Analysis of Clostridium botulinum toxigenic types A, B, and E for fatty and carbohydrate content.
Lyophilized, 48-hr log-phase vegetative cells were extracted with chloroform-methanol (2:1, v/v) and ethanol-ether (3:1, v/v) and then saponified with methanolic KOH. Gas-liquid chromatography of the methyl esters of extractable fatty acids revealed distinctive "pattern profiles" of Clostridium botulinum toxigenic types "A," "B," and "E." C. perfringens type "A" and Escherichia coli strain "B" were also studied in a similar manner and were found to give pattern profiles which were distinct even from those obtained for the C. botulinum microorganisms. Amino sugar content of the five microorganisms was determined by using a Beckman amino acid analyzer. The molar ratio of glucosamine to that of galactosamine was found to be of further assistance in distinguishing the individual microorganisms.
Serological Studies of Clostridium botulinum Type E and Related Organisms
Clostridium botulinum type E antigens prepared from washed cells by either Formalin treatment or heating at 100 C were used for immunizing rabbits. Agglutination tests showed that high levels of antibody were produced by both types of preparations. Flagellar antigens were highly strain-specific, whereas the somatic antigens were sufficiently similar to produce complete cross-agglutination. One toxigenic strain produced toxigenic and nontoxigenic progeny which were physiologically and antigenically identical in all other respects. Other nontoxigenic strains whose growth, physiological, and morphological characters were identical to type E and strains which had some physiological differences completely cross-agglutinated with type E strains via the somatic antigen. Neither type of antiserum agglutinated other clostridia against which they were tested except for C. acetobutylicum. This reaction seems to be due to a nonspecific anamnestic response and does not appear to be related to the immunizing strains. The nontoxigenic strains studied seem to have no greater antigenic differences from type E strains than the type E strains have from each other.
Clostridium Botulinum Type E
Two cultures of Clostridium botulinum were sent to this laboratory in March, 1936, by Dr. L. Bier of the Bacteriologic Institute at Dniepropetrowsk, Ukraina, U.S.S.R. Toxin-neutralization tests had suggested a new type. The original cultures were toxic for mice in doses of 0.001 cc. and antitoxin of types A, B, C and D failed to protect against the the toxin. The organism is a Gram positive, motile, granular, pleomorphic rod and forms oval, subterminal spores which swell the rods very slightly. In liver-agar shake-cultures small, disc-like colonies with or without small polar fluffs are formed. Colonies on the surface of glucose blood plates are non-hemolytic, greyish, translucent, smooth and flat with a marked tendency toward confluence. These cultures are not ovolytic and do not liquefy coagulated egg-white even after a month's growth. In beef heart medium there is slight reddening of the meat and a large volume of gas is evolved. Brain is not blackened or digested. In milk, slight acid is produced but the casein is not attacked. Slight liquefaction of gelatin occurred after 23 days. The peptolytic properties are extremely low and the Sörenson figure was 2.00 after 21 days' growth. Dextrose, levulose, maltose, sucrose, arabinose, xylose and adonite are fermented with little or no gas production in a medium composed of 0.3% Liebig's extract, 0.5% Difco peptone, and 1% carbohydrate. Lactose, rhamnose, galactose, dextrin, raffinose, glycerin, salicin, mannite, inulin and dulcite are not fermented. The thermal resistance of the spores is extremely low. Spores were destroyed in a suspension in buffer solution (pH 7.4) containing 5 million spores per cc. after heating at 100°C. for 2 minutes or 80° C. for 6 minutes. A suspension containing 50 million spores per cc. failed to show growth after 5 minutes at 100°C. or after 40 minutes at 80°C.