Groups of guinea pigs were vaccinated by the intracutaneous route with cultures of BCG grown in a liquid medium containing Tween 80 and the soluble fraction of heated human serum. After the cultures had been stored at 4°C. for various periods of time, the antigenic response was compared with that of another group of guinea pigs receiving standard BCG vaccine prepared by the conventional technique.

The local lesions occurring at the site of injection of cultures in Tween-serum filtrate medium were more severe than those produced by the standard vaccine. It was shown that this difference was probably due to the much larger number of viable bacilli in the former preparations. A marked degree of sensitization could be produced with culture dilutions containing as few as 10 viable units (single bacilli or small clumps). Slightly larger doses of BCG led to the highest degree of tuberculin allergy detectable by the technique employed. Further increases in the dose of vaccine failed to alter the level of sensibility when the animals were tested with tuberculin 5 weeks after vaccination.

The same degree of sensitization was achieved by vaccination with 0.1 cc. of either the standard vaccine or any of the fresh or stored cultures in Tween-serum filtrate medium. It was shown that these doses contained numbers of living bacilli far greater than the minimal number required to induce maximal sensitization.

Under the conditions used, the guinea pigs vaccinated with cultures of BCG (fresh or stored) grown in the Tween-serum filtrate medium exhibited a marked degree of resistance to subcutaneous infection with virulent tubercle bacilli.

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