APPLIED MICROBIOLOGY, Feb. 1968, p. 335-339 Copyright @ 1968 American Society for Microbiology Vol. 16, No. 2 Printed in U.S.A. Factors Influencing the Survival and Revival of Heat-treated Escherichia coli A. D. RUSSELL AND DIANN HARRIES Welsh School of Pharmacy, University of Wales Institute of Science and Technology, Cardiff, Britain Received for publication 23 October 1967 Maximal revival of heat-damaged Escherichia coli occurred in nutrient media containing 0.8 to 1.0% (w/v) of Difco yeast extract. Vitamins did not appear to be involved in the recovery process. The situation with amino acids was less clear-cut, and, although certain of these may be essential for revival, proof for this is as yet inconclusive. Replica plating, in which colonies (from cells which had survived a heating process) on a rich medium were replicated onto minimal agar, revealed that no auxotrophic mutants had been formed as a result of heat treatment. Ba cteria which were heated in 1 % (w/v) yeast extract were killed more slowly than those heated in water. In recent years, there have been several reports dealing with the revival of bacteria damaged by chemical and physical means (9, 16). The composition and ph of the recovery medium and the temperature of incubation affect the recovery of bacteria damaged by phenol (9, 12), heat (1, 8, 15, 19; F. E. Nelson, Bacteriol. Proc., p. 40, 1956), and irradiation (2, 18). Thermally injured bacteria require an enriched medium for optimal revival (8, 15), and Amaha (3) found that the apparent extinction time of spores of Bacillus natto was doubled when broth containing yeast extract was used in place of broth alone as a subculture medium. A preliminary report from this laboratory, however, showed that different yeast extracts affected revival to varying degrees (8). The present study was thus undertaken to investigate the nature of those factors, present in supplemented media, which were responsible for the revival of heat-damaged Escherichia coli. In addition, the thermally induced death of bacteria suspended in yeast extract has been compared with that of cells in water. MATERIALS AND METHODS Organism. Details of the strain of E. coli, growth of cultures, preparation of washed suspensions coning 2 X 106 to 3 X 106 (or, in some experiments, about 4 X 108 to 5 X 108) viable cells/ml, and the heating procedure were described previously (8, 17). Viable-counting procedure. Samples of heated and unheated suspensions were removed, when required, and serially diluted in sterile water; 1-ml portions were then transferred to the recovery medium as rapidly as possible (20). Viable counts were carried out by this pour-plate method, since, in contrast to the situation with phenol-treated bacteria (9, 10, 12), for heated bacteria this technique is more satisfactory than the surface-viable method (1, 4, 8). Media used were: (i) nutrient agar (Oxoid, Ltd., London, England) containing (per liter) Lab-Lemco Beef Extract, 1 g; yeast extract (Oxoid), 2 g; peptone, 5 g; sodium chloride, 5 g; and agar, 15 g; (ii) "nutrient agar no. 2" consisting of nutrient broth no. 2 (Oxoid), containing (per liter) Lab-Lemco beef extract, 10 g; peptone, 10 g; and sodium chloride, 5 g; solidified with 10 g of lonagar no. 2 (Oxoid); (iii) a synthetic medium (J. R. L. Walker, Ph.D. Thesis, University of Leeds, England, containing (per liter) potassium dihydrogen phosphate, 20 g; ammonium sulfate, 1 g; magnesium sulfate, 1 g; and glucose, 3.6 g; solidified with 1% (w/v) lonagar no. 2. When required, media were supplemented with yeast extract (Difco). The ph of all media was 7.4. Plates were set up in quintiplicate, and were incubated at 37 C for 48 hr, after which the colonies were counted. No additional colonies developed on further incubation. Vitamins were purchased from the Laboratory Chemicals Group, British Drug Houses, Poole, Eng- 335 land, and amino acids from T. J. Sas and Son, Ltd., Holborn, London, England. Concentrated solutions were prepared and were sterilized by autoclaving (amino acids) or filtration (vitamins). Sufficient amounts of these solutions were included in synthetic agar to give the desired concentration. The amounts of amino acids and vitamins incorporated into the synthetic medium were based on the amounts present in a "typical" batch of Difco yeast extract. The amounts used (per 10 ml of agar) were: arginine, 1 mg; aspartic acid, 5 mg; glutamic acid, 6.5 mg; glycine, 2.5 mg; histidine, 1 mg; isoleucine, 3 mg; leucine, 3.5 mg; lysine, 4 mg; methionine, 1 mg; phenylalanine, 2 mg; threonine, 3.5 mg; tyrosine, 0.5 mg; valine, 3.5 mg; pyridoxine, 2 ug; biotin, 0.1 MAg; thiamine, 0.3 Mg;
336 APPL. M ICROBIOL. RUSSELL AND HARRIS nicotinic acid, 28,ug; and riboflavine, 2,g. In viable- 7 counting procedures, five plates of each medium were made, and colonies were counted as described above. Analyses of variance were made by the two-way 6 variance test, and the t test was used where necessary. Differences in colony counts within a medium were not significant (P = 0.05), but, where stated, were significant (P = 0.05) between media. 5 Replica plating. Replica plating, to determine whether auxotrophic mutants had been formed, was carried out by the method of Lederberg and Lederberg _ 4 (13). Colonies, from heated cells, on supplemented \ agar [i.e., synthetic agar containing 1% (w/v) yeast extract] were replicated onto minimal agar by means 3X of sterile velvet. Heat treatment of cells in the presence of yeast ex- > tract. Yeast extract was used in these experiments at a cr concentration of 1% (w/v). The ph was adjusted 2- before use to conform to that of the water used. The initial number of viable bacteria per milliliter was about 2 X 108 to 3 X 108 in all cases. For the viable counts, samples from suspensions at 50, 55, or 60 C in water or yeast extract were removed and serially diluted in sterile water; 1 -ml portions were transferred,_,_,_. to petri dishes, to which were added 10 ml of nutrient 2 agar (Oxoid) containing 1% (w/v) yeast extract Hours (Difco). The plates were incubated at 37 C for 48 hr, um and the colonies were counted. FIG. 1. Effect ofrecovery mediwn on viable counts of No additional colonies developed on continued suspensions of Escherichia coli heated at 50 C. Reincubation at 37 C. covery media: nutrient agar (0) and nutrient agar containing 1% yeast extract (0). RESULTS Effect of supplemented media on revival. Difco thus included in recovery media to act as an yeast extract, at a concentration of 0.8 to 1.0% arbitrary standard in certain other experiments. (w/v), enhanced revival of heat-damaged cells of Higher colony counts were also obtained with the strain of E. coli used (Table 1). Figure 1 heated cells in nutrient agar no. 2 than in nutrient shows the survival curves of heated bacteria in agar (Fig. 2). Differences in colony counts benutrient agar with and without added yeast ex- tween media were significant. Nutrient agar no. 2 tract. Differences in colony counts between media is richer in certain nutritional constituents than were significant. nutrient agar. Nutrient agar no. 2 containing A 1 % (w/v) concentration of yeast extract was yeast extract also gave higher colony counts than nutrient agar (Fig. 3), and the differences between media were again significant. However, no further TABLE 1. Effect of concentration of Difco yeast enhancement of revival occurred in agar no. 2 extract in nutrient agar on the colony counts of containing yeast extract than in agar plus extract unheated (50 C, I hr) suspensions of Escherichia or in agar no. 2 alone. coli The shape of these time-survivor curves indi- Colony countsa cates that cells of unequal thermal resistance are Concn (%, w/v) of present (7, 11). Before heating After heating Effect of vitamins on revival. The superiority of Difco yeast extract over the Oxoid brand in 0 243 25 enhancing revival of thermally injured E. coli 0.2 231 86 cells (8) could be due to the fact that the former 0.4 244 85 contains a more complete range of amino acids 0.6 237 87 and a greater number of vitamins. Accordingly, 0.8 266 150 the addition to synthetic agar of those five vita- 1.0 240 151 mins -present in Difco yeast extract was investia Viable counts per milliliter may be obtained gated. The vitamins were included singly and in by multiplying colony counts by 104 for unheated combiation, but no enhancementof recoveryover suspensions and by 102 for heated suspensions. the control (all vitamins absent) was observed. E
VOL. 16, 1968 SURVIVAL AND REVIVAL OF HEAT-TREATED E. COLI 337 Replica plating. Al colonies produced on synthetic agar containing 1% yeast extract from heatdamaged (50 C, 1 hr) cells could be replicated onto synthetic agar, indicating that no auxotrophic mutants had been formed. Heat treatment of cells in the presence of yeast extract. Bacteria suspended in yeast extract were killed less rapidly at 50, 55, and 60 C than those held in water at the corresponding temperatures (Table 4). It is considered unlikely that yeast extract carried over from the heated suspension into the subculture medium would account for the increased viable counts since (i) a considerable degree of dilution would be effected before a sample was plated into agar, (ii) increases in concentration of yeast extract above 1 % had no further effect on revival. The protective effect of yeast during the heating process would not appear g3-02 Hou rs FIG. 2. Effect ofrecovery medium on viable counts of suspensions ofescherichia coli heated at 50 C. Recovery media: nutrient agar (0) and nutrient agar no. 2 (0). Similarly, despite some fluctuations in the colony counts, the addition of vitamins singly and in combination to synthetic agar containing Vitamin Free Casamino acids (Difco) has always given statistically significant and lower colony counts than those obtained with synthetic agar containing Difco yeast extract, e.g., as shown in Table 2. The Vitamin Free Casamino product itself stimulated recovery, but not to the same extent as did yeast extract. Effect of amino acids on revival. The concentration of each amino acid incorporated into synthetic agar was identical to that given in an analysis of a "typical" batch of yeast extract. Colony counts were made in synthetic agar with and without added yeast extract, in synthetic agar to which was added the full complement of amino acids used, and in synthetic agar containing 12 amino acids (one, in turn, being omitted from the list). The results are shown in Table 3. 5 '4- U\\ Z 0~~~~~~~ * 3\ Hours FIG. 3. Effect ofrecovery medium on viable counts of suspensions ofescherichia coli heated at 50 C. Recovery media: nutrient agar (0) and nutrient agar no. 2 containing 1% yeast extract (X). TABLE 2. Effect of vitamins, alone and in combination, on the revival of heated suspensions (50 C, I hr) of Escherichia colia Type of agar Added vitamins Colony count Synthetic None 12 Synthetic + CA None 53 Synthetic + YE None 287 Synthetic + CA BPRNT 63 B 38 P 52 R 34 N 35 T 54 a B, biotin; P, pyridoxine; R, riboflavine; N, nicotinamide; T, thiamine; CA, Casamino Acids (Vitamin Free), 1% (w/v); YE, yeast extract, 1% (w/v). Viable counts per milliliter may be obtained by multiplying colony counts by 102.
338 RUSSELL AND HARRIS APPL. MICROBIOL. TABLE 3. Effect of combinations of amino acids in synthetic agar on revival of heated (50 C, I hr) suspensions of Escherichia coli Amino acid omitted Viable count/ml Argininea... 2.2 X 102 Aspartic... 4.6 X 102 Glutamic... 3.5 X 102 Glycine... 4.0 X 102 Histidine... 1.5 X 102 Isoleucine... 1.1 X 102 Leucine... 3.2 X 102 Lysine... <1 X 102 Methionine... <1 X 102 Phenylalanine... 1.3 X 102 Threonine... <1 X 102 Tyrosine... 1.8 X 102 Valine... 2.7 X 102 Allb... 2.4 X 102 All... 41 X 102 None... 2.8 X 102 a The L form was used. All other amino acids (except glycine) were DL. b Synthetic agar alone. c Synthetic agar containing 1% (w/v) yeast extract. TABLE 4. Decrease in viability in bacteria suspended in water and 1% (w/v) yeast extract at different temperatures Temp (C) Time (min) Viable count/mla of bacteria suspended in Water 1% yeast extract 50 60 3 X 105 4.5 X 106 55 30 2.5 X 104 3.3 X 105 60 15 1.3 X 104 3.0 X 105 a Initial number of viable bacteria per milliliter in each case was 2.6 X 108. to be peculiar to this organism, for it has previously been found (5) that the exponential timesurvivor curve for Bacillus stearothermophilus spores heated in a killed-yeast suspension was less steep than for spores heated in water. DiscUSSION It has been shown in these experiments that Difco yeast extract appears to stimulate repair mechanisms in a proportion of thermally injured E. coli cells. It has not been possible to elucidate what factor(s) is responsible for this revival. Moreover, it must be remembered that the numbers of cells involved are small. However, vitamins are not implicated in the recovery process, a conclusion that is similar to that reached for frozen and stored suspensions of Aerobacter aerogenes and E. coli (14) and for irradiated suspensions of E. coli (18). The omission of methionine, lysine, and threonine [the first two of which were also found to be necessary for the revival of frozen and stored cells (14)] from the synthetic recovery medium (Table 3) resulted in decreased survival, whereas the inclusion in synthetic agar of all those amino acids present in Difco yeast extract did not enhance recovery to the same extent as synthetic agar containing yeast extract. However, one puzzling feature of the results is that, whereas a "typical batch" of Casamino Acids (Vitamin Free, Difco) has a similar amino acid composition to yeast extract, the former product does not enhance recovery to the same extent (Table 2). The addition of the five vitamins to the Casamino Acids product in synthetic agar, to give an amino acids plus vitamin mixture equivalent to that of yeast extract, likewise did not give a recovery equal to that of synthetic agar containing yeast extract. Thus, it is not possible to come to any firm conclusions concerning the amino acid requirements of heat-damaged cells. It may, therefore, be concluded that the yeast extract provides an unknown factor(s) which is essential for the damaged cell to survive or to manufacture new cell constituents, or for both processes. Preliminary experiments have been carried out in which the yeast extract was fractionated by means of Sephadex columns. However, although the incorporation of the various fractions into the recovery medium affected the revival of heated cells, we have not been able to reach any firm conclusions as to what is responsible for increased revival. Moreover, nutrient agar no. 2, which is richer in nutrients than nutrient agar, also stimulated recovery. The addition of yeast extract to agar no. 2 did not enhance recovery to an even greater extent (Fig. 3). Thus, the problem remains as to whether yeast extract is providing a specific, at present unidentified, factor, or whether revival takes place in any suitably nutritional (i.e., "nonspecific") medium. Although the experiments on revival reported in this paper refer to cells heated at 50 C, the stimulating effect of Difco yeast extract on the revival of cells heated at higher temperatures (55 and 60 C) has also been observed. Replica plating indicated that no auxotrophic mutants, i.e., bacteria requiring a growth factor not required by the unheated cells, had been formed as a result of heat treatment. Bacteria which were heated at 50, 55, and 60 C in the presence of yeast extract were destroyed less rapidly than cells held in water at the corresponding temperature. Although an extracellular
VOL. 16. 1968 SURVIVAL AND REVIVAL OF HEAT-TREATED E. COLI 339 accumulation of ribonucleic acid (RNA) normally occurs with E. coli cells heated in water (17), it is possible that this leakage is prevented or reduced with cells in the extract; proof for this hypothesis is as yet lacking. However, Demain (6) has found that yeast extract markedly increased cell yield in, but decreased leakage of RNA from, B. subtilis. LITERATURE CmED 1. ALLWOOD, M. C., AND A. D. RUSSELL. 1966. Some factors influencing the revival of heat-damaged Staphylococcus aureus. Can. J. Microbiol. 12: 1295-1296. 2. ALPER, T., AND N. E. GILLEs. 1958. 'Restoration' of Escherichia coli strain B after irradiation: its dependence on suboptimal growth conditions. J. Gen. Microbiol. 18:461-471. 3. AMAHA, T. 1952. The effect of sugars in the subculture medium on the survival time of Bacillus natto. J. Agr. Chem. Soc. Japan 26:306-313. 4. BAIRD-PARKER, A. C., AND E. DAVENPORT. 1965. The effect of recovery medium on the isolation of Staphylococcus aureus after heat treatment and after the storage of frozen and dried cells. J. Appl. Bacteriol. 28:390-402. 5. COOK, A. M., AND R. J. GILBERT. 1965. The effect of yeast cells in the heating medium on the heat resistance of Bacillus stearothermophilus spores. J. Pharm. Pharmacol. 17:20S-21S. 6. DEMAIN, A. L. 1966. Amino acid control of ribonucleic acid excretion in Bacillus subtilis. Biochem. Biophys. Res. Commun. 24:39-43. 7. HANSEN, N.-H., AND H. RImEMANN. 1963. Factors influencing the heat resistance of non-sporing bacteria. J. Appl. Bacteriol. 26:314-333. 8. HARRES, D., AND A. D. RUSSELL. 1966. Revival of heat-damaged Escherichia coli. Experientia 22: 803-804. 9. HARRiS, N. D. 1963. The influence of the recovery medium and the incubation temperature on the survival of damaged bacteria. J. Appl. Bacteriol. 26:387-397. 10. HARRIS, N. D., AND M. WHITEFIELD. 1963. A lethal effect on damaged bacteria associated with the counting technique. Nature 200:606. 11. INGRAHAM, J. L. 1962. Temperature relationships p. 265-296. In I. C. Gunsalus and R. Y. Stainer [ed.] The bacteria, vol. 4. Academic Press, Inc., New York. 12. JACOBS, S. E., AND N. D. HARRIS. 1960. The effect of environmental conditions on the viability and growth of bacteria damaged by phenols. J. Appl. Bacteriol. 23:294-30. 13. LEDERBERG, J., AND E. M. LEDERBERG. 1952. Replica plating and indirect selection of bacterial mutants. J. Bacteriol. 63:399-406. 14. MAcLEoD, R. A., L. D. H. SMmrH, AND R. GELI- NAS. 1966. Metabolic injury to bacteria. I. Effect of freezing and storage on the requirements of Aerobacter aerogenes and Escherichia coli for growth. Can. J. Microbiol. 12:61-72. 15. 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. 16. RUSSELL, A. D. 1964. Damaged bacteria: recovery and revival. Lab. Pract. 13: 114-122. 17. RUSSELL, A. D., AND D. HARR.Es. 1967. Some aspects of thermal injury in Escherichia coli. Appl. Microbiol. 15:407-410. 18. STAPLETON, G. E., A. J. SBARRA, AND A. HOLLAEN- DER. 1955. Some nutritional aspects of bacterial recovery from ionizing radiations. J. Bacteriol. 70:7-14. 19. STILES, M. E., AND L. D. Wim-ER. 1965. Thermal inactivation, heat injury and recovery of Staphylococcus aureus. J. Dairy Sci. 48:677-681. 20. WLLis, B. A. 1957. Resistances of vegetative bacteria to moist heat. J. Pharm. Pharmacol. 9: 864-875.