Cultivation 403 



spinal fluid, the blood of the patient or the purulent exudation upon 

 or in the removed brain or spinal cord. 



As has been remarked, the life of the culture is brief so that daily 

 transplantation may be required to keep the culture growing. But 

 once the culture has been isolated, the blood medium may no longer 

 be necessary, sheep-serum dextrose agar-agar being easier to pre- 

 pare and just as satisfactory. Experience indicates that the greatest 

 longevity of a generation of cocci may be secured by the employment 

 of a medium suggested by.Bordet and Gengou for the cultivation 

 of Bacillus pertussis, which Roos ("Jour, of Bacteriology," 1916, 

 vol. I, No. I, p. 67) makes as follows: 



1. a. Potato peeled, cut into small pieces and washed for two 



hours in running water loo grams. 



b. Water containing 4 per cent, double distilled glycerin free 



from acid 200 c.c. 



c. Mix and autoclave for 40 minutes. 



d. Allow to stand over-night and strain through cheese-cloth. 



2. a. Mix in an Erlenmeyer flask. 



Potato extract as made above So cc. 



0.6s per cent, sodium chloride solution 150 cc. 



Agar-agar S grams. 



b. Heat in an Arnold sterilizer until the agar-agar is melted. This requires 

 from one-half to one hour. 



3. Tube without filtering and sterilize in an autoclave for about 40 minutes. 



4. When wanted for use, melt the medium, cool to about 4S°C., and then add 



5 per cent, of sterile, defibrinated horse's blood. 



Upon this medium Roos succeeded in keeping the meningococcus 

 alive for as long as four weeks. 



Colonies. — When grown upon blood agar-agar plates, the col- 

 onies develop only upon the surface appearing larger or smaller 

 according to circumstances. When the culture is pure and the 

 colonies not crowded, they may attain a diameter of two or three 

 millimeters. They are uniformly creamy white and look soft. Col- 

 onies close together become confluent When touched they are 

 found to be viscid. Small colonies viewed under a low-power lens 

 appear regularly rounded, finely granular or transparent. The 

 colonies are usually colorless by transmitted light, but may be uni- 

 formly slightly yellowish. 



Vital Resistance. — The organism is soon killed by drying, by 

 exposure to the sun, and by quite moderate variations of tempera- 

 ture. It succumbs to very high dilutions of most germicides in a 

 very short time. 



The thermal endurance of the organism is very slight. It is 

 killed in five mmutes at 6o°C. 



Agglutination. — ^When animals are immunized by repeated in- 

 jections of the Diplococcus intracellularis, their blood-serum and 

 body-juices become agglutinative. Such serums carefully titrated 

 and kept in the laboratory are indispensible for the identification 

 of the coccus in fresh culture. The serums have an agglutinating 

 power that varies from i : 500 to i : 3000. 



