396 IMMUNOLOGY 



may be necessary to use another wire for getting the sputum off the 

 loop. To each bit of sputum an equal quantity of the undiluted 

 rabbit serum* (Type I serum to one, and Type II serum to the 

 other) and a loopful of standard alkaline methylene blue was 

 added. A special deep, large, hollow-ground glass slide, big enough 

 to cover both drops, was used; this special slide is convenient but 

 not essential. The edges of the slide are smeared with petrolatum, it 

 is placed over the cover slip, and the preparation is inverted. The 

 examination is made with the oil-immersion lens and an artificial 

 blue light. Although the reaction occurs almost immediately, it is 

 best to delay the examination for about two minutes to allow for 

 proper diffusion of the serum. 



''It is important to observe that ordinarily, in the hanging drop 

 preparation, pneumococci in sputum show no capsules; occa- 

 sionally a faint halo of light but without any definite outline may 

 be seen about the organism. However, in the type-specific mix- 

 tures of sputum and serum one finds the pneumococci surrounded 

 by peripheral zones of characteristic appearance and distinct out- 

 line. This peripheral zone consists of a refractile substance which 

 does not take the stain and which may be described as having a 

 ground-glass appearance; the organism within it is stained blue. 

 The size of this zone of 'quellung' varies in different sputums 

 with organisms of the same type; generally, Type II pneumococci 

 presented the larger zone. It is important to stress, however, that 

 it is not so much the size of the zone as its characteristic appearance 

 which determines a positive reaction. It is also necessary to 

 state that this reaction does not depend on agglutination of tlie 

 pneumococci, although this occasionally occurs in the sputum, but 

 on the appearance of the individual organisms; furthermore, this 

 appearance is so characteristic that the finding of even a single 

 diplococcus which shows it is sufficient to diagnose the type, as 

 shown in the accompanying illustrations." (Figs. 18 and 19.) A 

 more extensive discussion of pneumococcus typing is given by 

 Kolmer and Boerner (1941, p. 447). 



Typing of Hemolytic Streptococci has already been discussed 

 in Chapter XIX. It will be recalled that Lancefield, by means of 

 immune sera, has been able to divide the hemolytic streptococci 



♦Polyvalent serum representing known types is used before the final typing 

 is done with individual specific type serum. 



