Immunity 479 



about twenty minutes, by which time the bodies of the pneumococci are usually 

 dissolved. It may be well at this point to test the reaction of the fluid. If acid, it 

 should be neutralized by the addition of a drop or two of an alkaline solution. 

 One-half of a cubic centimeter of the bile solution is mixed with an equal volume 

 of each of the immune serums corresponding to the types of pneumococci known. 

 That corresponding to the coccus tested, causes a delicate precipitate in the 

 medium. 



2. The Method by Agglutination. — When no ox bile is available, the fluid culture 

 cleared of blood-corpuscles by centrifugation is pipetted into small tubes, 0.5 

 cc. each, and each receives 0.5 cc. of the type immune serums. As the pneu- 

 mococci have not been dissolved, they agglutinate with the serum of their own 

 type. 



Krumwiede and Valentine* recommend the following method also 

 based upon the specific precipitation of the pneumococcic antigen 

 by the homologous type serum: 



From 3 to 10 cc. of the sputum, depending upon the amount available, is 

 poured from the sputum container into a test-tube. This is placed in boiling 

 water for several minutes or longer until a more, or less firm coagulum results, 

 which will occur if the specimen be a suitable one. The coagulum is then broken 

 up with a heavy platinum wire or glass rod, and saline is added. Just enough 

 saline should be added so that, on subsequent centrifuging, there will be suiEcient 

 fluid to carry out the test. If too much saline is added the resulting antigen 

 may be too dilute. 



In some instances little or no saline is necessary as sufficient fluid separates 

 from the coagulum. 



After the addition of saline, the tube is again placed in boiling water for a 

 few moments to extract the soluble antigen from the coagulum, the tube being 

 shaken several times during heating. 



The broken clot is then thrown down by the centrifuge and the clear superna- 

 tant fluid used for the test. To hasten the appearance of the reaction and to 

 obtain a reaction even should the antigen be dilute, the antigen is layered over 

 the "type" serums, using the latter undiluted. 



Two-tenths cubic centimeter of the three "type" serums are placed in narrow 

 test-tubes and the antigen added from a capillary pipet with a rubber teat. If 

 the tubes containing the serums are tilted, and the antigen dropped slowly on the 

 side of the tube just above the serum, no difficulty will be encountered in obtaining 

 sharp layers as the undiluted serum is sufficiently high in its specific gravity. The 

 tubes are then placed in a water-bath at so-5S°C. and observed after several 

 minutes. If a fixed type was present in the sputum, and should the sputum 

 have been rich in antigen, a "definite contact ring is seen in the tube contain- 

 ing the homologous serum. With sputum less rich in antigen, the ring may 

 develop more slowly, and it will be less marked. Some experience is necessary 

 in detecting the less marked contact rings and in differentiating these from an 

 apparent ring which may be confusing if one of the serums is darker in color giv- 

 ing them a sharper contrast with the supernatant antigen. The true ring is 

 more or less opaque, and this quality can be seen by tilting the tubes and looking 

 at the area of contact against a dark back-ground. 



Immunity. — Pneumonia in human beings terminates by crisis as 

 though from a supply of antitoxin or other immunizing agent sud- 

 denly liberated. Ordinarily the pneumococci are not taken up by 

 the leukocytes, but immediately following the crisis the leukocytes 

 become and seem to remain actively phagocytic and distend them- 

 selves with the organisms. Recovery is followed by immunity from 

 further infection by pneumococci of that particular type by which 

 the patient was infected, but not from the other types. Thus^are 

 explained the frequent "relapses" of the disease. 



* "Jour. Amer. Med. Assoc," 1918, lxx, p. 513- 



