628 APPENDIX: LABORATORY METHODS 



(2) For colony counts and for studying the activities of pneumococ- 

 cal colonies in blood agar, the usual procedure is to dilute the culture 

 to an extent that will provide well-isolated colonies, using peptone 

 water as a diluent; measured amounts of the diluted culture, usually 0.5 

 cc, are transferred to duplicate Petri dishes, and as soon as possible, 

 liquid blood agar at a temperature of 45° to 50° is poured into the 

 dishes. The contents are mixed by gentle rotary motions of the plates, 

 care being taken to avoid splashing or bubbling of the medium. 



Blood Cultures (adapted from Zinsser and Bayne-Jones 1579 ) 



Blood is drawn in the usual manner, observing surgical precautions 

 and strict aseptic technique. After the blood is drawn, it should be im- 

 mediately transferred to the proper medium, for which the usual infu- 

 sion agar (as referred to under blood agar) and Pneumococcus bro^h 

 are equally satisfactory. 



At least six tubes of agar should be melted and immersed in water at 

 about 45°. Before blood is mixed with the medium, the agar should be 

 cooled to about 41°. The blood is added to the tubes in varying quanti- 

 ties, ranging from 0.25 to 1.0 cc. each, in order that different degrees 

 of concentration may be obtained. Mixing is accomplished by the usual 

 dipping and rotary motion, the formation of air-bubbles being thus 

 avoided. The mouth of each test tube should be flamed before pouring 

 the contents into the plates. 



Three flasks of broth suitable for development of Pneumococcus, 

 each containing 100 to 150 cc. of medium, should be inoculated with 

 varying quantities of blood — at least one of the flasks containing the 

 blood in high dilution. It may be advisable to place in the flasks of 

 broth, in addition to glucose, 1 gram of powdered calcium carbonate to 

 inhibit the action of acid on the pneumococci. 



For quantitative work, accurately measured amounts of blood should 

 be seeded in blood agar, preferably placing the blood in the Petri 

 dishes, pouring in the melted and cooled agar, and mixing with a gentle 

 rotary motion. 



///. Type Determination 

 Mouse Method (Sabin 1208 ) 



One cc. of a fresh sample of sputum is injected intraperitoneally into 

 a mouse. From 3 to 4 hours later, some of the peritoneal fluid is ob- 

 tained by puncture with a glass capillary. A glass slide is marked off 



