APPLICATION OF COMPLEMENT FIXATION 195 



highly satisfactory if placed in amber ampoules in a refrigerator. If 

 considered advisable preservatives may be added such as 0.5 per cent, 

 phenol or 50 per cent, glycerol. If the serum is of high titer it may 

 be preserved by desiccation, particularly if frozen and dried in a vacuum 

 desiccator. Noguchi has obtained good results by drying the serum in 

 filter paper. The filter paper is subsequently cut into strips and titrated 

 by cutting measured lengths of the strips. We have found that this 

 does not permit of sufficiently accurate titration and also that the titer 

 is not well maintained. 



Preservation of Erythrocytes. If kept in a cool place without freezing, 

 sheep erythrocytes show slight hemolysis in a few days and well-marked 

 hemolysis in about a week. The cells oi other animals show variable degrees 

 of fragility, those of the dog being especially fragile. Various methods of 

 preserving sheep erythrocytes for the Wassermann test have been studied 

 by Reimann in this laboratory. The methods of particular value are preserva- 

 tion with formalin (Bernstein and Kaliski) and with the solutions of Rous 

 and Turner. For formalization the sheep blood is allowed to run directly 

 into formalin solution in the proportion of 0.5 c.c. of 40 per cent formaldehyde 

 solution to 400 c.c. blood. The blood is then defibrinated by shaking with glass 

 beads, preserved in the refrigerator and before use washed three times with 

 saline for use. The method of preservation as worked out by Rous and 

 Turner is carried out in the following manner: The sheep is bled directly into 

 Locke's solution containing i per cent, sodium citrate, in the proportion of 

 i part of blood to 4 parts of solution. The corpuscles are separated by rapid 

 centrifugalization and carefully washed three times in Locke's solution containing 

 0.25 per cent, gelatin. The cells are then placed in ampoules in a layer not 

 more than 2 mm. in depth and covered with saccharose-Locke solution to a 

 depth of about 2 cm.; the ampoules are sealed and stored at a temperature of 

 5 C. to 6 C. Just prior to use the cells are washed with .85 per cent, saline 

 to remove the saccharose solution, and proper dilution effected with saline. 

 Strict asepsis is to be observed. 



THE SOLUTIONS 



Locke-sodium citrate solution : 



Sodium citrate 10 grams 



Sodium chloride 9.2 grams 



Sodium bicarb 0.05 gram 



Potassium chloride o.i gram 



Calcium cholride o.i gram 



Aq. dest. q.s. ad 1000 c.c. 



Locke-gelatin solution: 



Gelatin 2.5 grams 



Sodium chloride 9.2. grams 



Sodium bicarb .* 0.05 gram 



Potassium chloride o.i gram 



Calcium chloride o.i gram 



Aq. q.s. ad 1000 c.c. 



The Locke and saccharose solutions are sterilized separately and used in 

 the proportion of 2.8 c.c. of the saccharose solution and 7.5 c.c, of the 

 Locke's solution. 



Saccharose solution : 



Saccharose 103.0 grams 



Aq. q.s. ad 1000 c.c. 



Locke's solution: 



Sodium chloride 9.2 grams 



Sodium bicarb 0.05 gram 



Potassium chloride o.i gram 



Calcium chloride o.i gram 



Aq. q.s. ad 1000 c.c. 



