EXPLANTING AND CULTIVATING 429 



(3) Stopper the flask and shake the contents vigorously for 1 minute. 



(4) Add 0. 5 mg. of phenol red. This will cause the medium to become 

 slightly purple, when the albumen is present, so that it will be the easier 

 to see the white explants. 



b. Ringer-Agar component : 



(1) Place from 0. 13 to 0. 15 grams of powdered (USP XI) Agar in a small 

 Erlenmeyer flask along with 30 cc. of chick Ringer's solution. The Agar 

 may be increased to 0. 2 or 0. 4 grams, in which case a much firmer 

 medium that is more easily handled, will result. 



(2) Bring the Ringer-Agar mixture to a slow boil over a small flame of the 

 Bunsen burner (or in a water bath). Agitate constantly to prevent the 

 Agar from sticking or charring. After the Agar is completely dissolved, 

 cool the mixture slowly down to 40° to 45*-'C. 



(3) Add to this Ringer-Agar 20 cc. of the Ringer -Albumen mixture. Exclude 

 the foamy portion. Gently shake to mix the two media. 



c. The medium : 



When the two components are mixed the medium is completed. Pour approx- 

 imately 2 cc. of this mixture into each previously prepared, sterile, watch 

 crystal which is supported in a cotton moat in a Petri dish. Cover the Petri 

 dish and allow the medium to gel (about 30 minutes to 1 hour) before moving 

 the dishes. 



2. Saline-agar "A": 



a. Make up 100 cc. of unbuffered chick "Ringer's" ("A" on preceding page). 



b. Add 0. 25 grams of powdered Agar (USP XI). 



c. Sterilize by boiling (gently) or autoclaving. Avoid charring the Agar. 



d. Make up a stock solution of 1% NaHCOj, sterilize by filtration (if possible) 

 through a Berkefeld filter, and then saturate with COo. 



e. Cool the saline solution down to 40°C. and add 1 to 2 cc. of the sterile bi- 

 carbonate solution. 



f. With sterile pipette place 2 cc. of the medium in culture dishes, where it 

 will slowly set to form a soft gel. 



3. Saline-Agar "B" : (See White, 1946) 



a. Add the following salts to 100 cc. of double distilled water. 



NaCl 0. 83 grams 



KCl 0. 037 grams 



Ca(N03)2- H2O 0. 021 grams 



MgS04 0. 027 grams 



Fe(N03)3- 9H2O 0. 00014 



b. Add 0. 25 grams of Agar, gently boil to dissolve Agar (avoid charring). 



c. Prepare the buffer separately, and sterilize it before adding to the medium. 



Na2HOP^- I2H2O 0. 0145 grams 



KH2PO4 0. 0026 grams 



NaHC03 0. 055 



d. When the saline-agar is cooled to 40°C. add the buffer. 



e. It is advisable to add 0. 001 gram percent of phenol red. The pH range is 

 generally between 7. 5 and 8. 9. 



4. Saline-agar plus yolk-albumen extract: 



a. Place the entire contents of two fresh, unincubated eggs in a sterile 500 ml 

 Erlenmeyer flask. Stopper and shake vigorously to homogenize. 



b. Centrifuge portions of the above at 2-3000 rpm for about an hour. This much 

 more concentrated extract makes it possible to cultivate unincubated blasto- 

 derms, heretofore almost completely refractory, with almost 100% development 



