﻿310 STRONG. 



cultures which already were of a low grade of virulence. 114 Hetseh has ex- 

 perimented extensively with the question of the artificial attenuation of the 

 plague organism. He found that prolonged growth of the bacilli at temperatures 

 ranging from 40° to 45° C. caused no attenuation of the surviving organisms. 

 Cultivation of the germ in an insusceptible animal such as the frog (in its 

 dorsal lymph sac) also caused no lowering of the virulence. After experiment- 

 ing with a number of chemical substances, particularly with various staining 

 reagents, he found that the most satisfactory means of attenuation consisted 

 of cultivating the organism in flasks of bouillon to which increasing amounts of 

 alcohol were gradually added. 



I have been successful in attenuating two strains of pest bacilli by 

 employing the method recommended by Hetseh. My cultures in the 

 alcoholic bouillon, in addition, were frequently grown at a temperature 

 of from 41° to 43° C. The strains used for the experiments were 

 those designated as "Pest Virulent" and "Pest Avirulent Manila/' The 

 organism was first inoculated into a 50 cubic centimeter flask containing 

 bouillon; after forty-eight hours' growth at 30° C. the alcohol was added 

 and the flask placed at a temperature of 41° to 43° C. for a period usually 

 of three weeks. Cultures from the flask were then inoculated upon 

 agar for many generations, a fresh one being made each day, sometimes 

 for one or two weeks, and the organism usually cultivated at the same 

 high temperature. Beginning with 0.1 cubic centimeter of absolute 

 alcohol and 50 cubic centimeters of bouillon, the amount of the former 

 was gradually increased in successive cultures up to 5 cubic centimeters 

 in 50 cubic centimeters of bouillon. In some instances the alcohol in 

 the flask was increased each week and in others every few days. The 

 organisms in the flask containing the larger amounts of alcohol fre- 

 quently perished entirely and in other instances, when inoculations were 

 made from them upon agar only, a few isolated colonies developed in 

 the medium. If the former took place, the cultures were obviously lost 

 and the last step of the attenuation had then to be repeated with the 

 previous transplant of the more virulent organism. 



This method of attenuation does not give quick results, and as 

 Hetseh admits, it is frequently not satisfactory for diminishing the 

 virulence of the plague organism. Moreover, it sometimes appears to 

 be very inefficient in bringing about this change when the strains are of 

 very great virulence. However, it would appear to be the most successful 

 method which has yet been discovered. The time during which the 

 experiments were carried on in the attenuation of the strain "Pest 

 Avirulent Manila" was a little over seven months, and in the strain 

 "Pest Virulent" about seventeen months. 



114 Ztsclir. f. Eyg. u. Infectionskrankh., Leipz. (1904), 48, 443. 



