Effect of Sodium Chloride and ph on the Outgrowth of Spores of Type E Clostridium botulinum at Optimal

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1 APPLIED MICROBIOLOGY, Jan., 1966 Copyright 1966 American Society for Microbiology Vol. 14, No. 1 Printed in U.S.A. Effect of Sodium Chloride and on the Outgrowth of Spores of Type E Clostridium botulinum at Optimal and Suboptimal Temperaturesi W. P. SEGNER, C. F. SCHMIDT, AND J. K. BOLTZ Metal Division Research and Development, Continental Can Company, Inc., Chicago, Illinois Received for publication 23 July 1965 ABSTRACT SEGNER, W. P. (Continental Can Co., Inc., Chicago, Ill.), C. F. SCHMIDT, AND J. K. BOLTZ. Effect of sodium chloride and on the outgrowth of spores of type E Clostridium botulinum at optimal and suboptimal temperatures. Appl. Microbiol. 14: The sodium chloride inhibition of spore outgrowth of four strains of type E Clostridium bolulinum was determined in a Trypticase-peptoneglucose (TPG) medium. At 16, 21, and 30 C, spores of three strains required 5.0% and one strain 4.5% salt for complete inhibition during 1 year of incubation. At 8 and 10 C, spores of the four strains required 4.5% salt for definite inhibition. Salt concentrations slightly lower than those providing inhibition tended to extend spore outgrowth time at low temperatures. The minimal permitting outgrowth of type E spore inocula was affected by the concentration of reducing compound present in the system. When either 0.02% sodium thioglycolate or 0.05% L-cysteine hydrochloride was used, outgrowth at 30 and 8 C occurred at much lower levels than when 0.2% thioglycolate was added. At 30 C, spores of one strain showed outgrowth in TPG medium as low as 5.21 with an inoculum of 2 million spores per replicate tube. At a 10-fold higher inoculum, the same strain showed outgrowth at 5.03 in one of five replicate tubes. At 8 C, spore outgrowth of the four strains occurred at 5.9, but not at 5.7, in TPG medium containing L-cysteine hydrochloride. As a result of much experimentation, the maximal sodium chloride level and the minimal level permitting spore outgrowth of Clostridium botulinum types A and B have been adequately determined. However, little data are available regarding either the salt or inhibition of type E spores, or the extension of spore outgrowth time by either of these factors at low temperatures of incubation. Pedersen (7) determined the concentration of sodium chloride inhibiting outgrowth of spores of two type E strains at 30 C. Based on an incubation time of 30 days in a peptic digest liver broth, he observed growth in 3.9%, but not in 4.1 %, salt. At a salt concentration below that showing inhibition, a delay in spore outgrowth time was observed. It is generally accepted that outgrowth of spores of the proteolytic types A and B C. botulinum is inhibited at 4.6 (5, 11). However, the lowest 1 Presented in part at the 64th Annual Meeting of the American Society for Microbiology, Washington, D.C., 3-7 May permitting outgrowth of spores of type E C. botulinum has not been documented as well. After the occurrence of a type E outbreak in home-pickled herring in 1949, Dolman et al. (2) inoculated herring with a young type E culture isolated from the outbreak and incubated the fish at room temperature until toxin was produced. The remainder of the experiment is described as follows: "Portions of the foregoing herring, of known high toxicity, were 'pickled' according to the housewife's formula. These preparations were also kept at room temperature and periodically subjected to bacterial counts and lethal potency tests. The pickling fluid exhibited a high, and to all intents undiminished toxicity, for several weeks. Meanwhile, the Type E organisms only slowly declined in numbers, and indeed multiplied intermittently, despite the low range of 4.5 to 4.8. (A parallel experiment, in which pickling fluid was saturated with sodium chloride, gave similar results.)" More recently, Dolman and lida (3) reported growth of the VH strain at an even lower : 49

2 50 SEGNER, SCHMIDT, AND BOLTZ APPL. MICROBIOL. "Moreover, the Vancouver ('VH') strain produced appreciable amounts of toxin within a week when grown either in pickled herring ( ) at 23 C, or in fresh herring at 6 C." Meyer and Kietzmann (6) observed growth of types A, B, and E strains in a brain infusion medium at 5.25 to 5.30, but not at lower values. Furthermore, when fresh herring was inoculated and held 7 days in a pickling solution at 2 C, until an equilibrium of 5.0 to 5.2 was reached, and was then stored at room temperature, no toxin was detected. In view of the conflicting data available, it is extremely difficult to judge the minimal permitting outgrowth of spores of type E C. botulinum. The purpose of this investigation was to determine the highest concentration of sodium chloride and the lowest permitting outgrowth of spores of selected strains of type E C. botulinum. An additional objective was to explore the possible interaction of temperature with salt and with as a means of extending the time of outgrowth of type E spores at temperatures below 10 C. MATERIALS AND METHODS Spore suspensions. Cultures of the Beluga, 8E, Alaska, and Minneapolis strains were used to prepare type E spore suspensions. Suspensions were prepared and standardized according to the procedures described by Schmidt, Nank, and Lechowich (8). Media. The basal medium (TPG) consisted of 5% Trypticase, 0.5% peptone, and 0.4% glucose. The desired weight of sodium chloride, calculated on a weight basis, was dissolved in the medium, and the was adjusted to 7.0. The medium was dispensed in 200-ml quantities into 8-oz screw-cap bottles and autoclaved at 121 C for 15 min. Sterile concentrated sodium thioglycolate (20%) was added to the medium just prior to use to give a final concentration of 0.2%70. TABLE 1. Since the bottles containing the medium were weighed before and after sterilization, the balance of the water lost by evaporation was restored to the nearest gram by the addition of sterile deionized water. TPG medium was also used to determine the effect of on the outgrowth of type E spore inocula. The medium was buffered to eliminate minor fluctuations of the adjusted levels. To each 100 ml of doublestrength TPG (10% Trypticase, 1% peptone, and 0.8% glucose) was added 20 ml of 0.67 M potassium dihydrogen phosphate, plus 80 ml of deionized water. Therefore, the final medium consisted of M phosphate-buffered single-strength TPG. Concentrated HCI was added to the approximate end point, followed by diluted HCl to the desired. A model 76 expanded scale meter (Beckman Instruments, Inc., Fullerton, Calif.) and a magnetic stirring device were used in the adjustments. The adjusted medium was transferred in 200-ml quantities to screwcap bottles and sterilized. Media of 5.0 or lower were autoclaved for 5 min at 121 C, and those above 5.0 were autoclaved for 10 min at this temperature. Either sterilized sodium thioglycolate or L-Cysteine hydrochloride was added to give respective concentrations of 0.02 and 0.05% just prior to the use of the medium. In addition to TPG medium, liver broth was utilized to determine the effect of on spore outgrowth time. Liver infusion medium was prepared as recommended in the National Canners Association Laboratory Manual (10). It was adjusted to various levels, bottled, and autoclaved as described above. At inoculation and at several periods during incubation, the of uninoculated TPG medium and liver broth medium was determined. All values cited are those obtained after autoclaving. Inoculation and outgrowth techniques. Either 16 by 125 or 16 by 150 mm sterile screw-cap tubes were inoculated with 0.2 ml of a preheated (60 C, 13 min) spore inoculum equivalent to 2 million viable spores. With the use of a five-tube replicate test per variable, the inoculated tubes were poured with about 10 ml o medium, sealed with sterile melted Vaspar, and tem Extension of outgrowth time of spores of the Minneapolis strain at several sodium chloride concentrations and temperatures NaCI _. 8 C 10 C 16 C 21 C 30 C 37 C 0 9 (7-9) 5 (4-7) (3-4) (14-16) (21-23) 14 8 (7-9) (32-47) 19 (18-21) 11 (9-14) 6 4 (3-6) > >365 > (16-30) 25 (13-48) 8, 13, 20 (>365) 5.0 >365 >365 >365 * Ranges are given in parentheses; where no range is shown, each of five tube replicates gave the same outgrowth time. Where fewer than five tubes showed growth, the outgrowth time for each tube is indicated.

3 VOL. 14, 1966 INHIBITION OF SPORE OUTGROWTH OF C. BOTULINUM 51 pered in either cold tap water or ice water when incubation at low temperatures was desired. Each tube was dried and carefully examined for entrapped air bubbles. Converted to the nearest degree centigrade, tubes were incubated at 46 F (8 C), 50 F (10 C), 60 F (16 C), 70 F (21 C), 85 F (30 C), and 98 F (37 C). During incubation, tubes were examined on a Monday, Wednesday, and Friday schedule. Toxin assays. The presence or the absence of type E toxin was determined according to the procedure described by Schmidt et al. (8) with the exception that 0.5% rather than 1.0% trypsin was used. RESULTS NaCl inhibition of spore outgrowth. The term outgrowth is used here to indicate the first appearance of gas in Vaspar stratified tubes and a change in turbidity of the culture medium. Table 1 shows the average and range of outgrowth times of spores of the Minneapolis strain at various sodium chloride levels and at several incubation temperatures. A delay in outgrowth time occurred with increasing salt concentration and decreasing temperature. At equivalent salt TABLE 2. Sodium chloride inhibition of spore outgrowth offour type E strains at various temperatures Strain Incubation temp Per cent NaCl concn Growth Grwh growth* No Beluga 8, 10, 16, 21, E, Alaska 8, and Minn 16, 21, * Based upon 365 days of incubation and an inoculum of 2 X 106 spores per replicate tube. TABLE 3. Thio- glycolate levels, the outgrowth times were substantially shorter at 30 C, which is considered optimal for growth, than at lower temperatures. Occasionally, at the salt level just below that showing complete inhibition, fewer than five replicate tubes exhibited outgrowth. Spores of the Beluga, 8E, and Alaska type E strains gave outgrowth times similar to those shown in Table 1. Salt levels necessary for complete outgrowth inhibition at each temperature based upon 1 year of incubation are summarized in Table 2. The sodium chloride level for inhibition at low temperatures was not much less than that at 30 C. In fact, spores of the Beluga strain required 4.5% salt for outgrowth inhibition at temperatures from 8 to 30 C. At 37 C, the inhibitory concentration of salt was somewhat less than that at lower incubation temperatures. The preceding data show the salt tolerance of type E spore inocula preheated at 60 C for 13 min in aqueous suspension and then inoculated into TPG medium at various sodium chloride concentrations. The effect on outgrowth time of preheating type E spore inocula in TPG medium at various sodium chloride levels also was determined. At comparable temperatures between 16 and 37 C, spores preheated in the presence of salt showed approximately the same outgrowth times at equivalent sodium chloride levels as did spores preheated in aqueous suspension. However, at 8 and 10 C, spores preheated in salt showed slightly longer outgrowth times at comparable salt levels, but no difference in the level of sodium chloride for complete inhibition. After 1 year of incubation at 21 and 30 C, several tubes containing spore inocula preheated in the presence of sodium chloride were selected to determine the extent of spore germination in TPG medium at 5.0% salt. The tubes were centrifuged, and the spore sediments were examined by Effect of sodium thioglycolate conicentration on the tolerance of type E spore inocula at 8 C in TPG medium (33-49) 32 (23-35) 26 (23-30) 45 (23-67) (11-13) 13 (11-15) >212 >212 >212 > (13-18) 21 (13-25) 16 (13-20) (15-25) 18 (15-25) 24 (18-32) 21 (18-25) , 139 (>212) 139, 172, 172 (>212) > (>212) * Average and range of outgrowth time shown at levels 6.4 to 6.0. At 5.8, the outgrowth time for each individual tube is shown. Outgrowth times at 5.8 and 6.0 in the presence of 0.2% thioglycolate were more than 212 days.

4 52 SEGNER, SCHMIDT, AND BOLTZ APPL. MICROBIOL. use of dark-phase contrast microscopy. Spores of the four strains incubated at 21 C were predominantly refractile, as were spores of the Minneapolis strain incubated at 30 C. However, more than one-half of the Beluga, 8E, and Alaska spores incubated at 30 C were phase-dark, indicating germination. Each of the tubes examined yielded positive subcultures producing type E toxin when inoculated into TPG medium and incubated at 30 C. Inhibition of spore outgrowth by. In preliminary outgrowth experiments involving as a variable, 0.2% sodium thioglycolate was added as a reducing compound to the -adjusted medium. Suspecting that there might be an interaction between thioglycolate and in the inhibitory effects observed, we reduced the concentration of thioglycolate to 0.02%. This was the lowest concentration of thioglycolate that could be used to achieve adequate reduction of the medium for type E growth. Table 3 presents the outgrowth times at 8 C of type E spore inocula in TPG medium containing TABLE 4. Effect of on type E spore outgrowth time at 8 C in TPG medium containing 0.05% L-cysteine hydrochloride (12-19) 13 (12-14) 17 (14-19) 15 (14-16) (16-21) 24 (19-26) 18 (16-19) (19-26) 27 (26-30) 44 (21-75) 34 (26-40) 5.7 >212 >212 >212 >212 * Range is given in parentheses; no range indicates that the five replicate tubes gave the same outgrowth time. 0.2 and 0.02% sodium thioglycolate. At 6.4, 0.2% thioglycolate markedly delayed spore outgrowth time and completely inhibited outgrowth at 6.2 for 212 days of incubation. However, the use of 0.02% thioglycolate permitted outgrowth of spores of three strains as low as 5.8, but with a considerable delay in outgrowth time. The inhibitory effect of 0.2% thioglycolate was considerably greater at 8 C than at 30 C. In TPG medium containing 0.2% thioglycolate, outgrowth at 30 C was obtained within 4 to 5 days of incubation at 5.6, whereas reduction of the thioglycolate concentration to 0.02% permitted type E spore outgrowth in 1 to 2 days at this. Substitution of 0.05 % L-cysteine hydrochloride for 0.02% thioglycolate gave comparable outgrowth times at 8 C at approximately the same levels (Table 4). Outgrowth of spores of the four type E strains was observed at 5.9 during an incubation period of 3 to 5 weeks. Definite inhibition was obtained at 5.7 up to 7 months of incubation at 8 C. Data in Table 5 show the lowest permitting outgrowth of a 2 million spore inoculum at 30 C. Spores of the Beluga strain exhibited outgrowth as low as 5.21, whereas spores of the 8E, Alaska, and Minneapolis strains showed outgrowth at 5.31, but not at 5.21, in the presence of sodium thioglycolate. With 0.05% L-Cysteine hydrochloride, outgrowth was not observed below Table 6 shows the minimal permitting outgrowth at 30 C of a 20 million spore inoculum level and compares the sensitivity of unheated to mildly preheated inocula. Regardless of the somewhat faster outgrowth times of unheated spores of the Beluga strain at 5.22, only one tube inoculated with preheated spores gave outgrowth at The production of type E TABLE 5. Minimal permitting type E spore outgrowth at 30 C in TPG medium containing 0.02% sodium thioglycolate or 0.05% L-cysteine hydrochloride Reducing compound Thioglycolate (3-17) , 3, 3, 5 (>250) 17 (7-33) 10 (7-14) 30 (21-38) , 19, 24, 40 (>250) >250 >250 > >250 L-Cysteine (10-21) 7 (5-10) 10 (5-12) 5.24 >250 >250 >250 >250 * Inoculum = 2 X 106 spores per tube. No range indicates that the five replicate tubes gave the same outgrowth time; outgrowth time for each tube is shown where fewer than five tubes gave outgrowth.

5 VOL. 14, 1966 INHIBITION OF SPORE OUTGROWTH OF C. BOTULINUM 53 TABLE 6. Comparative sensitivity at 30 C ofunheated and mildly preheated type E spores Treatment 5.41 Preheated t Unheated Preheated 29, 29, 54 (>365) > 365 > 365 > 365 Unheated 12 (9-14) 18 (>365) >365 > Preheated 83 (>365) >365 Unheated > 365 * Based upon an inoculum of 20 X 106 spores per tube and outgrowth in TPG medium containing 0.02% sodium thioglycolate. Outgrowth time for each tube is shown where fewer than five tubes gave outgrowth. t Preheated = 60 C, 13 min. TABLE 7. Minimal permitting type E spore outgrowth at 30 C in liver broth Outgrowth time* (days) >270 >270 >270 > >270 >270 >270 > >270 >270 >270 >270 * Inoculum = 2 X 106 preheated (60 C, 13 min) spores per tube; five-tube replicate set per variable. toxin at the lower levels was confirmed with type-specific antitoxin. Freshly prepared liver broth at various levels was inoculated with spores of the four type E strains and incubated at 30 C. Liver broth was employed to eliminate the necessity of adding a reducing system as required for type E growth in TPG medium, thereby eliminating the possible interaction of even low concentrations of either sodium thioglycolate or L-cysteine hydrocloride with. As shown in Table 7, spore outgrowth of each of the four type E strains was observed at 5.44 after 2 days and at 5.22 after 3 days of incubation. Liver broth adjusted to 5.01, 4.80, and 4.58 failed to show outgrowth during 9 months of incubation. After this length of incubation, several tubes at 5.01 were centrifuged, and the sediments were examined by use of phasecontrast optics to ascertain the extent of spore germination. Approximately 70% of the Beluga and 8E spores were phase-dark, whereas somewhat less than 50% of the Alaska and Minneapolis spores appeared germinated. Occasionally, vegetative cells considerably smaller than typical type E cells were seen in wet-mount preparations. The absence of aerobic growth on streaked agar plates (Difco, Plate Count Agar) appeared to discount the cells as contaminants, and suggested that they represented the partial development of type E cells. None of the culture supernatant fluids showed type E toxin with or without trypsin digestion. Subcultures of the centrifuged sediments showed rapid growth and gas production at 30 C and the presence of type E toxin. DIscussIoN It is generally accepted that approximately 10 % sodium chloride (calculated as per cent brine concentration) is adequate to prevent growth and toxin production from spore inocula of C. botulinum types A and B (1, 4, 9). Data presented here indicate that type E C. botulinum possesses a level of salt tolerance considerably below that recognized for types A and B. At salt concentrations below those showing definite inhibition, there is a moderate extension of spore outgrowth time at low incubation temperatures. Although 4.0% salt had no major effect on delay in outgrowth time at 30 C, at 8 C it produced a three- to fourfold extension. The results illustrate that salt concentrations which give no effect at temperatures near optimum can display a significant extension of outgrowth time when incubated at low temperatures. There is considerable evidence that type E C. botulinum possesses a tolerance within the range reported for types A and B. It is conceded that certain food substrates may support type E growth and toxin production at somewhat lower levels than selected laboratory culture media. Nevertheless, it appears unlikely that growth and toxin production will occur below 4.8, the

6 54 SEGNER, SCHMIDT, AND BOLTZ APPL. MICROBIOL. lowest permitting growth of types A and B (5, 10). There is a marked interaction of with low temperatures of incubation in the delay in visible growth and gas production in TPG medium. However, fish homogenates have not shown this phenomenon of interaction (Segner, Boltz, and Schmidt, unpublished data). Therefore, it is advisable to suspect growth and toxin production of type E C. botulinum in food products at low temperatures at levels normally permitting growth at 30 C. ACKNOWLEDGMENT This investigation was conducted under contract AT(11-1) 1183 with the Division of Biology and Medicine of the U.S. Atomic Energy Commission. LITERATURE CITED 1. ANDERTON, J. I Pathogenic organisms in relation to pasteurized cured meats. Scientific and Technical Surveys No. 40, p The British Food Manufacturing Industries Research Association. 2. DOLMAN, C. E., H. CHANG, D. E. KERR, AND A. R. SHEARER Fishborne and Type E botulism: two cases due to home-pickled herring. Can. J. Public Health 41: DOLMAN, C. E., AND H. IIDA Type E botulism: its epidemiology, prevention and specific treatment. Can. J. Public Health 54: GREENBERG, R. A., H. J. SILLIKER, AND L. D. FATrA The influence of sodium chloride on toxin production and organoleptic breakdown in perishable cured meat inoculated with Clostridium botulinum. Food Technol. 13: INGRAM, M., AND R. H. M. ROBINSON The growth of Clostridium botulinum in acid bread media. Proc. Soc. Appl. Bacteriol. 14: MEYER, V., AND U. KIETZMANN Probleme des Verderbens von Fischkonserven in Dozen. Veroeffentl. Inst. Meeresforsch. Bremerhaven 5: PEDERSEN, H The survival of Clostridium botulinum in curing brines. Proc. Intern. Symp. Food Microbiol., 2nd, p SCHMIDT, C. F., W. K. NANK, AND R. V. LECHOWICH Radiation sterilization of food. II. Some aspects of the growth, sporulation, and radiation resistance of spores of Clostridium botulinum Type E. J. Food Sci. 27 : TANNER, F. W., AND F. L. EVANS Effect of meat curing solutions on anaerobic bacteria. 1. Sodium chloride. Zentr. Bakteriol. Parasitenk. Abt. II 88: TOWNSEND, C. T., I. I. SOMERS, F. C. LAMB, AND N. A. OLSON A laboratory manual for the canning industry. National Canners Association Research Laboratories, Washington, D.C. 11. TOWNSEND, C. T., L. YEE, AND W. A. MERCER Inhibition of the growth of Clostridium botulinum by acidification. Food Res. 19: Downloaded from on April 7, 2019 by guest