630 APPENDIX: LABORATORY METHODS 



Krumwiede Method (Krumwiede and Valentine 762 ) 



A small amount of sputum (3 to 10 cc.) is transferred to a test tube 

 and placed in boiling water for a few minutes until coagulation occurs. 

 The coagulum is broken up with a heavy platinum wire or glass rod and 

 sufficient saline solution added to give just enough fluid after centrifug- 

 ing. The suspension is again placed in boiling water for a few minutes 

 to extract the soluble antigen from the coagulum, and is shaken several 

 times during the heating process. The mixture is centrifuged and the 

 clear supernatant fluid used for the test. 



For the test, 0.2 cc. amounts of undiluted typing serum are placed in 

 narrow test tubes and the antigen carefully layered over the serum. The 

 tubes are placed in a water-bath at 50° to 55° and examined after sev- 

 eral minutes. A definite contact ring occurs in the tube containing ho- 

 mologous serum if the sputum is rich in antigen. The true ring is more 

 or less opaque and in the majority of positive reactions is evident in less 

 than 10 minutes. The success of the test depends upon the quality of 

 the sputum. If coagulation does not occur, there is little use in continu- 

 ing the test (see Chart 1). 



Neufeld Quellung Method (Beckler and MacLeod 96 ) 



There are variations in the application of the Neufeld Quellung phe- 

 nomenon to the determination of pneumococcal types in sputum sam- 

 ples. The one given here is the technique used in the Bacteriological 

 Laboratory of the Massachusetts Department of Public Health. 



Upon receipt of the sputum at the laboratory, stained liquid mounts 

 of the specimen are mixed with undiluted rabbit antiserums (Types I 

 to XXXII). Combinations of monovalent antiserums (rabbit) are used 

 instead of making separate preparations of the sputum with each of the 

 thirty-two monovalent serums. The combinations of serums used are: 



Type I 



A, Types II, IV, V, and VII 



B, Types III and VIII 



C, Types IX, XI, XIII, and XV 



D, Types Via, VIb, XVII, and XVIII 



E, Types XII, XIV, XVI, and XXVIII 



F, Types X, XIX, XX, and XXI 



G, Types XXII, XXIII, XXIV, and XXV 



H, Types XXVII, XXIX, XXX, XXXI, and XXXII 



Nine loopfuls of sputum are placed approximately one inch apart on a 

 9 by 2 inch glass slide; to each drop are added 2 loopfuls of the anti- 



