II. ISOLATION OF PNEUMOCOCCUS 627 



with a sterile capillary pipette, spread on a slide, stained by Gram's 

 method, and examined microscopically to determine whether there is an 

 abundant growth of Pneumococcus present. If there is an abundant 

 growth of Pneumococcus alone, the mouse is killed and the determina- 

 tion of type carried out. If the growth is only moderate, or if other or- 

 ganisms are present in any quantity, additional time must be allowed 

 until subsequent examination of the peritoneal exudate shows an abun- 

 dant growth of Pneumococcus. It should be emphasized that undue 

 haste in killing the mouse is time lost in the end. 



b. Mouse necropsy. As soon as the mouse is killed or dies, the peri- 

 toneal cavity is opened under sterile precautions and cultures are made 

 from the exudate in plain broth and on one-half of a blood-agar plate. 

 Films are made and stained for microscopic examination by Gram's 

 stain and Hiss's capsule stain. The peritoneal exudate is then washed 

 out by means of a sterile glass pipette with 4 to 5 cc. of sterile salt solu- 

 tion, the washings being placed in a centrifuge tube. Cultures are then 

 made from the heart's blood in plain broth and on the other half of the 

 blood-agar plate. The essential steps in the procedures given above are 

 shown in the accompanying Chart 1. 



Plating Methods (Zinsser and Bayne-Jones 1579 ) 



Although the methods used in preparing and using blood agar vary, 

 the following illustrate accepted procedures: 



a. Preparation of blood agar 



(1) Melt 100 cc. of meat infusion or Douglas' agar (pH 7.4 to 7.8) 

 in a flask. 



(2) Allow to cool to 45° to 50°. 



(3) Add 5 cc. of sterile defibrinated rabbit or horse blood. 



(4) Mix and pour into Petri dishes or transfer into tubes for slants 

 as desired. 



(5) Incubate for 24 hours at 37° to test sterility. 



b. Use of blood agar 



(1) Material to be cultured on blood agar for the isolation or pre- 

 liminary identification of Pneumococcus may be streaked on the surface 

 of blood-agar plates, either by platinum loops or wires, or by placing a 

 drop of the material on the surface of the medium and then spreading 

 the inoculum over the surface with a bent, sterile capillary pipette. Di- 

 lutions of the material in peptone water or broth may be made prior to 

 streaking, depending upon the source. 



