EXPERIMENTAL CHICK EMBRYOLOGY l4^ 



k. Add 0.5 ng- of phenol red. This will cause the medium to hecome slightly 

 purple, when the alhumen is present, so that it will be the easier to see 

 the white explaxits . 



B. Binder- Agar component : 



1. Place from O.I5 to 0.15 grams of powdered (USP Xl) Agar in a small Erlen- 

 meyer flask along with 50 cc. of chick Ringer's solution. The Agar may 

 be increased to 0.2 or 0.**- grams, in which case a much firmer medium that 

 is more easily handled, will result. 



2. Bring the Blnger-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 kO° to 45°C. 



5- Add to this Ringer-Agar 20 cc. of the El nger- Albumen mixture. Exclude 

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



C. The medium : 



When the two components are mixed the medium Is completed. Po\ir ap- 

 proximately 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 ajid allow the medium to gel (about 50 minutes to 1 hour) be- 

 fore 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^ NaHCO^, sterilize by filtration (if possi- 

 ble) through a Berkefeld filter, and then saturate with CO^. 



e. Cool the saline solution down to 4o°C. and add 1 to 2 cc. of the sterile 

 bicarbonate solution. 



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

 it will slowly set to form a soft gel. 



5. Sallne-Agar "B" : (See White, 19if6) 



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



NaCl 0.85 grams 



KCl 0.057 grams 



Ca(N05)2'%0 0.021 grams 



MgSOij^ 0.027 grams 



Fe(N0,),-9B20 O.OOOli^ 



b. Add 0.25 grams of Agar, gently boll to dissolve Agar (avoid charring). 



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

 medium. 



Nag HPOj^ -12320 0.01^5 grams 



K^POi^ 0.0026 grams 



NaHCOj 0.055 



d. When the saline-agar is cooled to Uo°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 • 



k. Saline-agar plus yolk-albumen extract : 



a. Mix the entire contents of a fresh, unincubated egg with 50 cc. of saline- 

 agar "A" (above). Shake well In an Erlenmeyer flask. 



b. Centrifuge the mixture at 2000 R.P.M. for 50 minutes to 1 hour. 



c. Draw off some of the supernatant fluid and mix with an equal volume of 

 saline-agar "A" kept at 55° to Uo°C., on a warming stage. 



d. Place 2 cc. of this mixture In each of the previously prepared culture 

 (watch crystal) dishes. 



(The amounts of yolk and of albumen may be varied to determine their 

 relative value in growth. ) 



