Simplified In Vitro Identification Test

BYRD, THOMAS R. (Harvard University School of Public Health, Boston, Mass.), AND H. L. LEY, JR. Clostridium tetani in a metropolitan area: limited survey incorporating a simplified in vitro identification test. Appl. Microbiol. 14:993-997.1966In a limited survey, three toxigenic and one nontoxigenic strains of Clostridium tetani were isolated from 18 environmental samples from metropolitan Boston. No C. tetani was found in 100 samples of human feces, 20 samples of dog feces, and two samples of horse feces. A simple modification of the halo precipitin test was studied in conjunction with the mouse lethality test for tetanus toxigenicity and was found to be a useful, although not a wholly definitive, technique. The literature is replete with suggestions that Clostridium tetani is virtually universally distributed in the environment, presumably as a result of contamination by the feces of domestic animals and man (10). Although Smith (15) concluded that the organism has many habitats in nature, including the soil, the idea of the horse as the prime source of the organism is still widely prevalent. Early work in this country, especially that of Bauer and Meyer (1) and Gilles (2), tended to exonerate the horse and implicate man and soil. A review of the literature revealed no substantive survey data on the distribution of tetanus spores in feces or the environment in this country since 1937. The low incidence of clinical tetanus, the apparent low level of immunization, and the relative scarcity of sources of massive fecal contamination (such as horses) suggested that the prevalence of C. tetani in the metropolitan Boston environment might be significantly less than that predicted by the literature. Consequently, a limited study was undertaken to determine both the approximate level of contamination of the environment with toxigenic strains of C. tetani and possibly the probable source of such contamination. The study was also designed to evaluate or develop techniques adaptable to mass studies of anaerobic organisms in a field situation. IPresent address: U.S. Naval Support Activity, Box 43, APO San Francisco, Calif. MATERIALS AND METHODS Isolation media. Cooked Meat Medium (Difco) was prepared according to the manufacturer's directions and used on the day of preparation. Yeast extractblood-agar and sorbic acid-polymixin B sulfate-thioglycolate medium (SAPB) were prepared as described by Wetzler et al. (17), except that Difco dehydrated media and defibrinated horse blood were substituted. Chloral hydrate-sodium azide inhibitory medium (CHSA) was prepared as recommended by MacLennan et al. (9), with the use of yeast extract-bloodagar as a base. Dextrose proteose No. 3 (DP-3) Agar. A 40-g amount of Difco Dextrose Proteose No. 3 Agar and 7 g of Difco Agar were dissolved in 1 liter of distilled water by heating to boiling and were dispensed in 8-ml amounts into tubes (16 by 150 mm). After being autoclaved for 15 min at 121 C, the media were incubated at 37 C overnight to check for sterility and then were stored at room temperature. Tetanus antitoxin. Equine antitoxin without preservative (Massachusetts Institute of Laboratories lot No. LA 308) containing 1,660 Lf (limit of flocculation) per ml was used for halo tests. Equine antitoxin with preservative (lot No. LA 117 P, from the same source) containing 2,000 antitoxin units per ml was used for passive immunization of control mice. Reference strains oforganisms. Two reference strains of toxin-producing C. tetani were used. Reference strain S-2 is the strain used for tetanus toxoid production by the Massachusetts Institute of Laboratories and was identified as their strain D-7. Reference strain S-3 was obtained from the American Type Culture Collection, accession no. 9441. Another strain (designated S-1 by us) was received from K. F. Girard of 993 on Jne 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom