APPLIED MICROBIOLOGY, Feb. 1970, p. 338-344 Copyright © 1970 American Society for Microbiology

Vol. 19, No. 2 Printed in U.S.A.

Radiation Treatment of Foods II. Public Health Significance of Irradiation-Recycled Salmonella' N. A. EPPS2 AND EDMUND S. IDZIAK Department of Microbiology, Macdonald College, McGill University, Montreal, Quebec, Canada

Received for publication 31 October 1969

Salmonellae resistant to gamma irradiation were developed by repeated irradiation and subculturing in a nutrient broth-yeast extract medium. Few differences were noted in the biochemical characteristics of parent and resistant cultures; however, microculture studies revealed variations in morphology and in cell division patterns. A considerable decrease in pathogenicity for day-old chicks was apparent with resistant cultures, but their phenol-water extracts were as toxic as parent material for 10-day chick embryos. Five serial chick passages did not reverse the reduced pathogenicity or aberrant morphology of a resistant Salmonella typhimuriun culture. Results of phage typing of both parent and serially irradiated S. typhimurium were inconclusive, whereas the 0-1 genus-specific phage lysed all parent serotypes tested but only one of the serially irradiated cultures. Agglutination of parent S. typhimurium cells with their homologous rabbit antiserum was unaffected by prior absorption with resistant strains. The results indicate that radiation recycling altered Salmonella into strains of lesser public health significance.

That radiation-resistant strains of different pathogenic organisms might be a problem in food preservation by ionizing irradiation has been suggested by several authors (4, 12, 14). Erdman et al. (4) isolated, after repeated sublethal irradiation-growth cyclings, radiation-resistant strains of several different pathogenic organisms. Some alterations in biochemical characteristics, in phage patterns of Staphylococcus aureus, as well as an indication of reduced virulence in Salmonella were also very briefly reported (I. E. Erdman et al., Can. J. Public Health, vol. 53, p. 37, 1962). Mainly because of the inability to identify the resistant strains by standard methods (not described in the report), they concluded that these organisms may constitute a greater public health hazard. Licciardello et al. (10) and Idziak and Incze (7) indicated that the possibility of such repeated cyclings occurring in industry would be extremely minimal. Furthermore, Idziak and Incze (7) had no difficulties in identifying the repeatedly irradiated and subcultured Salmonella isolates which had doubled in their radiation resistance. Data is presented in this paper which resolves the difficulties of identification of radiationgrowth cycled Salmonella and shows that the radiation-cycled organisms are considerably less

virulent, thus refuting any increased hazard to public health. MATERIALS AND METHODS

Salmonella anatum ESIO, S. blockley MCC601, S. enteritidis ES413, S. give ES586, S. infantis MCC605, S. manhattan ES875, S. montevideo MCC599, S. pullorum ES876, S. senftenberg ES877, S. typhimurium ES878, ES879, and ES881, and S. worthington MCC603 were obtained from the Food and Drug Directorate Research Laboratories, Ottawa. The Paracolobactrum arizona EP1551 was isolated from turkey poults. The cultures were maintained in the lyophilized state and on nutrient agar-0.3% yeast extract (NAYE) slants under sterile paraffin oil at room temperature. All media were Difco products; chemicals were reagent grade. Development of resistance. Stock slant cultures were grown on NAYE for 24 hr at 37 C. Duplicate transfers were then made to 50 ml of nutrient broth-0.3% yeast extract (NBYE) in 6-oz screw-cap bottles. After 24 hr at 37 C, 10-ml samples in screw-cap vials (12 by 70 mm) were irradiated in a Gammacell 220 [dose rate approximately 0.7 mrad/hr; (7)]. The cultures were irradiated 12 times at either 0.2 or 0.18 mrad with 24-hr incubation periods at 37 C in 50 ml of NBYE between each irradiation. The resulting population was then streaked on NAYE for isolation of individual colonies (SI strains). Further recycling at 0.25 and 0.30 mrad was done as mentioned above and yielded the SII and SIII strains, respectively. All isolated cultures were I Issued as Macdonald College Journal Series 607. stored on NAYE under paraffin oil. 2 Present address: Department of Microbiology, University of For D1o determination, NAYE slant cultures were Guelph, Guelph, Ontario, Canada. 338

VOL. 19, 1970

RADIATION TREATMENT OF FOODS. II

incubated overnight in 10 ml of NBYE. A 4-ml amount (8 ml in the case of S. pullorum) was then transferred to 250 ml of NBYE in 500-ml flasks and incubated for 5 hr at 37 C with occasional shaking. Five 10-ml samples from each culture were dispensed into the radiation vials and placed in the Gammacell 220. Rack positions were identified so that the location of each sample would be the same in replicate ex-

339

ated Salmonella cultures were inoculated into day-old chicks (28 to 34 g). The chicks were arranged in groups of 10 with a combined weight of approximately 300 g. They were brooded in Hotpack environmental chambers at 30 i 1 C; feed and water were supplied ad libitum. The feed was a commeicial chick starter without antibiotics but containing amprolium. Deaths were recorded twice daily to 10 days at which time the posures. survivors were sacrificed. The weights of survivors When the prescribed dose was reached, the contents were also recorded at 7 or 10 days postinoculation. of the vials were immediately diluted to 10-5 in 0.85% S. typhimuriwn ES878 parent and SII cells were also saline. When all samples had been irradiated, any heat-killed prior to inoculation of large numbers into necessary higher dilutions for five-tube most-proba- the chicks. The effect of chick passage on infectivity ble-number determinations were made. A 10-ml and virulence of S. typhimurium was also assessed. amount of each dilution was then added to 10 ml of Cultures isolated after each of five successive chick double-strength NBYE (final NaCl concentration, passages were injected into day-old chicks and mor0.5%). Results were recorded after 24 and 48 hr at tality was recorded. 37 C. Endotoxin. Phenol-water extracts from 48-hr culNutritional, biochemical, and morphological char- tures of S. typhimuriwn ES878 parent and SIII cells acterstics. The variation in response at 37 C to a were prepared by a modification of the method devariety of chemical compounds by 50 representative scribed by Westphal and Jann (13). Parent and SIII colonies from each of the parent and serially irradi- cells (4.5 g, dry weight) in 200 ml of saline were exated cultures was determined. Response of several of tracted with an equal volume of 90% phenol for 30 these isolates to different antibiotics was also assessed. min at 68 C with constant stirring. The mixture was The morphological stability of the cultures during cooled and centrifuged, and the water layer was reirradiation cycling was monitored microscopically. moved. The phenol layer and insoluble residue were Microculture techniques at 37 C were employed to ob- extracted twice more and the aqueous layers were comserve multiplication (6). The retention of the observed bined. These were dialyzed against distilled water and aberrant morphological forms and their resistance the residuum was lyophilized. These lyophilized exto normal host defense mechanisms were tested by tracts were serially diluted and inoculated into the chick passage. Approximately 106 cells were injected chorioallantoic membrane of 10-day chick embryos. into the yolk sac of day-old chicks, with recovery after After 24 hr at 38 C, the eggs were broken and embryo 1 week from fecal swabs and liver tissue by using viability was determined (11). tetrathionate-broth (37 C for 24 hr) and BrilliantGreen-agar isolation methods (2). The cultures were RESUILTS subjected to five consecutive chick passages, with With irradiation cycling at 0.2 mrads, the duprepresentative cultures being retained after each passage. Morphological and growth characteristics of licate S. pullorum and S. montevideo cultures were each of the isolations were then determined. The pres- eliminated after three exposures; P. arizona cultures were eliminated after four exposures. One ence of any indigenous Salmonella was monitored. Serology. All wild-type cultures were typed sero- replicate each of S. enteritidis and S. blockley logically prior to any experimental manipulation. During serial irradiation, apparent rough and smooth TABLE 1. D1o values for various Salmonella colonies were isolated and tested with acriflavine and 0 antigen group-specific antisera. D,o values (krads) S. typhimuriwn ES878 Parent and SIII cells were Species used in agglutination tests with antisera from rabbits Wild-type strains SII strainsa against Roschka-prepared Parent and SIII cell antigens (3). Rabbit antiserum against S. typhimurium ES878 was also absorbed separately by 24-hr NAYE S. anatum ........... 50-54 72-74 Parent and SII1 cells suspended in 0.3% phenolized S. blockley .......... 45-50 saline. The latter were centrifuged, and 0.5 ml of the S. enteritidis ......... 24-30 65-68 S. give ............... cell mass was added to a 1 :10 dilution of the parent 40-43 85-90 antiserum. The mixtures were incubated at 48 C for 2 S. infantis ........... 26-30 62-65 hr with occasional stirring and were then centrifuged. S. manhattan ........ 18-20 The filtrates were collected and used in agglutination S. montevideo ... 28-32 tests with parent cells. S. pullorum .......... 30-34 54-58 Phage typing of parent and serially irradiated S. S. senftenberg . ..... 55-60 .......23-26 typhimurium cultures was performed at the Labora- S. typhimurium....... 30-34 63-67 tory of Hygiene, Ottawa, with phages made available S. worthington ....... 45-47 by E. S. Anderson. Several cultures were also checked a SIT strains were obtained after 12 irradiationfor lysis by using the 0-1 phage of Felix and Callow growth cycles at 20 krads followed by another 12 (5). Alteration in infectivity. Parent and serially irradi- at 25 krads. ...

340

EPPS AND IDZIAK TABLE 2. Altered biochemical

responses

APPL. MICROBIOL.

of individual members of the parent and serially

irradiated populations of various Salmonella species and Paracolobactrum arizonaa S. anatum

S.

enter-

S.

give

ilidis

Determination

ESIO

ES1l

0/0 0/0 0/0

0/0 1/0 0/0 0/0 0/0 1/0 0/0 0/0 0/0 0/0

...................

4/0 3/0

3/0 3/0

Ss.....................

0/0

0/0

Dextrose............... Dulcitol ............... Lactose ................ Mannitol............... Sucrose ................ Citrate................. H2S.................... Indole, motility, urease. BGA, BGS, MCA...... BS..................... DCA .................. MM

5/0 5/0 0/0 5/0

0/0 1/0 2/0

ES413 5/0 3/0

S. infantis

ES586 MCC 605

S. pul-

ES876

0/0

0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

0/0

0/0

0/0 0/0 0/0 1/0 0/0 0/0 0/0 0/0 0/0 0/0

0/0

7/2

3/1

1/0 0/0

1/0

0/0 0/0

4/0

0/0

0/0 0/0

3/0

6/0

0/0 0/0 0/0 0/0 0/0 1/0

0/0 1/0 0/0 0/0

50/0 27/1 27/0

1/2

P. arizona

S. typhirnuriunm

lorum

ES881

ES878

EP1551

1/0 8/0 29/0 0/0 2/0 15/0 0/0 0/0 0/0 0/0 4/0 3/6 0/0 0/0 0/0 5/0 1/1 10/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

23/0 8/0

0/0 0/0 0/0 0/0 0/0 0/0

ES879

ES880

0/0 2/0

0/0 2/0

1/0 0/0 0/0

0/0 9/0 0/0 11/0

0/0 0/0 0/0 0/0 8/0

0/0 0/0

a Values represent number of clones out of 50 (irradiated/unirradiated) with altered reactions. Serially irradiated populations (SIII) were obtained after recycling 12 times at 0.2 mrads, 12 times at 0.25 mrads, and finally 12 times at 0.3 mrads. Abbreviations: BGA, Brilliant Green agar; BGS, Brilliant Green sulfapyridine agar; MCA, MacConkey agar; BS, bismuth sulfite agar; DCA, deoxycholate citrate agar; MM, minimal medium; SS, Salmonella-Shigella agar.

failed to initiate growth after seven exposures, TABLE 3. Effect of repeated irradiation on the antibiotic resistance of several Salmonella speciesa whereas both replicates of S. anatum, S. give, S. senftenberg, S. typhimurium ES878, and S. WildRadiation-cycled worthington survived 12 exposures. S. pullorum Concn type Antibiotic e es wit with reac(pUg/mi) and P. arizona did survive 12 repeated exposures srecie alterecd responsec tionb at 0.18 mrads. There is at least a threefold difference in the S None 10 . radiation resistance of the wild-type Salmonella Chloramphenicol. Plus streptomysp. (Table 1) (i.e., D1o of 18 krads for S. manS 10 None cin .......... S None 100 hattan to 50 to 54 krads for S. anatum). Upon Colistin ... S None 75 recycling, the radiation resistance of the surviving Neomycin ......... S None 50 Nitrofurazone ...... population always increased, but not uniformly Penicillin Sd 3 IU None G ........ (Table 1). S None Terramycin ........ 5, 10 10 Results of preliminary biochemical tests with Nitrofurazone ..... R S. iyphimurium" S. blockley0 10 Rf Streptomycin. gross cultures of S. anatum, S. blockley, S. enteritidis, S. give, S. senftenberg, S. typhimurium, and S. worthington isolated after 12 growth-irradiation cycles at 0.2 mrads were similar to the parent cultures except for delayed reactions in some instances. A more detailed study of single-cell isolates indicated that after recycling beyond 0.2 mrad only S. enteritidis and two strains of S. typhimurium (ES880 and ES881) contained a large percentage of cells altered in one or another response (Table 2). With the exception of some S. typhimurium isolates against nitrofurazone (10 lg/ml) and streptomycin sul-

fate and S. blockley against streptomycin sulfate, several other antibiotics were unaltered by irradiation cycling at the 0.2-mrad level (Table 3). As a result of the irradiation recycling, Salresponses to

Tylosin ............ Vancomycin ........

10 10

R R

S. typhimuriumg None None

a S. anatum ES10, S. blockley MCC601, S. enteritidis ES413, S. give ES586, S. senftenberg ES877, S. typhimurium ES878, S. worthington MCC603. b S, sensitive; R, resistant. c Salmonella were recycled 12 times at 0.2 mrads. d Variable reaction was obtained with S. typhimurium. e One of six isolates was sensitive. f Sensitive reaction was obtained with S. senftenberg. 9 One of 5 isolates was sensitive. A Three of seven isolates were sensitive.

monella were found to: (i) exhibit abnormal separation of daughter cells (Fig. im, n, o, p); (ii) form elongated cells with accompanying minicells (Fig. la, c, d, e, f, h); (iii) form larger than nor-

VOL. 19, 1970

RADIATION TREATMENT OF FOODS. IX

341

FIG. 1. Cellular morphology of radiation-resistant strains of S. anatum SI (a), S. blockley SI (b), S. enteritidis SI (c), S. pullorum SI (d), S. senftenberg SI (e), S. typhimurium ES878SI (f, g), S. worthington SI (h); modes of replication of S. typhimurium ES878 (i), S. blockley SI (j), S. typhimurium ES878 SI (k, 1); mode of replication of S. typhimurium ES878 SII (m-p); and morphological stability of S. typhimurium ES878 SII (q) after one to five successive chick passages (r to v, respectively). X 4,000.

mal "coccoid" types, often irregularly shaped polyvalent, and group B typing sera to the origi(Fig. lb, g); and (iv) form both filaments and nal SII isolate. With increasing radiation cycling, the cultures "coccoid" forms (Fig. lf, g). These were not terminal forms because they replicated (Fig. li, tended to become rough, as evidenced by colony j, k, 1), and they have retained these altered morphology, agglutination in 0.002% aqueous morphological forms during 3 years of main- acriflavine, and nonuniform turbidity in broth tenance. Repeated chick passage of the altered culture. Nevertheless, all radiation-cycled progeny S. typhimurium ES878 SIT strain did not result in were agglutinated by their wild-type 0 antisera. any alteration or selection back to the original Antisera from adult chickens against S. typhiparent form (Fig. lq, r, s, t, u, v). The SII isolate murium ES878 Sll antigen agglutinated, to varyafter five successive chick passages was identical ing degrees, both parent and homologous cells, in its biochemical response, agglutination in as determined by slide agglutination tests. The

342

EPPS AND IDZIAK

parent and SIII S. typhimurium strains were typed as S. typhimurium (4, 5, 12: i-1, 2). The parent cells prepared by the Roschka method were either more immunogenic or more effectively agglutinated by their homologous sera than similarly prepared SIII cells (Table 4). Parent antisera effectively agglutinated SIII cells. In addition, absorption experiments indicated that SIII cells did not remove, or removed very slightly, the agglutinating activity of parent antiserum against their homologous antigen (Table 5). Phage typing was inconclusive. One parent S. typhimurium was typed as 2b (poor) and another

APPL. MICROBIOL.

as type 1, whereas two others were untypable. Nearly all serially irradiated cultures were reported to be "rough-therefore untypable" except one which was "Lmtypable-slow growing culture." All parent cultures showed plaques or confluent lysis with the 0-1 phage, whereas plaques were observed with only one serially irradiated culture (i.e., S. infantis). TABLE 5. Agglutination of parent Salmonella typhimurium ES878 cells with parent antiserum after absorption with parent and radiationresistant SIll cells Serum dilution&

Treatment of parent antiserum

TABLE 4. Agglutination of parent Salmonella typhimurium ES878 and its radiation resistant-mutant SIIl Antigena Antiserum Parent

SIII

Parent .4 Sill .0

4F

1

Degree of agglutination: 0 to 4 (from none to heavy); F, fine clumps.

1/20 1/40 1/80 1/160 1/320

None .................. Heated at 48 C for 2 hr Heated at 48 C for 2 hr and absorbed with parent cells ............ Heated at 48 C for 2 hr and absorbed with SIII cells ..................

4 4

4 4

4 4

4 3

2 1

0

0

0

0

0

4

4

3

2

0

aDegree of agglutination: 0 to 4 (from none to heavy).

TABLE 6. Mortality in chicks inoculated with parent and irradiation-resistant mutants of various Salmonella Organism

S. anatum S. enteritidis S. infantis

Typea

p

No. of mortalities from 10 injected chicks at dilution Total 10of basal inoculum day mortalities of SO chicks I" I10 10-3 10-4 10-' 10-6 10' 10' 10' 10

SIII

1.2 X 109 1.2 X 108

1

1

0

0

0

p

SI"I

1.2 X 10' 1.2 X 10'

1

0

0

1

0

p

1.5 X 109 1.7 X 109

7

0

0

1

0

0

10 1 1

SI"I S. typhimurium

No. of cells in basal inoculum (0.5 ml)

p SIl SII1b

SI12 SII3

S114

SI"l SIII

1.8 X 10' 1.2 X 109 7.4 X 109 4.5 X 109 6.0 X 108 5.8 X 10' 5.5 X 10' 4.6 X 10l

2

6 6

4 8 7

0

5

6 2

3

2 2 3

0

0

2

1

1

0

0

0

0

2

2

0

1 1

0

0

0

0

1

3

1

0

5

3

8

2

1

6

4

4

4

24 8

10

10

10

5

45 8 14 13 9 7 7 0

0

Control (0.5 ml saline) Control (no injection) a I

9 2

1

p, parent or wild type; SII and Sll, radiation-resistant strains. Subscript indicates the number of times the SII strain was passaged through chicks.

20 2

2 0

VOL. 19, 1970

RADIATION TREATMENT OF FOODS. II

TABLE 7. Mortality in chick embryos inoculated with phenol-water extracts of Salmonella typhimurium ES878 and its radiationresistant mutant SIll Dose per embryo

(pg)

512 256 128 64 32

Number of deaths per 10 embryos inoculated witha

ES878

ES878-SIII

9 9 7 6

9 10 9 10 7

4

a No deaths occurred from inoculation with saline control. Total deaths among 50 chicks inoculated with ES878, 35; from ES878-SIII, 45.

Preliminary experiments indicated that upon challenge by various wild-type Salmonella sp., day-old chicks were found to be more sensitive than mice although morbidity was noted with all mice receiving high concentrations of cells. Without exception, the serially irradiated and more radiation-resistant strains were less virulent to day-old chicks than was their parent stock (Table 6). Five successive chick passages of S. typhimurium ES878 SIT (SII, to SII5) did not result in a reversion to the original high virulence of the parent strain. No general correlation between numbers of bacteria and virulence could be established. All the surviving chicks inoculated with viable parent or radiation-resistant strains gained less weight than did uninoculated controls during the 10-day postinoculation period. Salmonella were isolated from the intestinal contents and liver tissues of inoculated animals. Heat-killed S. typhimurium ES878 parent did not kill chicks up to 7 days postinoculation, thus eliminating intoxication as the cause of death after injection of large numbers of parent cells. There was also no evidence of morbidity as a result of this latter treatment. The results of inoculating 10-day chick embryos with the crude parent and SIII phenol water extracts indicated that they were of equal potency (Table 7). All the dead embryos exhibited extensive hemorrhages as well as a breakdown of the vascular system.

DISCUSSION In the development of resistance in Salmonella by repeated irradiation and subculturing, the level of irradiation employed may vary with the serotype, as shown by the loss of both cultures in some cases and only one or none of the duplicates in other cases. Differences in radiation resistance

343

between species was evident, indicating that the more radiation-resistant types should be employed if dose rates are to be calculated for Salmonella elimination from a particular product. The biochemical tests with "gross" parent and irradiated cultures indicated that 12 cycles at 0.2 mrad did not induce sufficient gross changes to prevent identification of the cultures by routine methods. These results are in agreement with those of Idziak and Incze (7) who reported that recycled Salmonella and Staphylococcus aureus cultures were identifiable by standard Food and Drug Directorate methods. Some of our cultures recovered as single-cell isolates after repeated irradiation and subculturing beyond 0.2 mrads did contain a measureable proportion of cells which gave responses different from the parent type. A selection of these colonies for subsequent screening would, of course, result in a negative or doubtful test for Salmonella. This would agree with the observations of Erdman et al. (Can. J. Public Health, vol. 53, p. 37, 1962) that radiation-recycled Salmonella would not be detected as such. The same must also be true for a lesser number of wild-type single-cell isolates. It was only with deoxycholate citrate agar (DCA), however, that all the isolated resistant clones exhibited a response different from the parental type. DCA was also found by Licciardello et al., (9) to give poor recovery of irradiated Salmonella. Nevertheless we are certain that the use of an appropriate selective medium and the utilization of "gross" cultural techniques would eliminate difficulties in the identification of serially irradiated Salmonella. There is no doubt that the observed morphological changes, also observed by Licciardello et al. (10), would lead to problems in identification, especially since even after five successive chick passages and subculturing over a 3-year period these cultures did not revert to their wild-type form. The fact that more than one aberrant form can be obtained from a single wild-type strain does not alleviate the problem. However, most, if not all, Salmonella detection methods do not make use of microscopic examination of the isolates as a criterion for a positive test, but rather they make use of 0-antigen agglutination reactions. The observed tendency towards roughnessloss of lipopolysaccharide terminal carbohydrates-based on colony morphology differed from that of J. J. Previte (personal communication, 1967). He attributed this difference to the fact that he used a modified Brain Heart Infusion agar which would reduce the ability to detect roughness on the basis of colony morphology when compared to growth on the nutrient agar-yeast

344

EPPS AND IDZIAK

APPL. MICROBIOL

extract used in our experiments. The results of upon radiation recycling, may acquire characthe absorption experiments (Table 5) indicated teristics which would make them potentially more that the Salmonella typhimurium ES878 SIII cells of a hazard to public health appear to be unhad indeed progressed considerably towards founded. roughness; nevertheless, this culture could still be ACKNOWLEDGMENTS identified as S. typhimurium on the basis of its 0 Financial assistance was received from Atomic Energy of and H antigens. A. Taylor (personal communica- Canada, Ltd., and from the Qudbec Agricultural Research Countion, 1968) suggested the use of the term "partially cil. degraded" to describe such a culture. Kauffmann Technical assistance provided by Linda Richardson (Cross(8) stated that the only satisfactory method for field) is greatly appreciated. detecting rough forms is rabbit immunization LITERATURE CITED followed by testing the sera for 0 agglutination. H. I., W. D. Fisher, A. Cohen, and A. A. Hardigree. In our experiments (Table 4) a weak response was 1. Adler, 1967. Miniature E. coli cells deficient in DNA. Proc. Nat. obtained with the S. typhimurium SIII cultures, Acad. Sci. U.S.A. 57:321-326. and microscopically visible agglutination did 2. Byrne, A. F., M. M. Rayman, and M. D. Schneider. 1955. Methods for the detection and estimation of numbers of occur. Salmonella in dried eggs and other food products. Appl. This alteration in the lipopolysaccharide (LPS) Microbiol. 3:368-372. might be considered to be in some way responsible 3. Edwards, P. R., and W. H. Ewing. 1962. Identification of for reduced pathogenicity in day-old chicks Enterobacteriaceae, p. 110. Burgess Publishing Co., Minneapolis. (Table 6) if it were not for the fact that crude I. E., F. S. Thatcher, and K. F. MacQueen. Studies phenol water extracts from wild-type and serially 4. Erdman, on the irradiation of microorganisms in relation to food irradiated cultures were equally toxic. It is more preservation. II. Irradiation resistant mutants. Can. J. probable, therefore, that the amount of toxic Microbiol. 7:207-215. material produced per unit of cell mass must be 5. Felix, A., and B. R. Callow. 1943. Typing of paratyphoid B bacilli by means of Vi bacteriophage. Brit. Med. J. 2:4308different. Quantitative extraction and chemical 4310. analyses of purified LPS from wild-type and re- 6. Hoffman, H., and M. E. Frank. 1961. Form and internal sistant Salmonella, now in progress, would elucistructure of cellular aggregations in early Escherichia coli microcultures. J. Gen. Microbiol. 25:353-364. date this phenomenon. Also, since the resistant E. S., and K. Incze. 1968. Radiation treatment of strains were readily isolated from the test ani- 7. Idziak, foods. I. Radiation of fresh eviscerated poultry. Appl. mal 10 days postinoculation, it would appear Microbiol. 16:1061-1066. that as a result of the recycling treatment the 8. Kauffman, F. 1966. The bacteriology of the Enterobacteriaceae. The Williams & Wilkins Co., Baltimore. Salmonella sp. have become relatively innocuous J. J., J. T. R. Nickerson, and S. A. Goldblith. parasites on the test animal. However, it is not 9. Licciardello, 1967. Elimination of Salmonella in poultry with ionizing known whether the parasites multiplied to any radiation. Presented at a panel meeting on "Elimination of great extent within the animal host. harmful organisms from food and feed by irradiation." sponsored by the International Atomic Energy Agency at Phage-typing experiments were inconclusive Zeist (The Netherlands) 11-17 June, 1967. because difficulty was experienced with both wild- 10. Licciardello, J. J., J. T. R. Nickerson, S. A. Goldblith, C. A. type and resistant strains. The 0-1-specific phage, Shannon, and W. W. Bishop. 1969. Development of on the other hand, certainly differentiated beradiation resistance in Salmonella cultures. Appi. Microbiol. 18:24-30. tween wild-type and serially irradiated Salmonella R. T., and L. Thomas. 1956. The lethal effect of endo(except S. infantis), a result in disagreement with 11. Smith, toxins on the chick embryo. J. Exp. Med. 104:217-231. that of A. von Seefried (personal communication, 12. Thormley, M. J. 1963. Microbiological aspects of the use of 1968) who found that serially irradiated Salradiation for the elimination of salmonellae from foods and feeding stuffs, p. 81-106. In Radiation control of salmoneUae monella were equally and effectively lysed. The in food and feed products. Int. At. Energy Agency Bull. reason for this discrepancy in results is not known. 13. Westphal, O., and K. Jann. 1965. Bacterial lipopolysacIn conclusion, Salmonella mutants or selectants charides. Extraction with phenol-water and further appliobtained in our studies, by repeated irradiation cations of the procedure. In R. L. Whistler (ed.), Methods in carbohydrate chemistry. 5:83-91. Academic Press, Inc., treatment at sublethal doses of gamma energy in New York. all instances exhibited reduced virulence to test 14. Wright, S. J. L., and F. C. Hill. 1968. The development of animals and were identified as Salmonella. On the radiation-resistant cultures of E. coll by a process of basis of these results, statements that Salmonella, "growth-irradiation" cycles. J. Gen. Microbiol. 5197-106.