Tubercle bacilli were grown in the presence of different concentrations of tween 80, ranging from 0.05 to 2.1 per cent. Equal numbers of viable bacteria from these cultures were compared in infection experiments in the mouse. The average survival time of the mice was used as a criterion for the virulence of the bacilli. High tween concentrations in the culture medium caused a reduction of the bacterial virulence. The reduction was slight in bacterial suspensions from cultures with tween 80 ranging from 0.05 to 1.0 per cent, but considerable in cultures with 2.1 per cent tween.
Bacteria grown in the presence of 2.1 per cent tween gave rise to the same number of colonies, in vitro, as bacteria grown in ordinary media. Their oxygen uptake was increased as compared with that of bacilli grown in media containing less tween.
Virulent bacteria grown in the presence of high amounts of tween 80 decolorized methylene blue in a test in which organisms from the same virulent strain but cultured without tween, or with only small proportions of the detergent in the medium, did not reduce the dye. A positive methylene blue test is typical of non-virulent tubercle bacilli and of saprophytic mycobacteria.
Essentially the same changes occurred when virulent tubercle bacilli were grown in the presence of 0.5 µg./ml. of para-formacetanilide thiosemicarbazone (TBI). This small amount of the substance was not sufficient to prevent the growth of bacteria, or to reduce the number of viable cells in a culture, but it reduced the virulence of the bacteria considerably and rendered them capable of decolorizing methylene blue.
Cord factor, a lipid constituent of virulent bacteria which is toxic for mice, was shown to be present in filtrates from cultures of virulent bacteria when the media contained 2 per cent tween 80, but no such material could be recovered from culture filtrates containing the usual 0.05 per cent tween. On the other hand, no toxic material could be extracted from bacteria grown in the presence of 0.5 µg./ml. TBI.