III. TYPE DETERMINATION 629 



into four parts, and a minute drop of the peritoneal fluid is expelled on 

 each one of the four partitions. The first is smeared with saline solution 

 for control, and the others with a loopful of 1:10 dilution of Type I 

 and of Type II, and a 1:5 dilution of Type III diagnostic serums, re- 

 spectively. These dilutions of serum are chosen largely to eliminate 

 group agglutinins. Thin smears are made, allowed to dry, and fixed by 

 passing the slide through a flame; they are then stained for from 20 to 

 30 seconds with a fuchsin solution (10 cc. saturated alcoholic solution 

 of basic fuchsin plus 90 cc. of water) or any other available stain. The 

 stain is washed off in water or 20 per cent copper sulfate solution, and 

 the smears are examined with the oil immersion lens. If a specific ag- 

 glutination reaction is observed in one of the smears with diagnostic se- 

 rum, the organism is of that type. If no reaction occurs in any of the 

 smears and numerous pneumococci are clearly seen, a diagnosis of 

 Group IV is suggested. When it is desired to know whether the organ- 

 ism is one of the fixed types of Group IV (especially those for which 

 concentrated antiserums are available) a similar procedure is carried 

 out with the homologous immune serums. 



Bacteria other than pneumococci in the sputum as well as avirulent 

 forms of pneumococci may occur in clumps in the peritoneal exudate ; 

 but these differ in appearance from those produced by specific agglu- 

 tination and can be distinguished further by their occurrence in the sa- 

 line control smear. Unless a fresh sample of sputum is used, many of 

 the organisms will have undergone autolysis, and therefore more time 

 must be allowed for growth. Since the mouse is not killed, another type 

 determination can be made if the first should show insufficient organ- 

 isms, and then after the death of the mouse, the type may be confirmed. 



In the case of Type III pneumococci, sufficient organisms are usu- 

 ally present even two hours after injection. The appearance of the spe- 

 cific reaction with Type III pneumococci, primarily on account of the 

 larger size of the capsule, differs somewhat from that obtained with 

 pneumococci of other types ; the organisms are farther apart in the ag- 

 glutinated clumps which occur in mucoid strands. When diagnostic se- 

 rums for pneumococci of types formerly in Group IV are used, a dilu- 

 tion must be chosen that fails to show any cross-agglutination with any 

 other type. 



The peritoneal exudate may contain too many organisms four hours 

 after inoculation or after death of the mouse, so that it may be neces- 

 sary to dilute the fluid in the capillary tube with saline solution in order 

 to obtain a correct typing. This method is illustrated in Chart 1. 



