634 APPENDIX: LABORATORY METHODS 



The intensity of the reaction may vary from an almost imperceptible 

 cloud to a heavy, flocculent precipitate. The reaction may occur in some 

 instances immediately on mixing the urine and serum, or it may require 

 incubation in the water-bath at 37° for one hour. Prolonged incubation, 

 however, must be avoided, since bacterial growth may obscure the test. 

 In the precipitin reaction it is essential that all the reagents used, in- 

 cluding the immune serums, should be water-clear. In case the reaction 

 is negative or so faint as to be indefinite with the whole urine, the fol- 

 lowing method of concentrating the urine may be employed: 



b. Concentrated urine for precipitin test. Twenty-five cc. or more of 

 a 24-hour specimen of urine, with the addition of a few drops of acetic 

 acid, are evaporated to a volume of about 5 cc, filtered through paper 

 to remove any precipitate of albumin that may occur, and the filtrate is 

 added to 8 to 10 volumes of 95 per cent alcohol. The precipitate which 

 forms is collected by centrifuging and rapidly dried to remove the ex- 

 cess of alcohol, and the residue extracted with 2 to 3 cc. of salt solution, 

 which redissolves the specific substance. Any undissolved material is re- 

 moved by centrifuging, and the clear salt solution extract is used for 

 the precipitin test. Chart 3 shows the procedure. 



IV. Isolation of Several Components of Pneumococcus 



Medium for the Production of Culture of Standard Maximal 

 Density, Virulence, and Polysaccharide Content (O'Meara and 

 Brown 1031 ) 



Witte peptone 10.0 grams 

 Difco proteose peptone, Fairchild, Parke-Davis, or Neopeptone 40.0 grams 



Glucose 5.0 grams 



Sodium chloride 5.0 grams 



Sodium bicarbonate 5.0 grams 



Potassium phosphate (K 2 HP0 4 ) 2.0 grams 



Thioglycollic acid 0.1 grams 

 Distilled water to 1000.0 cc. 



The Witte peptone and sodium chloride are dissolved in water and 

 the reaction of the solution adjusted to pH 8.0. The sodium bicar- 

 bonate, glucose, potassium phosphate, and extra peptone (usually Difco 

 proteose) are then added and when they have dissolved the broth is 

 clarified by passing through filter paper. Thioglycollic acid is finally 

 added and the medium sterilized by candling. During the entire course 

 of preparation the temperature of the medium is not allowed to rise 

 above 50°. 



