THE TECHNIQUE OF SERUM REACTIONS 329 



a 1 per cent alcoholic solution of cholesterin is added. These various 

 portions are then diluted with 5 parts of saline as described below, and 

 set up against a standard extract. At least two such extracts should be 

 used in each test. 



EXTRACT DILUTION. The alcoholic cholesterinized extract is diluted 

 with 5 parts of saline as follows: The required amount of extract is 

 placed in an Erlenmeyer flask ; to it is rapidly added from a burette 

 an equal volume of saline. It is shaken gently and allowed to stand 

 10 minutes, then the remaining 4 volumes of saline are rapidly run 

 in. It is again shaken and is ready for use. 



SERUM. Serum should be as fresh as possible. Three or four days 

 is not too old. A slight degree of hemolysis does not interfere. Before 

 use in the test, it is heated for y 2 hour at 55 to 56 C. and should 

 not be used sooner than 3 hours after heating. Sachs and Georgi 21 

 recommend that spinal fluids should be used undiluted in amounts of 

 1 c.c. and 0.5 c.c. 



Saline. 0.85 per cent sodium chlorid in distilled water. Should 

 be sterile and as fresh as possible. 



Test. 0.1 c.c. serum is diluted with 0.9 c.c. saline and to this is 

 added 0.5 c.c. extract dilution. On each serum a control should be 

 set up consisting of 0.1 c.c. serum -f- 0.9 c.c. saline + 0.5 c.c. of 95 per 

 cent alcohol, diluted 1 :6 with saline. Each extract dilution should be 

 controlled by a tube containing 0.5 c.c. extract dilution + 1 c.c. saline. 



The tubes are thoroughly shaken and placed in the incubator at 

 37.5 C. for 20 hours. A preliminary reading may now be made; 

 then the tubes are placed at 14 to 18 C., or in the ice-box for 20 

 hours, and the final and decisive readings are taken. 



The reactions present appearances similar to macroscopic bacterial 

 agglutination or precipitin reactions, and are read with the naked 

 eye, the positives showing varying amounts of precipitates and the 

 negatives remaining opalescent as at the beginning of the test. Sus- 

 picious tests are centrifuged at moderate speed for a few minutes, and 

 are proved positive or negative by the fact that in the positives after 

 centrifuging a few definite white compact flocculi can be shaken from 

 the bottom of the tube, whereas the negatives, at most, show a slight 

 grayish sediment which disperses on shaking. The serum controls 

 should remain clear. If a precipitate does occur, the serum is unsuit- 

 able and another specimen must be obtained. The extract controls 

 should remain diffusely opalescent, and should show no precipitate. 



21 Sachs-Georgi, Med. Klinik, No. 33, 1918, 805; Parker and Haigh, un- 

 published. 



