390 Cerebro-spinal Meningitis 



Flexner* found that the difficulties of cultivation were greatly- 

 reduced by the employment of sheep-serum instead of human 

 serum. Sheep-serum water was prepared according to the method 

 of Hiss (sheep-serum i part, water 2 parts, sterilized in the auto- 

 clave) and mixed with a beef-infusion agar-agar containing 2 per 

 cent, of glucose. The quantity of sheep-serum need not exceed }^q 

 to Ho of the volume of the agar-agar It is added to the sterile 

 melted agar, which is afterward slanted in test-tubes or allowed to 

 congeal on the expanded surface of i6-ounce Blake bottles when 

 mass cultures are to be used. There is nothing characteristic 

 about the cultures. The cocci grow only at the temperature of 

 the body, attain only a sparse development, and form a more or 

 less confluent line of minute, rounded, grayish colonies which are 

 easily overlooked upon opaque media like blood-serum. The 

 general characteristics of the growth are not unlike those of the 

 pneumococcus, streptococcus, and gonococcus. 



Colonies. — When grown upon agar-agar plates, the deep colonies 

 scarcely develop at all, appearing under the low-power lens as 

 minute, irregularly rounded, granular masses. The surface colonies 

 are larger, and consist of an opaque yellowish-brown nucleus about 

 which a fiat, rounded disk spreads out. The edges may be dentate; 

 the color is grayish or yellowish near the center, becoming less 

 intense as the thin edges are reached ; the structure is finely granular. 



Vital Resistance. — ^The vitality of the culture is low, and the 

 cocci die quickly. It becomes necessary, therefore, when studying 

 the organism to transplant it frequently — Parkf says every two 

 days. FlexnerJ found that they do not survive beyond two or 

 three days and that transplantations do not succeed unless con- 

 siderable quantities of the culture are placed upon the surface of 

 the fresh medium, showing that many of the organisms were already 

 dead. This is confirmed by the microscopic appearance of the 

 cultures. Those sixteen to twenty-four hours old stain sharply 

 and uniformly ; on the second day many of the cocci show irregularities 

 of size and staining, and after several days no normal-looking cocci 

 can be found. It was found, however, that in carefully preserved 

 cultures of certain strains a few cocci might survive for many 

 months. Vitality is preserved longest when the cultures are kept 

 in the thermostat and not taken out when grown, to be kept at room 

 temperature or in a refrigerator. The addition of a small quantity 

 of a calcium salt favors prolonged vitality and will sometimes main- 

 tain it for four or five weeks in cultures that would otherwise die 

 in a few days. Sodium chlorid is injurious to the cocci. Flexner 

 attributed the autolysis of the cultures to an enzyme. 



The organism is soon killed by drying, by exposure to the sun, 



* "Jour. Experimental Med.," 1907, ix, p. 105. 



t "Bacteriology in Medicine and Surgery," 1899, p. 362. 



t Loc. cit. 



