U52 



EXPERIMENTAL CHICK EMBRYOLOGY 



EXPLANTING AND CULTIVATING EARLY CHICK EMBRYOS IN VITRO* 



Spratt (19't7, 19^) has shovm that the bacteriolytic property of egg albiimen used In 

 hla various culture media makes it unnecesaary to include the elaborate sterilization pro- 

 cedures used in the classical tissue culture 

 methods. Tap water can be used in the place of 

 distilled water when egg-albumen is included In 

 the medium. When the egg-albumen is not in- 

 cluded, the glassware and the instruments are 

 dry sterilized and the solutions are autoclaved. rl'v''.'-' ■"■"."'■;... 



Preparation of the equipment : 



1. Wash, rinse (in hot running water), 

 and set aside the glassware and in- 

 struments to dry on a clean towel. 



2. Prepare moist-chamber culture dishes 

 as follows: Place a moist cotton 

 ring in a Petri dish, set a watch 

 crystal with concave side up in the 

 center of the petri dish, and replace 

 the cover of the dish. All parts 

 must have been previously sterilized. 

 (Method of Fell and Eoblson, I929 

 and Waddington, 1952.) 



Preparation of the culture media : 



Physiologically balanced "Ringer's" for 

 chick embryos : 



A. Unbuffered isotonic salt (Spratt, 

 1947) 



NaCl 0.9 grams 



KCl 0.01+2 grams 



CaClg O.O2I+ grams 



Doubly distilled 



water 100.0 cc. 



B. Buffered isotonic salt (Bomanoff, 



191+5) , 



NaCl! 0.86^ 



KCl [ 0.051^ 



CaCl ! . 02^ 



MgClg • 6^0. . 01^ 



K^PO^ 0.02^ 



Na2KP01+'12B^0 0.08^ 



Glucose 0.2^ 



Made up in glass distilled water. 



■■■';>■ 





mr' 



va? 



Camera luci 

 hour-old, 1 

 terlor port 

 blastoderm 

 0.4 ram. pes 

 result, was 

 verbatim th 

 sallne-agar 

 tie practlc 

 late many s 

 symmetrical 

 morphogenes 



da drawing o 

 iving explan 

 ion of a sho 

 which was tr 

 terlor to th 

 obtained aft 

 e method out 



albumen med 

 e, the stude 

 imilar cases 



and essentl 

 is. 



f a typical 10- 

 t of the an- 

 rt head-process 

 ansected about 

 e node. This 

 er carrying out 

 lined for 

 la. With a lit- 

 nt can accumu- 



of beautifully 

 ally normal 



From Spratt l^k^ 

 Science. 106:U52 



(Note: Romanoff found that this medium 

 respiration of all embryonic tie 

 change. ) 



could sustain a nearly constant rate of 

 sues tested, without providing any pH 



1, Ringer -albumen -Agar medium : This medium is made up from 2 components, as fol- 

 lows. 

 A. Ringer- albumen component : 



1. Separate the yolk from the white (albumen) of a fresh, unincubated egg. 



2. Add the albumen to 50 cc. of ordinary chick Ringer's in a 500 cc. Erlen- 

 meyer flask, previously sterilized. 



5. Stopper the flask and shake the contents vigorously for 1 minute. 



* The author is Indebted to Dr. N. T. Spratt, Jr. for these procedures, and for helpful 

 suggestions in the organization of this exercise on the chick embryo. 



