Terrestrial strains of Clostridium botulinum type C, designated 468 and 571, were used to screen various media for growth and sporulation at 30 C. Of the various formulations tested, only egg meat medium fortified with 1% additions of yeast extract, ammonium sulfate, and glucose (FEM medium) gave good growth and satisfactory sporulation. FEM medium was used to recover four marine type C isolates from inshore sediments collected along the Atlantic, the Gulf of Mexico, and the Pacific coasts of the United States. The isolation techniques involved repeated transfer of cultures showing type C toxin in FEM medium and purification by a deep tube method. The medium used for purification was beef infusion-agar supplemented with 0.14% sodium bicarbonate and 0.1% L-cysteine hydrochloride. L-Cysteine was adopted in preference to sodium thioglycolate, because some lots of the latter were definitely inhibitory for growth. The addition of bicarbonate markedly increased viable spore counts of both the marine and terrestrial strains. Various cultural and biochemical characteristics of the marine and the terrestrial strains were compared. With the exception of some variations in their fermentation patterns, both groups showed similar characteristics. Of 23 fermentable compounds tested, the terrestrial strains attacked only glucose and mannose. The marine strains fermented glucose, mannose, galactose, and ribose actively; dextrin, inositol, maltose, and melibiose were weakly fermented. MATERIALS AND METHODS Origin of terrestrial strains. Strains 468 and 57.1 were obtained from the National Canners Association Laboratory at Berkeley, Calif. They were originally isolated by A. R. Prevot from animals reportedly dead from type C botulism. Sources of marine sediments. Various marine sedi-1017
SEGNER, W. P. (Continental Can Co., Inc., Chicago, Ill.), C. F. SCHMIDT, AND J. K. BOLTZ. Effect of sodium chloride and pH on the outgrowth of spores of type E Clostridium botulinum at optimal and suboptimal temperatures. Appl. Microbiol. 14:49-54. 1966.-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 pH 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 pH levels than when 0.2 % thioglycolate was added. At 30 C, spores of one strain showed outgrowth in TPG medium as low as pH 5.21 with an inoculum of 2 million spores per replicate tube. At a 10-fold higher inoculum, the same strain showed outgrowth at pH 5.03 in one of five replicate tubes. At 8 C, spore outgrowth of the four strains occurred at pH 5.9, but not at pH 5.7, in TPG medium containing L-cysteine hydrochloride.
Minimal growth temperatures of four marine and two terrestrial strains of Clostridium botulinum type C were determined in a laboratory culture medium, fortified egg meat medium (FEM), and in ground haddock. The inoculum equaled 2 x 106 viable spores per tube with five-tube replicate sets. The spores were preheated in aqueous suspension at 71 C for 15 min prior to inoculation to reduce toxin carry-over. Similar results were obtained in both substrates. Both the marine and the terrestrial strains grew at 15.6 C, but only the terrestrial strains grew at 12.8 C. None of the strains grew at 10 C during prolonged incubation. The sodium chloride tolerance and the pH sensitivity of the marine and the terrestrial strains were determined at 30 C. The basal medium consisted of beef infusion broth. The inoculum level equaled 2 x 106 unheated spores per replicate. Growth was inhibited at salt concentrations from 2.5 to 3.0%. The terrestrial strains were more pH-sensitive than the marine strains. Whereas the terrestrial strains failed to grow below pH 5.62, three of the marine strains grew at pH 5.10, but not at pH 4.96, during extended incubation. One marine strain grew at pH 5.25, but not below. FEM and proteose peptone-Trypticase-yeast extract-glucose medium permitted the production of high levels of botulinum toxin among four media tested. Toxin produced by the marine and terrestrial strains showed no increase in toxicity after incubation with trypsin.
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