III. TYPE DETERMINATION 633 



serum, that is, the first drop is mixed with Type I antiserum, the sec- 

 ond drop with combined serum A (II, IV, V, and VII), the third drop 

 with B, et cetera. The preparations are stained with Loeffler's alkaline 

 methylene blue (2 drops to each mixture) and are covered at once with 

 cover-slips to prevent drying. Examination is made with the oil immer- 

 sion lens, with the light dimmed. When a positive reaction occurs, which 

 is usually within a few minutes, there is a decided swelling of the cap- 

 sule of the pneumococcus present. The swollen capsule is of a light 

 greenish-gray color, is much less translucent than one that is not swol- 

 len, and has a definite outline which is one of the most characteristic 

 features of a positive reaction. In the preparations in which no reaction 

 is evident, the capsule of the pneumococcus appears as a halo of re- 

 fracted light. In all preparations the body of the pneumococcus stains a 

 definite blue. If the reaction is observed in drop 1, then the pneumococ- 

 cus present is Type I and can be reported immediately ; if the reaction 

 is observed in drop 3, for example, the test is repeated using 2 drops of 

 the sputum mixed with Type III and Type VIII undiluted monovalent 

 rabbit serums, respectively; if the reaction is observed in drop 9, five 

 loopfuls of the sputum are mixed with Type XXVII, XXIX, XXX, 

 XXXI, and XXXII undiluted monovalent serums, respectively. Should 

 no reaction be seen on the first examination, the preparations are re- 

 examined at the end of 30 minutes. 



When dealing with sputums containing many Type III organisms, it 

 is necessary occasionally to dilute the sputum with salt solution before 

 any Quellung of the pneumococcus capsule becomes evident. In such in- 

 stances, when the regular Neufeld technique is used, the organisms form 

 large masses surrounded by much precipitated material, and no definite 

 swelling of the capsules can be seen. However, upon dilution of the 

 sputum and repetition of the test, the individual diplococci will usually 

 show a typical, positive reaction. 



The age of the specimen of sputum makes little difference in relation 

 to the success with which the Neufeld method is applied. Positive reac- 

 tions can be demonstrated on sputum 48 hours or more after collection 

 from the patient. Chart 2 shows the steps in the technique. 



Urine Test (Dochez and Avery 321 ) 



a. Unconcentrated urine. A specimen of urine is obtained as soon as 

 possible and cleared by centrifuging. The clear urine is mixed in quan- 

 tities of 0.5 cc. each with equal amounts of antipneumococcic serum of 

 the different types in a series of agglutination tubes. 



