664 APPENDIX: LABORATORY METHODS 



It has been found advisable to have available a supply of buffered glu- 

 cose solution to administer intravenously to the occasional animal suf- 

 fering severe systemic shock after having been bled. A minimum of 20 

 cc. of blood is removed from each rabbit and placed in 50 cc. centrifuge 

 tubes, and slanted until coagulation has occurred. After standing for a 

 few hours at room temperature, the clots are broken, and the tubes of 

 blood stored overnight at 4°. The next morning the serum is drawn off, 

 centrifuged, pooled, and preserved with 0.3 per cent (final concentra- 

 tion) of tricresol. 



d. Testing of serum. The pooled serum of each type is tested for 

 type-specific Neufeld reactions and for heterologous reactions with 

 known closely related types. For these tests, mice are injected intra- 

 peritoneally with cultures of the types of Pneumococcus desired, and 

 the peritoneal exudate washed out with saline solution about six hours 

 after inoculation. Neufeld tests are set up in the usual manner, and if 

 satisfactory results are obtained and no cross-reactions observed, the 

 serum may be either filled immediately into separate containers, or 

 added to the stock supply of serum of the respective types. 



e. Dilution of serum. It is possible, after high titered serums are ob- 

 tained, to obtain satisfactory Neufeld reactions with type-specific se- 

 rums diluted with normal rabbit serum. The degree of dilution must be 

 determined by tests, taking into consideration the amount of dilution 

 which may occur in making the pools used in routine type determination 

 in the diagnostic laboratory. 



Note: Since diagnostic serums have been made subject to Federal li- 

 cense, the method of testing prescribed by the National Institute of 

 Health (Minimum Requirements for Pneumococcus Typing Serum, 

 Jan. 17, 1939) has superseded that described in paragraph "d" 

 above. The method is also described by Dr. Bernice E. Eddy, Public 

 Health Reports, 55 (9):347, Mar. 1, 1940. 



E. S. R. 

 L. A. B. 

 March, 19^1 



