To observe the effect of 30°C storage on microorganisms on irradiated rice, unpolished and polished rice harvested in Japan were used as the samples. The storage life of polyethylene-packed rice containing 14-15% moisture was extended by 3-4 times by means of 0.2 Mrad irradiation. The number of molds on the irradiated rice grains was not increased for 3-4 months under this condition. When the moisture in the rice sample was 17%, more than 0.4 Mrad was necessary to extend its storage life.
The species of molds which can propagate on storage-rice grains containing 17% moisture were mainly Aspergillus glaucus group and Penicillium citrinum series, where Asp. restrictus, Asp. vercicolor, Asp. candidus, etc. were minor ones. When moisture content was 16%, Asp. glaucus group and Asp. restrictus were observed. Only the halophilic species of Asp. restrictus was the species of molds which propagated on the rice sample containing 14-15% moisture.
When a sheet of porous material, e.g. craft paper bag, was used to pack rice, the storage life of rice containing the moisture of less than 15% was extended by 3-4 times by means 0.2 Mrad irradiation, the storage conditions being 30°C 75% relative humidity. In this case the number of molds on rice was not increased for 3-4 months under this condition.

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Herring homogenates were inoculated with 10⁵ spores of Clostridium botulinum type E per gram, irradiated at 0, 0.1 and 0.3 Mrads and then stored at 20°C or 10°C. Total volatile base, total bacterial counts, and oxydation-reduction potential were periodically examined and the relation between these indices in irradiated samples was compared with that in unirradiated ones.
Toxin production in unirradiated samples occurred so rapidly that there seems to be no significant difference between the time for development of toxin and the spoilage time, whilist toxin production in irradiated samples began to occur before the appearance of signs of spoilage. In addition, more toxin was produced in irradiated samples than in unirradiated ones.
The fact that radiation pasteurization apparently enhances the growth and toxin production of Clostridium botulinum type E in herring homogenates was further discussed from the view of oxydation-reduction potential.

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An intermediate radical, CH₂OH, was produced in aqueous methanol solution containing nitrous oxide by γ-irradiation.Yields of ethylene glycol and formaldehyde, the major and the minor product from CH₂OH, respectively, changed on the addition of some solutes. Cysteine lowered the both product yields to zero even at a low concentration of 5×10^-5M. Oxygen of low concentrations (2.5 7.5×10^-5M) changed effectively the major product from ethylene glycol to formaldehyde as shown in Fig. 4. k(CySH+CH₂OH)/k(O₂+CH₂OH) was calculated as 0.5 from the straight line in Fig.5.
Ascorbic acid (5×10^-5M) lowered ethylene glycol yield to 48%, cystine (10^-3M) to 15%, methionine (10^-3M) to 31%, histidine (10^-3M) to 42%, tryptophan (10^-3M) 46%, tyrosine (10^-3M) to 77%, phenylalanine (10^-3M) to 73%, hypoxanthine (10^-3M) to 37%, adenine (10^-3M) to 52%, uracil (10^-3M) to 20%, thymine (10^-3M) to 10%, cytosine (10^-3M) to 49%, rutin (10^-3M) to 23%, pyrogallol (10^-3M) to 41%, and gallic acid (10^-3M)to 78% of the control as shown in Fig. 6 and listed in Table I.
These results suggest that the reactions of the secondary radicals such as CH₂OH perform an important role in material change of foods irradiated with γr rays.
(Abstracted from the original paper which appears in Agr. Biol. Chem. 33, 250 (No.2), 1969).

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In this study, the authors investigated the radiolysis of sulfur-containing amino acids, cysteine and methionine, in aqueous glucose solution to constract models like foods and to elucidate the radiation chemistry of foods.
Cysteine depressed the carbonyl yield produced from glucose to zero at a concentration of one-tenth of glucose.Methionine of one-tenth concentration of glucose lowered the carbonyl yield to about 16% of those from the solution dissolved glucose only (Table I).If methionine acts only as ·OH scavenger, that of one-tenth concentration of glucoseis calculated to suppress carbonyl yield at about half level.This difference may be explained to some extent by the reaction of methionine with radicals secondarily produced from glucose.
When 10^-3M cysteine solution was irradiated with the addition of glucose at the concentration of ten-fold of cysteine, the G-value of products produced from cysteine were similar to those from 10^-3M cysteine solution. However, in the case that glucose was added at the concentration of a hundred-fold, the high value of G(-cysteine) and disagreement of material balance of sulfur was observed(Table II and III).
These facts suggest that secondary radicals produced from cysteine may react with glucose and/or secondary radicals produced from glucose, resulting that cysteine converted to unknown products which are not listed in the table.
From the results using e^-_aq scavenger, it was concluded that oxidation to methionine sulfoxide and cleavage to α-aminobutyric acid was caused mainly by ·OH and e^-_aq attack, respectively (Table IV and V).

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Glucose aqueous solution 50mM was irradiated at room temperature with γ-rays from Co-60 source under nitrogen atmospheres (total dose 2 Mrad). Detections of degradation products were carried out by thin-layer chromatography (TLC) and gas chromatography (GLC) and many spots of degradation products were found on chromatograms (Fig. 1 and 2).Some of these products were positive to 2, 4-dinitrophenylhydrazine and aniline hydrogen phthalate reagent, and many spots were due to unidentified compounds. One (C-1) of these products was obtained as a colorless plates by silica gel column chromatography using ethylacetate-methanol (4:1v/v) and C-1 afforded m.p. 147-8°C, α²⁰_D+9.2, and molecular formula C₇H₁₄O₆.From IR, NMR (Fig.5) and mass spectra of C-1 acetate, C-1 was identified as methyl 2-deoxygluconate. However, it is difficult to deduce that the methyl ester was directly formed by γ-irradiation. It seems likely that glucose degradated to 2-deoxygluconic acid or its lactone by γ-irradiation and this was taken place methanolysis in its separation procedure to give methyl ester. From GLC (Fig. 2 peak 3), it was suggested that 2-deoxygluconic acid is one of main γ-radiolysis products of glucose.

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Effect of gamma-irradiated sugar solutions on the growth of E. coli and Ps. fluorescence was investigated by comparison of time delay in the log-phase growth with the addition of irradiated sugar solution on the basal synthetic medium (Table 1).
The results obtained with glucose and fructose are shown in Tables 3 and 4. In either sugars, a marked inhibitory effect on the growth of bacteria was observed even with 0.1 M rad when the irradiation was done under aeration, and the effect was considerably weakened by the heating at 120°C after irradiation. While with the irradiation under air free condition, the effect was found only in fructose with the doses above 0.1 M rad, but it was not so clear in glucose even with 1.0 M rad. And interestingly, in this case the heat treatment diminished the effect of fructose and, on the contrary, enhanced that of glucose.
Among the various sugars tested here, (shown in Table4 and 5), xylose, ribose, arabinose, and gluconic acid exhibited a stronger inhibitory effect than that of glucose, on the other hand, galactose, mannose, and glucuronic acid showed only a weak or no effect. The tendency is almost the same with different conditions of irradiation (in air or in N₂) and microorganism.
From the results obtained in this work, it seems to say that the toxicity of irradiated sugar solutions may not bearised from a simple factor but the inhibitory effect maybe caused by several factors of different type in toxicity and contribution of each factor will be dependent on the experimental conditions such as testing sugar, irradiation condition, and after treatment.
The chemical investigations about isolation and identification of toxic factors in each sugar are in progress and will be presented in elsewhere.

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The aim of this study is to get preliminary observation about the influences of γ-irradiation on egg white protein.
The development of odour from egg white was recognized in irradiation of 0.05 Mrad γ-ray at 0°C and 25°C (Table 1), while thining of thick white was recognized in 0.2 Mrad at -78°C, in 0.1 Mpad at 0°C and 0.05 Mpad at 25°C. With reference to electrophoretic behaviour of irradiated egg white, peak area of ovomucoid-globlin fraction was increased gradually corresponding to the increase of irradiated doses and was separated into two peaks at 1.0 Mrad in 45°C (Fig.1). With the increase of irradiated doses, released amounts of non-protein nitrogen and hexose from egg white, preparated ovalbumin and ovomucoid were increased as shown in Table 2 and Fig. 2. Released amounts of hexose from ovalbumin got rise in higher temperature probably because of its heatunstability. Starch gel electrophoretic patterns of migrated protein bands became broader corresponding to the increase of doses (Fig. 3). When egg white was irradiated, changes of anti-tryptic action and lytic action were not observed, but in preparated ovomucoid and lysozyme their biological activities were decreased with the increase of irradiated doses (Fig. 4 and 5).

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Headspace gas composition would contribute information that might help clarify the deterioration of canned foods and the aspect of can corrosion. The change of headspace gas induced by γ-irradiation in cans containing 5'-IMP solutions was investigated by gas chromatography. Solutions containing 5'-IMP were sealed in plain cans (#2, for baby food) and irradiated with a 5, 000 curie cobalt-60 source at the dose rate of 2, 0 × 10⁵ r/hr. The gas samples were injected into an equipment, Shimadzu GC-1B, packed with silica gel and molecular sieve 5A. The carrier gas was argon.
The effect of irradiation at elevated doses is shown in Table 1. The higher the dose was, the lower the level of remaining 5'-IMP was. In the case of 5'-IMP solution (Table 2), O₂ content in headspace gas decreased and H₂ content increased with increasing γ-ray dose. In canned distilled water (Table 3), a little increase in H₂ gas was observed with increasing γ-ray dose. O₂ gas vanished during the storage for 90 days at 25°C During the storage of irradiated 5'-IMP solutions, the formation of NH₃-N and the dissolution of Sn were negligible (Table 4). Remarkable amount of H₂ gas was formed by irradiation at 4.5 Mrad (Table 5), but it vanished gradually during the storage for 90 days. In the irradiated solutions of 5'-IMP packed with glucose, sucrose, casamino acid or NaCl, the formation of NH₃-N, the dissolution of Sn and Fe were negligible (Table 6). Remarkable amount of H₂ gas was formed in the headspace of canned and irradiated 5'-IMP solutions packed with glucose, sucrose or NaCl (Table 7).

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In thepreviousreport effects ofgamma-irradiation on the gelation of fish actomyosin were investigated. It has been demonstrated that a remarkable change from sol to gel of actomyosin solution differs among the various conditions such as the concentration of actomyosin or KCl as a solvent, the irradiation dosages, the presence of oxygen or nitrogen, and the addition of oxidants or an antioxidant. Moreover, from the results of polarographic observation of protein waves and the protein solubility in centrifugal preparation of irradiated actomyosin, it has been suggested that the gel formation is probably due to the interaction of sulfhydryl groups and a higher macromolecule of actomyosin formed during irradiation.
In this paper to obtain some aspects on the mechanism of the gel formation of irradiated actomyosin, the sulfhydryl-disulfide exchange reactions between thiamines and actomyosin were ascertained by paper chromatography, paper electrophoresis and fluorometry as follows.
1) The bound thiamine with protein and free thiaminewere produced by the reaction of thiamine disulfide (TDS) with actomyosin at pH 7.3, 37°C for 60 minutes, but not formed by the reaction with the actomyosin treated with SH-masked reagents such as p-chloromercuribenzoate or iodo-acetic acid (Figs. 1 and 2). Therefore, it is supposed that the above reaction is caused by the SR-SS exchange between the sulfhydryl group of actomyosin and the disulfide group of TDS.
2) Thiol-type thiamine was unreactive at pH 7.3 because of the probable uncleavage of thiol on the thiazole ring whereas TDS reacted easily with actomyosin regardless of irradiation dosages (Figs. 3 and 4).
3) In the pH ranges 8-9 the reactivity of irradiated actomyosin and thiol-type thiamine increased extremely with alkalifing pH and reached constantly above pH 10 (Fig. 5), while at pH 10 the amounts of bound thiamine increased almost linearly with increasing irradiation dosages up to about 100, KR (Fig. 6).
4) The reactivity of thiol-type thiamine with the irradiated actomyosin, in the presence of oxygen, in which case the actomyosin was little gelled, was higher than that in the presence of nitrogen (Fig. 7).
From these results, it is suggested that the interchange from thiol to disulfide bond in the actomyosin molecule will occurs, while it is doubtful if such interchange is of great importance for the maintenance of specific gel formation by irradiation.

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The present study was undertaken to investigate the effect of gamma irradiation on the plant pigments.Perilla Ocimoides was mainly used for this experiment, which contained anthocyans, chlorophylls and carotenoids.
Fresh or dehydrated leaves and extracted solution with solvents were irradiated with the dose of O-150 Krad from Cs-137 gamma source, followed by the determination of three pigments by the colorimetry. The results obtained were as follow:
1) Anthocyan of fresh leaves decreased with the dose, and that of the dehydrated one did not significantly decreased. The sigmoidal curve shown Fig. 2 might suggest that perilla could regenerate anthocyan with a small doseirradiation.Anthocyan in H₂O-extracted solution was much more stable than that in HCl:MeOH-extracted solution.(Fig. 3) H₂O-extracted solution might contain some unknown protective material to gamma irradiation of anthocyan.
2) Chlorophylls in fresh leaves such as perilla and spinach were rather stable to gamma irradiation. Stability of chlorophyll a was nearly the same magnitude as chlorophyll b, and both did not depend upon water content of leaves. (Fig. 4) This might be due to the fact that chlorophylls found in chloroplast together with fat soluble materials such as lipids and carotenoid. (Fig. 5)
3) Gamma irradiation at the dose of 50-100 Krad led to an increase of total carotenoid content in fresh leaves, and this dose decreased as the perilla grew older. (Fig. 6)Thin layer chromatography indicated that carotenes decreased, but xanthophylls increased with the dose of irradiation.(Fig. 8) It would appear that a small dose of gamma radiation might bring about the accumulation or formation either by disturbing biogenesis of carotenoids by radiolysis of carotenoids and their related compounds.

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Potato tubers, sweet potato roots and carrot roots were irradiated with cobalt-60 gamma rays, and then cut into thin disks, which were incubated aerobically at 30°C under high humidity. The effects of gamma radiation on metabolic changes in the tissues in response to cutting were investigated. The polyphenol content in potato tissues increased as compared with the nonirradiated sample. However, the increase in polyphenol content in response to cutting was lower in the irradiated sample. In the case of sweet potato and carrot tissues, the in crease in polyphenol content in response to cutting was significantly stimulated in the irradiated sample. In sweet potato tissues irradiated with 90 krads, the increase in polyphenol content was correlated with the increase in phenylalanine ammonia-lyase. The effect of radiation on peroxictase development in sweet potato tissues was similar to that of polyphenol production. No significant effect on o-diphenol oxidase development was observed in sweet potato tissues. Even though gamma radiation stimulated the production of polyphenols and peroxidase development in sweet potato tissues in response to cutting, formation of lignin-like substance was inhibited by radiation.

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The experiment was carried out to determine the radiation- induced flavor of some horticultural products (apple pulp, apple jam and dried bananas), which were bleached at the doses of 0.5-2.0 Mrad by gamma rays.
Gas chromatographic analyses indicated the increasing of volatile carbonyl compounds: acetaldehyde, n-propylaldehyde and n-butylaldehyde in irradiated apple pulp;n-propylaldehyde and acetone in irradiated apple jam;acetaldehyde and acetone in irradiated dried bananas (Fig. 1-3 and Table 1). Ultraviolet absorption spectra of the distillate obtained by water vapor distillation of apple pulp, apple jam and dried bananas also showed the increase of carbonyl compounds in irradiated samples (Fig. 4-6).In the distillate of irradiated apple pulp, the large amount of carbonyl compound as acetaldehyde was detected (Table 2). Organoleptic evaluation showed the intensive off-flavor in apple pulp irradiated at the dose of 2.0 Mrad, but not in other irradiated products (Table 3-5).

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The study was dealt with the changes of chemical constituents and qualities in grape juice, apple juice and Satsuma orange juice which were irradiated at the sterilizing dose levels of gamma radiation.
Anthocyanins in grape juice were sensitive to gamma rays (Fig. 1 and Table 1) and β-carotene in irradiated Satsuma orange juice was relatively stable (Fig. 2). Irradiated apple juice became brown slightly (Fig. 3 and Table 2). The qualities of these juice products deteriorated with high doses (Table 3 and 4). The deterioration of Satsuma orange juice was slightly prevented by the addiation of propyl gallate (Table 5). Addition below 50mg% of ascorbic acid was effective to the protection of the deterioration of Satsuma orange juice and apple juice, but not above 100mg% of ascorbic acid (Table 4). The retention percent of ascorbic acid content in irradiated juice was high concentration of ascorbic acid than in low concentration of ascorbic acid (Fig. 4). There were no differences between the irradiated and unirradiated samples in titratable acidity and reducing sugar content up to the doses of 1.0-2.0 Mrad (Table 6).

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Citrus Unshiu (Japanese mandarin orange “ Satsuma”) is particularly susceptible to gamma ray irradiation and the adverse effects on flesh flavor and peel appearance were found when fruit was subjected to 50 Krad or more.The work has conducted on shallow irradiation (surface radurization) of Citrus Unshiu by cathode ray to prevent the off-flavor.
Pasteurizing effect on Pen.digitatum inoculated to lower shoulder of fruit was decreased with the time from inoculation to irradiation even the inoculated fruits were stored at 5°C before irradiation. (Table 1, 2) With higher dose softening of peel and the destruction of oil-gland developed during the post-irradiation storage and finally the browning of peel appeared.These phenomena depended on the storage time from the harvest to the radiation treatment;deterioration was more severe with the shorter storage after harvest. (Table 3, 4) The browning was accelerated at room temperature but repressed at 5°C storage.The browning was not affected by curing treatment (RH: 75-80%, 5-7°C for 3 weeks) immediately after harvest. (Table 5)
There was same pasteurizing effect by the cathode ray of 1.0 and 0.5 Mev. (Table 3) Pannel test detected the deteriorated flavor change with gamma ray irradiation over 50 Krad and the acceptability was lost over 100 Krad, but the shallow irradiation with 1.0 Mev of cathode ray did not change the flavor acceptability up to 250 Krad. (Table 4)
Analytical data of fruit flesh and juice showed no significant differences between irradiated and nonirradiated samples after the long term storage. (Table 6) Shallow irradiation of 100-300 Krad appeared to cause some advantages in score of acceptability after the long term storage. (Table 7)

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Amylase, protease and ribonuclease activity in endosperm of germinating rice seed after γ-irradiation was examined. γ-irradiation promoted the amylase and protease activity at early germination stage with exogenous gibberelli acid-3 (GA₃). In embryotess half seed incubated with GA₃, the amylase activity was not promoted by γ-irradiation.

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Irradiation of brown rice was carried out with cobalt-60 γ-rays at the doses of 1 to 20×10⁴ rad, and changes in quality after irradiation were investigated in the following points:
1) The activities of α- and β-amylases, catalase in brown rice were hardly decreased by the irradiation performed here, but they were rather slightly increased at the doses of 5×10⁴ rad or lower. (Figs. 1, 2 and 3)
2) No change in the amount of free reducing sugar in brown rice due to irradiation was observed with the doses up to 20×10⁴ rad. (Fig. 4)
3) In the cooking test of rice polished after irradiation, intensity of iodine coloration with water soluble fraction and amount of wayer-soluble substances were apparently increased with the doses above 5×10⁴ and 10×10⁴rads, respectively. (Fig. 5)
4) In organoleptical evaluation of cooked rice, the difference between unirradiated rice and irradiated one could be regarded as significant at the dose of 20×10⁴rad. (Table 1)
5) Germination of brown rice was not so affected by the irradiation, but the growth of sprout and root after germination was markedly inhibited at the higher doses employed here. (Fig. 6)
6) There were no significant differences in the above mentioned effects of irradiation on rice by changing dose rate and by fractional irradiation at a given total dose, but the low dose rate irradiation seems to be somewhat effective for catalase activity and organoleptical test. (Table 2-5)

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This study was made to determine the effect of processing temperature, and storage temperature on the quality and shelf life of cooked horse-mackerel irradiated with cobalt-60 gamma rays.
Fresh fillets of horse-mackerel with skins were packed in pouches of cellophane laminated with polyethylene (0.0625 mm) under air or under a vacuum with automatic heat sealer. The size of pouches were 6×12 cm. Packed fillets were divided into five groups: 1) was irradiated at 0.20 Mrads, 2) was cooked in a water 80 to 85°C for 15 minutes, 3) was cooked in a water at 80 to 85°C for 15 minutes, 4) was irradiated at 0.20 Mrads after cooked for 5 minutes, and 5) was irradiated at 0.20 Mrads after cooked for 15 minutes. Those samples were stood at 12 to 15°C.
From the results of organoleptic tests (Table 1), chemical and bacteriological examinations (Tables 2 and 3), indicated that the storage life of the first group which was irradiated on the raw state was 2 weeks, and the second group which was cooked only showed 3 weeks of storage life, and 5 weeks in the third group. On the effect of irradiation after cooking, the fourth group showed 8 weeks of storage life, and 8 to 11 weeks in the fifth group.
In the next experiments, the shelf life of fillets of horse-mackerel during storage at 30°C were estimated. As the package material, pouches of aluminum laminated with polyethylene were used. The overall thickness of aluminum-polyethylene foil was 0.0715 mm, and size of pouch was 6×12 cm. The fillets were packed in pouches under air or under a vacuum. And packed fillets were divided into three groups: 1) was cooked in a water 85 to 90°C for 30 minutes, 2) was irradiated at 0.20 Mrads after cooked for 30 minutes, and 3) was cooked for 30 minutes after irradiated at 0.20 Mrads. Those samples were stood at 30°C.
For packaging with aluminum-polyethylene foil, the first group showed about one week of storage life, and the second group and the third group showed about two months of storage life either in air-packed or in vacuum-packed. (Table 4, Fig. 1 and Table 5).

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Commercially produced castilla which is a kind of cake baked of the mixture of refined flour, sugar, maltose and chicken eggs becomes to have plenty of growth of moulds at the end of about twenty days preservation at room temperature after production. To attempt the prolongation of its storage life at room temperature, gamma irradiation was applied to them. Optimum dose without unacceptable changes in its flavor and taste was found to be in the range of 300-500 Krad for the commercially produced cakes. By administration of this dosage, the cakes could be preserved for forty days at room temperature without any visual growth of moulds. Decoloration occurred with the increase of dosage, but the color of the cakes of irradiated at 300 Krad dose was acceptable. No significant differences between the growth rate and feed consumption of mice fed with cakes irradiated at 500 Krad at a level of 90% in the basic diet and mice fed with unirradiated diet were observed in three months feeding studies. Separated moulds which grew on the surfaces of commercially produced cakes and identified to be in the groups of Penicillium, Aspergillus and Cladosporium were inoculated artificially in varied concentration on the hollowed surface of cakes, packaged, irradiated at varied dose and then preserved at room temperature for varied period. By this in vivo test, it was found that preservation periods of irradiated cakes could be predicted by the relation of the concentration of moulds before and after irradiation and the irradiation doses. On the other hand, to avoid practical unexpected contamination, sanitary conditions of the manufacturing factories were inspected. As a result, fingers of two female operators packaging the cakes were found to be contaminated of plenty of bacteria and moulds. Recommendation to enforce frequent hand-washing or wearing clean hand-gloves was given to the maker, because the dose required to radurisation of the products depends on the pre-irradiation contamination of them.

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By the use of color radiofieldgraphy which had been developed by authors, figures of dose-effect distribution of gamma irradiated samples which had oxidation-reduction dyes in an agar gel were observed. The results are as follows:
1) Outer or internal surface faced to air of the irradiated samples had less irradiation effects compared to other parts; that effect was called “Barrier effect” or “ Rand effect”.
2) Isodose figures of the sample which was rotated around its central axis while being irradiated were found to be akin to the original figure of sample, and the closer to its center, the more its irradiation effect.
3) Sample which was rotated around its central axis, revolved around gamma source, and brought up and down in parallel with the axis of the source pencils while being irradiated was observed to have strangely unsatisfactory figure of dose-effect distribution although such irradiation method was expected to give ideal figure of uniform doseeffect distribution for the practical industrial irradiation process.

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Decoloration by gamma rays of aqueous solutions of potassium copper chlorophyllin, sodium copper chlorophyllin, potassium iron chlorophyllin, sodium iron chlorophyllin, riboflavin and cochineal and of olive oil solution of β-carotene was observed. β-carotene in olive was found to be the most radioresistant and sodium copper chlorophyllin in water was the most radiosensitive among them. Generally the natural food colors were found to be more radioresistant than the coal-tar food colors. Changes in color-hue of β-carotene and cochineal were brought about with the increase of irradiation dose. Acute toxicity of the irradiated natural food colors was tested; the irradiated aqueous solution of potassium copper chlorcphyllin were found to be toxic to Japanese goldfishes as test animals, whereas unirradiated solution as control did not give toxicity.

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Ronsivalli et al. (Food Tech., 20, 1074, 1966) have devised a method to determine the bacterial permeability of plastic films, in which, pouches of various plastic films are filled with suitable bacterial growth medium, sealed hermetically, sterilized by means of γ ray, and immersed in a highly contaminated bath of spoiling fishes slurry at low temperature for a long period. The pouches are then removed from the contaminated bath and incubated. After incubation is complate, contents of the pouches are inspected for any visual evidences of microbial growth-e.g., turbidity, gas formation, etc. If a medium in a pouch remain sterile, the pouch materials is judged to be impermeable to the bacteria contained in the bath.
The authors of the present paper adopted Ronsivalli's method to the plastic films shown in Table I. Seven or eight pouches (7.8×7.8 cm) of each film filled with the growth medium of Difco-Bact-Trypton and a few dry pease which had been previously sterilized by heating at 120°C. for 15 min., and heat-sealed by an impulse sealer, were subjected to the irradiation by γ ray of ⁶⁰Co at 4.5 Mrad for the purpose of cold sterilization, followed by immersion in a contaminated medium with Cl. Pasteurianum of 10⁷/cc concentration. After removal from the contaminated bath, the pouches are incubated at 35°C. for 7 days, and then inspected as to the gas formation and turbidity. The anaerobic Cl. Pasteurianum has been proved by the authors to grow rapidly in the aerobic Difco-Bact-Trypton medium.
In the preliminary test, one of eight pouches showed the bacterial contamination, which, probably due to the failure of heat-sealing. Therefore the operaters became more careful for the heat-sealing of pouches.
In the first experiment carried out, with greater care of heat sealing. All of the test pouches were observed to remain sterile.
The same result was obtained from the second experiment.
Thus, it is safe to say that the plastic films are impermeable to the bacteria.
The films as pouch material were tested with respect to UV transmittance, (a) before irradiation by γ ray (eg. the original film), (b) after irradiation, keeping in air, (c) after irradiation, keeping in contaminated growth medium. And, as the result, any noticeable differences between (b) and (c) were not detected (Fig. 2-5).

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