DIME? ANQ NB‘IEEEECT EFFEC'FS 0? Mflfié MDEATEQN QR YEAS'E’ CELLS Tho-st: §cw {'Em Dog-m at? M. 5-. MCHEGAN STATE UKWERSETY Noemi Diaz -= Santiago £967 THES|8 ‘g—u- A_A _\ LIBRARY Michigan State University 1 cu, ABSTRACT DIRECT AND INDIRECT EFFECTS OF GAMMA RADIATION ON YEAST CELLS by Noemi Diaz-Santiago Saccharomyces cerevisiae var. ellipsoideus (Hansen) Dekker. Candida krusei (Castellani) Berkhout. and Rhodo- torula glutinis (Fresenius) Harrison, were irradiated in liquid media and their ability to form colonies after ir- radiation was measured (direct effects). D values. in Krad. for g, cerevisiae var. ellipsoideus. Q. krusei. and B. lu- tinis when irradiated in a) demineralized water were: 65. 43. and 65 respectively; b) in synthetic growth medium: 95. 60. and 50 respectively; and c) in apple juice: 125. 68. and 73 respectively. When the resistance of the afore— mentioned yeasts was studied in synthetic medium the pH of which varied in the range of 3.0 to 6.0. it was found that the lower pH favored their radiation resistance. Repeated Noemi Diaz-Santiago exposure of Q, krusei to doses of 50-200 Krad resulted in lowering the radiation resistance of this organism. Synthetic growth media containing either glucose or sucrose as carbon source and sterilized by either heat or filtration were irradiated at the levels of l. 2. 3. and 4 Mrad. These media were subsequently inoculated with .§. cerevisiae var. ellipsoideus. Q, krusei. E. glutinis. IQ. tropicalis. and g, cerevisiae and the outgrowth of these yeasts was determined (indirect effects). A signifi- cant reduction of outgrowth was observed for all of these organisms at the dose of 4 Mrad. regardless of the sugar used or the sterilization treatment prior to irradiation. Irradiated media containing sucrose affected more adversely the outgrowth of the yeasts under study. ‘g. cerevisiae var. ellipsoideus ATCC 560 did not grow on sucrose contain- ing media which were exposed even to the level of l Mrad. Filter-sterilized fresh apple juice and canned apple juice exposed to 4 Mrad did not support yeast growth to the same extent as juice irradiated at 0. l. 2. and 3 Mrad. DIRECT AND INDIRECT EFFECTS OF GAMMA RADIATION ON YEAST CELLS BY Noemi Diaz-Santiago A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1967 To Dr. Robert C. Baker. Professor of Poultry Science at Cornell University ACKNOWLEDGMENTS The author expresses her sincere appreciation to her major professor, Dr. Pericles Markakis,for his guidance and constructive criticism during her graduate work. The author also appreciates the help and interest of Professors C. L. Bedford and Dr. E. S. Beneke in the prepar— ation of this manuscript. Special thanks are due to Dr. E. S. Beneke for providing some of the yeast cultures and to Dr. R. C. Nicholas for his encouragement and guidance while carrying on this research. The author is grateful to the personnel of Phoenix Memorial Laboratory, University of Michigan, for treating the samples in their radioactive source and to the United States Atomic Energy Commission for providing the financial support of this project. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS. . . . . . . . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . . . . . . . Vii LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . ix INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . 4 METHODS AND MATERIALS. . . . . . . . . . . . . . . . . 6 l. Organisms. . . . . . . . . . . . . . . . . . . 6 2. Growth media . . . . . . . . . . . . . . . . . 6 3. Preparation of the sample. . . . . . . . . . . 7 4. Irradiation. . . . . . . . . . . . . . . . . . 8 a. Yeast cells suspended in liquid media. . . 8 1) Water. . . . . . . . . . . . . . . . . 8 2) Citrate buffered synthetic medium. . . 8 3) Difco Yeast Nitrogen Base——glucose synthetic medium . . . . . . . . . . 9 4) Canned apple juice . . . . . . . . . . 9 b. Irradiation of liquid media free of yeast cells. . . . . . . . . . . . . . . 10 c. Re-irradiation of yeasts surviving certain levels of radiation. . . . . . . . . . . 10 5. Survival assay . . . . . . . . . . . . . . . . 11 iv Table of Contents/cont. Page RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 14 A. Direct effects of gamma radiation on yeast cells. . . . . . . . . . . . . . . . . 15 l. Saccharomyces cerevisiae var. ellipsoideus 15 a) Irradiation in demineralized water . . 15 b) Irradiation in synthetic medium. . . . 16 c) Irradiation in apple juice . . . . . . 18 d) Irradiation at various pH levels . . . l9 2. Candida krusei . . . . . . . . . . . . . . 19 a) Irradiation in demineralized water . . 19 b) Irradiation in synthetic medium. . . . 22 c) Irradiation in apple juice . . . . . . 22 d) Irradiation at various pH levels . . . 22 3. Rhodotorula glutinis . . . . . . . . . . . 22 a) Irradiation in demineralized water . . 22 b) Irradiation in synthetic medium. . . . 24 c) Irradiation in apple juice . . . . . . 24 d) Irradiation at various pH levels . . . 24 4. Re—irradiation of g, krusei cells surviving certain levels of gamma radiation. . . . 25 B. Indirect effects of gamma radiation on yeast cells. . . . . . . . . . . . . . . . . 31 1. Q, krusei . . . . . . . . . . . . . . . . 31 a) Irradiation of synthetic media . . . . 31 b) Irradiation of apple juice . . . . . . 35 2. S, cerevisiae var. ellipsoideus. . . . . . 38 a) Irradiation of synthetic media . . . . 38 b) Irradiation of apple juice . . . . . . 4O Table of Contents/Cont. Page 3. R, glutinis. . . . . . . . . . . . . . . . 40 a) Irradiation of synthetic media . . . . 40 b) Irradiation of apple juice . . . . . . 44 SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . . 46 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . 50 REFERENCES . . . . . . . . . . . . . . . . . . . . . . 55 vi Table 1. Percent survival of g, krusei after first exposure to gamma radiation in yeast nitrogen base growth medium . . . . . . . . 2. Q, krusei survivors from 50 Krad gamma radiae tion in yeast nitrogen base growth medium 3. g, krusei survivors from 100 Krad gamma radia— tion in yeast nitrogen base growth medium . 4. g, krusei survivors from 200 Krad gamma radia— tion in yeast nitrogen base growth medium . 5. g, krusei survivors from 300 Krad gamma radia— tion in yeast nitrogen base growth medium . 6. Percent outgrowth of g, krusei grown on both irradiated heat-sterilized and filter- sterilized yeast nitrogen base-glucose. . . 7. Percent outgrowth of g, krusei grown on both irradiated heat—sterilized and filter- sterilized yeast nitrogen base-sucrose. . . 8. Analysis of variance of Q, krusei outgrowth on YNP—glucose and YNB-sucrose media . . . . . 9. Radiation treatment means of actual counts/ml comparison for Q, krusei grown on irradiated media . . . . . . . . . . . . . . . . . 10. Percent outgrowth of g, krusei grown on both LIST OF TABLES irradiated filter—sterilized and canned apple juice . . . . . Vii Page 29 29 29 3O 31 33 33 34 35 37 List of Tables/cont. Table 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Percent outgrowth of g, krusei grown on both irradiated filter-sterilized and canned apple juice . . . . . . . . . . . . . . . . . Percent outgrowth of g, cerevisiae var. ellip- soideus grown on both irradiated filter- sterilized and heat-sterilized yeast nitrogen base-glucose. . . . . . . . . . . . . . . . . Analysis of variance of S. cerevisiae var. ellipsoideus outgrowth data . . . . . . . . . Radiation treatment means of actual counts/m1 comparison for g, cerevisiae var. ellipsoideus grown on irradiated media . . . . . . . . . . Percent outgrowth of g, krusei, g, cerevisiae var. ellipsoideus and R, glutinis grown on irradiated canned apple juice . . . . . . . . Percent outgrowth of R, glutinis grown in both irradiated filter-sterilized and heat— sterilized yeast nitrogen base—glucose. . . . Percent outgrowth of R, glutinis grown on both irradiated filter-sterilized and heat— sterilized yeast nitrogen base-sucrose. . . . Analysis of variance of 3, glutinis outgrowth on YNB—glucose and YNB-sucrose media. . . . . Radiation treatment means of actual counts comparison for R, glutinis grown on irradiated media. . . . . . . . . . . . . . . Percent outgrowth of yeasts grown on heat— sterilized yeast nitrogen base~sucrose medium exposed to 4 Mrad of gamma radiation. . . . . viii Page 37 39 39 4O 41 41 42 42 43 45 LIST OF FIGURES Figure Page 1. Survival curve of S, cerevisiae var. ellipsoideus irradiated in various suspension media. . . . . . . . . . . . . . . l7 2. Survival curve of S, cerevisiae var. ellipsoideus irradiated in 0.02 M citrate buffered synthetic growth media at various pH levels . . . . . . . . . . . . . . . . . . l9 3. Survival curve of S, krusei irradiated in various suspension media. . . . . . . . . . . 21 4. Survival curve of S, krusei irradiated in .002 M citrate buffered synthetic growth media at various pH levels . . . . . . . . . . . . . . 23 5. Survival curve of S, glutinis irradiated in various suspension media. . . . . . . . . . . 25 6. Survival curve of R, glutinis irradiated in 0.02 M citrate buffered synthetic growth media at various pH levels. . . . . . . . . . 26 ix DIRECT AND INDIRECT EFFECTS OF GAMMA RADIATION ON YEAST CELLS INTRODUCTION The food supply has been of important consideration in the develOpment of civilization. The history of man is the history of his search for food and his attempts to pre- serve some of his harvest. Before man knew what caused his food to spoil. he was busy trying to prevent it. The principal methods for keeping edible food available are: drying. salting and smoking. pickling and fermentation. refrigeration and freezing. sterilization and pasteuriza- tion. sugar concentrates and chemical preservatives. The possibility of radiation preservation of foods was recognized in general terms in the early 1900's. Re- search in the mid-1940's revealed the potential of the pro- cess. Pasteurization research was undertaken by the Atomic Energy Commission in the 1950's. believing this process would be of benefit to the consumers. Thus. the use of ionizing radiation became the most recent step in the preservation of foods. When an atom or a molecule is bombarded by radiation having sufficient energy to remove electrons. the neutral atom or molecule is converted to a positively charged particle or ion. This ionization causes an alternation of vital macromolecules which may result in destruction of bacteria and other or— ganisms. Radiation preservation of foods is accomplished in two ways: Pasteurization. which is achieved by doses from 100 to 600 Kilorads. and sterilization. which requires doses between 2 and 5 Megarads. The purpose of this study has been to investigate the effects of gamma radiation from a Cobalt-60 source on the yeasts: Saccharomyces cerevisiae var. ellipsoideus. Candida krusei and Rhodotorula_glutinis. These yeasts have been involved in the spoilage of fresh fruits. fruit juices. pickles. dairy products. and frozen foods. ‘S. cerevisiae var. ellipsoideus and S, krusei fer- ment fruit juices and beverages from apples. grapes. and citrous fruits (mostly orange). .3- glutinis has been reported as one of the chief spoilage agents of frozen peas. and frozen oysters (9). Sour milk or cream and pickles develop pink spots due to the presence of this yeast. This study was divided into two parts. In the first part. the yeasts were irradiated in various suspension media and their survival was determined. In the second part. the media were preirradiated. subsequently innoculated with yeast and their growth was measured. The objective of the latter work was to study the possible formation of toxic substances by irradiation. using yeasts as test organisms. LITERATURE REVIEW Yeast cells have frequently been used as test ob- jects for the biological effects of ionizing radiation (5. 10. 18. 21. 24. 26-28). A number of studies have been made which illustrate the fact that while most of the microor— ganisms are destroyed largely by direct action. which is the target theory. indirect effects of radiolysis products are also important in the destruction of most Species (1. 6. 8. 12. 25. 29). James and Werner (18) made an excellent review of the radiobiology of yeasts in which they considered the various effects of radiation and the many factors which modify these effects. The commercial feasibility of pasteurization of fruit juices through irradiation is of world-wide interest. It is known that. generally. fruit juices are excellent media for the growth of yeasts. In the last few years attention has been given to irradiation effects which result in abnormal plant. animal or bacterial cell responses when grown on irradiated mater— ial (8. 25. 6. 16. 30). Choppra £3 £1- (8) observed cytotoxic (chromosomal aberrations) effects in plant materials (barley and onion seeds) grown on orange or apple juice. which had been ir— radiated with 200 Krad of gamma rays. Holsten and coworkers (16) have reported inhibition of growth in carrot cells due to cytotoxic substances pro- duced by gamma irradiation of the sucrose of the growth medium. Toxicity to mammalian cells in vitro. as shown by growth inhibition produced by glucose and fructose in the culture media which had been exposed to levels of gamma radiation from 2 to 5 Mrad. was demonstrated by Berry 2; _i. (6). The bactericidal action of gamma irradiated glucose was reported by Molin and Ehrenberg (25) using Pseudomonas species. Chromosomal aberrations in vitro were observed by Shaw and Hayes (30) when human lymphocytes came in con- tact with sucrose solution preirradiated to 2 Mrad of gamma radiation. The use of irradiated sterilized sucrose as a bactericidal agent in the canning industry has been sug- gested by Kiss §£_Sl, (19). To the knowledge of the author. no studies have been reported on the effects of irradiated media on yeast cells. ME THODS AND MATE RIALS Organisms The following yeasts were used in this study: Saccharomyces cerevisiae var. ellipsoideus ATC 560 (Hanson) Dekker. Candida krusei ATC 2159 (Castellani) Berkhout. and Rhodotorula qlutinis ATCC 2527 (Fresen— ius) Harrison. Growth media a. Yeast Nitrogen Base-~Glucosg The synthetic medium used for the stock cultures was the same used for the survival experiments. This is the medium which Etchells and coworkers (11) called Synthetic Agar B and prepared (33) as follows: Difco yeast nitrOgen base (DYNB) solution. to which glucose was added to reach a concentration of 4%. and a 3% agar solution were separately heat- sterilized (1210C. for 17 min.) and mixed at equal volumes just prior to plating. The ready to use medium had a concentration of 2% glucose. 1.5% agar. and a pH of 5. b. Yeast Nitrogen Base--Sucrose The same procedure for the preparation of yeast nitrogen base--glucose was followed but sucrose was used instead of glucose. Preparation of the samples The cultures received from the American Type Cul- ture Collection were transferred twice to the DYNB- glucose slants before starting the irradiation experi— ments in order to adapt them to the new growth medium. A third transfer was made on similar agar slantsand after three days of growth at room temperature the yeast cells were harvested as follows. Demineralized water containing Triton X-100 was poured into the tubes and the surface of the slant was scraped with a wire loop. The cells were then centrifuged and suspended in similar water. This process was repeated twice more. The sus- pension contained in a 50 ml round bottom centrifuge tube was shaken with sterile glass beads to disperse any clumps present. A Petroff-Hausser bacteria counter was used for direct microscopic count and to check for uniformity of cell dispersion. The suspension was brought to a final concentration of approximately 10 cells per m1. 4. Irradiation 3. Yeast cells suspended in liquid media Gamma radiation from a 10.000-curie Co6O source located at the Phoenix Memorial Laboratory of the University of Midhigan in Ann Arbor was used for this study. The washed suspension containing sterile glass beads was shaken with a Lab-Line super mixer until a uniform cell dispersion was obtained. One ml portions of the suspension were transferred into sterile test tubes containing 2 m1 of the liquid medium in which the irradiation was carried out. Four different media were used for this pur- pose: 1) Water. Harvested cells as described previously were irradiated in water demineralized by means of a Bantam demineralizer. Triton X-100 at a concentration of 0.01% was added as a wetting agent. 2) Citrate buffered synthetic medium. Citrate buffered DYNB--glucose was prepared to obtain a nutritive medium at a definite pH. 3) The final buffer concentration was 0.02 M. Difco Yeast Nitrogen Base--glucose synthetic medium. Double strength DYNB--g1ucose medium was heat sterilized. The pH was 5.0 after the sterilization. Canned applegjuice. The canned apple juice (brand Blossom Queen) used had no sugar or preservatives added to it. The pH was 3.40. The cell suspensions were irradiated at dose levels varying from 0 to 300 Kilorads. The temperature during irradiation was 48oF. The test tubes were held straight by inserting them in a styrofoam block cut to fit the circum- ference of the Co60 cage. The distance of the tubes from outside the center well in cm was calculated on the basis of the source calibra— tion curve prepared by the University of Michi— gan personnel and allowed the absorption of the desired dose in one hour. 10 b. Irradiation of liquid media free of yeast cells The following media were irradiated before they were inoculated with yeast cells. filter-sterilized--DYNB--4% glucose heat-sterilized--DYNB--4% glucose filter-sterilized-—DYNB--4% sucrose heat-sterilized--DYNB-—4% sucrose filter-sterilized fresh apple juice canned apple juice These media were aseptically transferred into 100 m1 test tubes and exposed to irradiation for 16 hours at the corresponding distances from the center well in order to absorb l. 2. 3. and 4 Megrads. c. Re-irradiation of yeasts surviving certain levels of irradiation Candida krusei cells grown and harvested as des- cribed previously were suspended in DYNB--glucose medium and exposed to 0. 50. 100. 150. 200. and 300 Krads of gamma radiation. A composite inoculum from several survivors' colonies was used to prepare DYNB --glucose agar slants. Survivors of only four of the original six doses were used for this: 50. 100. 200. and 300 Krad. After three days of growth the 11 cells were harvested and prepared for a second ex- posure to gamma rays. The suspensions from the sur- vivors of each of the selected doses were then ex- posed to the whole range of 0 to 300 Krad. The same procedure was repeated once more as indicated in Scheme 1. Per cent survivors was obtained for each dose using the non-irradiated controls for 100% sur- vival. Survival assay The irradiated samples (either cell suspensions or free-of-cells media) were kept at 38oF overnight. The tubes containing the cells were well mixed with a Lab— Line super mixer. Appropriate water dilutions were made to obtain suitable colony counts per plate. At least triplicate samples were plated on DYNP--glucose agar for each of 2 to 3 dilutions. One ml of cell suspension was mixed with the growth medium before it was solidified in the Petri dish. The irradiated free—of—cells media corresponding to each dose were mixed with an equal volume of 3.0% agar just before plating. and one ml of unirradiated suspen— sion of approximately 103 cells per ml was dispersed in 12 :odumaomu mEEow mo momOU cfimuuwu mo muo>fi>usw ammsux moflvcmo mo cofiumfivmuufilwm .H mEmLom 03.8.— Ou. o Suzanna-ca. n A60uxv one- 57. 60. 58. 46. 45. 79. From this table it is apparent that the reduction of out— growth on the heat-sterilized and irradiated sucrose medium was statistically significant (P=0.001) for all organisms tested. .S. cerevisiae var. ellipsoid- eus was particularly sensitive to the combined treat- ment of the growth medium. SUMMARY AND CONCLUSIONS The direct and indirect effects of gamma radiation on the survival of several food spoilage yeasts were inves— tigated. This study consisted of two parts. In the first part. the direct effects were investigated by exposure of yeast cells to gamma radiation up to 300 Krad in the follow- ing suspension media: 1. demineralized water. 2. synthetic growth medium (yeast nitrogen base double strength). 3. canned apple juice. 4. synthetic media (YNB) adjusted to pH levels of 3.0. 4.5. and 6.0 with a citrate buffer. The yeasts studied were: Saccharomyces cerevisiae var. ellipsoideus ATCC 560. Rhodotorula glutinis ATCC 2527. and Candida krusei ATCC 2159. The D values in Krad were calculated from the survival curves in demineralized water. in synthetic growth medium. and in apple juice and were: 65. 95. and 125 for S. cerevisiae var. ellipsoideus; 43. 60. and 68 for S, krusei; 65. 50. and 73 for S. glutinis. for the 46 47 three media respectively. Survival curves showed a resistant tail for the three species. The higher percent survival was observed when the yeasts were irradiated suspended in apple juice. Demineralized water was the poorest suspension medium for the survival of the cells. S, cerevisiae var. ellipsoid— .§2§_showed higher resistance to irradiation in all the media used as compared to S. glutinis and S, krusei. The lower pH (3.0 - 4.5) favored the survival of all these species. Thrice repeated irradiation of S. krusei survivors at the dose range of 50 to 200 Krad resulted in lowering of the radiation re- sistance of this organism. At 300 Krad re-irradiation re- sulted in increase of the resistance of this organism to radiation. In the second part of this study media without cells were exposed to doses of l. 2. 3. and 4 Mrad of gamma radia- tion and subsequently tested for the support of growth of six different yeasts. The following observations were made: 1. Irradiated media containing sucrose affected more adversely the outgrowth of most of the yeasts under study. This was significant to the 1% probability level. 48 Heat sterilization prior to irradiation of the media accentuated the inhibition of outgrowth of most of the yeasts studied. .S. cerevisiae var. ellipsoideus ATCC 560 did not grow on either heat or filter-sterilized sucrose containing media which were exposed even to the level of l Mrad of gamma radiation. No significant difference was found between irradi— ated filter-sterilized apple juice and irradiated canned apple juice as growth media for S, krusei. A significant suppression of growth was observed when S. krusei was grown on either canned or filter— sterilized apple juice pre-irradiated to 4 Mrad. No significant difference in outgrowth was found between S. glutinis. S, krusei. and S. cerevisiae var. ellipsoideus when they were grown on irradiated apple juice. A significant outgrowth reduction was repeatedly ob- served when the yeasts were grown on media pre— irradiated to 4 Mrad regardless of the species 49 grown. the nature of the media (synthetic or natural) or the sterilization treatment prior to irradiation. APPENDIX 51 TABLE I.--Percent survival for S, cerevisiae var. ellipsoid— eus irradiated in various suspension media. Dose Demineralized Synthetic Growth Canned Krad Water Medium* Apple Juice % Survivors 0 100 100 100 50 15 22 41 100 3.3 10 13 150 0.53 8.3 7.8 200 0.34 3.4 4.3 300 0.28 1.8 3.0 *Yeast nitrogen base double strength. TABLE II.--Percent survival of S. cerevisiae var. ellipsoideus irradiated in 0.02M citrate buffered synthetic growth media at various pH's. pH 3.0 4.5 6.0 Dose Krad % Survivors 0 100. 100. 100. 50 52.06 36.59 15.75 100 34.33 17.43 6.43 150 17.47 10.53 2.06 200 10.85 3.96 1.68 300 5.23 1.33 0.27 52 TABLE III.--Percent survival of S, krusei irradiated in various suspension media. Dose Demineralized Synthetic Canned Krad Water Growth Medium Apple Juice % Survivors 50 8.6 20. 37. 100 0.42 2.4 2.5 150 0.02 0.28 0.35 200 0.002 0.19 0.12 300 0.001 0.01 0.005 TABLE IV.--Percent survival of Q, krusei irradiated in 0.02 M citrate buffered synthetic growth medium at various pH's. Dose pH Krad 3.0 4.5 6.0 % Survivors 50 17. 15. 8.0 100 2.2 1.50 1.3 150 0.23 0.86 0.17 200 0.07 0.10 0.04 300 0.005 0.009 0.002 53 TABLE V.-—Percent survival of S. glutinis irradiated in various suspension media. Dose Demineralized Synthetic Canned Krad Water Growth Medium Apple Juice % Survivors 50 12. 11. y 26. 100 3.7 0.84 2.7 150 0.15 0.11 0.18 200 0.04 0.02 0.05 300 0.003 0.004 0.02 TABLE VI.--Percent survival of S. glutinis irradiated in 0.02 M citrate buffered synthetic growth medium at various pH's. Dose . pH Krad 3.0 4.5 6.0 50 20. 16. 2.2 100 0.58 1.6 0.87 150 0.30 0.16 0.02 200 0.03 0.05 0.007 300 0.004 0.005 - 54 TABLE VII.--D values in Krad for S, cerevisiae var. ellip- soideus. S, krusei. and S. glutinis irradiated in various suspension media. Demineralized Media Apple Yeast Water Synthetic Juice Medium S, cerevisiae var. ellipsoideus 65 95 125 Q, krusei 43 60 68 .3. glutinis 65 50 73 REFERENCES Alper. T. Hydrogen peroxide and the indirect effect of ionizing radiation. Nature. 162. 615 (1948). Effects on subcellular units and free-living cells. In: Errera. M. and Forssberg. A. (Eds.). Mechanisms in Radiobiology. Academic Press. N. Y.. pp. 353-412 (1961). . and Gillies. N. E. Restoration of Eschirichia coli Strain B after irradiation. Its dependence on suboptimal growth conditions. J. Gen. Micro— biol.. LS. 461 (1958). Bacq. Z. and Alexander. P. Fundamentals of Radiobiology. Pergamon Press. N. Y.. 1961. Beam. C. A.. Mortimer. P. K.. Wolfe. R. G.. and Tobias. C. A. The relation of radioresistance to bud- ding in Saccharomyces cerevisiae. Arch. Biochem. BiOthS.I £21 110 (1954). Berry. R. J.. Hills. P. R.. and Trillwood. W. Demonstra- tion of a cytotoxic agent in gamma-irradiated carbohydrate solutions. Int. J. Rad. Biol.. 2:6. 559 (1965). Bridges. A.. Olivo. J. P.. and Chandler. V. Relative radioresistances of microorganisms to cathote rays. II. Yeasts and molds. Appl. Microbiol.. 4, 147 (1956). Choppra. V. L.. Natarajan. A. T.. and Swaminathan. M. S. Cytological effects observed in plant material grown on irradiated fruit juices. Radiation Botany. S. l (1963). 55 10. ll. 12. 13. 14. 15. 16. 17. 18. 19. 56 Cook. A. H. (Ed.). The Chemistry and Biology of Yeasts. Academic Press. N. Y.. 1958. Elkind. M. M. and Beam. C. A. Variations of the bio- logical effectiveness of X~rays and alpha part- icles in haploid S, cerevisiae. Radiation Res.. S, 88 (1955). Etchells. J. L.. Bell, T. A., and Jones, I. D. Morphology and pigmentation of certain yeasts from brines and cucumber plants. Farlowia. ge3. 265 (1953). Frey. H. E.. and Pollard. E. C. Ionizing radiation and bacteria. Nature of the effect of irradiated medium. Radiation Res. SS, 668 (1966). Goldblith. S. A. Wholesomeness of irradiated foods: Past history. present status. international as— pects and future outlook. Food Technol. 39,2. 93 (1966). Radiation Sterilization of Food. Nature. 2 0. 433 (1966). Hollaender. A. (Ed.). Radiation Protection and Recovery. Pergamon Press. N. Y.. pp. 1-392 (1963). Holsten. R. D.. Sugii. M.. and Steward. F. C. Direct and indirect effect of radiation on plant cells: their relation to growth and growth induction. Nature SSS, 850 (1965). Ingram. M. Yeasts in food Spoilage. In: Cook. A. H. (Ed.). The Chemistry and Biology of Yeasts. Academic Press. N. Y.. pp. 603-631 (1958). James. A. P. and Werner. M. M. The radiobiology of yeast. Radiation Botany. S. 359 (1965). Kiss. 1.. Falkas. J.. Andrassy. E.. and Beczassy. K. The sterilization of sugar by ionizing radiations and investigations related to the use of steril- ized sugar in the canning industry. A paper pre- sented in the 2nd International Congress on Food Science and Techbology. Warsaw. August 1966. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 57 Korogodin. V. I. Forms of inactivation of yeast cells by ionizing radiations. Biofizika S;2. 206 (1958). Laser. H. Some observations on irradiation effects in yeast. Radiation Res.. SS. 471 (1962). Lea. D. E. Actions of Ionizing Radiations or Living Cells. Cambridge University Press. N. Y.. 1955. L6floth. G. Toxic effects of irradiated foods. Nature. 211. 302 (1966). Miller. J. J.. Kingsley. V. V.. and Ramirez. C. Com— parative sensitivity to gamma radiation of yeast mitosis and meiosis. Radiation Botany. S. 383 (1965). Molin. N.. and Ehrenberg. L. Anti-bacterial action of irradiated glucose. Int. J. Rad. Biol.. S;3. 223 (1964). Mortimer. R. K. Factors controlling radiosensitivity of yeast cells. Fundamental aspects of radio— sensitivity. Brookhaven Symp. Biol.. i4. 62 (1961). O'Brien. R. T. Radiation sensitivity studies on related fermenting and respiring yeasts. Radiation Botany. ‘l. 61 (1961). Patrick. M. H.. Hayes. R. H.. and Uretz. R. B. Dark re- covery phenomena in yeasts. Radiation Res. SS. 144 (1964). Pollard. E. C.. Ebert. J. M.. Miller. C.. Kolacz. K.. and Barone. T. F. Ionizing radiations: Effect of irradiated medium in synthetic processes. Science L41. 1045 (1965). Shaw. M. W. and Hayes. E. Effects of irradiated sucrose on the chromosomes of human lymphocytes in vitro. Nature 211. 1254 (1966). 31. 32. 33. 58 Stapleton. G. E. Factors modifying sensitivity of bac- teria to ionizing radiations. Bacteriol. Rev. .;2. 26 (1955). Stuart. B. 0.. and Stannard. J. N. Radioprotection of yeast by cystamine. Radiation Res. SS:3. 609 (1966). Wickerham. L. J. Taxonomy of yeasts. U. 8. Dept. Agr. Tech. Bull. 1029. l (1951). AN STAITE UNIVERSITY LIBRARIIE I|II|III| II II IIII IOII..II III