 per cent beef extract and stored for 5 days at -18 C. When the ph of the beef extract was varied between ph 5 and ph 8 by the addition of 0.04 M phosphate buffer, the least number of injured cells of P. fluorescens was found at the extremes of the ph range, i. e., at ph 5 and ph 8 where deathwas greatest and, conversely, the greatest number of injured cells were present at ph 6 and ph 7 where death was least extensive. Nature of the cold injury. To obtain information on the specific nature of the cold injury, two experimental approaches were used. In one, an attempt was made to identify the substances in trypticase soy agar responsible for the recovery of injured cells. In the other approach, various growth factors were added to the minimal agar to try to make it more suitable for the growth of injured cells. Trypticase soy agar contains 1.5 per cent trypticase, a pancreatic digest of casein, and 0.5 per cent phytone, a papaic digest of soya meal. A small amount of NaCl and, of course, agar are also present. The efficacy of trypticase and of phytone individually for recovery of injured cells was determined and also that of nutrient agar. Trypticase soy and minimal agar served as controls. The results obtained with P. fluorescens

184 STRAKA AND STOKES [VOL. 78 TABLE 5 Effect of plating medium on recovery of Pseudomonas fluorescens after exposure to cold Plating Agar Population Recovered* Expt 1 Expt 2 Trypticase soy... 42 51 Glucose-mineral salts (minimal) 19 28 Nutrient... 14 Phytone, 2%... 17 Trypticase 2%... 43 52 1..44 0.5%..32 0.25%..29 * After freezing in 0.5 per cent beef extract and storage at -18 C for 1 day. are given in table 5. These data show clearly that only trypticase is active. Recovery of cells is as great with 2 per cent trypticase as with the complete trypticase soy medium. Cell recovery, however, decreases progressively as the trypticase concentration is lowered so that at the 0.25 per cent level, the recovery is no better than with minimal agar. Phytone, even at a 2 per cent level is completely inactive so that cell recovery is the same as with minimal agar. Nutrient agar is also inactive. The addition of 2 per cent trypticase to minimal agar made the latter as good a recovery medium as trypticase soy agar. On the basis that the activity of trypticase might be due to its content of amino acids, acid hydrolyzed casein (vitaminfree) enriched with cystine and tryptophan was tested in minimal agar at concentrations as high as 2 per cent. It was inactive. Also, a mixture of B-vitamins and of purines and pyrimidines singly and in combination with acid hydrolyzed casein were entirely inactive. These results appear to rule out at least the common growth factors as active agents in the recovery of cold injured cells. In addition to trypticase, two other enzymatic digests of casein, N-Z-case and N-Z-amine, type B, were also fully active when tested at a 2 per cent concentration in minimal agar. Negative results, however, were obtained with N-Z-amine, types A and E, both also enzymatic digests of casein, with Hy-ease, an acid digest of casein, and with edamin, an enzymatic digest of lactalbumin. All of these preparations are Sheffield products. The above results suggest that the activity of trypticase and the other enzymatic digests of casein may be due to specific peptides present in them. This would explain the negative results with acid hydrolyzed casein in which there would be very little or no peptides. The inactivity of some of the enzymatic digests of caseins may be due to the absence of the specifically required peptides or to too extensive a hydrolysis of the casein. Peptides may be required for the resynthesis of essential enzymes or other proteins destroyed by exposure of the bacterial cells to low temperatures. Haines (1938) has suggested and has provided supporting evidence that denaturation and subsequent flocculation of cellular protein occurs in frozen bacteria and that this process is one of the maj or factors in the death of the bacteria. DISCUSSION There is not much known with certainty about the mechanism of injury and death of microbial cells at low temperatures. Mechanical destruction due to the formation of ice crystals in the cells is often postulated although the supporting evidence is scanty and there are good data against it (Haines, 1938; Weiser and Osterud, 1945; Mazur et al., 1957). The possibility of metabolic injury to bacteria due to cold is frequently mentioned in the early literature and receives support from our investigations. Similar, but less direct evidence has been presented by Gunderson and Rose (1948) and by Hartsell (1951). They noted that when coliform bacteria, salmonellae, and other bacteria were stored in foods at subzero temperatures for months, the bacteria became progressively less able to grow on selective agar media as compared to growth on nonselective, highly nutritious media. The concept of metabolic injury by cold is in harmony with data on injury to bacteria by heat, ultraviolet irradiation, and toxic chemicals. The experiments of Curran and Evans (1937) indicate that bacteria which survive destruction by the above physical and chemical agents are more exacting in their nutritive requirements than unexposed cells. Similar data for heat treated bacteria have been presented by Nelson (1943). Also, the more recent experiments of Heinmets

1959] INJURY OF BACTERIA AT LOW TEMPERATURES 185 and co-workers (1954a, b) on reactivation of E. coli, injured by ultraviolet irradiation, heat, and chemicals, with metabolites of the Krebs cycle indicate metabolic injury although the validity of these particular experiments has been questioned (Chambers et al., 1957; Hurwitz et al., 1957). SUMMARY Metabolic injury to bacteria at subzero temperatures has been demonstrated with several species of the genus Pseudomonas and with Escherichia coli. Cold injury is manifested by an increase in nutritional requirements. The injured cells can no longer grow on a simple, glucose-salts agar medium but can develop on a rich, complex medium, trypticase soy agar. Injured cells may constitute as much as 40 per cent of the bacterial population. The percentage varies with time and temperature of storage and with the nature and ph of the suspending fluid. Recovery of injured cells on trypticase soy agar is due to the activity of the trypticase component, an enzymatic digest of casein. Other similar casein digests were active but acid hydrolyzed casein was inactive. Peptides may be the active substances in the enzyme-digested casein and may be required by injured cells for resynthesis of essential proteins denatured by the subzero temperatures. REFERENCES BUTTERFIELD, C. T. 1932 The selection of a dilution water for bacteriological examinations. J. Bacteriol., 23, 355-368. CHAMBERS, C. W., TABAK, H. H., AND KABLER, P. W. 1957 Effect of Krebs cycle metabolites on the viability of Escherichia coli treated with heat and chlorine. J. Bacteriol., 73, 77-84. CURRAN, H. R. AND EVANS, F. R. 1937 The importance of enrichments in the cultivation of bacterial spores previously exposed to lethal agencies. J. Bacteriol., 34, 179-189. GUNDERSON, M. F. AND RoSE, K. D. 1948 Survival of bacteria in a precooked, fresh-frozen food. Food Research, 13, 254-263. HAINES, R. B. 1938 The effect of freezing on bacteria. Proc. Roy. Soc. (London), 124B, 451-463. HARTSELL, S. E. 1951 The longevity and behavior of pathogenic bacteria in frozen foods. The influence of plating media. Am. J. Public Health, 41, 1072-1077. HEINMETS, F., LEHMAN, J. J., TAYLOR, W. W., AND KATHAN, R. H. 1954a The study of factors which influence metabolic reactivation of the ultraviolet inactivated Escherichia coli. J. Bacteriol., 67, 511-522. HEINMETS, F., TAYLOR, W. W., AND LEHMAN, J. J. 1954b The use of metabolites in the restoration of the viability of heat and chemically inactivated Escherichia coli. J. Bacteriol., 67, 5-12. HURWITZ, C., RoSANO, C. L., AND BLATTBERG, B. 1957 A test of the validity of reactivation of bacteria. J. Bacteriol., 73, 743-746. MAZUR, P., RHIAN, M. A., AND MAHLANDT, B. G. 1957 Survival of Pasteurella tularensis in gelatin-saline after cooling and warming at subzero temperatures. Arch. Biochem. Biophys., 71, 31-51. NELSON, F. E. 1943 Factors which influence the growth of heat-treated bacteria. I. A comparison of four agar media. J. Bacteriol., 45, 395-403. STRAKA, R. P. AND STOKES, J. L. 1957 Rapid destruction of bacteria in commonly used diluents and its elimination. Appl. Microbiol., 5, 21-25. WEISER, R. S. AND OSTERUD, C. M. 1945 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. Bacteriol., 50, 413-439.