626 APPENDIX: LABORATORY METHODS 



following medium devised by Hiss is used, a positive reaction being in- 

 dicated by the production of acid and the coagulation of serum protein. 

 Clear beef serum is added to 2 or 3 volumes of distilled water. Heat the 

 mixture for 15 minutes in an Arnold sterilizer at 100° to destroy fer- 

 ments present in the serum. Add 5 per cent aqueous litmus solution to 

 a concentration of 1 per cent or an amount sufficient to give a deep blue 

 color. Add inulin (C.P.) to the serum water to a concentration of 1 per 

 cent. The inulin solution may best be sterilized by autoclaving at 15 

 pounds pressure for 15 minutes. Sterilize the inulin serum water by the 

 fractional method at 100°. 



Author's Note: An alcoholic solution of bromcresol purple is preferable to 

 the litmus solution as an indicator. 



//. Isolation of Pneumococcus 

 Mouse Method (Rockefeller Monograph No.'7 36 ) 



a. Mouse inoculation. A small portion of the sputum, about the size 

 of a bean, is selected and washed through three or four changes of ster- 

 ile salt solution in sterile Esmarch or Petri dishes to remove surface 

 contaminations. When the sputum is too friable or when the specimen is 

 relatively free from secondary organisms, this washing process may be 

 omitted. In either event, the kernel of sputum selected is transferred to 

 a sterile mortar, ground up, and emulsified with about 1.0 cubic centi- 

 meter of sterile bouillon or salt solution, added drop by drop, until a 

 homogeneous emulsion is obtained that will readily pass through the 

 needle of a small syringe. With a sterile syringe, 0.5 to 1.0 cc. of this 

 emulsion is inoculated intraperitoneally into a white mouse. Pneumococ- 

 cus grows rapidly in the mouse peritoneum, while the majority of other 

 organisms rapidly die off, with the exception of Friedlander's bacillus, 

 Bacillus influenzae, and occasionally Micrococcus catarrhalis, Staphylo- 

 coccus, and Streptococcus. Pneumococcal invasion of the blood stream 

 also occurs early. Bacillus influenzae, if present, likewise invades the 

 blood stream ; other organisms, as a rule, do not. The time elapsing be- 

 fore there is sufficient growth of Pneumococcus in the mouse peritoneum 

 for the satisfactory determination of type varies with the individual 

 case depending upon the abundance of pneumococci in the specimen of 

 sputum and the virulence and invasiveness of the strain present. It may 

 be from 5 to 24 hours, averaging 6 to 8 hours with the parasitic fixed 

 Types I, II, and III. As soon as the injected mouse appears sick, a 

 drop of peritoneal exudate is removed by means of peritoneal puncture 



