In the preceding paper in this series (1), the fractionation of “cord factor” derived from tubercle bacilli and the purification of the toxic component of the factor were described. It was shown that the latter represents but a small frac tion of the total extract and that the yield of this material is poor. As the work progressed, the need for better sources of active material became more urgent, and methods were developed to improve the rendering of extraction. The present paper describes such various procedures.“Cord factor” had first been obtained by superficial extraction of young cul tures of virulent tubercle bacilli (2). This method furnished a crude product which was adequate for certain animal experiments which required only small amounts of material (2, 3), but it became unsatisfactory when larger quantities had to be at hand for chemical analysis of the extracts. Bacterial cultures older than ap proximately two weeks which had a more luxuriant growth gave greater yields upon extraction with petroleum ether, but the relative toxicity of the extracts dropped as the cultures grew older and larger doses had to be injected to produce the same effects. Since the toxic component was of particular interest, this pro cedure was obviously no solution to the problem. Equally unsuccessful were at tempts to improve the yield by extracting the bacilli more completely with petroleum ether in a Soxhlet apparatus. While more material was obtained by this method, the proportion of the toxic principle did not increase correspond ingly. The previous observation was confirmed that the active material which appeared to be located at the surface of the bacterial cell was easily obtainable by superficial petroleum ether extraction of short duration, but that any attempt at improving the yield would have to be made through different and more thor ough methods of extraction.

It had been found that treatment with petroleum ether was not very injurious to the bacillus. The organisms were able to multiply in vivo as well as in vitro after they had been suspended in petroleum ether for long periods of time (2). The first question that arose was whether the tubercle bacillus still contained toxic material after extraction with petroleum ether. Previous observations sug gested that the material removed by this solvent is a secretory product released by the bacillus into the surrounding medium, provided the organisms are sus pended in a lipophilic environment where “cord factor” is sufficiently soluble. Indeed, it was possible to recover material with “cord factor” toxicity from