Bacteriology of leprosy. V. Further isolation results

Results obtained from cultivation experiments employing glycerin veal agar slants, minced chick embryo medium, and chick tissue cultures are reported. A total of eleven nodules from seven patients were used in the experiments, and 311 tubes and flasks were inoculated, from which 190 successful cultures were obtained . The cultivated organisms were, in every case, acid-sensitive and acid-resistant forms appeared. Young cultures showed a preponderance of acid-fast forms in the tissue media. As the cultures aged the numbers of acid-resistant organisms became progressively less. On transferring some of the growth from an old culture to fresh tissue media the same picture was repeated. MOrphologically and tinctorially the organisms appear to be similar to those previously isolated from patients in Honolulu, and their characteristics as regards acid-fastness are similar. It is believed that the results represent a confirmation of those previously reported in the first paper of the present series.

Bacteriology of leprosy. IV. Influence of environment on the phenomenon of acid-fastness

Four nonacid-fast diphtheroids isolated from human nodules and rat granuloma are acid-fast in tissue culture and in minced embryo medium but not on the usual laboratory media. Minced liver, kidney and spleen of adult rabbits and guineapigs served as satisfactory substitutes for minced chick embryo medium for the production of acid-fast forms. The property of adult and embryonic tissues to induce the acid-fast stage of the diphtheroids is not destroyed by heating or removed by filtration. Therefore, neither living nor dead tissue is necessary for the production of acid-fast forms. Acid-fast forms can be produced in serum. The organisms are capable of assuming the acid-fast stage in a medium composed of beef heart and cholesterol. It is concluded that for the production of acid-fast forms from these organisms two factors are essential: (a) The medium must be of such composition as to foster the growth of the organisms to the stage a which they are capable of becoming acid-fast. (b) Cholesterol or some other substance must be supplied in the medium when this stage is reached. A change in the lipoid metabolism of these organisms is believed to take place concurrently with the change in morphology as followed by microscopic observation. A group of microorganisms of the order Eubacteriales inoculasted into minced tissue medium and also into 0.5 percent cholesterol beef heart medium did not show any acid-fast forms. The phenomenon of acid-fastness appears to be restricted to members of the order Actinomycetales.

On the cultivation of an acid-fast organism from leprous lesions in tissue cultures and other media

Chick tissue cultures of Carrel were used as the culture medium for the isolation of the organisms of human and rat leprosy. An acid-fast organism together with a diphtheroid were isolated from four human nodules and one rat granuloma. When transfers were made to artificial culture media only the blue-staining diphtheroid multiplied. After the primary isolations on chick tissue cultures, minced chick embryo medium furnished and excellent substrate for the cultivation of the organisms. As with chick tissue cultures, the minced embryo medium gave rise to acid-fast and non-acid-fast organisms. A pure culture of the diphtheroid when inoculated into chick embryo medium gave acid-fast and non-acid-dast organisms. The diphtheroid and the acid-fast rods are apparently different growth phases of the same organism. The organisms were acid-fast in tissues and non-acid-fast on laboratory media. The tinctorial characteristics varied, depending upon the living condition of the tissue. In vigorous, actively growing tissue the organisms were strongly acid-fast. As the tissue became less vigorous the acid-fast property wass less pronounced and, finally, as the tissue died, only non-acid-fast diphtheroids were seen. It is believed that human and rat leprosy are caused by one and the same organism.