266 PROCEEDIXGS OF THE INDIANA ACADEMY OF SCIENCE. 



water (see figure I) until it gave no test for ehJorides, this required twent3'- 

 four to forty-eight hours. The same results may be obtained in a compara- 

 tively short time by dialyzing in warm (.50°C.) running distilled water, thus 

 not only cutting down the amount of distilled water used and time reqmred 

 but also the degree of bacterial development. The dialyzed filtrate was 

 diluted to one liter with distilled water, plus 2 per cent peptone; 0.5 

 per cent sodium chloride; .01 per cent calcium chloride; 1 per cent normal 

 sodium carbonate and 2 per cent agar. One or 1 J^ cc. of this agar was 

 placed in each tube and sterilized in an autoclave by heating to 105°C. for 

 fifteen minutes. This dialyzed nutrient medium was also diluted with two 

 volumes of defibrinated rabbit blood. 



In order to ascertain the relative value of these media a series of cultures 

 were carried out . For this purpose the blood of an infected rat was transferred by 

 means of a Pasteur capillary pipette, bent at right angles (figure II) to usually 



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Figure II — Pasteur Capillary pipette. Full Size. 



twelve tubes of each medium and im-ubated at 25°C. in an almost horizontal 

 position. Six comparative trials were thus made. The results of these ex- 

 periments were decidedly favorable to the dialyzed mediimi. since 80 per cent 

 of the tubes gave a positive growth. In case of the original Xovy-MacNeal 

 medium which was the least favorable, only 25 per cent of the tubes gave 

 positive results. On the modified Nicolle medium 48 per cent of the tubes 

 were positive. The bean and pea medium revealed the presence of cultural 

 forms in 53 per cent of the tubes (chart I). 



No advantage was found by increasing the blood constituent to three 

 parts to one of agar whereas, if it were decreased to one part no growth oc- 

 curred. Also no benefit resulted by altering the amount and kind of alkali. 

 Inoculated tubes of the various media were placed in atmospheres of different 

 gases, such as hj'drogen, nitrogen, and carbon dioxide, but all such attempts 

 proved to be total failures, therefore, it seems that the ordinary aerobic 

 conditions are best. 



HaA^ing apparently a satisfactory nutrient agar it seemed advisable to 

 improve the blood constituent of the medium. Various attempts were made 

 in this direction. Defibrinated rabbit blood was transferred to sterile centri- 

 fuge tubes and centrifugated for ten minuted at about 8,000 revolutions per 

 minute. The serum was then drawn ofif by means of a Pasteur bulb 



