109 767 {HESIS LIE-RARY ‘ Michigan State University THERMAL INJURY AND RECOVERY. OF SACCHAROMYCES CEREVISIAE Y25 by Thomas Ray Graumlich A DISSERTATION Submitted to Michigan State University‘ in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1978 ABSTRACT THERMAL INJURY AND RECOVERY OF SACCHAROMYCES CEREVISIAE Y25 BY Thomas Ray Graumlich The purpose of this investigation was to determine the influence of thermal stress on Saccharomyces cere- visiae Y25. Viability and respiration were measured in resting cells of g. cerevisiae subjected to heating at 56 C for 0 to 5 minutes. Immediately after heating plate counts on potato dextrose agar (PDA) were up to 1.5 log cycles lower than those on plate count agar (PCA). The proportion of the population affected was related to the severity of heat-stress. Delayed plating after storage at 22 C in distilled water resulted in increased plate counts on both PCA and PDA. Cannibalistic growth studies and recovery in the presence of growth inhibitors re- vealed increased plate counts during the first 12 hours of storage were related to recovery from injury rather than cryptic or cannibalistic growth. Recovery was pre- vented by storage at 4 C or in the presence of 2,4-dini- trophenol but was not prevented by storage in the pres— ence of cycloheximide, chloramphenicol, hydroxyurea, or actinomycin D. Reduced recovery of thermally injured Thomas Ray Graumlich cells on PDA in comparison to PCA was related to glucose concentration. Recovery on a minimal medium (MM) was also related to glucose concentration, however, recovery on MM containing filter-sterilized glucose was consider- ably higher than recovery on MM containing steam-steri- lized glucose. Respiratory activity of thermally stressed cells re- flected the severity of the heat-stress. The endogenous respiration was approximately 40 ul/mg/hr for cells heated for 2 minutes at 56 C as compared to 2 ul Oz/ml/hr for nonheated cells. There was a distinct decrease in respiration after 1 to 3 hours, but after 20 hours the respiration rate of heated cells was less than that of nonheated cells. Along with the abnormal rates of endo- genous respiration, respiratory quotients of cells were altered after heat stress. Addition of 2,4-dinitrophenol Stimulated OZ-uptake in nonheated cells but decreased Oz-uptake of heated cells. Due to the high rate of endogenous respiration, addition of glucose resulted in no substantial change in the rate of respiration of heated cells. However, glucose caused a delay in the characteristic decrease in respiration observed in heated cells. DEDICATION To my parents. ii ACKNOWLEDGMENTS I would like to express my sincere appreciation to Dr. Kenneth E. Stevenson for his guidance and patient encouragement during the course of the investigation and preparation of this dissertation. Appreciation is also expressed to the members of my graduate guidance committee: Dr. L. G. Harmon, Dr. C. M. Stine, Dr. E. S. Beneke, and Dr. D. R. Heldman. The assistance and encouragement of Ms. Marguerite Dynnik and my fellow graduate students is gratefully acknowledged. Finally, I wish to express my appreciation to the Department of Food Science and Human Nutrition for pro- viding the financial assistance and facilities to make this study possible. iii TABLE OF CONTENTS List of Tables . . . . . . . . . . . . . . . . . . List of Figures . . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . Sensitivity to Environmental Conditions . NaCl and water activity. . . . . . . . pH 0 O O O O O O O O O I O O O O O O 0 Temperature C O O O O O O O O O O O O Alterations in Growth and Cell Characteristics Nutritional requirements . . . . . . . . . . Growth and morphology . . . . . . . . . . . Subcellular Alterations . . . . . . . . . . . . Metabolic activity . . . . . . . . . . . . . Disruption of membranes and cellular organ- ization . . . . . . . . . . . .‘. . . . Nucleic acids . . . . . . . . . . . . . . . smary . O C O O O O O C O O O O O O O O O O 0 MATERIALS AND METHODS . . . . . . . . . . . . . . Organism and Cultural Conditions Thermal Stress . . . . . . . . Enumeration of "Viable" Cells Factors Affecting Viability . Composition of media . . . Temperature of storage . . Temperature of incubation Heat-killed cells . . . Metabolic inhibitors . Manometric Measurements . O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 'Cell Leakage . . . . . Statistical Analyses . iv Page vi viii moo \Imm (11wa w H Page RESULTS......................23 Recovery of Thermally Stressed Cells of Saccharo- myces cerevisiae Y25 on PCA and PDA . . . . . 23 Media Composition . . . . . . . . . . . . . . . 27 Cannibalistic Growth . . . . . . . . . . . . . . 30 Recovery of Heat-Injured Cells . . . . . . . . 36 Effect of Metabolic Inhibitors on Recovery of Heat-Stressed Cells . . . . . . . . . . . . . 39 Effects of Temperature on Recovery . . . . . . . 47 Storage temperature . . . . . . . . . . . . . 47 Incubation temperature . . . . . . . . . . 47 Respiration of Heat-Stressed Cells . . . . . . . 51 Leakage of Intracellular Constituents . . . . . 58 DISCUSSION 0 O O C O O O O O O O O O O O O C O O I 61 Recovery of Heat-Stressed Cells on PCA and PDA . Effect of Media Composition on Recovery . . . . 62 Recovery versus Growth . . . . . . . . . . . . . Recovery of Heat-Injured Cells . . . . . . . . 65 Effect of Inhibitors on Repair . . . . . . . . . 66 Effect of Temperature on Recovery . . . . . . . 68 Respiration of Heat-Stressed_Cells . . . . . . . 69 Cell Leakage . . . . . . . . . . . . . . . . . . 74 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 76 LIST OF REFERENCES . . . . . . . . . . . . . . . . 78 LIST OF TABLES Table Page 1. Information concerning metabolic inhibitors utilized in this investigation . . . . . . . 20 2. Plate counts of heat-stressed Saccharomyces cerevisiae on plate count agar (PCA): potato dextrose agar (PDA), PDA containing 0.1 M NazHPO4, and PCA containing 2.0% glucose . . 28 3. Plate counts of heat-stressed Saccharomyces cerevisiae on plate count agar (PCA), potato dextrose agar (PDA), and laboratory-prepared potato dextrose agar (LPDA) containing 0.1 or 2.0% glucose . . . . . . . . . . . . . . 29 4. Plate counts of heat-stressed Saccharomyces cerevisiae on minimal media (MM) containing 02 to 6.0% glucose 0 O O O I O O O O O O I 31 5. Plate counts of heat-stressed Saccharomyces cerevisiae on plate count agar (PCAT, potato dextrose agar (PDA) and minimal media (MM) containing 2.0% filter-sterilized glucose or 2.0% steam-sterilized glucose . . . . . . . 32 6. Plate counts of heat-stressed Saccharomyces cerevisiae on plate count agar (PCA), potato dextrose agar (PDA), and PDA supplemented with 0.50% yeast extract or 0.25% tryptone . 33 7. Growth at 22 C of unheated cells of Saccharo- myces cerevisiae in suspensions of heat- killed cells. Heat-killed cells were pre- pared by heating 3.5 x 1°7.§- cerevisiae cells/ml for 30 minutes at 56 C. . . . . . . 35 8. Plate counts on plate count agar (PCA) and po- tato dextrose agar (PDA) of heat-stressed Saccharomyces cerevisiae stored in water with or without 5 mg/ml of chloramphenicol. . . . 42 vi Table . Page 9. Plate counts on plate count agar (PCA) and potato dextrose agar (PDA) of heat-stressed Saccharomyces cerevisiae stored in water with or without 5 mg/ml chloramphenicol and 10 ug/ml cycloheximide . . . . . . . . . . . 43 10. Plate counts on plate count agar (PCA) and potato dextrose agar (PDA) of heat-stressed Saccharomyces cerevisiae stored in water with or without 0.10 mg7ml actinomycin D . . 44 11. Plate counts on plate count agar (PCA) and 'potato dextrose agar (PDA) of heat-stressed Saccharomyces ceregisiae stored in water with or without .075 M hydroxyurea . . . . . 45 12. Plate counts on plate count agar (PCA) and potato dextrose agar (PDA) incubated at 20, 25, 30, and 35 C of heat-stressed Saccharo- myces cerevisiae stored for 24 hours at 22 C in water . . . . . . . . . . . . . . . . . 50 13. Plate counts on plate count agar (PCA) and potato dextrose agar (PDA) of heat-stressed Saccharomyces cerevisiae immediately after heating and after 20 hours of storage in water at 22 C . . . . . . . . . . . . . . . 53 14. Rates of endogenous 0 -uptake in water at 30 C for nonheated an heat-stressed Saccharo- myces cerevisiae . . . . . . . . . . . . . . 54 15. Respiratory quotients (R.Q.) in water at 30 C for endogenous respiration of nonheated and heat-stressed Saccharomyces cerevisiae . . . 56 16. Quantitation of materials which absorb at 260 and 280 nm in Saccharomyces cerevisiae sus- pensions heated at 56 C for 0-5 mi utes. The suspensions contained 1.8 x 10 cells/ ml . . . . . . . . . . . . . . . . . . . . . 60 vii LIST OF FIGURES Figure Page 1. Plate counts of heat-stressed Saccharomyces cerevisiae on plate count agar (PCA) and potato dextrose agar (PDA). Cells were heated at 56 C . . . . . . . . . . . . . . 24 2. Comparison of recovery of heat-stressed Sac- charomyces cerevisiae on plate count agar. Cells were heated at 56 C . . . . . . . . 25 3. Growth of Saccharomyces cerevisiae in water and in suspensions of heat-killed cells at 22 C. Heat-killed cells were prepared by heating 3. 5 x 107 S. cerevisiae cells/ml at 56 C for 30 minutes . . . . . . . . . . 34 4. Growth of Saccharomyces cerevisiae heated at 56 C for 3 minutes and‘stored at 22 C in water undiluted or diluted 10"2 with var- ious concentrations of heat-killed cells. The heat-killed Sells were prepared by heating 3.5 x 10 S. cerevisiae cells/m1 at 56 C for 30 minutes and were used as storage media at 0, 30, 60 or 100% . . . . 37 5. Survivor curves of Saccharomyces cerevisiae heated at 56 C and plated on plate count agar (PCA) and potato dextrose agar (PDA) immediately and after storage in water at 22 C for 24 hours . . . . . . . . . . . . 38 6. Effect of storage in water at 22 C on plate counts of heat-stressed Saccharomyces cere- visiae. Cells were heated at 56 C and— plated on plate count agar (PCA) and pota- to dextrose agar (PDA) . . . . . . . . . . 40 7. Effect of storage at 22 C in water with or without 1.0 ug/ml cycloheximide on plate counts of heat-stressed Saccharomyces cere-. visiae. Cells were heated at 56 C and plated on plate count agar (PCA) and pota- to dextrose agar (PDA) . . . . . . . . . . 41 viii Figure Page 8. Effect of storage at 22 C in water with or without 2,4-dinitrophenol (DNP) on plate counts of heat-stressed Saccharomyces cerevisiae. Cells were heated at 56 C for 1.5 or 3.0 minutes and plated on plate count agar (PCA) and potato dextrose agar (PDA)...................46 9. Effect of storage in water at 4 C or 22 C on plate counts of heat-stressed Saccharomyces cerevisiae. Cells were heated at 56 C for 1.5 or 3.0 minutes and plated on plate count agar (PCA) and potato dextrose agar (PDA) . . . . .‘. . . . . . . . . . . . . . 48 10. Effect of incubation at 20, 25, 30, or 35 C on plate counts of heat-stressed Saccharomyces cerevisiae. Cells were heated for 1.5, 3.0, or 4.5 minutes at 56 C and plated on plate count agar (PCA) and potato dextrose agar (PDA) . . . . . . . . . . . . . . . . . . . 49 ll. Endogenous respiration at 30 C of heat-stressed Saccharomyces cerevisiae in water. Cells were heated for 0, l, or 2 minutes at 56 C. 52 12. Effect of addition of 0.10 mM 2,4-dinitrophenol (DNP) on endogenous respiration at 30 C of heat-stressed Saccharomyces cerevisiae in water. Cells were heated for 0, l, or 2 minutes at 56 C and DNP was added after 30 minutes . . . . . . . . . . . . . . . . . . 57 13. Effect of addition of 1.0 mM glucose on respir- ation at 30 C of heat-stressed Saccharomyces cerevisiae in water. Cells were heatedIfor 0, l, or 2 minutes at 56 C and glucose was added after 30 minutes . . . . . . . . . . 59 ix INTRODUCTION Over the past decade food microbiologists have become increasingly aware of the implications of sublethal injury to microorganisms with respect to the interpreta- tion of data from the microbiological examination of food (Ordal, 1971; Hobbs and Olson, 1971; Busta, 1976). With continued emphasis being placed on food safety and quality as illustrated by the adoption of microbiological stan- dards for foods, methods of microbiological analyses must undergo further scrutiny to ensure the best possible and most representative evaluation of the microbial popu- lation. Although bacterial injury and recovery has been, and continues to be, investigated extensively, injury and re- covery of yeasts and molds has received leSs attention. Investigations concerned solely with injury and recovery of yeasts and molds are few; however, a number of other studies contain data pertaining to this area. One example concerns the use of acidified potato dextrose agar (APDA) which is recommended for the enumer- ation of yeasts and molds in many food products (APHA, 1976). Acidified potato dextrose agar gave lower esti- mates of fungal populations in foods than media which were nearer to neutrality in pH and contained antibiotics or dyes to inhibit bacterial growth (Skidmore and Ko- burger, 1966; Mace and Koburger, 1967; Koburger, 1970, 1971, 1972, 1973; Ladiges st 31., 1974). Undefined en- vironmental stresses were assumed to result in a suble- thally injured fungal population sensitive to low pH. However, Koburger and Farhat (1975) reported use of PDA plus 100 mg/l each of chloramphenicol and chlortetracy- cline gave estimates of fungal populations similar to several other media, including plate count agar, malt agar, and mycophil agar. One specific factor often considered important in consideration of environmental sensitivity of microorgan- isms is heat-stress. Nelson (1972) reported maximum recovery on PDA of heat-stressed yeast required adjust- ment of PDA to pH 8. Recovery of injured yeast popula- tions at pH values above and below pH 8 varied and was markedly reduced with some strains. Stevenson and Richards (1976) found that pH was not the major factor in differences observed in plate counts of thermally stressed yeasts obtained on‘APDA, PDA, and plate count agar (PCA). Plate counts on APDA and PDA were equivalent and those counts were substantially lower than plate counts on PCA. Obviously, several questions remain con- cerning the recovery of thermally injured yeasts. The purpose of this investigation was to further study thermal injury and recovery of Saccharomyces cerevisiae Y25. LITERATURE REVIEW Manifestations of thermal injury reported to occur in yeasts and molds may be divided into several cate- gories (Stevenson and Graumlich, 1978). These categories include increased sensitivity to environmental conditions, alterations in growth and cell characteristics, and sub- cellular alterations. Sensitivity to Environmental Conditions As a result of thermal injury, some fungi exhibit increased sensitivities to NaCl, water activity, pH and temperature. NaCl and Water Activity. Fries (1969) reported thermally stressed cells of gphiostoma mutiannulatum and Rhodotorula glutinis were sensitive to NaC1 and other inorganic salts containing C17 or Br-. The sensitivity, expressed as decreased recoveries on media containing C1- or Br-, was partially reversed by D- and L-histidine as well as some other imidazole compounds. Later, Fries (1972) found exposure to 2,4-dinitrophenol resulted in a similar sensitivity to halogens. Tsuchido gt 31. (1972a) also found Candida utilis had an increased sensitivity to NaCl as a result of thermal stress. Candida utilis was normally able to grow on Czapek-Dox medium containing 7% NaCl. However, sublethal thermal stress (45 C for 10 min) resulted in a 90% reduction in viability on Czapek-Dox medium + 7% NaCl as compared to Czapek-Dox medium. Delayed plating following storage in culture medium, phosphate buffer, or distilled water for 5 to 6 hours allowed restoration of salt tolerance. Inhibition of recovery by 8-azaadenine and cycloheximide suggested RNA synthesis and protein synthesis were necessary for recovery. Tsuchido 33 al. (1972b) and Shibasaki and Tsuchido (1973) reported that although sorbic acid did not prevent recovery of salt tolerance, thermal destruction was greater in the pres- ence of sorbic acid. Inhibitions of protein synthesis and respiratory activity were noted when sorbic acid was present during storage. Adams and Ordal (1976) investigated the effects of thermal stress on Aspergillus parasiticus and reported decreased viability of conidia on solid media containing 10% NaCl. Reduced viability was also noted on MPN enum- erations in liquid media at lowered water activity (Aw). Storage at an Aw of 0.92 prevented recovery when NaCl, glycerol and sucrose were used as solutes to control Aw. Gibson (1973) also reported an increased sensitivity to Aw after thermal injury. Thermal destruction of S3322: aromyces rouxii and Torulopsis globosa was studied at high sucrose or sucrose + glucose concentrations. After heating,these osmophilic yeasts appeared to require less osmophilic growth conditions. pH. The effect of pH on recovery of thermally stressed yeast was investigated by Nelson (1972). Ten species of yeast were subjected to sublethal temperatures and recovered on PDA adjusted to a wide range of pH values (2 to 10). Maximum recovery for all species was at pH 8, except g. utilis, which had maximal recovery at pH 10. Recoveries at pH 3.5 ranged from 1 to 100% of recoveries at pH 8 for the various species. The results would seem to correspond with several investigations concerning the effects of pH on the recovery of yeast and molds from foods. Skidmore and Koburger (1966, Mace and Koburger (1967), Koburger (1970, 1971, 1972, 1973), Jarvis (1973), and Ladiges et 31. (1974) reported that acidified media gave lower estimates of fungal popula- tions in foods than media which have pH values nearer to neutrality and which incorporate dyes or antibiotics to inhibit bacterial growth. Temperature. In a series of articles Fries (1963, 1964, 1965, 1970, 1972; Fries and Soderstrom, 1963), re- ported thermosensitivity of fungal growth due to thermal injury. Exposure of a number of fungi, Ophiostoma, Rhodotorula, Dipodascus, Exobasidium, and Tilletiopsis, to sublethal temperatures resulted in inability to grow at temperatures slightly below their normal maximum temperature for growth. Cells exposed to 2,4-dinitro- phenol or UV light demonstrated similar thermosensitivity. Incubation at lower temperatures resulted in full re- covery of injured populations. Gibson (1973) reported similar findings in yeast populations exposed to supramaximal temperatures. In- jured cells of S. rouxii and T. globosa were reported to have lowered optimal growth temperatures. Several investigators have noted storage of injured populations at low temperatures prevented recovery from thermal injury. Schenberg-Frascino (1972) reported storage at 4 C prevented recovery of viability in ther- mally stressed yeast. Baldy £3 a1. (1970) also reported recovery from thermal injury was prevented in conidia of Penicillium expansum by storage at 0 C, although re- covery occurred during storage at 23 C. Alterations in Growth and Cell Characteristics In addition to greater sensitivity to extreme en- vironments, thermally injured fungal populations were reported to have increased nutritional requirements, as well as altered growth and morphology. Nutritional Requirements. Yeast growth at supra- optimal temperatures was dependent on nutrient concentra- tion. Sherman (1959a) reported growth of S. cerevisiae was limited by the concentration of yeast extract in the medium. When cultured in yeast extract plus 4% glucose broth, the yeast was unable to grow at 40 C in the broth containing 0.5% yeast extract, but grew in broth containing 1.0% yeast extract. Van Uden and Madeira- Lopes (1975) reported growth of S. cerevisiae was depen- dent on glucose concentration and approached the optimum temperature of growth with decreasing glucose concentra- tions. Thermally stressed yeast also have increased nutritional requirements. Exposure of Candida nivalis, an obligate psychrophile, to temperatures above its max- imum temperature of growth of 20 C resulted in losses in viability as well as in injury to surviving cells (Nash and Sinclair, 1968). Injury was demonstrated by reduced viability on a minimal medium as compared to growth on a complex medium and recovery of cells was enhanced by the presence of glutathione or thioglycollate. Supplementa- tion of the minimal medium with yeast extract reduced the ability of glutatione or thioglycollate to enhance re- covery. Stevenson and Richards (1976) reported differ- ences in recovery on PCA and PDA of thermally stressed. S. cerevisiae which apparently resulted from differences in nutrient composition rather than pH. Acidification of PCA to the pH of PDA (5.6), did not resolve differences in recovery. Growth and Morphology. Longer lag periods before resumption of growth were noted in many fungal popula- tions after thermal stress. Organisms in which extended lag periods were observed include: Typhula idahoensis, T. incarnata, and T. trifoli (Dejardin and Ward, 1971); Sclerotinia borealis (Ward, 1966a and 1968b); Aspergillus parasiticus (Adams and Ordal, 1976); T. globosa and S. rouxii (Gibson, 1973); Cryptococcus s2. (Hagen and Rose, 1961); and Candida spp. (Evison and Rose, 1965; Meyer, 1975). The extended lag period was generally proportion- al to the time and temperature of exposure (Hagen and Rose, 1961; Evison and Rose, 1965; Ward, 1966a and 1968b; Dejardin and Ward, 1971). The increased lag periods were attributed to time necessary for repair of thermosensi- tive components or systems within the cells. Exposure to supramaximal temperatures also caused a change in morphology. A thickening of older hyphae and loss of normal coordination of hyphae within colonies occurred when Sclerotinia borealis was exposed to tem- peratures above its maximum for growth (Ward, 1968b). Thermal destruction of the more sensitive hyphal tips apparently resulted in loss of apical dominance. De- jardin and Ward (1971) noted atypical growth with devel- opment of fan-shaped sectors after exposure of Typhula gpp. to sublethal temperatures. The obligately psychro- philic yeast Leucosporidium stokesii formed enlarged cells and buds when exposed to temperatures above the maximum for growth (Silver gt 21., 1977). Subcellular Alterations Indications of the effect of thermal injury at the subcellular level have also been obtained. Changes in metabolic activity, disruption of cell membranes and or- ganization, and thermosensitivity of nucleic acids were reported. Metabolic Activity. Decreased respiratory activity associated with thermal injury was reported in a number of investigations on fungi (Bacter and Gibbons, 1962; Evison and Rose, 1965; Sinclair and Stokes, 1965; Ward, 1968b; Dejardin and Ward, 1971; Baldy st 31., 1970; Spencer, 1972; Shibasaki and Tsuchido, 1973; and Meyer, 1975). Decreased fermentative activity was also found in fungi exposed to supramaximal temperatures (Sinclair and Stokes, 1965; Sinclair and Grant, 1967; Grant gt a1., 1968). One of the more intensive studies of thermal injury and recovery of fungi was conducted by Meyer (1975). An association between active metabolism and thermal injury was demonstrated in Candida P25, an obligate psychrophile, after exposure to 30 C. Cells respiring exogenous ma- terials were less resistant to thermal injury than those respiring endogenous materials, regardless of their physiological state. Heat injury affecting endogenous respiration was irreversible when cells were heated in the presence of glucose but was reversible when heated in the absence of glucose. The presence of glucose during storage did not affect recovery. In contrast, Baldy §t_al. (1970) reported glucose inhibited recovery from thermal injury during storage 10 of heat-stressed conidia of P. expansum. Conidia heat stressed in water at 54 C for up to one hour were ob- served to recover viability up to 20-fold during storage in water at 23 C for three days. The presence of glu- cose, potassium phosphate, ammonium or sodium acetate, sodium azide, 2,4-dinitrophenol, and sodium or potassium salts of pyruvate and acids from the tricarboxylic acid cycle prevented recovery. Malate, citrate, succinate, and acetate stimulated respiration in unheated condidia and inhibited respiration in heated conidia. Other thermosensitive metabolic activities have been reported for fungi. Baxter and Gibbons (1962) observed decreased alcohol dehydrogenase activity and reduced up- take of glucosamine in a psychrophilic Candida sp. after exposure to supramaximal temperatures. Hagen and Rose (1961, 1962) reported synthesis and uptake of amino acids, and synthesis of a-oxoglutarate decreased after thermal stress. Protein synthesis was also sensitive to heat (Sinclair and Grant, 1967; Nash gt 31., 1969; Spencer, 1972). Thermosensitivity of protein synthesis in L. stokesii was correlated with thermolability of a number of aminoacyl-tRNA synthetases and soluble enzymes in- volved in formation of ribosomal bound polypeptide chains (Nash et 31., 1969) as well as thermal instability of ribosomes (Nash and Grant, 1969). Heat-damaged ribosomes were deficient in binding of tRNA. Recently, Silver 33 31. (1977) reported that the maximum temperature of 11 growth for S. stokessi was due to temperature-sensitive inhibition of DNA synthesis. RNA synthesis was inhibited at a slightly higher temperature. Disruption of Membranes and Cellular Organization. Thermal injury to fungal cell membranes was reported in a number of investigations. Leakage of intracellular constituents after thermal stress was reported in S. multiannulatum (Fries, 1972), A. parasiticus (Adams and Ordal, 1976), S. utilis (Tsuchido 25‘21., 1972ab; Shi- basaki and Tsuchido, 1973; Rudenok and Konev, 1973), S. cerevisiae (Rudenok and Konev, 1973; Hagler and Lewis, 1974), Candida EE- (Spencer, 1972; Meyer, 1975), S. navalis (Nash and Sinclair, 1968), Leucosporidium frigi- Q m and S. stokessi (Spencer, 1972). The environmental sensitivity described previously has been attributed to thermally induced membrane damage _ by some investigators. Nash and Sinclair (1968) con- sidered damage to a "permeability barrier" responsible for altered nutritional requirements of thermally stressed S, nivalis.' Likewise, Fries (1969, 1970) con- cluded sensitivity to C17 and Br- in heat-shocked cells of S, multiannulatum and S. glutinus resulted from mem- brane damage. Rudenok and Konev (1973) observed what they termed "self-protection" of S. cerevisiae and S. utilis cells from thermal injury. High concentrations of cells released sufficient intracellular materials during heat treatment to provide increased thermal 12 resistance, presumably due to protection of cell mem- branes. Low concentrations (<0.01 mM) of nonpolar aro- matic and heterocyclic amino acids also provided a similar protection from thermal injury. However, in con— trast to the finding of Fries (1969, 1970), histidine did not provide significant protection during heating. Hagler and Lewis (1974) reported heat stress of S. EEEEI visiae in the presence of glucose resulted in membrane damage as demonstrated by extracellular ATPase activity and loss of maintenance of sorbose gradients. The effect was noted uniquely with utilizable sugars and was inhibited by Ca++ or inhibitors of sugar utilization. Other indications of thermally disrupted cellular organization have been described. Meyer (1975) found thermally stressed Candida P25 had extensive ultrastruc- tural changes including aggregation, alteration, and loss of mitochondria along with the appearance of numerous large vacuoles. Arnold and Lacy (1977) reported exten- sive membrane damage in heat-killed cells of S. EEEEI visiae. Nucleic Acids. Evidence of nucleic acid sensitivity to thermal treatment was reported in several investiga- tions. Sherman (1956, 1959b) found growth at supraop- timal temperatures or exposure to lethal temperatures resulted in increased prOportions of respiratory-defi- cient cells (petite mutants) of S. cerevisiae. Respira- tory-deficiency was attributed to inactivation of l3 cytochrome oxidase and the.Self-replicating cytoplasmic units responsible for its production. Other investiga- tors have since demonstrated the cytoplasmic factor was actually mitochondrial DNA (Mounolou EE.E£-r 1966; Nagley and Linnane, 1970). Schenberg-Frascino and Moustacchi (1972) reported recovery from lethal and muta- genic effects resulting from heat treatment of a haploid strain of S. cerevisiae. Increased viability and repair of cytoplasmic mutations (petite mutants) and nuclear mutations (canavanine resistance) occurred in injured cells stored at 28 C in agitated distilled water. Re- pair was inhibited by storage at 4 C or by inhibitors of protein synthesis. The degree of injury was dependent on the physiological state of the cells; exponential cells were much more susceptible to injury than station- ary phase cells. Bullock and Coakley (1976) also found evidence re- lating thermal sensitivity of DNA to cell physiology. Heat sensitivity of synchronous cultures of Schizosac- charomyces pombe was highest during nuclear division. They concluded decreased viability resulted from thermal damage of DNA. Additional evidence of thermal injury was provided by Parry and Zimmerman (1976). They found increased numbers of monosomic colonies after heat treatment of a diploid yeast which was capable of monitoring mitotic non-disjunction through phenotypic expression of a set of 14 coupled and recessive markers. Expression of the markers through monosomic colony formation required a post-treat- ment growth period in a non-selective medium suggesting repair of injury. SUMMARY Thermal injury in yeasts and molds was reported to result in increased sensitivity to environmental condi- tions, alterations in growth and cell characteristics, and subcellular alterations. Environmental sensitivity was apparent through altered sensitivity to NaCl and Aw, pH and temperature. Increased nutritional requirements and altered cellular and colony morphology were also noted after thermal stress. Subcellular manifestations of thermal injury were reported to include altered metabolic activity, membrane damage and damage to nucleic acids. MATERIALS AND METHODS Organism and Cultural Conditions Saccharomyces cerevisiae Y25, a diploid yeast, from the culture collection of the Michigan State University. Food Microbiology Laboratory, was utilized in all exper- iments. Stock cultures were maintained on YM (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, and 1.0% glu- cose) agar slants at 4 C. 7 Growth from a YM agar slant incubated at 25 C for 24 hours was used to inoculate 500 ml of’YM broth in a 1- liter erlenmeyer flask. After incubation for 4 days at 25 C and 200 rpm on a Model G-25 gyratory shaker (New Brunswick Scientific Co.; New Brunswick, N.J.), the cells were harvested by centrifugation for 10 minutes at 1500 x g, washed 3 times by centrifugation with 200 m1 of dis- tilled water, and suspended in 50 ml of distilled water. The resulting cell suspensions were held at 4 C until use, normally less than 1 hour later, at which time they were equilibrated to room temperature (22 C) by immersion in tap water. Thermal Stress Cells of S. cerevisiae were subjected to thermal stress utilizing the flask method described by the Nation- al Canners Association (1968). After preliminary 15 16 experiments indicated the destruction rates of S. EEEEI visiae Y25 at various temperatures in water, a temperature of 56 C was chosen for subsequent experiments. Five millimeters of cell suspension were added to 245 ml of water preheated to 56 C in a 500-ml screw-capped erlen- meyer flask. Temperature was maintained by immersing the flask in a water bath heated by a Bronwill Model 20 Con- stant Temperature Circulator (Bronwill Scientific; Roch- ester, N.Y.). The flask was stabilized by large metal washers and sufficient water was added to the bath to maintain a level which was within one inch of the screw cap. The contents of the flask were mixed with a mag- netic stirring bar to help provide a uniform temperature and to maintain suspension of the yeast. Heated cell suspensions were withdrawn at appro- priate times from 0 to 5 minutes by pipetting. The samples were placed in 16 x 125-mm screw-capped test tubes and cooled by immersion of the tubes in cold tap water. The samples were plated immediately, or, for delayed plating, were held at room temperature in test tubes on a New Brunswick Model TC-6 rotating drum which provided aeration and maintained the yeast in suspension. Enumeration of "Viable" Cells Viability of the unheated and heated cell suspen- sions was determined from plate counts on plate count agar (PCA; Difco Laboratories; Detroit, Michigan), l7 potato dextrose agar (PDA; Difco), and a minimal medium (MM) composed of 0.67% yeast nitrogen base (Difco), 0.2- 6.0% glucose, and 1.5% agar. The samples were serially diluted in water and duplicate pour plates were prepared of the appropriate dilutions. The media were tempered to 45 C before pouring the agar plates. Colonies were counted after incubation at 25 C for 5 to 6 days. Factors Affecting Viability A number of environmental variables and metabolic inhibitors were evaluated for their influence on recovery of heat-stressed cells. The following variables were tested: composition of media, temperature of storage, temperature of incubation, presence of heat-killed cells, and metabolic inhibitors. Composition of media. Compositional differences between PCA and PDA were explored with regard to the re- covery of heat-injured cells. PDA was supplemented with components of PCA such as 0.5% yeast extract or 0.25% tryptone. The pH of PDA was adjusted by addition of 0.1 HPO M Na to a final pH of 6.6. The pH was measured on a 2 4 Beckman Research pH Meter (Beckman Instruments Inc.; Fullerton, California) by immersion of a combination pH electrode into solidified media at room temperature. Laboratory-prepared potato dextrose agar (LPDA) was also utilized (Van der Walt, 1970). Glucose concentration of the laboratory-prepared medium was 2.0% as described for 18 PDA or 0.1% as in PCA. In addition, recovery of heat- stressed cells on PCA and PDA was compared to recovery on MM adjusted to several concentrations of glucose ranging from 0.2 to 6.0%. For MM, glucose and agar were normally steam sterilized; however, in some experiments glucose was filter-sterilized along with the yeast nitro- gen base. Temperature of Storage. Thermally stressed cells were diluted 1:10 in distilled water immediately after heating. The samples were divided with one portion held at room temperature and 150 excursions/minute on an Eberbach no. 6000 reciprocating shaker (Eberbach Corpora- tion; Ann Arbor, Michigan) and the other at 4 C and 160 rpm on a New Brunswick Model V gyratory shaker. Un- stressed cells were treated in a similar manner to serve as controls. The samples were plated at appropriate intervals from O to 24 hours. Temperature of Incubation. Heat-stressed cells and unheated controls were plated immediately after heating and after storage for 24 hours at 22 C. Duplicate plates of PCA and PDA inoculated with appropriate dilutions from each sample were incubated at 20, 25, 30 and 35 C for 5 to 6 days. Heat-killed Cells. A set of experiments was de- signed to determine the extent of cannibalistic growth in various suspensions. Cell suspensions containing approximately 3.5 x 107 cells/ml were heated for 25-30 19 minutes at 56 C to produce heat-killed cells. The heat- killed cell suspensions were inoculated with unheated cells, held in loosely-capped 16 x 125-mm test tubes at room temperature on a New Brunswick RT-6 rotating drum, and plated at intervals up to 21 days. Unheated cells were used as controls. For comparison, cell suspensions heated at 56 C for 3 minutes were held undiluted, or, diluted into various concentrations of heat-killed cells, and were plated at similar intervals. Metabolic Inhibitors. Several metabolic inhibitors were utilized to determine their effect on the recovery of heat-stressed cells. Various concentrations of some of the inhibitors were added to a yeast nitrogen base (0.67%) plus glucose (0.5%) broth to determine the mini- mum inhibitory concentrations (MIC) of inhibitors as described by Spooner and Sykes (1972). The media were inoculated with S. cerevisiae and growth was measured for 24 hours by direct cell counting utilizing a Model ZBI Coulter Counter (Coulter Electronics, Hialeah, Flori- da). Information concerning the metabolic inhibitors is given in table 1. Heat-stressed cell suspensions and controls were divided so that a comparison of recoveries with and with- out inhibitors could be made. Appropriate amounts of the inhibitors were added immediately after heat stress to yield the desired final concentration. Serial dilu- tion of samples before plating prevented inhibition 20 cowumuucoocoo wuouwbflncw ESEfl:«zm mHE\mE oa.o mammbushm dzm .ou Havaamno mamwm o cwoaaocfluo< AamwupcO£ooufiEv HE\mE o.v mammbucmm samuoum .oO HMUHEocU memfim HooflcwbdsmuoHnu mHE\m:o.H mfimobucmm cflwuoum .oo Gnome: mpflEfixmnoHomu coaumuOQHoo mz mho.o mammbucwm €20 HMOHEmnoon .m.D mouswxoupam coflumamuonmmond o>fluoofixo :oflumuomuoo Hocmbm SE oa.o Amwadsoocav HmoflEanOflm .m.D Iouuflcwplv.m cowumuucwocoo cofluflbflbcH monsom HouflnfibcH uo muwm .coHumm Iflumm>Cfl was» as wouflaflus muouwbwncfl oflaonmuwe mcflcuoocoo coaumEH0mcH .H wanna 21 interference with growth in the various media. Manometric Measurements Respiration of heat-stressed and nonstressed cells suspended in water at 30 C was studied by conventional manometric techniques (Umbreit et al., 1972) using a Gilson Differential Respirometer, model GR-l4 (Gilson Medical Electronics, Inc.; Middleton, Wisconsin). In order to provide cell concentrations sufficient for measurement of respiration, the thermal stress procedure was modified. Eleven milliliters of cell suspension were added to 99 ml of water preheated to 56 C in a 250-ml erlenmeyer flask and samples were withdrawn after 1 and 2 mdnutes. In some experiments heat-stressed and non- stressed cells were washed by filtration on a Nucleopore membrane filter (Nucleopore Corp.; Pleasanton, Califor- nia) with 0.4OAUm pores, and suspended in water. Alter- nately, heat-stressed and nonstressed cells were added directly to flasks after apprOpriate dilution. Each flask contained 2.0 ml of yeast suspension (2.0-4.5 mg/ ml dry weight). The center will contained 0.2 ml of 20% KOH or water and a folded 2 x 2-cm strip of Whatman #1 filter paper. Glucose (11 umoles or 2.2 umoles in 0.2 ml) or DNP (0.2 ml of 1073M) was sometimes added from the side arm. In some experiments DNP was added immed- iately after heating. The gas phase was air. A dry weight-turbidity curve was utilized to determine the dry 22 weight of the yeast. Production of CO2 was determined by the direct method (Umbreit SE El°r 1972). Cell Leakage Cell suspensions heated from O'to 5 minutes at 56 C were measured for leakage of materials absorbing at 260 and 280-nm. The suspensions were centrifuged at 3000 x g for 10 minutes and the supernatant was decanted. Ab- sorbance was measured on a Beckman DB-G Spectrophoto— meter. Statistical Analyses Student's t test, linear regression, and correlation coefficients were calculated as detailed by Snedecor and Cochran (1967). RESULTS Recovery of Thermally Stressed Cells of Saccharomyges cerevisiae Y25 on PCA and PDA Sampling times of O, 1.5, 3.0 and 4.5 minutes were normally employed for studying the thermal injury and destruction of S. cerevisiae Y25 at 56 C in water. Fig- ure 1 summarizes plate counts immediately after heating on PCA and PDA compiled during the course of the study. Analysis of recovery data by linear regression indicated slopes of -0.62 and -O.93 for PCA and PDA, respectively. Comparison of the regression coefficients by the student t-test indicated the difference between them was highly significant (P<0.001); the correlation coefficients of the survivor curves were -0.88 and -0.90 for recovery on PCA and PDA, respectively. Comparison of plate counts on PCA and PDA by student t—test revealed no significant difference (P>0.10) be- tween the two media for recovery of unheated cells. How- ever, plate counts of cells heated for 1.5, 3.0, and 4.5 minutes at 56 C were significantly different (P<0.001). A further comparison of plate counts on PCA and PDA of heat-stressed cells is presented in Figure 2. If plate counts on PCA and PDA were equal, one would expect a plot of log10 PCA counts vs. loglo PDA counts for re- covery of heat-stressed cells to have a slope of 1.0 and 23 LQG CFU/ML Figure 1. Plate 24 counts of heat-stressed Saccharo- myces cerevisiae on plate count agar (PCA) and potato dextrose agar (PDA). Cells were heated at 56 C. 8 )- §I \ \ ‘7 ' \\\ ‘\ o \\ \ o 6 ’ \ . \( 0 \ \ \ 1 \ \\ 5 ' \ \ \ \\ e-T. 4 » x \\ \ 3 .. ——— PDA {.5 3:0 4 4.5 TIME (MINUTES) Figure 2. LOG PDA RECOVERY 25 Comparison of recovery of heat-stressed Saccharomyces cerevisiea on plate count agar (PCA) and potato dextrose agar (PDA). Cells were heated at 56 C. LOG PCA RECOVERY 26 an intercept of 0. Analysis of the results by linear regression gave a slope of 1.3, an intercept of -0.45 and a correlation coefficient of 0.96. Plate counts of unheated (0 minute) samples were not included in the analysis of data in Figure 2. Variations in recovery of heat-stressed cells on PCA and PDA were observed between experiments. Slight differences in temperature and sampling times, coupled with the rapid destruction rate of S. cerevisiae Y25 at 56 C in water probably contributed to this variation. For example, after heating for 4.5 minutes there would be a llO-fold difference in survivors on PDA between 56.5 C and 55.5 C using a z-value of 5 (Stevenson 33.31., 1975). Differences in culture sensitivity to heat be- tween experiments also may have contributed to variations in recovery of heat-stressed cells. Despite varying rates of destruction, predictable differences were ob- served with different levels of destruction as shown in Figure 2. One of the primary reasons for choosing 56 C was the high rate of destruction which allowed rapid sampling times over a wide range of destruction. Al- though sufficient data were collected to compare average recoveries on PCA and PDA at the various sampling times, in other experiments such as inhibition studies where fewer replications were conducted, data from individual experiments are presented. All studies were conducted at least twice for verification of results. 27 Media Composition Several aspects of media composition were investi- gated to determine their affect on the recovery of heat- injured cells. These included pH, variations in glucose concentrations, and supplementations of PDA with PCA components. Neutralization of PDA with NazHPO4.had no apparent effect on recovery of heat-stressed cells (Table 2). The final pH values of PCA, PDA, and PDA + 0.1 M NaZHPO4 were 6.9, 5.4, and 6.6, respectively. One major difference in composition between PCA and PDA is the glucose concentration. Potato dextrose agar contains 2.0% glucose and PCA contains 0.1% glucose. Supplementation of PCA with glucose to a concentration of 2.0% resulted in decreased recovery of heat-stressed cells and the counts obtained were intermediate between those obtained on nonsupplemented PCA and PDA (Table 2). In another experiment laboratory-prepared potato dextrose agar (LPDA) containing 0.1% glucose supported recoveries of heat-stressed cells similar to recoveries on PCA, while LPDA containing 2.0% glucose supported recoveries of heat-stressed cells similar to those obtained with commercially prepared PDA (Table 3). The pH of LPDA with 0.1% glucose was 6.9 and LPDA with 2.0% glucose had a pH of 6.6. Recovery of heat-injured cells on MM was also 28 6.6 sec . d m m m h m.m mam mo.m 3.". omé 32m o.m mos 36 mod mmK mA 34. mmé. 3;. mmfi o AHE\amo moat lease . o mmoosam o vommmmz 0 mm as wo.m + com 2H.o + «on nmuoo mooMEoumsoomm pmmmouumlumms mo mucsoo wumam .N manna 29 as.m vo.m om.m mm.m c.m mw.s mm.» mo.» om.s m.H mm.» oo.s mm.s mm.s o m om.e Hm.m mm.q em.m o.m qa.o o~.s mm.m mo.s m.H me.s cm.» 05.5 mm.s o o Aas\smo moss lease Aunt Ammoosam wo.mv Ammoosaw wH.ov 6 mm um some «can and rum mmmuumaummm, wmmuoum .OmoosHm wo.m no H.o mcwcwmucoo Amumo moomaonmboomm ommmwuumlumm: mo mucsoo wumHm .m wanna 30 related to glucose concentration. Decreasing recoveries of heat-injured cells were obtained on MM with increasing concentrations of glucose (Table 4). However, MM pre- pared with steam-sterilized glucose gave reduced recovery of heat-injured cells in comparison to MM prepared with filter-sterilized glucose (Table 5). Recovery of heat- injured cells on MM containing filter-sterilized glucose was similar to recovery on PCA. The pH of MM containing 2.0% glucose when filter-sterilized was 5.5 and 5.2 when steamesterilized. Supplementation of PDA with yeast extract or tryp- tone did not increase recovery of heat-stressed cells (Table 6). Cannibalistic Growth Variation in plate counts of nonheated cells stored in water for 21 days was minimal while growth occurred during storage in solutions containing heat-killed cells (Figure 3). The heat-killed cells were present at a concentration equal to concentrations employed in re- 7 cells/ml) and growth over covery experiments (3.5 x 10 the 21-day period reached final numbers which were 3 to 6% of the original heat-killed cell concentration. Growth was not observed during the first 12 hours after inoculation, whereas between 12 and 24 hours the in- creases in viable counts averaged 69 to 75% (Table 7). In contrast, heat-stressed cells which had not 31 o~.v h~.v wm.v mo.v m.v om.m mm.m vm.m ma.o o.m eo.n HH.h HH.~ om.h m.H Hm.h Hm.h em.> hm.» 0 NH mm.~ Hm.~ ma.m om.m m.e ma.e mm.v em.¢ w~.m o.m mm.w m¢.o vs.m vo.h m.H mm.n mv.h hm.h om.h o o lasxomo sou. lasso Anne .3 SJ o.~ I~.|o 0 mm um a mmoosam w 22 mmmuumlumm: momuoum .mmoooao wo.m o» ~.o mcflsflmucoo Azzv paces Hmeflcfle so omamw>mumo mwomEOHMBOUMm Umwmouumuumo: mo mucsoo madam .v OHDMB 32 mm.m o~.m ma.v AH.> oc.s om.w m¢.s am.» am.» no.4 mo.e om.m mm.o Hm.m ~m.m ma.a ms.> ma.s Aas\amo moqv .wmoosam pmuwafluovmlfimmum wo.~ no mmoosam pmuflafiumumlumuafim wo.m mcflcflmucoo Azzv MAUOE Hmfiflcwfi was Amuoo mmoNEonmnoomm pmmmmuumlummz mo nussoo madam mmoosam omuflawuoumluwuawm omoosam ponwaflu om.m oo.h mm.h ma.¢ mm.w mm.h m no: mumom mm.m mm.m mm.m Ha.o o.m mm.o Hm.e mm.o mo.s m.a n n n mm.s o «N Ion.~v Aom.~c nimm.~v as.v o.m om.m mo.o mo.m ms.m m.H n u an Hm.a o o AHE\amo moss Isaac rune uomuuxm IIIIIII 0 am an .mIMMImm mcoudmue + and “mums + <66 oumo moomEoumnoomm pmmmmnumuumms mo mucsoo mumHm .m manna Loe CFU/ML 34 Figure 3. Growth of Saccharomyces cerevisiae in water and in suspensions of heat-killed cells at 22 C. Heat killed cells were prepared by heating 3.5 x 107 S. cere- visiae cells/ml at 56 C for 30 minutes. 7b 6- ’ ,w ‘ o o 5 D o o C o o 4 " C Suspended in water C o O Suspended in 3.5 x 107 heat- killed cells 3- o o 2_ n n L L 1 l 1_ 1 1 n O 4 8 12 16 20 TIME (DAYS) 35 Table 7. Growth at 22 C of nonheated cells of Saccharomyces cerevisiae in suspensions of heat-killed cells. Heat-kille were prepared by heating 3.5 x 10 cerevisiae cells/m1 for 30 minutes 56 C. Incubation Time (hr) 0 6 12 (Log CFU/ml) l4- 4.59a .15 4.58 i .16 4.60 i .08 4.83 3.50a i .11 3.51 i‘.09 3.55 t .06 3.72 aInoculum levels were approximately 10"3 and as concentrated as the heat-killed cells. cells S. at 24 l+ .26 t'.21 10' 36 undergone complete thermal destruction showed much larger increases in plate counts during the first 24 hours of storage (Figure 4). Cells heat-stressed for 3 minutes at 56 C were held undiluted or diluted lOO-fold into various concentrations of heatekilled cells. In- itially the PCA count was approximately 3 loglo- cycles lower than the original numbers of 3.5 x 107 cells/m1. Plate counts during 20 days of storage for undiluted heat-stressed cells or heat-stressed cells diluted into 100% heat-killed cells approximated numbers reached by growth of unheated cells on heat-killed cells, that is, 3 to 6% of the original concentration. However, growth of heat-stressed cells diluted into 60, 30 or 0% heat- killed cells was lower than would be anticipated if growth was in proportion to the total (live and dead) cell concentration. Nonetheless, plate counts of both undiluted or diluted cells increased approximately 10- fold during the first day after heat stress. Recovery of Heat-Injured Cells The effect of delayed plating on a survivor curve for S. cerevisiae Y25 heated at 56 C in water is shown in Figure 5. Differences between recovery of heat- stressed cells on PCA and PDA were quite apparent when samples were plated immediately after heating. After 24 hours of storage, plate counts on both PCA and PDA had increased and the differences in recoveries on the LOG CFU/ML (21 I,» I} 37 Figure 4. Growth of Saccharomyces cerevisiae heated at 56 C for 3 minutes and stored at 22 C in water undiluted or diluted 10"2 with various concentrations of heatekilled cells.‘ The heat-killed cells were prepared by heating 3.5 x 107 S. cerevisiae cells/m1 at 56 C for 30 minu- tes and were used as storage media at 0, 30, 60 or 100%. . Undiluted O 10"2 dilution O A fr“ v ‘6‘) 6 0 O e—C>—~ ,. -—43 30 O 4 8 I2 16 20 TIME (DAYS) Q) 01 LOG CFU/ML 38 Figure 5. Survivor curves of Saccharomyces cere- visiae heated at 56 C and plated on plate count agar (PCA) and potato dextrose agar (PDA) immediately after storage in water at 22 C for 24 hours. 3 (.i \\ '\ '\ \\\ ‘\ o \ \\ \\ \ \ \ \ \\ \ \ \\ . \Y C) (3 14C3LHQS \§1\\ o 24 HOURS \ —— PC A \ --- PO A \o L J I In 1 2 3 4 5 TIME (MINUTES) 39 two media were reduced considerably. The differences in recovery of heat-stressed cells between PCA and PDA were reduced substantially during the first six hours of storage (Figure 6). Plate counts on PCA and PDA increased considerably during the first 6 hours of storage with progressively smaller increases occurring during 6 to 12 hours and from 12 to 24 hours of storage. Effect of Metabolic Inhibitors on Recovery of Heat- Stressed Cells Repair of heat-injured cells was not inhibited during storage in the presence of cycloheximide (Figure 7), chloramphenicol (Table 8), chloramphenicol plus cyclo- heximide (Table 9), actinomycin D (Table 10), and hydrox- yurea (Table 11). Apparently protein synthesis, RNA synthesis and DNA synthesis were not required for re- covery of colony—forming ability of heat-injured cells. These experimental conditions also preclude increases in viable counts during storage resulting from cannibalistic growth since the concentrations of hydroxyurea, cyclo- heximide and actinomycin D were high enough to inhibit growth. Storage in the presence of DNP prevented recovery of colony-forming ability of heat-injured cells (Figure 8). Viability of unheated cells was not affected by the presence of 0.1 mM DNP. Potato dextrose agar plate L06 CFU/ML Figure 6. 40 Effect of storage in water at 22 C on plate counts of heat-stressed Saccharo- myces cerevisiae. Cells were heated at 56 C and plated on plate count agar (PCA) and potato dextrose agar (PDA). PCA o 1.5 minutes PDA U 3 minutes A 4.5 minutes ......._———='=-'=Q 6 12 18 2217 TIME (HOURS) L06 CFU/ML Figure 7. 41 Effect of storage at 22 C in water with or without 1.0 ug/ml cycloheximide on plate counts of heat-stressed Saccharo- myces cerevisiae. Cells were heated at 56 C and plated on plate count agar (PCA) and potato dextrose agar (PDA). O D Water 0 I 1 ug/ml cycloheximide PCA Q . 1.5 minutes PDA U I 3 minutes J 6 12 16 24 TIME (HOURS) 42 Table 8. Plate counts on plate count agar (PCA) and potato dextrose agar (PDA) of heat-stressed Saccharomyces cerevisiae stored in water with or without 5_mg/m1 of chloramphenicol. Storage Time (hr) Heat-Stress Stored with Medium 0 6 at 56 C Chloramphenicol (Log CFU/ml) (fiin) O - PCA 7.38 7.40 PDA 7.40 7.32 + PCA 7.40 7.42 1.5 - PCA 5.99 6.91 PDA 5.15 6.86 + PCA 5.90 6.72 PDA 5.15 6.52 3.0 - PCA 3.69 5.23 PDA (2.60)a 5.04 + PCA 3.77 5.15 PDA (2.48) 4.92 aData represent estimated counts calculated from plates containing <30 colonies. 43 Table 9. Plate counts on plate count agar (PCA) and potato dextrose agar (PDA) of heat-stressed Saccharomycgg cerevisiae stored in water with or without 5 mg/ml chloramphenicol and 10 pg/ m1 cycloheximide. Stored with Storage Time (hr) Heat-Stress Chloramphenicol Medium 0 6 at 56 C + Cycloheximide (Log CFU/ml) (min) 0 - PCA 7.51 7.58 PDA 7.53 7.56 + PCA 7.51 7.52 1.5 - PCA 6.57 7.04 PDA 6.11 6.67 + PCA 6.45 7.04 PDA 6.11 6.72 3.0 - PCA 5.30 5.56 PDA 4.31 5.26 + PCA 5.08 5.67 PDA 4.26 5.18 44 Table 10. Plate counts on plate count agar (PCA) and potato dextrose agar (PDA) of heat-stressed Saccharomyces cerevisiae stored in water with or without 0.10 mg/ml actinomycin D. Storage Time (hr) Heat-Stress Stored with Medium 0 6 at 56 C Actinomycin D (Log CFU/ml) (min) 0 - PCA 7.53 7.51 PDA 7.49 7.57 + PCA 7.42 7.46 PDA 7.42 7.51 1.5 - PCA 7.15 7.28 PDA 6.69 7.11 + PCA 7.18 7.18 PDA 6.70 7.08 3.0 - . PCA 5.92 6.08 PDA 4.82 5.76 + PCA 5.92 6.08 PDA 4.85 5.78 4.5 - PCA 4.23 7.78 PDA 2.89 4.18 + PCA 4.30 4.72 PDA 2.85 4.11 45 Table 11. Plate counts on plate count agar (PCA) and potato dextrose agar (PDA) of heat-stressed Saccharomyces cerevisiae stored in water with or without .075 M hydroxyurea. Storage Time (hr) Heat-Stress Stored with Medium 0 6 at 56 C Hydroxygrea ' (Log CFU/mI) (min) 0 - PCA 7.34 7.32 PDA 7.36 7.32 + PCA 7.36 7.34 1.5 — PCA 6.30 7.04 PDA 5.78 6.90 + PCA 6.30 6.76 PDA 5.81 6.60 3.0 - PCA 4.04 5.28 PDA (2.00)a 5.15 + PCA 4.08 5.30 PDA (2.60) 5.15 aData represent estimated counts calculated from plates containing <30 colonies. L06 CFU/ML Figure 8. 46 Effect of storage at 22 C in water with or without 0.10 mM 2,4-dinitrophenol (DNP) on plate counts of heat-stressed Saccharomyces cerevisiae. Cells were heated at 56 C for 1.5 or 3.0 minutes and plated on plate count agar (PCA) and potato dextrose agar (PDA). O [3 Water I I 0.10 mM DNP -— PCA O . 1.5 minutes PDA U I 3 minutes 6 12 16 24 TIME (HOURS) 47 counts of heat-stressed cells which were stored in the presence of DNP did not increase or increased slightly during storage. Plate counts on PCA of heat-stressed cells stored in 0.1 mM DNP decreased during storage. Effects of Temperature on Recovery Storage Temperature. Storage at 4 C prevented re- covery of colony-forming ability of heat-injured cells (Figure 9). Plate counts on PDA of heat-stressed cells decreased during the first 6 hours of storage, and then increased slightly through 24 hours of storage. The PCA counts of heat-stressed cells held at 4 C remained con- stant during storage. Incubation Temperature. Little difference was noted in PCA or PDA plate counts of unheated cells of S. 92527 visiae Y25 when incubated at 20, 25, 30, or 35 C. How- ever, plate counts of heat-stressed cells were affected by incubation temperature (Figure 10). Plate counts on PCA of heat-stressed cells were not substantially affec- ted by temperature, but plate counts on PDA decreased with increasing temperature and the effect was in pro- portion to the severity of the heat treatment. For ex- ample, differences between log PDA counts at 20 and 35 C were 0.44 and 1.31 after heating for 1.5 and 4.5 minutes, respectively. Storage for 24 hours at 22 C prior to plating allowed restoration of colony-forming ability at all temperatures (Table 12). L06 CFU/ML 48 Figure 9. Effect of storage in water at 4 C or 22 C on plate counts of heat-stressed Sac- charomyces cerivisiae. Cells were heated at 56 C for 1.5 or 3.0 mdnutes and plated on plate count agar (PCA) and potato dextrose agar (PDA). O E] Stored at 22 C . I Stored at 4 C PCA O O 1.5 minutes .u... PDA D I 3 minutes 4.. 3 o 1’ 1’ 1’ \~ - ‘54 J- 4’” J , 6 ’12 ’18 24 TIME (HOURS) Figure 10. LOG CFU/ML (I) (II 49 Effect of incubation at 20, 25, 30, or 35 C on plate counts of heat-stressed Saccharomyces cerevisiae. Cells were heated for 1.5, 3.0, or 4.5 minutes at 56 C and plated on plate count agar (PCA) and potato dextrose agar (PDA). PCA O l . 5 minutes [3 3 . 0 minutes .__.. PDA A 4 . 5 minutes c: 4—4:—— -—cr—~ 2_1j c>--«-~<>e~ .1_. __a\ R— ‘9‘ N U.\\ Ar—';:>IIJEI _{>.““-n3 5‘ T}N._m_u_{3\\ \4 \. 7“\ 45~“\ I] “‘»s ZL‘\‘ 5‘ ‘“\ \A\ - “~. . . ‘79 2O 25 3O 35 TEMPERATURE 50 66.6 66.6 66.6 66.6 <66 56.6 66.6 66.6 46.6 «on 6.6 64.6 64.6 64.6 66.6 666 64.6 66.6 66.6 66.6 «om 6.6 66.5 44.5 64.5 66.5 466 64.5 64.5 66.5 66.5 4om 6.4 I4E\omo 6666 16466 1111. 1111. 1111 o 66 66 66 66 66 cm 664662 66646616666 ADV musumummfime coflu6b=oc4 .umumz cm 0 mm as 64:0: 6N How omuoum mmflmfl>mumo mmomeoumnoomm pmmmwuumlummn mo 0 mm 0:6 .om .mm .om um Umu6bso:6 Aénmv “mom mmouuxmo oumaom 6cm Adomv 4666 unsoo muMHQ co mucsoo wu64m .N4 OHQMB 51 Respiration of Heat-stressed Cells Differences in endogenous OZ-uptake between heat- stressed and nonheated cells of S. cerevisiae Y25 were noted. Oxygen-uptake of cells of heat—stressed at 56 C for l or 2 minutes was considerably higher than that of nonheated cells (Figure 11). Cells heated for 1 minute at 56 C had a high initial uptake of 02 which declined shortly after measurements were started. Cells heat- stressed for 2 minutes at 56 C also had a high initial rate of O -uptake which continued through the first hour 2 of measurement. The prolonged high rate of Oz-uptake was associated with thermal injury as indicated by re- duced recovery of heat-stressed cells on PDA and PCA (Table 13). Plate counts on PCA and PDA of unheated cells and cells heated for 1 minute were similar, but plate counts of cells heated for 2 minutes were initially lower on PDA in comparison to controls. When cells were heat-stressed for 2 minutes and stored for 20 hours, plate counts on PCA and PDA were similar. Table 14 summarizes endogenous Oz-uptake rates of unheated and heated cells for up to 4 hours after heating. Nonheated cells had an initial Q02 (pl Oz/mg dried yeast/ hr) of 2.0 which declined slightly during the four hours to a 002 of 1.8. Cells heat-stressed for 1 minute had an average initial Q02 of 40.6 which declined to 7.5 during the second hour and declined gradually to 5.4 during the fourth hour of measurement. Cells heated for 2 minutes 52 Figure 11. Endogenous respiration at 30 C of heat— stressed Saccharomyces cerevisiae in water. Cells were heated for 0, 1, 0r 2 minutes at 56 C. p1 02/M6 CELL DRY WEIGHT 4C)1 O Unheated [j 1 minute 32‘. . 2 minutes 24‘ 1 r- 6 - I II II . . ’ 1| 1I 8' __43 _ln.43._{3__<)-(>-<>-<> O A 4 4 J 4 n 4 O 10 20 30 4O 50 60 TIME (MINUTES) 53 Table 13. Plate counts on plate count agar (PCA) and potato dextrose agar (PDA) of heat- stressed Saccharomyces cerevisiae imme- diately after heating and after 20 hours of storage in water at 22 C. Storage Time (hr) Heat-Stress Medium 0 20 at 56 C (Log CFU/ml) (min) 0 PCA 8.15 8.13 PDA 8.16 8.15 1 PCA 8.13 8.16 PDA 8.10 8.15 2 PCA 7.99 8.06 PDA 7.81 8.04 54 Table 14. Rates of endogenous Oz-uptake in water at 30 C for nonheated and heat-stressed Saccharomyces cerevisiae. Time after Heating (hr) Heat—Stress at 56 C 0 1 - 2 2 - 3 3 - 4 (min) 002 (ul Oz/mg dried yeast/hr) O 2.0 1.9 1.8 1.8 1 40.6 7.5 6.4 5.4 2 39.7 33.5 16.7 6.0 55 had an initial 002 of 39.7 which declined to 33.5 during the second hour and then gradually declined to 6.0 during the fourth hour. Although not shown in Table 14, it was interesting to note that after 20 hours of storage, en- dogenous OZ-uptake of heated cells was actually lower than unheated cells. The Q02 of unheated cells was 2.0 and of cells heated for 1 and 2 minutes was 0.8 and 0.9, res- pectively. Respiratory quotients (R.Q.) of heat-stressed cells were also different from unheated cells (Table 15). The respiratory quotient is defined as the ratio of CO2 pro- duced/O2 consumed. Unheated cells had R.Q. values at or near 1.00 during the first 3 hours of measurement. Cells heat-stressed for 1 minute had an initial R.Q. of 0.70 associated with high initial rates of OZ-uptake. The R.Q. of cells heat-stressed for 1 minute then increased to 0.91 by the third hour and was 0.93 during the fourth hour. In contrast, cells heat-stressed for 2 minutes had an initial R.Q. of 1.04 which dropped to 0.72 during the second hour and increased to 0.95 and 0.93 during the third and fourth hours. Oxygen-uptake in response to the presence of DNP was also measured in unheated and heated cells (Figure 12). Rates of oxygen-uptake in unheated cells and cells heat-stressed for 1 minute increased when DNP was added. However, oxygen-uptake in cells heat-stressed for 2 min- utes declined when DNP was added. The results were 56 Table 15. Respiratory quotients (R.Q.) in water at 30 C for endogenous respiration of non- heated and heat-stressed Saccharomyces cerevisiae. Time after Heating (hr) Heat-Stress at 56 C 0 l - 2 2 - 3 3 - 4 (min) R.Q. (ul COZ/Ul 02) 0 1.00 .98 1.00 .80 l .70 .78 .91 .93 2 1.04 .72 .95 .93 pl 02/M6 CELL DRY WEIGHT Figure 12. 5C) 4() 3C) t V V 57 Effect of addition of 0.10 mM 2,4- dinitrophenol (DNP) on endogenous respiration at 30 C of heat-stressed Saccharomyces cerevisiae in water. Cells were heated for 0, l, or 2 min- utes at 56 C and DNP was added after 30 minutes. ' — Water u_-— DNP 20 3O 40 50 60 TIME (MINUTES) 58 similar when DNP was added immediately after heating. Addition of glucose (2.2 umoles or 11 umoles) stimu- lated oxygen-uptake in unheated cells and cells heat- stressed for 1 minute, however, little stimulation in rate of oxygen-uptake was observed in cells which were heat-stressed for 2 minutes (Figure 13). This latter re- sult probably was due to the relatively high rate of 02- uptake in cells heat-stressed for 2 minutes. In addition, after 4 hours the total Oz-uptake resulting from addition of glucose was more than 2 times greater for cells heat- stressed for 2 minutes than it was for nonheated cells or cells heat-stressed for 1 minute. Leakage of Intracellular Constituents Leakage of materials absorbing at 260 and 280 nm was minimal after heating for 5 minutes at 56 C (Table 16). The supernatant from unheated cells had an absor- bance of 0.047 at 260 nm and 0.037 at 280 nm. The super- natant from a cell suspension heated for 5 mdnutes at 56 C had an absorbance of 0.097 at 260 nm and 0.063 at 280 nm. pl 02/M6 CELL DRY WEIGHT Figure 13. 401 30 T 20 lOr 59 Effect of addition of 1.0 mM glucose on respiration at 30 C of heat-stressed Saccharomyces cerevisiae in water. Cells were heated for 0, 1, or 2 min- utes at 56 C and glucose was added after 30 minutes. Water - 2 -—-—- Glucose . o Jilg> 2 ’9' ’0 ' . .’ . ’ o //O/ ’9’ p/ 0’ / . o | «0“‘0’0 C .. :’o 0 . C 4O 60 TIME (MINUTES) Table 16. Heat-Stress 60 Quantitation of materials which absorb at 260 and 280 nm in Saccharomyceg cerevisiae suspensions heated at 56 C for 0-5 minutes. The suspensions con- tained 1.8 x 108 cells/ml. Absorbance of Supernatant at 56 C at 260 nm at 280 nm (fin) 0 .047 .037 l .070 .055 2 .070 .055 3 .087 .065 4 .090 .060 5 .097 .063 DISCUSSION Investigations of thermal injury and recovery of microorganisms have typically utilized minimal or stress media for the demonstration of thermal injury. Although APDA is recommended for the enumeration of yeasts and molds from food products (APHA, 1976), thermal injury in Saccharomyces cerevisiae Y25 was demonstrated by reduced plate counts on PDA as compared to PCA. The injury was repairable during storage in water at 22 C and was dis- tinguishable from cryptic or cannibalistic growth. Recovery of Heat-Stressed Cells on PCA and PDA Even though plate counts of nonheated cells on PCA and PDA were similar, thermal stress at 56 C resulted in reduced colony-forming ability on PDA, as compared to PCA, for a substantial portion of the heat-stressed yeast population. The proportion of survivors affected was related to the severity of heat treatment. In addition, the intercept of -0.45 of the log10 PCA plate counts vs. loglo PDA plate counts indicated an initial heat-induced sensitization to growth on PDA when cells had been heated for 1.5 minutes or more. Previous reports have attri- buted reduced recovery on PDA to pH sensitivity of yeasts and molds which had been subjected to environmental stress. Nelson (1972) reported maximum recovery of 61 62 heat-stressed (51 C for 20 min) S. cerevisiae near pH 8; recovery of heat-stressed cells was markedly reduced on PDA with pH values above and below this pH. A pH sensi- tivity was also noted in yeasts and molds isolated from food products (Skidmore and Koburger, 1966; Mace and Ko- burger, 1967; Koburger, 1970, 1971, 1972, 1973; Jarvis, 1973; Ladiges EE.El-' 1974). Recently, Koburger and Farhat (1975) reported use of non-acidified PDA (pH 5.6) plus antibiotics gave recoveries of fungi from foods similar to PCA plus antibiotics. Effect of Media Composition on Recovery Stevenson and Richards (1976) concluded differences in recovery of heat-stressed S. cerevisiae Y25 on PCA and PDA were not based on pH since acidification of PCA to pH 5.6 did not alter differences in recovery between the two media. In addition, PDA and APDA gave similar re- coveries despite differences in pH. The results of this investigation confirm and extend these observations. Recovery on PDA neutralized to pH 6.6 or on LPDA with 2.0% glucose (pH 6.6) did not appear to differ from recovery on PDA. Also, supplementation of PDA with yeast extract or tryptone did not increase recovery. In contrast, supplementation of a minimal medium with yeast extract improved recovery of thermally injured Candida nivalis (Nash and Sinclair, 1968). One medium component found to influence recovery of 63 thermally injured cells in this investigation was glucose. Decreasing the glucose concentration improved recovery on LPDA. Similar effects due to glucose concentration were noted on recovery of heat-stressed cells using MM con- taining 0.2 to 6.0% steam-sterilized glucose. However, recovery of heat-stressed cells on MM with 2.0% filter- sterilized glucose (pH 5.5) was considerably higher than recovery on MM with 2.0% steam-sterilized glucose (pH 5.2). The small difference in pH between the two media did not appear to account for the large differences in recovery. Plate count agar containing 2.0% glucose also gave reduced recoveries. Thus, it appears that inhibitory products produced during steam sterilization by the inter- action of glucose with other constituents of the medium were responsible for the reduced recoveries obtained with increasing concentrations of glucose (Tanner, 1944). 1 3 Baldy 2E 21' (1970) reported storage in 10- to 10- M glucose prevented recovery of sublethally heat-injured conidia of Penicillium expansum. No difference in re- covery of heat-stressed conidia immediately after heating was noted between PDA, ammonium acetate minimal medium, glucose-NH4 minimal medium (1.0% glucose) and a yeast hydrolysate-neopeptone medium (2.0% glucose). However, acetate was also reported to inhibit recovery during storage. The acetate inhibition and similar glucose concentrations of the other media may account for lack of differences in recovery among the media. 64 Hagler and Lewis (1974) reported exposure of yeast to glucose during or immediately after thermal stress at 44 C resulted in leakage of intracellular materials. Leakage in suspensions containing 0.4 to 10.0% glucose was similar but in a suspension containing 0.2% glucose leakage was reduced. Little or no leakage was measured when yeasts were suspended in water and exposed to simi- lar temperatures. They concluded thermal injury to the yeast cytoplasmic membrane was enhanced in the presence of glucose. In contrast, Meyer (1975) found exposure to glucose after thermal stress to have no effect on recov- ery of Candida P25; however, thermal injury in the pres- ence of glucose was irrepairable. Recovery versus Growth Postgate (1967) suggested cryptic or cannibalistic growth may interfere with viability measurements of stressed organisms. In response to that suggestion, can- nibalistic growth of S. cerevisiae Y25 was studied in this investigation. With conditions and concentrations of cells similar to those utilized in recovery experiments, growth of unheated cells on heat-killed cells was found to occur during a 21-day period. However, growth did not occur during the first 12 hours after heating and slight growth occurred from 12 to 24 hours. In contrast, heat- stressed cells had much greater increases in plate counts during the same period of time. Thus, the large increases 65 in plate counts of heat-stressed cells observed during the initial 24 hours of storage were the result of re- pair of thermal injury rather than cannibalistic growth. Using exponential phase cells of S. cerevisiae, Schenberg-Frascino (1972) reported no cannibalistic growth or resorption of released materials was expected during storage of heat-stressed cells since the cells were washed prior to storage. Near maximal recoveries were noted within the first 48 hours of storage at 28 C in water. Baldy EE.E$° (1970) noted mycelium formation of heat- stressed S. expansum conidia during storage at 23 C in water if spore concentrations were greater than 107 spores/ml. Subsequently concentrations of 107 spores/ml were utilized for studies of recovery of colonyvforming ability during storage. Maximum recovery was observed after storage for 3 days. Recovery of Heat-Injured Cells Increases in plate counts up to lO-fold or more over those obtained upon immediate plating on PCA were noted within the first 6 hours of storage in water at 22 C. In addition, much of the difference in recovery between PCA and PDA was resolved during this time. Smaller increases in plate counts on PCA and PDA occurred between 6 and 12 hours and between 12 and 24 hours. Fries (1969, 1970, 1972) reported thermally induced 66 salt sensitivity of Qphiostoma multiannulatum and Rhodo- torula glutinis was repairable as demonstrated by in- creased plate counts after storage for several hours. Tsuchido SE 21- (1972a) also noted recovery from ther- mally induced salt sensitivity of Candida utilis, and the recovery was almost complete after 6 hours of storage. Recovery of colony-forming ability on "non-stress" media was reported to require longer periods of time. Baldy 2E 21- (1970) reported maximal recovery of colony- forming ability on PDA of heat-stressed conidia of S. expansum required 3 days. Plate counts of conidia heat- stressed at 54 C for 1 hour were observed to increase up to 20-fold over values obtained with immediate plating. Schenberg—Frascino (1972) stored heat-stressed cells of S. cerevisiae for up to 5 days after heating; however, near maximal recoveries were reached after 2 days of storage. Increases in plate counts during the first 24 hours of storage were up to loo-fold more than plate counts immediately after heating when a yeast extract- peptone-glucose agar (2.0% glucose) medium was utilized. Effect of Inhibitors on Repair Storage in the presence of cycloheximide, chloram- phenicol, hydroxyurea, or actinomycin D had no effect on the recovery of colony-forming ability of heat-stressed cells. Plate counts immediately after heating and during storage were not markedly affected by the presence of 67 some metabolic inhibitors. Apparently, protein synthesis, DNA synthesis, and RNA synthesis were not required for re- pair of thermal injury as demonstrated by differences in recovery on PCA and PDA or by increases of plate counts on both media during storage. ‘ Baldy 2E121- (1970) reported similar results con- cerning repair of sublethal thermal injury of S. expansum conidia. Recovery of colony-forming ability during storage of heat-stressed conidia was not affected by the presence of cycloheximide or 5-fluorouracil, inhibitors of protein synthesis and RNA synthesis, respectively. In contrast, Schenberg-Frascino (1972) found inhi- bition of protein synthesis by cycloheximide or fluoro- phenylalanine prevented recovery of heat-injured exponen- tial phase cells of a haploid strain of S. cerevisiae. Similar inhibition of repair was noted if-the yeast was incubated before heating with cycloheximide; however, the heat resistance of the yeast was considerably in- creased. The increased thermal resistance may have re- sulted from a shift from exponential to stationary phase due to the inhibition of growth by cycloheximide. In ‘addition, recovery of salt tolerance of S. utilis was prevented by inhibition of‘protein and RNA synthesis (TsuChido 2E.El-r 1972ab). Storage of thermally stressed S. utilis in the presence of cycloheximide or 8-azaadenine prevented recovery of salt tolerance. Storage of heat-stressed cells in the presence of 68 0.1 mM DNP was found in this investigation to prevent recovery from thermal injury. Plate counts on PDA of heat-stressed cells stored in the presence of DNP in- creased slightly or not at all and plate counts on PCA decreased. A similar inhibition of recovery by DNP was reported in heat-stressed conidia of S. expansum (Baldy EE.El'I 1970). Interestingly, Fries (1972) reported DNP induced salt sensitivity similar to that induced by heat- shock in S. multiannulatum. The salt sensitivity was interpreted as being a consequence of injury to cellular membranes, particularly those of the mitochondria, and to impaired oxidative phosphorylation. In a related investigation, Hagler and Lewis (1974) reported DNP in- creased glucose-induced leakage of intracellular consti- tuents of heat—stressed yeasts. Effect of Temperature on Recovery Storage at 4 C prevented recovery of colony—forming ability of heat-injured cells in this investigation. Plate counts on PDA of heat-stressed cells stored at 4 C decreased during the first 6 hours of storage and in- creased only slightly after 24 hours. Plate counts on PCA did not increase when heat-stresed cells were stored at 4 C. Similar results were reported by Baldy 2E.E£- (1970) and Schenberg-Frascino (1972). Storage at 4 C is used as an indicator of the involvement of metabolic ac- tivity in repair. Since storage at 4 C or in the 69 presence of DNP prevented recovery, metabolic activity was apparently required for repair of injury. Incubation temperature also influenced expression of thermal injury. Although plate counts of heat-stressed cells on PCA were not substantially affected by incuba- tion temperature, plate counts on PDA decreased as tem— peratures increased from 20 to 35 C. The influence of incubation temperature on PDA plate counts was also re- lated to the severity of the heat treatment. Since PDA plate counts of heat-stressed cells decreased with in- creasing temperature, this may reflect variations in degree and/or types of thermal injury. Fries (1963, 1964, 1970, 1972), Fries and Soderstrom (1963) and Gibson .(1973) have also reported thermosensitivity of fungal growth resulting from thermal injury. Fries (1964) dis- covered a similar thermosensitivity was induced by expos- ure of S. multiannulatum to DNP. He interpreted these results as indicating heat stress and DNP caused damage to mitochondrial membranes with an accompanying decrease in generation of ATP. Respiration of Heat-Stressed Cells Rates of endogenous respiration appeared to reflect thermal injury. Prolonged high rates of endogenous res- piration were related to evidence of differencial re- covery of heat-stressed cells on PCA and PDA. After 20 hours of storage when plate counts on PCA and PDA of 70 heat—stressed cells were similar, rates of oxygen-uptake were diminished to levels approximating or lower than those of unheated cells. Brandt (1941) reported a high endogenous oxygen- uptake in cells of S. cerevisiae heat-stressed at 50 C. Evidence of trehalose disappearance from cell reserves concomitant with high rates of oxygen-uptake was presen- ted. While attempts were not made to measure trehalose in this investigation, the initial respiratory quotient of cells heat-stressed for 2 minutes would appear to reflect carbohydrate utilization. Baldy EEYEl- (1970) reported endogenous Oz-uptake of nonheated and heated spores was similar; however, viabil- ity of heated spores measured for OZ-uptake was less than 1.0% of nonheated spores. In contrast, viability of heated cells utilized in this investigation was only slightly reduced in comparison to nonheated cells even though endogenous rates of OZ-uptake were drastically different. Meyer (1975) reported declines in endogenous 02- uptake during exposure to supramaximal temperatures which correlated with decreases in viability of heat-stressed Candida P25. Damage to respiratory activity was repair- able when cells were heat-stressed in the presence of glucose. In addition, decreases in viability were greater when cells were heat-stressed in the presence of glucose. 71 Changes in the respiratory quotient of heat-stressed cells may have reflected changes in endogenous substrate utilization or assimilation of substrates for repair of injury. Although the theoretical R.Q. is 1.0 for carbo- hydrates, 0.9 for amino acids and proteins, and between 0.7 and 0.8 for lipids (Geise, 1962), "oxidative assimi- lation" of substrates may result in less than theoretical oxygen-uptake (Dawes and Ribbons, 1962). Spiegelman and Nozawa (1945) concluded endogenous respiration of S. cerevisiae utilized carbohydrate re- serves. However, respiratory quotients of 0.99, 1.01, 0.92 and 0.72 were observed when respiration of S. cere- visiae was measured over a period of 7 hours. This in- vestigator found a similar trend since the R.Q. of un- heated cells declined to 0.80 after 3 hours. In contrast, heat-stressed cells were observed to undergo fluctuations of R.Q. Cells heat-stressed for 1 minute demonstrated no detectable injury with respect to difference in plate counts on PCA and PDA; however, the R.Q. was 0.70 in- itially and increased to 0.93 after 3 hours of storage. Furthermore, cells heat-stressed for 2 minutes, which sustained thermal injury as demonstrated by differences in plate counts on PCA and PDA, were observed to have a R.Q. of 1.04 immediately after heating. The R.Q. dropped to 0.72 during the second hour of storage, and then in- creased to 0.93-0.95 during the third and fourth hours of storage. 72 It would appear that cells heat-stressed for 2 min- utes may have incurred a loss of respiratory control similar to that observed with uncoupling of oxidative- phosphorylation. Since respiratory quotients of cells heat-stressed for l and 2 minutes return to values near 0.95 after being at values near 0.70 during storage, this may represent utilization of a different substrate during a portion of the repair process. The initial R.Q. of 1.04 plus the high 002 of cells heat-stressed 2 minutes then appears to be uncontrolled respiration which resulted from thermal injury similar to that prOposed by Fries (1972). Ward (1968a) postulated uncoupling of respira- tion as an explanation for anomalous respiratory activity in the presence of DNP and reduced incorporation of sub- strate of Sclerotinia borealis exposed to maximal temper- atures for growth. Obviously, without further evidence such as studies of substrate utilization and P/O ratios the above must remain as speculation. Further evidence of thermal injury was provided by the rates of oxygen-uptake observed in the presence of DNP. Addition of DNP to unheated cells or cells heat- stressed for 1 minute resulted in increased 02- uptake or typical uncoupler activity. On the other hand, oxygen- uptake in cells heat-stressed for 2 minutes was depressed by the addition of DNP. Membrane damage might also account for the decreased oxygen consumption in the presence of DNP for cells which were heat-stressed for 73 2 minutes. One possible explanation is that intracellu- lar concentrations of DNP were increased due to enhanced entrance of DNP into cells with damaged cytOplasmic mem- branes. Alternatively, low concentrations of DNP may uncouple oxidative phosphorylation of thermally injured mitochondria in a manner similar to that observed at higher concentrations of DNP with mitochondria of non- stressed cells.’ Lee (1970) determined that stimulation or inhibition of respiration of S. cerevisiae by DNP was dependent on DNP concentration, pH, and metabolic state of the cells. Inhibition of respiration was observed at high concentra- tions of DNP, 5 x 10.4 M or higher, and a pH of 5.0 or lower. Stimulation of respiration was observed at lower concentrations of DNP or at higher pH. The inhibition was also dependent on the metabolic state of the cells. Glucose-induced respiration of rapidly metabilizing cells was inhibited; however, glucose-induced respiration of starved cells was not affected. Oxygen-uptake in response to addition of glucose was related to thermal stress in this investigation. Al- though Oz-uptake due to glucose addition was not appre- ciably stimulated initially in cells heat-stressed for 2 minutes, the high rate of oxygen-uptake in those cells may have precluded or masked its effect. However, glu- cose did increase the total Oz-uptake of cells heat- stressed for 2 minutes in comparison to unheated cells 74 and cells heat-stressed for 1 minute. The increased con- sumption of 0 may reflect utilization of added glucose 2 to meet increased energy demands of heat-stressed cells. Alternatively, Hagler and Lewis (1974) reported glucose addition resulted in damage to cytoplasmic membranes after thermal stress. Thus, addition of glucose may have increased oxygen consumption due to further injury. Baldy EE.El- (1970) also reported glucose inhibited recovery from thermal injury in S. expansum conidia. Cell Leakage Although leakage of cellular constituents, and in particular UV-absorbing materials, is often associated with thermal injury, leakage of materials absorbing at 260 and 280 nm from cells in this investigation was min- imal after heating for 5 minutes at 56 qun water. It would appear that extensive damage to the cytoplasmic membrane was not present in thermally stressed cells; however, the amount of leakage in some suspending media may not represent losses in viability or thermal injury. Despite the decreased salt tolerance of thermally stressed S. utilis, Shibasaki and Tsuchido (1973) reported minimal leakage of materials absorbing at 260 nm after heating cells for 10 minutes at 55 C in phosphate buffer, al- though viability was reduced by a factor of 104; however, heating S. utilis at 60 C for 5 minutes resulted in con- siderably more leakage with a lOS-fold reduction in 75 viability. Hagler and Lewis (1974) reported leakage of yeast in the presence of glucose occurred at temperatures even below the maximum temperature for growth; yet leak- age in water was minimal at supramaximal temperatures despite reductions in viability. (Meyer (1975) noted similar findings upon exposure of the psychrophile Candida P25 to 30 C. CONCLUSIONS Sublethal thermal injury of heat-stressed Saccharo- myces cerevisiae Y25 resulted in reduced and differ- ential recovery on plate count agar (PCA) and potato dextrose agar (PDA). The proportion of the population affected was related to the severity of heat stress. Storage of heat-stressed cells at 22 C in water allowed recovery from injury. The plate counts of heat-stressed cells on both PCA and PDA increased during storage. Increases in colony-forming ability during the first 12 hours were related to recovery from injury rather than cryptic or cannibalistic growth as shown by cannibalistic growth studies and recovery in the presence of growth inhibitors. Recovery from thermal injury was prevented by storage at 4 C or in the presence of DNP, but not by storage in the presence of cycloheximide, chloramphenicol, hydroxyurea, or actinomycin D. Apparently protein synthesis, RNA synthesis, and DNA synthesis were not required for recovery of colony-forming ability of thermally injured cells. Reduced recovery of thermally injured cells on PDA in comparison to PCA was related to glucose concentra- tions in the media rather than pH. Recovery of ther- ‘ mally stressed cells on a minimal medium (MM, yeast 76 77 nitrogen base plus glucose) was also influenced by the glucose concentration and the method of sterili- zation. Recovery on MM with filter-sterilized glu- cose was improved in comparison to MM steam-sterilized with glucose. Respiratory activity of heat—stressed cells reflected the severity of heat stress and injury. Oxygen- uptake due to utilization of endogenous and exo— genous substrates was increased and respiratory quotients were altered after heat stress. In addi- tion, endogenous respiration of thermally stressed cells in response to addition of DNP was different than that of unheated cells. LI ST OF REFERENCES LIST OF REFERENCES Adams, G.H. and Z.J. Ordal. 1976. Effects of thermal stress and reduced water activity on conidia of Aspergillus parasiticus. J. Food Sci. 41: 547-550. APHA. 1976. Compendium of Methods for the Microbiologi- cal Examination of Foods. M.L. Speck, ed. Am. Publ. Health Assoc. Washington, D.C. Arnold, W.N. and J.S. Lacy. 1977. Permeability of the cell envelope and osmotic behavior in Sgccharomyces cerevisiae. J. Bacteriol. 131: 564-571. Baldy, R.W., N.F. 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