APPENDIX 



SPECIAL METHODS USED IN THE STUDY OF 

 PNEUMOCOCCUS AND IN THE PREPARA- 

 TION OF ANTIPNEUMOCOCCIC SERUM 



/. Media 



Pneumococcus Broth for All General Purposes (Rockefeller In- 

 stitute. Courtesy of Miss Dorothy Sloan) 



Beef heart 1 pound 



Peptone (Witte or Pfanstiehl) 10 grams 



Sodium chloride 5 grams 

 Tap water 1 liter 



The beef hearts are obtained directly from the slaughter-house and 

 after immediately removing all fat, the muscle is ground fine. For every 

 10 liters of water add 10 lb. of minced heart muscle and 500 cc. extra 

 of water. Stir and skim off fat rising to the surface. Repeat stirring and 

 skimming ten times. Place in refrigerator overnight, heat to 85° for 

 one-half hour, then filter through Prat-Dumas No. 50 or other coarse 

 filter paper. Add peptone and salt, reheat to boiling and add sufficient 

 N sodium hydroxide to bring the reaction of the medium to pH 8.2. Boil 

 for 5 minutes and filter immediately through coarse paper. Sterilize in 

 the Arnold sterilizer for one hour on each of 3 successive days. The 

 final reaction should be pH 7.8. 



For the production of massive amounts of pneumococci, the medium 

 in which the Pfanstiehl peptone is used is inoculated with 100 to 150 

 cc. of an 8-hour culture in plain broth for every 3,000 cc. of medium. 

 The maximal growth will have occurred in 8 to 10 hours under these 

 circumstances, but the yield of organisms is greatly increased by the 

 addition of 0.10 per cent dextrose after 8 hours' incubation. Following 

 the addition of dextrose, incubation is carried out for one to one and 

 one-half hour; longer incubation will render the medium too acid and 

 rapid autolysis will result. The resulting growth is immediately col- 

 lected by means of a specially constructed Sharpies centrifuge. 



Nutrient Broth for Mass Cultures for the Production of Solu- 

 ble Specific Substances (Courtesy of Dr. F. D. Hager) 



a. Preparation of infusion. Fresh horse meat is ground and mixed 



