EFFECT (33F LONIZENG RADIATEONS 0H FOQD DECAY FUNGE Thesis for the Beam a? M. S. mcmam STATE umveasm Maggy KapeFman 1965 LIBRARY "E Michigan State E University 1,? TH E816 ABSTRACT EFFECT OF IONIZING RADIATIONS ON FOOD DECAY FUNGI by Maggy Kopelman Spores of Asperqillus flavus, Asperqillus niqer, Botrytis cinerea, Penicillium s2, and Rhizopus stolonifer were exposed to gamma rays from Cobolt 60, under various conditions. The lethal effect of the irradiation was deter- mined. Spores of A, flavus were also exposed to 1 MeV elec— from a resonant transformer accelerator. A, flavus, A, niger and Penicillium sp, spores were found to have a D value in the range of 30 to 35 krad of gamma rays,whereas g, cinerea spores had a D value of 55 krad and g, stolonifer a D value of 100 krad. A, flavus and Penicillium sp, spores irradiated at pH 3, 4, 5, 6 and 7, showed almost no change in their radio- sensitivity. g, cinerea spores showed a distinct decrease in their radioresistance at the higher pH levels (6 and 7). Penicillium s2, spores irradiated in sucrose and dex- trose solutions (0 to 20%) showed no significant change in 'their radioresistance in comparison to irradiation in water. g, cinerea spores displayed higher radioresistance Maggy Kopelman When they were irradiated in 5 to 20 per cent sucrose solu- tion than in water. A, flavus spores showed an increase in their radioresistance to destruction by gamma rays, when the dextrose concentration of the suspension media increased from O to 40%. g, flavus spores were considerably more radioresistant when they were irradiated in the dry state than in water suspension. A comparison of gamma with cathode rays indicated no significant difference in their lethal effect on A, flavus spores irradiated in water. The age of Penicillium s2, spores was found to have a very significant effect on their radioresistance: the older the spores, the higher the sensitivity to gamma rays, after the age of 20 days. Strawberries inoculated with a g, cinerea spore suspen— sion at 5, 2 and 0 days before irradiation with 200 krad of gamma rays, showed that the longer the period between inoc- ulation and irradiation, the less the protection by irradi- ation against spoilage. The best protection was achieved when irradiation immediately followed the inoculation. EFFECT OF IONIZING RADIATIONS ON FOOD DECAY FUNGI BY Maggy Kopelman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1965 To my family ACKNOWLEDGEMENT The author is greatly indebted and appreciative of her major professor, Professor P. Markakis, for encouragement, guidance and constructive criticism during her graduate work. The author also wishes to convey her sincere apprecia— tion to Professor E. S. Beneke for his constructive help and for supplying some of the fungi cultures; to Professor I. J. Pflug and Professor 0. Mickelson for their consideration and interest in their review of this manuscript. The author especially wishes to express his gratitude to the United States Atomic Energy Commission for providing the financial support of this project. Thanks go to Professor B. S. Schweigert, Chairman, Food Science Department, for his interest and support of this program; to the personnel of Phoenix Memorial Laboratory, University of Michigan, for treating the samples in their radioactive source and to Mr. George Birk for his comments and assistance while carrying on this research. ii D value krad Mev ABBREVIATIONS The radiation dose (in krad) necessary to kill ninety per cent of the microorganisms. one thousand rad. Rad is a radiation unit that represents an energy absorption of 100 ergs per one gram of material. one million ev. ev is the energy gained by an electron in moving through a potential difference of one volt and is equivalent to 1.602 x 10-12 ergs. This symbol appears in the figures. It shows the upper and the lower limits of the distri— bution and its mean average. iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 4 METHODS AND MATERIALS . . .... . . . . . . . . . . 6 l. Organism 6 2. Growth media 6 3. Spore growing and harvesting 7 4. Preparation of samples for irradiation 7 (a) Cobalt-60 source 7 (b) Cathode ray source 8 5. Suspension media for spores during irradiation 8 (a) Water 8 (b) pH 8 (c) Dextrose 8 (d) Sucrose 8 (e) Dry state 8 6. Irradiation sources 9 (a) Cobalt-60 9 (b) Resonant transformer 9 7. Irradiation 10 (a) Cobalt—60 10 (b) Resonant transformer generator 10 8. Survival determination ll 9. An experiment on established infection 11 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 13 1. General observations _ 13 (a) Production of small colonies 13 (b) Delayed growth 14 2. Sporulation media 15 3. A. flavus . 16 (a) Irradiation in demineralized water 16 (b) Irradiation at various pH.levels 19 (c) Irradiation in dextrose solutions 19 (d) Irradiation in the dry state 24 4. A, niger 27 5. g, cinerea ' 29 iv (a) Irradiation (b) Irradiation (c) Irradiation 6. Penicillium sp, (a) Irradiation (b) Irradiation (c) Irradiation (d) Irradiation in in in in at in in demineralized water various pH levels sucrose solutions demineralized water various pH levels sucrose solutions dextrose solutions 7. Rhizopu§_stolonifer 8. Irradiated inoculated strawberries 9. General discussion (x1 food irradiation SUMMARY . . . . . . . APPENDIX 1 . . . . . REFERENCES . . . . . . Page 29 29 31 31 35 35 38 39 39 41 41 45 47 69 LIST OF TABLES Table Page 1. Effect of gamma rays on the viability of Aspergillus flavus spores irradiated in demineralized water . . . . . . . . . . . . . 47 2. ~Effect of cathode rays on the viability of Aspergillus flavus spores irradiated in demineralized water . . . . . . . . . . . . . 47 3. Effect of gamma rays on the viability of Aspergillus flavus spores irradiated in 0.075M citrate buffer of various pH . . . . . . . . . 48 4. Effect of cathode rays on the viability of Aspergillus flavus spores irradiated in 0.075M citrate buffer of various pH . . . . . 48 5. Effect of gamma rays on the viability of Aspergillus flavus suspended in dextrose solutions . . . . . . . . . . . . . . . . . . 50 6. Effect of cathode rays on the viability of Aspergillus flavus suspended in dextrose solutions . . . . . . . . . . . . . . . . . . 52 7. Effect of gamma rays on the viability of dry and wet spores of Aspergillus flavus . . . . . 54 8. Effect of cathode rays on the viability of dry and wet spores of Aspergillus flavus . . . 54 9. Effect of gamma rays on the survival of .Aspergillus niger spores suspended in demineralized water containing 0.01% Triton X-100 . . . . . . . . . . . . . . . . . . . . 56 10. Effect of gamma rays on the viability of Botrytis cinerea spores suspended in demineralized water containing 0.01% Triton X-100 . . . . . . . . . . . . . . . . . . . . 56 vi Table 11. 12. 13. 14. 15. 16. 17. 18. 19. Page Effect of gamma rays on the viability of Botrytis cinerea spores irradiated in 0.075M citrate buffer of various pH T, ,., , . 58 The Effect of gamma rays on the viability of Botrytis cinerea spores suspended in sucrose solutions . . . . . . . . . . . . . . 58 Effect of age of Penicillium s2, spores on their resistance to 50 krad of gamma rays . . 60 The effect of gamma rays on the viability of Penicillium s2, spores suspended in demineralized water containing 0.01% Triton X-100 . . . . . . . . . . . . . . . . . . . . 60 Effect of gamma rays on the viability of Penicillium s2, spores irradiated in 0.075M citrate buffer of various pH . . . . . . . . . 62 The effect of gamma rays on the viability of Penicillium g9, spores suspended in sucrose solutions . . . . . . . . . . . . . . . . . . 64 Effect of gamma rays on the viability of Penicillium sp, spores suspended in dextrose solutions. . . . . . . . . . . . . . . . . . . 64 Effect of gamma rays on Rhizopus stolonifer spores suspended in demineralized water containing 0.01% Triton X-100 . . . . . . . . 66 Effect of gamma rays on the spoilage of strawberries inoculated with Botrytis cinerea spores at different times before irradiation . . . . . . . . . . . . . . . . . 67 vii LIST OF FIGURES Figure Page 1. Effect of gamma rays on the viability of Aspergillus flavus spores irradiated in demineralized water . . . . . . . . . . . . . l7 2. Effect of cathode rays on the viability of Aspergillus flavus spores irradiated in demineralized water . . . . . . . . . . . . . l8 3. Effect of gamma rays on the viability of Aspergillus flavus spores irradiated in .075M citrate buffer of various pH . . . . . 20 4. Effect of cathode rays on the viability of Asperqillus flavus spores irradiated in .075M citrate buffer of various pH . . . . . 21 5. Effect of gamma rays on the viability of Aspergillus flavus spores suspended in dextrose solutions . . . . . . . . . . . . . 22 6. Effect of cathode rays on the viability of Aspergillus flavus spores suspended in dextrose solutions . . . . . . . . . . . . . 23 7. Effect of gamma rays on the viability of dry and wet spores of Aspergillus flavus . . 25 8. Effect of cathode rays on the viability of dry and wet spores of Asperqillus flavus . . 26 9. Effect of gamma rays on the viability of Aspergillus niger spores suspended in demineralized water . . . . . . . . . . . . . 28 10. 'Effect of gamma rays on the viability of Botrytis cinerea spores suspended in demineralized water . . . . . . . . . . . . . 30 11. Effect of gamma rays on the viability of Botrytis cinerea spores irradiated in .075M citrate buffer of various pH .. . . . . . . . 32 viii Figure 12. 13. 14. 15. 16. 17. 18. Page Effect of gamma rays on the viability of Botrvtis cinerea spores suspended in sucrose solutions . . . . . . . . . . . . . . . . . . 33 Effect of age of Penicillium s2, spores on their radioresistance (dose of irradiation 50 krad of gamma rays) . . . . . . . . . . . 34 Effect of gamma rays on the viability of Penicillium g2, spores suspended in demineralized water . . . . . . . . . . . . . 36 Effect of gamma rays on the viability of Penicillium s2, spores irradiated in .075M citrate buffer of various pH . . . . . . . . 37 Effect of gamma rays on the viability of Rhizopu§_stolonifer spores suspended in demineralized water . . . . . . . . . . . . . 40 Effect of gamma rays on the percent spoilage of strawberries inoculated with Botrytis cinerea 5, 2, and 0 days before irradiation . . . . . . . . . . . . . . . . . 42 Number of days needed for 50% spoilage vs. the number of days of inoculation before irradiation . . . . . . . . . . . . . . . . . 43 ix INT RODUCT I ON The discovery of ionizing radiation by Roentgen and Bequerel (32) in 1895 initiated research on its bacteri- cidal properties. The ionizing forms of radiation were the ones suggested for use in "cold sterilization." The ability to cause ionization of a receptor atom by ejection of an orbital electron, is the characteristic property of ionizing radiations. When an atom is ionized, the molecule of which it is a part almost certainly undergoes chemical changes. Some types of ionizing radiation cannot be applied to foods, since they have the further property of bringing about a nuclear transformation, which can form radioactive atoms. Only X-rays, gamma rays, beta rays and cathode rays remain for consideration, when they are used at energy levels which do not exceed 10—15 Mev. In this study the radiation forms used were gamma rays from Cobalt-60 and 1 Mev electrons generated in a General Electric resonant transformer. In the last few years, in addition to research on sterilization of foods by irradiation, the partial eradi- cation of food decay microorganisms, radio-pasteurization, has been explored. The radio-pasteurization of fresh foods, such as fruits, vegetables and fish, appears to be a very promising applih cation of irradiation. Fungi are one of the major group of decay organisms found on fruits and vegetables. The litera- ture concerning irradiation of food decay fungi is not very extensive. The objective of this study has been to investigate the effect of a number of different factors likely to affect the survival of fungi exposed to ionizing radiations. The factors studied were: presence or absence of water in the suspension medium, pH levels, and sucrose or dextrose concentrations in the suspension medium. The fungi studied were Aspergillus flavus, Aspergillus giggr, Botrytis cinerea, Penicillium g2, and Rhizopus @2211};- A, flavus was found to be a cause of poisoning in animals consuming moldy peanuts or cereals (41). A, gigg;_(black mold), is widely distributed in the air and soil. It is often found on exposed foods and causes their decay (l). g, cinerea was found by Powelson (28) to be responsible for 90%lof the decay losses of west coast strawberries. Penicillia are as common as the Aspergilli. They are also food decaying microorganisms (l). 3, stolonifer is one of the decay organisms found in peaches (4), strawberries, grapes, tomatoes, etc. The spores from the aforementioned fungi are all one- celled. The 3, stolonifer has sporangiospores, while all the other fungi have conidiospores. Both types of spores will be referred to later only as spores. LITERATURE REVIEW Attempts to sterilize with ionizing radiation date back to early researches on its bactericidal properties (29), soon after the discovery of X-rays, and the natural radio— activity by Roentgen and Becquerel respectively (32). In 1930 a French patent was issued to O. Wartfor for using ionizing radiation to preserve food (15). A strong inter— est in applying ionizing radiation to the preservation of foods began only around 1940, after the electron accelerators were developed. Brasch and Huber (8) were the first to report on the possibility of food preservation by accelerated electrons. During the past twenty years considerable inter- est has been expressed in the lethal effect of gamma and cathode rays on microorganisms as a means of full or partial eradication of food decay organisms. Only few of the early publications deal with fungi. Lea's classical book (20) is a summary of considerable studies done by him and others on the actions of radiations on living cells. Lea's book does not give any suggestions on the use of radiation as a practical means for destroying microorganisms for purposes of sterilization. Others how- ever, appeared to think in terms of such a purpose. Dunn (10) reported in 1962 his and his coeworkers' studies on the lethal doses of a large variety of micro- organisms, including a few molds, when exposed to X-rays and cathode rays. In 1955, Hannan's (15) excellent review of all phases and aspects of the radiation preservation of food appeared. Lethal doses of cathode and gamma rays of some fruit spoilage fungi was also determined by Bridges (9), Beraha and co-workers (5), Saravacos _§_§l, (31) and others. Today one of the most active groups in the study of the effect of ionizing radiation on food decay fungi is located at the University of California, Davis. Some of their publications deal with pure cultures of fungi irradi- ated with gamma rays (33, 36, 35, 25). Other papers of the same group report on the irradiation of the fruit itself with or without artificial inoculation with fungi (23, 22, 21. 26). METHODS AND MATERIALS 1. Organism Of the fungi studied here, Botrytis cinerea and Pepi: cillium s2, were isolated from moldy strawberries. Asperqillus flavus, Asperqillus giqgr_and Rhizopus sgg: lonifer (nigricans) were obtained from Dr. E. S. Beneke of the Department of Botany and Plant Pathology, Michigan State University. 2. Growth media (a) Difco Potato Dextrose Agar (P.D.A.) slants were used for maintaining the stock cultues. The same agar was used in the plates for counting colonies sub- sequent to irradiation. (b) V8 agar was used as a sporulation agar. It was prepared by centrifuging commercial V8 Vegetable Juice manufactured by Campbell Soup Company. The supernatant liquid was separated and adjusted to pH 6.5-7.0. Two percent Difco Bacto Agar was dis— solved in the separated supernatant and autoclaved in Roux type flasks for 20 minutes at 250°F. 3. Spore growing and harvesting A modified method of Bridges and co-workers (9) was used for the spore growing and harvesting. The fungus tested was transfered from the stock slant to V8 agar in the Roux type bottles. It was incubated at room temperature (25°C.) for two weeks, unless otherwise cited. The spores were harvested with sterile demineralized water containing 0.01% Triton X—100 as a wetting agent. Sterile glass beads were added to facilitate the harvesting. The spore suspension was fil- tered through sterile filter disks, to prevent micelia fila- ments from passing through. In order to eliminate clumping, the filtered suspension was shaken in an Erlenmeyer flask with glass beads. The suspension was centrifuged and washed twice by centrifugation with sterile demineralized water containing Triton X—100 and resuspended in it. 4. Preparation of sample for irradiation Depending on the radiation source used, the samples were prepared as follows: (a) Cobalt-60 source. The washed spore suspension was stirred with a magnetic stirrer having a sterile bar. One ml. portions of the suspension were trans- fered to sterile test tubes containing 3 ml. of the liquid in which the irradiation was carried out. (b) Cathode ray source. When the irradiation was carried out in the electron accelerator, the preparation procedure was the same, but the suspension was trans- fered to sterile petri dishes, and irradiated in them. 5. Suspension media for spores during irradiation (a) Water. All the fungal spores cited earlier were (b) (C) (d) (e) irradiated with gamma rays in water demineralized by means of a Bantam demineralizer. To this water 0.01% Triton X-100 was added as a wetting agent. pH. Citrate buffer was used to obtain suspending liquid of a definite pH. The final buffer concen- tration was 0.075 M, unless otherwise cited. Dextrose. Pure dextrose crystals (Baker reagent), were dissolved in demineralized water. The final concentrations used will be cited in the different cases. Sucrose. Pure sucrose crystals (Baker reagent) were dissolved in demineralized water. The final concen- trations will be cited when describing the individual experiments. Dry state. The spores were harvested in the same way as was described earlier in paragraph 3. The irradiation was carried out using gamma rays and cathode rays. One ml. portions of the spore sus- pension in demineralized water were dried in test tubes or Petri dishes under 27.5" of vacuum at 25°C. The test tubes were left in the drier for20 hours. The Petri dishes were dried for 4 hours under vacuum (27.5") and then placed in a desiccator overnight. 6. Irradiation sources All fungi were irradiated with gamma rays. A, flavus was irradiated with both gamma and cathode rays. (a) Cobalt-60 served as a source of gamma rays. The (b) Phoenix Radiation Facility and the Phoenix Memorial Laboratory sources at the University of Midhigan in Ann Arbor were used. The activity of the sources used was 2500 and 10000 curies respectively. A General Electric resonant transformer generator was used as a source of cathode rays. The electron beam had the energy of l Mev. The stated doses are at the surface. The maximum ionization occurs at .5 m"m water and the maximum penetration in 3.5 m"m of water. 7. Irra (a) (b) 10 diation Cobalt-60 source. The test tubes containing the fungal spores were placed in a styrofoam holder cut to fit around the cage of the Co-60 rods. The distance of the tubes from the cage was calculated on the basis of dosimetry performed by the Univer- sity of Michigan personnel, in order to expose the samples to the desired level of irradiation. The dose rate varied according to the source used and the distance of exposure. Resonant transformer generator. The Petri dishes containing the fungus spores were placed on a conveyor belt which carried the plates under the electron beam. The velocity of the conveyor was adjusted according to the dose desired and was based on available dosimetry data. Here again the dose rate was not the same when different doses were applied. During irradiation the Petri dish covers were removed and were immediately replaced following irradiation. Control plates containing sterile water indicated that the contamination using this technique was negligible. The irradiation was performed at room temperature. ll 8. Survival determination The irradiated spores were held overnight at a temper- ature of 400-420F. The test tubes and the Petri dishes con- taining the irradiated spore suspension were well mixed before diluting the plating with the aid of a vortex mixer. For plating, suitable water dilutions were made to give a count of 20—200 colonies per plate. In order to obtain the desired range, at least triplicate samples were run on P.D.A. at each of 2 to 3 dilution levels. The plates were incu- bated at room temperature (25°C.) for 10 hours to 3 days, according to the organism studied. The exact time will be cited later in each case. The number of survivors was determined by their ability to form colonies on the P.D.A. sub-culture. A Quebec Colony Counter was used for counting, with the exception of Rhizopus stOlonifer, where a binocular microscope was used. The Quebec Counter facilitated the counting of visible colonies as it has a light source, a low magnifying glass and a grided viewing surface. 9. An experiment on estabiished infection The effect of irradiation on established growth of the fungus Botrytis cinerea in fresh strawberries was studied in one experiment. 12 Strawberries were placed in small egg crate dividers with their tip upward. The strawberry tips were slightly wounded and inoculated with the aid of a wire brush which was dipped in a 106 spores/m1. suspension of the fungus spores. After inoculation, the crates were covered with a plastic bag to eliminate further contamination and to form a high humidity atmosphere. The inoculations were performed at intervals of 5, 2 and 0 days before irradiation. The strawberries were incubated at 52°F. following inoculation. For each inocu- lation time a control was used which was not irradiated. For each treatment 18 berries were used. Following the irradiation, the berries were stored at 52°F. The berries were checked for visual fungal growth over a period of 18 days. RESULTS AND DISCUSSION Most of the results were expressed as percent survival of organism. number of spores after irradiation number of spores before irradiation x 100 % survival = The percent survival was plotted on a logarithmic scale on the y-axis and the dose level or the solution concentrations were plotted arithmetically on the x-axis. The reason for this way of plotting is that generally the biological effects of irradiation are exponential functions of dose (20). The dose rate was not kept the same in all the experi— ments, as it was foundin the literature that there is no rate dependence of irradiation on the lethality of yeasts and bacterial spores exposed to cathode, gamma and X-rays (20, 39, 38, 17). They found that the mean lethal dose was the same, whether the irradiation was performed at a low intensity and spread over a prolonged time, or at high intensity for a short time. 1.. Generai.observation§_ (a) Production of small colonies All the tested fungi produced small colonies after irradiation. The number of small colonies among 13 14 survivors increased with increasing dose of irradi— ation, in an irregular manner. The small colonies did not reach the size of the regular colonies, even if they were held for longer incubation times. Laser found in 1964 (18) similar results when yeasts were exposed to X—rays. (b) Delayed growth A temporary inhibition of division and formation of visible colonies was observed among the spores that survived the irradiation. The duration of the delay increased with increasing dose. It is most apparent in the doses approaching the lethal dose. A similar retardation was reported by Lea (20) to be a general action of irradiation in a great variety of living cells. Demineralized water was used in all the liquid suspensions prepared for irradiation because it was shown by Dunn (11) that the presence of certain inorganic cations can affect the sensitivity to some extent. The first experiment was the isolation of some fungi from moldy strawberries. The fungi genera* were identified *Dr. Beneke assisted in the identification of the species. 14 survivors increased with increasing dose of irradi- ation, in an irregular manner. The small colonies did not reach the size of the regular colonies, even if they were held for longer incubation times. Laser found in 1964 (18) similar results when yeasts were exposed to X-rays. (b) Delayed growth A temporary inhibition of division and formation of visible colonies was observed among the spores that survived the irradiation. The duration of the delay increased with increasing dose. It is most apparent in the doses approaching the lethal dose. A similar retardation was reported by Lea (20) to be a general action of irradiation in a great variety of living cells. Demineralized water was used in all the liquid suspensions prepared for irradiation because it was shown by Dunn (11) that the presence of certain inorganic cations can affect the sensitivity to some extent. The first experiment was the isolation of some fungi from moldy strawberries. The fungi genera* were identified *Dr. Beneke assisted in the identification of the species. 15 using the Handbook of Barnett (3) on imperfect fungi. The species isolated were Alterneria qrisea, Alterneri§_ tenuis, Hgimintho sporium_§p,, Botrytis ciner§§_and Peri: cillium sp. Only the last two fungi were chosen for the irradiation work. 2. Sporulation media In order to find good Sporulation media, the following media were tested: (a) Potato dextrose agar (P.D.A.), Difco. (b) Corn Meal Agar plus 0.5% Yeast Extract (Difco). (c) Mycological Agar (Difco). (d) V8 Agar, prepared from either: (1) the whole V8 juice, or (2) the centrifuged juice in its natural pH (around pH 4), or (3) the centrifuged juice adjusted to pH 6.5-7.0. Of these the V8 media gave the best results in terms of luxuriant Sporulation. Among the V8 media (d-3) was pre— ferred, because it solidified uniformly and strongly. The results will be presented on a species basis. l6 3. A, falvus This fungus was irradiated with both gamma and cathode rays under various conditions. (a) Irradiation in demineralized water Two week old spores were suspended in demineralized water and exposed to O, 50, 100, 150, 200, 250 and 300 krad of gamma or cathode rays. The absolute numbers and percentage of surviving spores are given in Tables 1 and 2 of Appendix 1 for gamma and cathode rays, respectively. The percent survivals are pre- sented in graphic form in Figures 1 and 2. An approximate D value of 35-37.5 krad can be calculated, indicating that A, flavus spores are rather sensitive to irradiation. In this experiment the radiosensitivity of A, flavus spores was not influenced significantly by the nature of radiation (gamma or cathode rays). Available information indicates that differences in the biological effect caused by these two types of rays are not large, but if the condi- tions of irradiation are not exactly the same, as for example in the irradiation of spore suspension in test tubes vs. Petri dishes, in which the ratio of surface to depth differs, the effect may be different (l3, l4). l7 Ffpure 1. Effect of ramma rays on the viability of Aspergillus flavus spores irradiated in demineralized water, I- -~--.- u...— 2 l l T r 1 0 '3! E. 3’ (3 ID a .1 3’ A -2 .3 .34 L 1 J L O . 50 100 150 200 Irradiation dose , la'ad 18 Figure 2. Effect of cathode rays on the viability of As rgillus flavus spores irradiated in Log 2 survival -1 -2 an eraIized water. J 1 L I i l O 50 100 150 200 Irradi ati on dose , krad . 19 (b) Irradiation at various pH levels Two-week old spores of A, flavus were suspended in 0.075M citrate buffers at pH 3, 4, 5, 6 and 7, and exposed to 0, 50, 100, 150 and 200 krad of gamma and cathode rays. The absolute numbers and percentage of surviving spores are given in Tables 3 and 4 of Appendix 1. The percent survivals are graphically presented in Figures 3 and 4. It is apparent that pH has not an appreciable effect on the radio-sensitivity of the spores. Similar results were obtained by Edwards and coeworkers (12) who irradiated Bacillus subtilis spores with cathode rays. Alper and Gillies (2) found some dependence of the pH on the survival of irradiated E, ggii, The higher the acidity of the media, the higher was the number of survivors. (c) Irradiation in dextrose solutions Two-week old spores of A, flavus were suspended in dextrose solutions to give a final concentration of 0, 10, 20, 30 and 40 percent (w/v) of the sugar. The suspensions were exposed to gamma and cathode rays at different levels up to 250 and 200 krad, respectively. The results are given in Tables 5 and 6 of Appendix 1, and are illustrated in Figures 5 and 6. A, flavus spores suspended in dextrose solutions show a slight decrease in Log % survival 20 Figure 3. Effect of gamma rays on the viability of Aspergjllus flavus Spores irradiated in .075rM citrate buffer of various 25. 2M krad I T I 5U krad I I 0 I I too kr‘éd I -i I .. -2 I. lSO krad I .3 l l l i 3 Li S 6 7 Log % survival 21 Figure 4. Effect of cathode rays on the viability of Asperqillus flavus spores irradiated in .075 M citrate buffer of vail-us pH. 0 krad L, - 50 krad C) “* b 150 krad 0 Li. 1 l 3 4 5 6 7 pH Log 1 survival Figure 5. Effect of gamma rays on the viability of Asggrgillus flavus spores suspended in gextrose solutions. 2 ft on... v 50 ltrad 1 iOO krad .1 v I ‘50 rad I i. .2 " .3 _. ‘7' .1. l 0 i0 20 30 #0 % dext rose sol ut Ion. 22 23 Figure 6. Effect of cathode rays on the viability of Asperqillus flavus Spores su5pended in dextrose solutions. 2 1 I 0 |krad I I survival Log % 1 L ' 1 AL '3 o lo 20 3o ho % dextrose solution; 24 their sensitivity and this decrease is more apparent at the high doses. Several workers (27, 16, 40, 43) studied the effect of dextrose present in the growth media or in the suspension media used for irradiation, on the resistance of micro— organisms. They found also that there is a slight increase in resistance of the microorganisms to irradiation, in the presence of dextrose. Latarjet and Loiseleur (19), 1942, quoted by Nickson (27) and later Hollaender and Stapleton, 1953 (16) offered some explanations for its protective action. (d) Irradiation in the dry state Two-week old spores of A, flavus were suspended in demineralized water, containing 0.01%>triton X-lOO. A number of these suspensions were dried as described under Materials and Methods. The wet and dried suspensions were exposed to 0, 50, 100, 150, 200, 250 and 300 krad of gamma and cathode rays. The results of this experiment are summarized in Tables 7 and 8 of Appendix 1 and in Figures 7 and 8. It is apparent that the dried spores of A, flavus are considerably more resistant than the spores suspended in water. Bhattacharjee (7) obtained similar results when he Survival Log % -2 -4 Figure 7. 25 Effect of gamma rays on the viability of dry and wet Spores of ASperqillus flavus. —’ L l i IOO l50 200 Irradiation dose, krad 250 300 Log 1 Survival Figure 0. 26 Effect of cathode rays on the viability of dry and wet Spores of Asgergillus flavus. 2 i i I i 0 -i .2 - '3 -h i l I l I 50 '00 '50 200 250 Irradiation dose. krad 300 27 irradiated A, ggii_with X-rays. Stapleton and Hollaender (37) showed that the higher the water content of A, terreus spores, the lower the lethal dose. Several other investigators also demonstrated this striking increase of microorganism radioresistance when irradiated in dry state rather than in wet state. The explanation offered to this phenomenon is that the removal of free water decreases the indirect effect,* thus the radiosensitivity decreases too (7, 24, 44, 20). 4. A, niqgr_ Onedweek old spores of A, niqgr_were harvested, sus- pended at the concentration of 106 spores per ml. of de- mineralized water containing o.or% Triton X-100, and exposed to 0, 50, 100, 150, 200, and 250 krad of gamma rays. The results are presented in Figure 9 and summarized in Table 9 of Appendix 1. It is apparent that this fungus is quite sensitive to gamma rays. At 200 krad no growth could be observed. *Damage is caused to a microorganism when the radiation hits its sensitive area directly (Lea's Target Theory). When the cause of the damage is due to the ionization in the outside environment of the target, the effect is called an indirect effect. Log % survival 28 Figure 9. 'Effect of gamma rays on the viability ()f Aspergillus niggr spores suSpended in demineralized water. l T l l O -l -2 -3 l .L. l l l 0 50 lOO l50 Irradiation dose. krad 29 Saravacos and his co-workers (31) found that 250 krad is the lethal dose of A, niggr, The difference in the re- sults may be due to the suspension media during the irradi- ation and the size of the original population. In their experiment the fungus was irradiated on malt extract agar slants with an undetermined population of cells. 5. A, cinerea Nineteen-day old cultures of A, cinerea were harvested and the spores were irradiated in several suspension media. (a) Irradiation in demineralized water Aqueous spore suspensions containing 105-106 spores per ml. were exposed to gamma rays at the dose range of 0 to 300 .krad. The results are presented in Table 10 of Appendix 1 and in Figure 10. The figure is based on five separate experiments. The survival curve obtained was not a straight line. The results indicated a D value of about 55 krad at the straight part of the- line. The irradiation destroys only a small number of spores at the low doses, resulting in a lag which extends to 100-150 krad. (b) Irradiationat various pH levels spores of A, cinerea were suspended in 0.075M citrate buffers pH 3, 4, 5, 6 and 7 and exposed to gamma rays in 30 Figure I0. Effect of gamma rays on the viability of Botryris cinerea Spores suspended in demineraliged water. '5 > 'E a o In 3* Cl 0 .J -I J .2 l a 1 1. l 0 50 IOO 150 200 250 300 Irradiation dose. krad 31 the dose range of 0 td 300 krad. The results are presented in Table 11 of Appendix 1 and Figure 11. The results indi- cated that the lower the pH, in the range studied, the higher the percent survivors at all doses. As the dose increased, the number of survivors decreased more sharply at higher rather than at lower pH levels. (c) Irradiation in sucrose solutions Spores of A, cinerea were suspended in 0, 5, 10, 15 and 20 percent (w/v) sucrose solutions and exposed to gamma rays at the range of 0 to 300 krad. The results are summarized in Table 12, Appendix 1, and Figure 12. From these data it appears that a greater number of spores are likely to sur- vive radiation destruction in sucrose solution than in water. 'However, no clear differences among the various sucrose concentrations tested can be ascertained in regard to radi- ation protection of the spores. 6. Penicillium A2, In order to study the possibility of a change in the radioresistance of fungi spores, due to difference in their age, Penicillium ER: spores at the age of 9, 15, 19, 26, 40 and 60 days were irradiated with 50 krad of gamma rays. The results are summarized in Table 13, Appendix 1 and in Figure 13. The curve shows a very small reduction in the radio- Log % survival Figure II. Effect of gamma rays on the viability of Botrytis cinerea Spores irradiated in .075 M citrate buffer of various gfl. O'Frad 'q7 #— -h 33 Figure l2. Effect of gamma rays on the viability of gotrytis cinerea Spores suspended in sucrose solutions. Log % survival o krad re 300V“ .6 I 1 J N 5 IO I5 20 % sucrose (HVV). 34 m>mv .nouoem mo om< om on c: on , ON a. jII.1.1.ITI~.4._E_J_ 1.1! ll 1’ T ____pr._h__ .aw>mt mEEmm mo out; om co_um_omct_ mo onoov oucmum_moto_umc e_o;u co motoam .deE:____o_coa Co 0mm mo somewm .m. ot:m_m o— m— ON [OAIAJOS % 35 resistance of the spores up to the age of 20 days. At a still older age, the resistance of the spores to irradiation falls almost linearily. The age of the fungi spores chosen to work with was 14-19 days. Proctor and co-workers (30), working with bacteria, found that the culture age played a role in the radio— sensitivity of the organism, the old cells are the most radiosensitive. (a) Irradiation in demineralized water Aqueous sporegsuspensions containing 106-107 spores per ml., were exposed to gamma rays at the dose levels of 0, 50, 100, 150, 200, 250 and 300 krad. The results are presented in Table 14 of Appendix 1 and in Figure 14. The survival curve is based on four separate experiments. Colony counting, two days after plating, indicated a D value of about 35 krad. (b) Irradiation at various pH levels Penicillium.§p, spore suspensions at pH levels of 3 to 6 produced using citrate buffers (0.075M), were exposed to gamma rays at the doses 0, 50, 100, 150 and 200 krad. The results are summarized in Table 15 of Appendix 1 and in Figure 15. Four separate experiments were conducted. Log % survival Figure lh. Effect of gamma rays on the viability of Penicillium Sp. Spores su5pended in demineralized water. 1 1 1 1 0 50 IOO ISO 200 Irradiation dose, krad 36 Log % survival 37 Figure 15. Effect of gamma rays on the viability of Penicillhnngp. spores irradiated in .075 M citrate buffer of various pH. ;g fIIIIlIEflII==IE====II========Ifi=----'-I-Ifi 0 krad 1 L: If + % 4.... P 50 krad we I O l 100 krad ‘ J. —l -2 150 krad 9) -3i: 1 I :T l_. 3 4 5 6 7 pH 38 These data indicate that the effect of pH on the radio- resistance of the spores is not pronounced. There might be a slight increase in the sensitivity at pH 6 over that at lower levels. The survivors at 200 krad were too few for any significant counting. (c) Irradiation in sucrose solutions Nineteen-day old Peniciiiium ER: spores were suspended in 0, 5, 10 and 15 percent (w/v) solutions of sucrose, and were exposed to 0, 50, 100 and 200 krad of gamma rays. The results are presented in Table 16, Appendix 1. From these data it appears that sucrose, at the concen- trations used, had little or no effect on the radio- resistance of the fungus spores. Another irradiation of 40 day old Penicillium ER: spores in sucrose solutions was performed. Spores suspended in 0, 10 and 20 percent (w/v) of the sugar were exposed to 0, 50, 100, 200 and 300 krad of gamma rays. The results are pre- sented in Table 16 (2) of Appendix 1. No data were reported for 200 and 300 krad doses, because no plate growth was observed under the conditions of plating used. The radioresistance of these spores in plain water and in the sugar suspensions is slightly lower than that observed in the previous experiment. The main reason may be the age of the spores used. 39 (d) Irradiation in dextrose solutions Forty-day old spores of Penicillium AR, were suspended in 0, 10 and 20 percent w/v of dextrose and were exposed to 0, 50, 100, 200 and 300 krad of gamma rays. The results are presented in Table 17 of Appendix 1. No data were reported for 200 and 300 krad, because no plate growth was observed under the conditions of plating used. The radioresistance of this spore in plain water is slightly lower than that observed in previous experiments. 7. Rhizopus stoionifer Fourteen-day old spores of the fungus were suspended in demineralized water containing o.or% Triton X-100, and were exposed to 0, 100, 150, 200, 250 and 300 krad of gamma rays. The survivals were counted after.20 to 24 hours of plating with the aid of a binocular. The reason for this way of counting is that colonies of this fungus older than 24 hours spread over adjacent colonies hindering the enumeration. The results are presented in Table 18 of Appendix 1, and graphed in Figure 16. Ahizgpg§_srolonifer is the most resistant fungus cheeked. The results obtained are quite similar to those reported in the literature (6, 31, 34), although a different procedure Log 1 survival 40 Figure I6. Effect of gamma rays on the viability of Rhizopus stolonifer spores suspended in demineralized water. i I l i l i l 0 SO i00 ISO 200 250 300 Irradiation dose, krad 41 of harvesting, irradiating and determining the survival was used. 8. Irradiated inoculategistrawberries Strawberries, inoculated with A, cinerea spore suspen- sion 5, 2 and 0 days before irradiation, were exposed to 0 and 200 krad of gamma rays, and were checked for visual spoilage for 18 days. The results are summarized in Table 19 of Appendix 1 and presented in Figures 17 and 18. It is apparent that the earlier the berries were inoculated before irradiation, the less protection against spoilage was achieved. The best protection against spoilage was at the zero (0) time of inoculation, as shown in Figure 18 in which the 50% spoilage was plotted vs. days elapsed between inoc- ulation and irradiation. 9. General digcussion on food irradiation Irradiation is a novel method of food preservation. It involves the use of gamma rays, cathode rays and X-rays. It differs from heat preservation procedures in that the temperature of the food does not increase appreciably. Radiation preservation of food might have some application in sterilization (pork), pasteurization (fruits, vegetables), disinfection (water), disinfestation (cereal) and sprout inhibition (potatoes). 142 _ co_ummpmte_ couem m>mo comsm_omtt_ touem m>mo co_um_omtt_ realm mime m. o. :_ ~— 14..— 4i _ _ _ _ _ _ _ _ _ _ _I _ _ o. m a a N o n. o. a. N. o. a o a _ N o p o a ,a _ _ _ _ _ _ _ ,4» _ .IL _ _ a _ a. _ _ _ _ _ _ _ _ .4 _ d _ A N o _ _ o _ _ 0. ON on an op 0m 8 om nu a9 oo. _ _ _ _ _ _ «PICI _ _I [Fla _ _ p _ _ _ _\ _ _ _ _ r _ _ _ _ name 0 name N .co_um_pmue_ oLOwon m>mp O can .N .m motoc_u m_u>tuom cu_3 ovum—:uOcm mo_tton36tum we ome__oam x org :0 mxmt mEEmm eo Supeem .m. ot:m_u o: abelgOOs Days for 50% Spoilage 43 Figure I8. Number of days needed for 50% Spoilage .1§.the number of days of inoculation before irradiation. Days of inoculation before irradiation 44 Irradiation extends the shelf life of fresh fruits over regular refrigeration methods. The fruit can reach its destination in a better condition while shipped long distances. It is predicted that irradiated fresh strawberries are going to be the first fruit to be distributed for public use. S UMMARY Survival curves for five species of food decay fungi were determined by exposing fungi spore suspensions in de- mineralized water to gamma rays. Asperqillus rlavus, Asperqillus giqgr and Penicillium A2, have almost the same sensitivity toward gamma rays, with D value in the range of 30 to 35 krad, whereas Botrytis cinerea has a D value of approximately 55 krad and Rhizopus atolonifgr, the most resistant fungus studied, has D value of approximately 100 krad. Asperqillus flavus and Peniciiium EB: spores irradiated in citrate buffer at pH 3, 4, 5, 6 and 7 showed almost no change in their radiosensitivity with pH. On the other hand, Botrytis cinerea spores showed a distinct decrease in their radioresistance at higher pH.levels (6 and 7) . Penicillium ER: spores irradiated in sucrose and dextrose solutions (0 to 20%) showed no significant change in their radioresistance. ,Botryti§_cinerg§.spores displayed a higher radioresistance when they were irradiated in 5 to 20 percent sucrose solution than in water. Asperqillus flavus spores showed an increase in their radioresistance to destruction by gamma rays when the dextrose concentration of the 45 46 suspension media increased from O to 40%. Dry spores of Asperqiilus flavus showed a considerable increase in their radioresistance when compared with spores irradiated in water. The effect of gamma and cathode rays on Asperqillus flavus spores was compared. It was found that when the irradiation is conducted in demineralized water, there is almost no difference between the two kinds of radiation. The age of Penicillium A2, spores was found to have a very significant effect on their radioresistance. After the age of 20 days, the older the spores, the higher the sensitivity to gamma rays. Strawberries inoculated with a Botrytis cinerg§_spore suspension at 5, 2 and 0 days before irradiation with 200 krad of gamma rays, showed that the longer the period be- tween inoculation and irradiation, the less the protection by irradiation against spoilage. The best protection was achieved when irradiation immediately followed the inoculation. APPENDIX 1 Table 1: Effect of gamma rays on the viability of Asperqillus ose 0 krad 50 krad 100 Exp. Cells % Cells % Cells No. per ml. survival per ml. survival per ml. 1 4.70 x 106 1.00 x 102 7.80 x 105 1.66 x 101 1.33 104 2 8.00 x 106 1.00 x 102 1.17 x 106 1.46 x 101 3.36 104 3 8.22 x 106 1.00 x 102 1.09 x 106 1.33 x 101 2.44 104 4 1.05 x 107 1.00 x 102 6.90 x 105 6.57 x 100 2.42 104 Table 2: -Effect of cathode rays on the viability of Asperqillus Dose 0 krad 50 krad 100 Exp. 4 Cells % Cells % Cells No. per ml. survival per ml. survival per m1. 3 l 1.98 x 106 1.00 x 102 .4.40 x 104 2 22 x 100 1.03 10 4 2 4.86 x 106 1.00 x 102 5.70 x 104 1.18 x 100 1.38 10 4 3 6.38 x106 1.00 x 102 1.62 x 105 2.54 £10O 1,97 '10 3 4 6.98 x 106 1.00 x 102 3.12 x 105 4 46 x 100 6.94 10 4 5 1.12 x 107 1 00 x 102 5.95 x 105 5 32 x 100 2.03 10 4 6 1.23 x 107 1.00 x 102 1.75 x 105 1.42 x 100 6.16 10 47 flavus spores irradiated in demineralized water *ekrad 150 krad 200 krad '% Cells '% ' Cells '% survival per m1: survival per m1. survival 2.83 x 10'1 6.60 x 102 1.40 x 10'2 7.50 x 100 1.60 x 10‘4 4.20 x 103 2.30 x 103 2.88 x 10'2 2.85 x 101 3.56 x 10-4 2.97 x 10‘1 8.60 x 102 1.05 x 10‘2 1.80 x 101 2.19 x 10"4 2.30 x 10'1 1.73 x 103 1.75 x 10’2 - - a flavus spores irradiated in demineralized water kradEI a 150 krad _200 krad % 3 Cells % Cells % survival per m1. survival per m1. survival 5.20 x 10'2 2.93 x 102 1.48 x 10'2 12.10 x 101 1.06 x 10'3 2.83 x 10"1 - - 5.13 x 10} 1.06 x 10"3 3.09 x 10"1 - - 1.14 x 102 1.79 x 10"3 9.94 x 10'2 1.94 x 103 2.78 x 10'2 4.45 x 101 6.38 x 10"4 1.81 x 10'1 2.86 x 103 2.55 x 10"2 1.20 x 102 1 07.x 10'3 5.00 x 10'1 - - 1.85 x 102 1.50 x10'3 48 Table 3: Effect of gamma rays on the viability of Asperqillus Dose 0 krad 50 krad pH Cells % Cells % per ml. survival per m1. survival 3 5 13 x 106 l 00 x 102 7 00 x 105 1.37 x 101 4 5 10 x 106 1.00 x 102 5.55 x 105 1.10 x 101 5 4.80 x 106 1.00 x 102 8.60 x 105 1.79 x 101 2 6 4.90 x 106 1 00 x 10 9 05 x 105 1.85 x 101 7 4.70 x 106 1.00 x 102 6.30 x 105 1.34 x 101 Table 4: Effect of cathode rays on the viability of Asperqillus Dose. 0 krad 50 krad Cells % Cells % pH per m1. survival per ml. survival 3 1.23 x 107 1.00 x 102 2.25 x 105 1.83 x 100 5* 8.19 x 106 1.00 x 102 2.34 x 105 2.86 x 100 7 9.55 x 106 1.00 x 102 6.28 x 105 6.55 x 100 *The data given are the mean of two separated experiments. flavus spores irradiated in(1075M citrate buffer of various pH 100 krad 150 krad Cells % Cells % per m1. survival per m1. survival 4 00 x 104 7 80 x 10'1 4.20 x 102 8 20 x 10'3 -1 2 -3 2 70 x 10 5 30 x 10 3.00 x.10 5 90 x 10 3.90 x 104 8.10 x 10'1 6.50 x 102 1 35 x 10’2 3 60 x 104 7 35 x 10'1 3.30 x 102 6 70 x 10'3 2.90 x 104 5 30 x 10’1 5.20 x 102 1.10 x 10’2 flavus spores irradiated in 0.075M citrate buffer of various PH 100 krad 150 krad Cells 96 Cells % per m1. survival per m1. survival 9.30 x 103 7.55 x 10‘2 3.11 x 103 2.52 x 10".2 3.96 x 104 4.84 x 10'1 1.41 x 103 1.58 x 10'2 9.50 x 103 9.95 x 10"2 8.00 x 103 8.36 x 10"2 50 Table 5: »Effect of gamma rays on the viability of Asperqillus Dose 0 krad 50 krad 100 krad Exp. 113::5e Cells % . cells % ' Cells % no. c0nc. per m1. sur- per ml. sur- per m1. sur- % . v1val_ v1val ? v1val 1 0 8.00x106 1.00x102 1.17x106 1.46x10l 3.36x104 4.20x10-l 10 7.80x106 1.00x102 1.49x106 1.91x10l 8.66x104 1.11x100 20 6.98x106 1.00x102 1.50x106 2.14x101 7.40x104 1.06x10O 30 7.18x106 1.00x102 1.76x106 2.47x10l 7.06x104 9.86x10"l 40 8.14x106 1.00x102 1.87x106 2.30x101 9.32x104 1.15x100 2 '0 8.22x106 1.00x102 1.09x106 1.33x10l 2.44x104 2.97x10_1 '10 6.92x106 1.00x102 1.62x106 2.34x10l 8.92x104 1.29x10o 20 6.62x106 1.00x102 1.79x106 2.7lx10l 8.38x104 1.27x10o 30 5.26x106 1.00x102 1.73x106 3.29x10l 5.98x104 1.14x100 40 5.52x106 1.00x102 2.79x106 5.05x101 1.04x105 1.88x100 flavus suspended in 51 dextrose solutions 150 krad 200 krad 250 krad Cells %. Cells % Cells % per m1. sur- per m1. sur- per m1. sur- vival vival vival 2.30x103 2.88x10"2 2.85x10l 3.56x10'4 5.00x10'1 6.25x10"6 5.00x103‘ 6.41x10'2 1.24x102 1.58x10’3 - - 3.76x103 5.37x10'2 5.55x101 7.94x10'4 3.50x10O 5.00x10'5 4.17x103 5.80x10'2 6.90x10l 9.59x10'4 - - 5.70x103 7.01x10‘2 2.96x102 3.64x10’3 1.60x101 1.97x10"4 8.60x102 1.05x10‘2 1.80x101 2.19x10"4 5.00x10"1 6.08x10'6 4.20x103 6.06x10'2 1.22x102 1.77x10—3 - - 2.50x103 3.78x10’2 8.04xlo1 1.21x10'3 4.54x103 8.64x10'2 6.28x10l 1.19x10'3 - - 6.12x103 1.11x10"l 8.15x10l l.47x10"3 1.3 x101 2.35x10’4 52 Table 6: Effect of cathode rans on the viability of Dose _ . 0 krad 50 krad Exp. Dex- Cells % Cells % no. trose per m1. survival per m1. survival conc. 1 0 4.83 x 106 1.00 x 102 5.70 x 104 1.18 x 100 10 7.65 x 106 1.00 x 102 1.56 x 105 2.03 x 100 20 7.17 x 10'6 1.00 x 102 2.23 x 105 3.11 x 100 30 7.82 x 106 1.00 x 102 9.53 x 105 1.22 x 101 40 1.02 X 10 1.00 X 10 2.39 x 10 2.34 X 10 2 O 1.23 X 107 1.00 X 102 1.75 X 105 1.42 X 100 10 8.38 X 106 1.00 X 102 2.31 X 105 2.76 X 100 20 1.15 X 107 1.00 X 102 4.01 X 105 3.49 X 100 30 1.08 X 107 1.00 X 102 7.72 X 105 7.15 X 100 7 2 6 1 40 1.18 X 10 1.00 X 10 1.80 X 10 1.52 X 10 53 A§perqillus flavus suspended in dextrose solutions 100 krad 200 krad Cells % Cells % per ml. survival per ml. survival. 1.38 104 2.86 10"1 5.13 x 101 1.06 10"3 8.98 103 1.17 10'1 4.20 x 102 5.50 10'3 5.35 104 7.46 10'1 3.85 x 102 5.36 10‘3 2.24 105 2.87 100 3.58 x 103 4.57 10'2 1.54 105 1.51 100 7.46 x 103 7.33 10'2 6.16 104 5.00 10'1 1.85 x102 1.50 10'3 8.78 104 1.04 100 1.64 x 102 1.96 10"3 1.19 105 1.03 10° 1.36 x 102 1-13. 10"3 2.38 105 2.20 100 2.12 x 103 1.96: 10'2 6.34 105 5.37 100 1.82 x 104 1.54. 10‘1 54 Table 7: Effect of gamma rays on the viability of dry and Wet spores Dry spores Dose' krad % % Per m1. survival Per ml. survival 6 2 2 O 8.00 x 10 1.00 x 10 6.24 x 10 1.00 x 10 50 1.17 X 106 1.46 X 101 2.43 x 106 3.89 X 101 100 3.36 x 104 4.20 x 10'1 8.62 x 105 1.38 x 101 3 -2 5 0 150 2.30 x 10 2.88 x 10 1.40 x 10 2.24 x 10 200 2.85 x 101 3.56 x 10'4 8.74 x 103 1.40 x 10"1 3 -2 250 --- -- 1.04 x 10 1.66 x 10 1 -3 300 —-- --- 9.00 x 10 1.44 x 10 Table 8: Effect of cathode rays on the viability of dry and Dose ._1_ Wet spores . Dry spores krad Per m1. ‘% survival Per m1. % survival 2 0 1.98 x 106 1.00 x 102 2.52 x 106 1.00 x 10 50 4.40 x 104 2.22 x 100 6.94 x 105 2.75 x 101 3 -2 5 1 100 1.03 x 10 5.22 x 10 6.60 x 10 2.62 x 10 150 2.93 x 102 1.48 x 10'2 1.35 x 105 5.36 x 10° 200 2.10 x 101 1.06 x 10"3 2.35 x 104 9.31 x 10"1 3 -2 250 --- -- 2.45 x 10 9.72 x 10 3 -2 300 --- -- 2.50 x 10 9.90 x 10 55 wet spores of Asperqillus flavus Wet spores Dry spores % % Per ml. survival Per ml. survival 8.22 x 10 1.00 x 102 6.84 x 106 1.00 x 102 1.09 x 106 1.33 x 101 2.60 x 106 3.83 x 101 2.44 x 104 2.97 x 10"1 7.84 x 105 1.09 x101 8.60 x 102 1.05 x 10’2 ‘1.30 x 105 1.90 x 10° 1.80 x 101 2.19 x 10‘4 2104 x 103 2.98 x 10'2 --- --- 6.36 x 102 9.31 x 10'3 --- --- 1.87 x 101 2.73 x 10(7-4 and wet spores of Asperqillus flavus. Wet spores ‘Dry spores Per m1. ‘% survival Per ml. ‘% survival 6.98 x 106 1.00 x 102 4.50 x 106 1.00 x 102 3.12 x 105 4.46 x 100 '1.42 x 106 3.06 x 101 6.94 x 103 9.94 x 10'2 6.52 x 105 1.45 x 101 1.94 x 103 2.77 x 10".2 7.46 x 105 1.66 x 101 4.45 x 101 6.37 x 10‘4 3.61 x 104 8.00 x10"1 -_- --_ 6.48 x103 1.44 x 10'1 -__ -__ 3.75 x 102 8.32 x 10‘3 56 Table 9: Effect of gamma rays on the survival of Asperqillus niger spores suspended in de- mineralized water containing 0.01% Triton X-100 Dose in Cells % krad per ml. survival 0 4.16 x 106 1.00 x 102 50 9.28 x 105 2.23 x 101 100 3.20 x 104 7.67 x 10"1 150 2.30 X 101 5.51 x 10-4 200 0 --- Table 10: .Effect of gamma rays on the viability of Botrytis Triton.X-100 Dose 0 krad 100 krad 150 krad Exp. Cells % Cells % Cells % no. per m1. survival per m1. survival per m1. survival 1 2.29x105 1.00x102 1.53x105 6.67x10l 7.72x104 3.37x101 2 7.10x104 1.00x102 4.20x104 5.92x101 ---- 3 1.45x105 1.00x102 7.90x104 5.45x10l ---- 4 1.53x106 1.00x102 1.11x106 7.25x10l --—- 2 5 1.20x106 1.00x10 9.10x105 7.60x101 -—-- 57 cinerea spores suspended in demineralized water containing 0.01% 200 krad 250 krad 300 krad Cells % Cells % Cells % per m1. survival per ml. survival per m1. survival 2.48x104 1.08x101 3.07x103 1.34x10O 1.75x102 7.64x10'2 1.59x104 2.24x10l --—— 2.98x102 4.20x10'1 1.12x104 7.74x10o ---- 1.00x102 6.90x10"2 1.20x105 7.85x10O ---- 2.40x103 1.60x10'l 1.20x103 1.00x10l ---- 6.00x103 5.00x10'l 58 Table 11: Effect of gamma rays on the viability of Botrytis levels Dose 0 krad 100 krad 150 krad H Cells % Cells % Cells % p per ml. survival per ml. survival per m1. survival 3 2.00x105 1.00x102 1.73x105 8.65x10l 1.27x105 6.35x101 4 2.27x105 1.00x102 1.67x105 7.25x10l 1.40x105 6.17x101 5 2.08x105 1.00x102 1.39x105 6.67x101 7.86x104 3.78x101 6 2.02x105 1.00x102 1.17X105 5.80x10l 6.26x104 3.10x101 7 2.41x105 1.00x102 7.40x104 3.07x10l 1.60x104 6.65x100 Table 12: The effect of gamma rays on the viability of Botrytis Dose 0 krad 100 krad Sucrose Cells 96 Cells % conc. per m1. survival per m1. survival 0 7.10 x 104 1.00 x 102 4.20 x 104 5.92 x 101 5 5.00 x 104 1.00 x 102 1.57 x 104 3.14 x 101 4 10 7.20 x 104 1.00 x 102 3.30 x 104 4.58 x 10 15 8.50 X 104 1.00 x 102 3.80 x 104 4.47 x 101 20 3.55 x 105 1.00 x 102 2.70 x 105 7.60 x 101 59 cinerea spores irradiated in 0.075M citrate buffer of various pH 200 krad 250 krad 300 krad Cells % Cells % Cells % per ml. survival per m1. survival per ml. survival 3.47x104 1.73x101 2.41X104 1.21X101 3.28x103 1.64X100 3.26X104 1.44x10l 2.34X104 1.04x101 2.57X103 1.13x10O 2.38x104 1.14x101 7.92x103 3.80x10O 1.65x103 7.93x10-1 3.50x103 1.73x100 8.50x10l 4.20x10'2 9.00x100 4.45x10'3 2.00x102 8.30xlO-2 1.00x10O 4.15x10'4 --- -—- cinerea spores suspended in sucrose solutions 200 krad 300 krad Cells % (Cells % per ml. survival per m1. survival 1 2 -1 1.59 x 10 2.24 x 10 2 98 X 10 4.20 X 10 1 3 0 4.50 x 10 9.00 x 10 2 25 x 10 4.50 x 10 l 4 1 1.80 X 10 2.50 X 10 1.59 X 10 2.21 X 10 2 4 1 8.50 X 10 1.00 X 10 1.77 x 10 2.08 x 10 6O Table 13: Effect of age of Penicillium AR, spores on their resistance to 50 krad of gamma rays Dose 0 krad 50 krad Age Cells % Cells % Days per m1. survival per m1. survival 9 2.50 X 106 1.00 x 102 3.90 x 105 1.56 x 101 15 3.90 X 106 1.00 X 102 5.80 x 105 1.49 x 101 19 2.74 x 106 1.00 x 102 4.00 x 105 1.46 X 101 26 1.05 x 107 1.00 x 102 1.20 x 106 1.14 X 101 40 7.70 x 106 1.00 x 102 2 91 x 105 3.78 x 100 60 3.70 x 10 1.00 x 10 No growth Table 14: The effect of gamma rays on the viability of 0.01% Triton X-100 Dose 0 krad 50 krad 100 Exp. Cells % Cells % Cells no. per m1. survival per ml. survival per m1. 1 2.50 x 106 1.00 x 102 3.50 x 105 1.40 x 101 2.92 X 103 2 4 2 1.45 x 107 1.00 X 10 1.74 X 106 1.20 X 101 3.33 X 10 2 3 3.90 x 106 1.00 x 10 5.80 x 105 1.49 x 101 9.10 x 103 3 4 1.05 x 107 1.00 x 102 1.20 X 106 1.14 X 101 4.10 x 10 61 Penicillium sp. spores suspended in demineralized water containing krad 150 krad 200 krad % Cells % Cells % survival per m1. survival per ml. survival 1.17 x 10'1 1.19 x 102 4.75 x 10'3 5.00 x 10° 2.00 x 10'4 2.30 x 10'1 - - 6.40 x 101 4.40 x 10'4 2.33 x 10'1 6.40 x 101 1.64 x 10"3 3.90 x 100 1.00 x 10'4 3.90 x 10"2 - - - - 62 Table 15: Effectcfifgamma rays on the viability of Peniciiiium Dose 0 krad 50 krad Exp. H Cells %) Cells % no. p per m1. survival per m1. survival 1 3 3.40 X 106 1.00 x 102 5.55 X 105 1.63 x 101 4 4.20 x 106 1.00 x 102 4.00 x 105 9.50 x 100 5 3.97 X 106 1.00 X 102 5.55 X 105 1.40 x 101 6 3.53 X 106 1.00 x 102 5.95 x 105 1.69 x 101 7 3.63 X 106 1.00 X 102 5.13 X 105 1.41 x 101 2 3 1.05 X 107 1.00 X 102 9.00 x 105 8.57 X 100 4 1.16 X 107 1.00 x 102 9.50 X 105 8.20 x 100 5 1.16 x 107 1.00 x 102 1.20 x 106 1.02 x 101 6 1.26 x 107 1.00 X 102 9.90 x 105 7.85 x 100 3 3 1.27 x 107 1.00 x 102 2.09 x 106 1.65 x 101 4 1.22 x 107 1.00 X 102 2.01 X 106 1.65 X 101 5 1.56 x 107 1.00 X 102 2.00 x 106 1.28 x 101 6 1.28 x 107 1.00 x 102 1.43 x 106 1.12 x 101 4 3 1.53 x 107 1.00 x 102 1.12 x 106 7.32 x 100 4 1.34 X 107 1.00 x 102 8.60 X 105 6.40 X 100 5 1.34 X 107 1.00 x 102 1.00 X 106 7.50 x 100 6 1.44 X 107 1.00 X 102 8.80 x 105 6.10 X 100 63 A2, spores irradiated in 0.075M citrate buffer of various pH 100 krad 150 krad Cells % Cells % per m1. survival per m1. survival 6.80 x 103 2.00 x 10'1 3.63 x 101 1.07 x 10'3 9.38 x 103 2.23 x 10'1 6.69 x 101 1.59 x 10'3 5.49 x 103 1.39 x 10'1 5.43 x 101 1.37 x 10"3 5.50 x 103 1.56 x 10'1 3.66 x 101 1.04 x 10'3 5.16 x 103 1.42 x 10'1 3.66 x 101 1.01 x 10"3 1.12 x 104 1.07 x 10'1 _ _ 1.30 x 104 1.12 x 10'1 - - 6.90 x 103 5.95 x 10"2 _ _ 3.20 x 103 2.54 x 10'2 - _ 1.71 x 104 1.35 x 10‘1 - _ 1.60 x 104 1.31 x 10"1 _ - 3.07 x 104 1.97 x 10'1 - _ 1.05 x 104 8.22 x 10"2 - _ 6.10 x 103 3.98 x 10'2 — - 9.50 x 103 7.10 x 10'2 - _ 8.40 x lo3 6.30 x 10"2 _ _ 4.60 x 103 3.20 x10'2 — - 64 Table 16: The effect of gamma rays on the viability of Dose 0 krad 50 krad Exp. %1 Cells % Cells % no. su- per m1. survival per ml. survival crose l O 3.01 x 106 1.00 x 102 4.00 x 105 1.33 x 101 6 2 5 1 Spores 5 6.60 x 10 1.00 x 10 7.30 x 10 1.11 X 10 age 6 2 5 1 19 10 5.20 x 10 1.00 x 10 7.50 x 10 1.44 x 10 days 6 2 5 I 1 15 5.10 x 10 1.00 X 10 7.00 X 10 1.37 x 10 2 0 7.70 x 106. 1.00 x 102 2.91 x 105 3.78 x 100 Spores 6 2 5 0 age 10 8.00 x 10 1.00 X 10 1.25 x 10 1.56 x 10 40 6 2 5 2 days 20 6.36 X 10 1.00 X 10 1.14 x 10 1.79 x 10 Table 17: Effect of gamma rays on the viability of Dose 0 krad 50 krad % Cells 96 Cells % Sucrose per ml. survival per ml. survival 0 0 7.70 X 106 1.00 x 102 2.90 x 105 3.78 X 10 10 8.00 X 106 1.00 x 102 1.30 x 105 1.63 X 100 20 6.40 x 106 1.00 x 102 1.10 X 105 1.72 x 100 65 Penicillium sp. spores suspended in sucrose solutions 100 krad 200 krad Cells 96 Cells % per ml. survival per m1. survival 1.49 x 104 4.95 x 10‘1 6.00 x 102 1.99 x 10"2 4 _ - 1.88 x 10 2.85 x 10 1 3.70 x 102 5.60 x 10 3 2.16 x 104 4.15 x 10'1 1.72 x 102 3.30 x 10"3 1.43 x 104 2.80 x 10'1 2.90 x 102 5.70 x 10"3 3 -2 1.10 x 10 1.43 x 10 No growth 2.90 x 102 3.62 x 10'3 - - 2.05 x 102 3.12 x 10’3 - - Penicillium _p, spores suspended in dextrose solutions 100 krad 150 krad. Cells 96 Cells ‘ % per m1. survival per m1. ' survival 3 -2 1.10 X 10 1.43 X 10 No growth 2.90 x 102 3.62 x10"3 - - 2.10 x 102 3.18 x10"3 _ _ 66 Table 18: Effect of gamma rays on Rhizopus stoloniigr spores suspended in demineralized water containing 0.01% Triton X-100 Exp. no. Dose in krad Cells per ml. % survival 1 0 6.76 105 1.00 102 100 5.66 105 8.36 101 150 4.48 105 6.64 101 200 '4.01 105 5.91 101 250 1.71 105 2.53 101 300 3.47 104 5.13 100 2 0 7.30 105 1.00 102 100 4.93 105 6.73 101 150 4.60 105 6.30 101 200 3.20 105 4.38 101 250 6.20 104 8.50 100 67 Table 19: Effect of gamma rays on the spoilage of strawberries before irradiation Days of inoculation 5 days 2 Dose 0 krad 200 krad 0 krad 22::r No. % No. % No. % irra- spoiled spoiled spoiled spoiled spoiled spoiled diation berries berries berries berries berries berries _1 13 72.2 1 5.6 0 0 2 16 89.9 4 22.2 9 50.0 4 18 100.0 14 77.8 17 94.4 7 all spoiled 18 100.0 18 100.0 11 - - all spoiled 16 88.9 14 _ — - - 16 88.9 18 - — — - 17 94.4 68 inoculated with Botrytis cinerea spores at different times before irradiation days 0 days 200 krad .0 krad 200 krad No. % spoiled spoiled berries berries No. % spoiled spoiled berries berries NO. 36 spoiled spoiled berries berries 1 5.6 1 5.6 4 22.2 12 66.7 16 88.9 16 88.9 17 94.4 O O 6 33.3 15 83.5 18 100.0 all spoiled 12 16 17 22.2 44.4 66.7 88.9 94.4 10. REFERENCES Alexopoulos, C. J., Introductory Mycology. John Wiley & Sons, Inc., New York, 1962. Alper, T. and Gillies, N.-E., Restoration of Escherichia 'coli Strain B After Irradiation: Its Dependence on Suboptimal Growth Conditions. J. Gen. Microbiol., iA, 461, 1958. Barnett, H. L., Illustrated Genera of Imperfect Fungi. Burgess Publication Co., 1956. Beraha, L., Ramsey, G. B., Smith, M. A., and Wright, W. R., Effects of Gamma Radiation on Brown Rot and Rhizopus.Rot of Peaches and the Causal Organisms. Phytopathology, AA, 354, 1959. Beraha, L., Smith, M; A. and Wright, W. R., Gamma Radiation Dose Response of Some Decay Pathogens. Phytopathology, Ag, 474,'1960. Beraha, L., Ramsey, G. B., Smith, M. A., wright, W. R., and Heiligman, F., Gamma Radiation in the Control of Decay in Strawberries, Grapes and Apples. Food Technol., ii, 94, 1961.’ Bhattacharjee, S. B., Action of X-Irradiation on E- Coli, Rad. Research, i5, 50, 1961. Brasch, A.’and Huber, W., Ultrashort Application Time .of Penetrating Electrons: A Tool for Sterilization and Preservation of Food in the Raw State. Science, 19.5.. 112, . 1947 . ~ Bridges, A. E.,'Olivo, J. P. and Chandler, V. L., Relative Resistance of Microorganisms to Cathode Rays. II. Yeasts and Molds. Applied Micrdbiol., A, 147,.1956. Dunn, C. G., Microbiological Aspects of Ionizing Radi- ations as a Means of Sterilization. Report prepared for the Industrial Liaison Canerence on Food Sterilization; 1952. 69 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 7O Dunn, C. G., Effects of Some Salts on Germicidal Action of High Voltage Cathede Rays Towards Micrococcus Pyogenes aureus. J. Bact., AA, 421, 1953. Edwards, R. B., Peterson, L. J. and Cummings, D. G., The Effect of Cathode Rays on Bacteria. Food Technol. A, 284, 1954. Goldblith, S. A., Proctor, B. E., Davison, S., Kan, B., Bates, C. J., Oberle, E. M., Karel, M. and Lang, A., Relative Bactericidal Efficiencies of Three Types of High Energy Ionizing Radiations. Food Res., AA, 659, 1953. Goldblith, S. A., Proctor, B.-E., Davison, S., Oberle, E. M., Bates, C. J., Kan, B., Hammerle, O. A., and Kusmeerek, B., How Processing Conditions Affect Microorganism Radioresistance. Nucleonics, iA, (1), 42, 1955. Hannan, R. S., Science and Technology of Food Preser- vation by Ionizing Radiation. Chemical Publishing Co., Inc., N.Y., 1956. Hollaender, A., and Stapleton, G. B.,-Fundamental Aspects of Radiation Protection from a Microbio- logical Point of View. Physiol. Revs., AA, 77, 1953. Howard, P., and Flanders, A., Physical and Chemical Mechanisms in the Injury of Cells by Ionizing Radiations. Adv. Biol. Med. Phys., A, 558, 1958. Laser, H., Production by X—rays of Petite Colonies in Yeast and Their Radiosensitivity. Nature, 203, 4942, 314, 1964. Latarjet, R., and Loiseleur, J., Compt. Rend. Soc. Biol., 136, 60, 1942. Quoted in reference N3, page 253. Lea, D.-E., Actions of Radiations on Living Cells. Cambridge University Press, N.Y., 1955. ’v 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 71 Maxie, E. C., Eaks, I. L. and Sommer, N. F., Some Physi- ological Effects of Gamma Irradiation on Lemon Fruit. Rad. Botany, 3, 405, 1964. Maxie, E. C., Nelson, K. E. and Johnson, F., Effect of Gamma Irradiation on Table Grapes. Proc. Amer. Soc. Hort. Sci., AA, 263, 1964. Maxie, E. C., Sommer, N. F., and Rae, H. L., Effect of Gamma Irradiation on Shasta Strawberries Under Marketing Conditions. Food Irrad., A, (l), 50, 1964. Moose, W. 8., Variation in Irradiation Effects on Micro- organisms in Relation to Physical Changes of Their Environment. J. Bacteriol., AA, 688, 1952. Nelson, K. E. and Eukel, W. E., The Fungistatic Effect of Ionizing Radiation on Botrytis cinerea in Pure Culture and ianable Grapes. Phytopathology, AA, 396, 1958. Nelson, K. E., Maxie, E. C. and Eukel, W., Some Studies in the Use of Ionizing Radiation to Control Botrytis cinerea Rot in Table Grapes and Strawberries. Phytopathology, AA, 475, 1959. Nickson, J. J., Ed., The Basic Aspects of Radiation Effects on Living Systems. Symposium on Radio- biology, Oberlin College, John Wiley & Sons, Inc., N.Y., 1950. Powelson, R. L., Initiation of Strawberries Fruit Rot Caused by Botrytis cinerea. Phytopathology, AA, 491, 1960. Prescott, S. C., The Effect of Radium Rays onthe Colon Bacillus, the Diphtheria Bacillus and Yeast. Science, N.S., AA, 246, 1904. Proctor, B.-E., Golblith,.S. A.,vaerle,«E. M., and Miller, W. C., Radiosensitivity of Bacillus sub- tilis, Under Different Environmental Conditions. Rad. Research, A, 295, 1955. 31. 32. 33. 34. 35. 36. 37. 38. 39. 72 Saravacos, G. D., Hatzipetrou, L. P., and Georgiadou, E., Lethal Doses of Gamma Radiation of Some Fruit Spoilage Microorganisms. Food Irrad., A, A6, 1962. Singleton, W. R., Nuclear Radiation in Food and Agri- culture. D. Van Nostrand Co., Inc., Princeton, N.J., 1958. Sommer, N. F., Creasy, M., Romani, R. J., and Maxie, E. C., Recovery of Gamma Irradiated Rhizopus stolonifer Sporangiospores During Autoinhibition of Germination. J. Cell. and Comp. Physiol., Ai, 93, 1963. Sommer, N. F., Creasy, M., Romani, R. J., and Maxie, .E. C., An Oxygen Dependent Postirradiation Restora- tion of Rhizopus stolonifer Sporangiospores. Rad. Res., AA, 21, 1964. Sommer, N.-F., Maxie, E. C. and Fortlage, R. J., Quantitative Dose-Response of Prunus Fruit Decay Fungi to Gamma Irradiation. Rad. Bot., A, 309, 1964. Sommer, N. F., Maxie, E. C., Fortlage, R. J. and Eckert, J. W., Sensitivity of Citrus Fruit Decay Fungi to Gamma Irradiation. Rad. Bot., A, 317, 1964. Stapleton, G. E. and Hollaender, A., Mechanism of Lethal and Mutagenic Action of Ionizing Radiations on Aspergillus terreus. J. Cell. Comp. Physiol., AA (Suppl. 1), 101, 1952. Stapleton, G. E., Factors Modifying Sensitivity of Bacteria to Ionizing Radiations. Bacteriol. Rev., 19.. 26, 1955. Tarpley, W., Manowitzb, I. J., and Horrigan, R. V., The Effect of High Energy Gamma Radiation from Kilocurie Radioactive Sources on Bacteria. J. Bact., AA, 305:-1953- 40. 41. 42. 43. 44. 73 Wainwright, S. D. and Nevill, A., Some Effects of Post Irradiation Treatment with Metabolic Inhibitors and Nutrients upon X-irradiated Spores of Strepto— myces. J. Bact., AA, 547, 1955. Wilson, B. J. and Wilson, C. H., Toxin from Aspergillus flavus Production on Food Materials of a Substance Causing Tremors in Mice. Science, 144 (3615), 177, 1964 0 Wood, T. H., Influence of Temperature and Phase State on X-ray Sensitivity of Yeasts. Arch. Biochem. Biophys., AA, 157, 1954. Wood, T. H., Inhibition of Cell Division. Rad. Res. (Suppl. 1), 332, 1959. Wood, T. H. and Randolph, 8., Dependence of X-ray Sensitivity of Yeast on Cellular Water Content. Rad. Res.,iA, 518, 1961. 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