STOCK CULTURES OF THE MENINGOCOCCUS 69 



6. Heat in Arnold sterilizer until agar is melted, requiring from thirty 

 minutes to one hour. 



3. Tube without filtering, and sterilize, in autoclave for about 

 forty minutes. 



4. When wanted for use, melt the agar, cool to about 45°C. and 

 add the desired amount of sterile defibrinated horse blood. 



The amount of blood to be added depends upon whether or 

 not the meningococcus has become accustomed to the medium. 



In transplanting from another medium to this potato blood 

 agar, a little difficulty may be experienced in getting the cultures 

 started upon the new substratum. For this reason a large 

 amount of the growth (not over twenty-four hours old) should 

 be transferred to the medium containing about 20 per cent of 

 defibrinated blood. In making the inoculation the culture 

 should be rubbed slightly into the surface. This is incubated 

 at 37.5°C. for about 2 days and then transplanted again to 

 the potato-extract-blood-agar containing just sufficient blood 

 to permit growth — that is, about 5 per cent. Subsequent 

 transplants need not be made more often than every thirteen 

 to fifteen days or longer, when kept at 37°C., provided that the 

 cultures do not become too dry. In the case of cultures paraf- 

 fined or sealed to prevent drying, a fair growth may be obtained 

 after six weeks. 



The meningococcus grows well at 37.5°C. At lower tem- 

 peratures it will remain alive for a considerable length of time, 

 although no growth occurs; viability may be retained at room 

 temperature apparently as long as, if not longer than, at incu- 

 bation temperature. Besides, maintenance at this temperature 

 renders paraffining or sealing less imperative for the prevention 

 of drying. Freshly transplanted cultures that were incubated for 

 twenty-four hours at 37.5°C., and then kept at room temperature, 

 showed fair growth after 4 weeks. Ice box temperature will 

 kill most strains of the meningococcus in a comparatively short 

 time. Cultures grown at 37.5°C. for twenty-four hours, and then 

 transferred to the ice box, grew well after five days, but after 

 ten days about twenty-five per cent failed to show any growth 

 at all, 50 per cent showed scanty growth, and only 25 per cent 

 a fair growth. 



