646 APPENDIX: LABORATORY METHODS 



(3) Mixing the Two Components (first step) : After the serum 

 dilutions have been properly prepared, quantitative pipettes are used 

 to deliver 1 cc. volumes into small agglutination tubes (10 cm. by 1 

 cm.) arranged in suitable racks. Then, with a quantitative delivery 

 pipette, 1 cc. of the chosen SSS dilution is added to each, and the 

 tubes are shaken separately to ensure thorough mixing. The tubes 

 are incubated for 2 hours in a 37° water-bath, in which the water 

 extends half-way up to the level of the mixtures in the tubes, and 

 then stored in the cold (4°) overnight. 



(4) Adding the Indicating Serum (second step) : The next morn- 

 ing, the precipitates are separated from the supernatant fluids by, 

 centrifugation for 3 or 4 minutes, preferably in the cold or at room 

 temperature. The supernatant fluids are drained into clean, clear ag- 

 glutination tubes and to them is added the indicating serum in the 

 amount previously found to be optimal, and each tube is shaken thor- 

 oughly. The mixtures are incubated at 37° for 2 hours, and then, 

 without disturbing the precipitates, allowed to settle for 2 hours at 

 room temperature before recording the end point. 



(5) Reading the End Point: For consistent and reliable results, 

 the end point is considered as that dilution next in series to the one 

 containing a well-formed disc precipitate. With some serums and 

 some SSS preparations, the end point is extremely definite, that is, of 

 two adjacent tubes in a series, one contains a disc precipitate, the 

 other contains not even a trace of flocculation. On the other hand, 

 there are instances when the mixtures next in series to the last disc 

 precipitate are not clear, but contain a fine sediment which becomes 

 a swirl when the tube is shaken. These fine precipitates are disre- 

 garded, and the tube following the last disc precipitate in the series 

 is taken as the equivalent end point. 



(6) Calculating the Unit Value: The greater the unit value of any 

 antibody preparation, the smaller is the volume required to combine 

 equivalently with a given dilution of specific carbohydrate. Hence, an 

 inverse proportion expresses the relationship between the antibody 

 content of an immune serum and the percentage dilution that repre- 

 sents its equivalent end point. The unit value of an unknown serum is 

 found from the proportion 



A:A' = B':B,or A = A 



B 



in which A equals the unit value of the unknown serum, A' the unit 



