55. EXPLANTING AND CULTIVATING 

 EARLY CHICK EMBRYOS IN VITRO 



Spratt (1947, 1948) has shown that the bacteriolytic property of egg albumen used in his 

 various culture media makes it unnecessary 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 included, the glassware and the instruments are dry sterilized and the solutions 

 are autoclaved. 



Preparation of the equipment: 



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

 aside the glassware and instruments to dry 



on a clean towel. "'.is'; [.'^V"-::n.. 



2. Prepare moist-chamber culture dishes as y^'H'!t-;::->i^i:>yiivKi-l'::-:','^'^::\ 



follows: Place a moist cotton ring in a ^''^'i''' /^^^\ '^"'^ftit '.•'-'■'■ 



Petri dish, set a watch crystal with con- Ov-/? . ' / *«C I ' '■&■:'■'' 



cave side up in the center of the petri dish, /vM •■ ■ \ \^t-- I •. ''^VJ 



.' •■••'HV':-. ■ V -Viv ■ -■ ^*-"\ 



and replace the cover of the dish. All ;--'..';Sv .. V-. Sij- / -;..■. 



parts must have been previously sterilized. /': vV "rA ' ■• *'\> \ 



(Method of Fell and Robison, 1929 and '^-^v.^'*'-'-^^---:.;^' -vr 



Waddington, 1932.) ^"""^^^^^ 



I I W 



Preparation of the culture media: ' ' I 



' I mm . I 



Physiologically balanced "Ringer's" for chick 



embryos: 



a. Unbuffered isotonic salt (Spratt, 1947) 



NaCl 0.7 grams Camera lucida drawing of a typical 10- 



KCl 0. 042 grams hour-old, living explant of the anterior 



CaCK 0. 024 grams ''°'''°" °^ ° '^°'^ head-process blosto- 



r-. i_i J- ^-11 J derm which was transected about 0.4 



Doubly distilled 



' mm. postenor to the node. This result 



^^ ^^ J-UU. U CC y^^g obtained after carrying out verba- 



b. Buffered isotonic sa lt (Romanoff, ""^ ""^ '""'""' outlined for saline- 



— — — agar-albumen media. With a little 



practice, the student can accumulate 



U. Ot> /o many similar coses of beautifully 



'^C'- 0. 031% symmetrical and essentially normal 



CaCl 0. 02% morphogenesis. 



MgClp-bH^O 0.01% 



^ i. C '" (From Spratt 1947 



KH2PO4 0. 02% Science. 106:452) 



Na2KP04- I2H2O. . . 0.08% 



Glucose 0. 2% 



Made up in glass distilled water. 



(Note: Romanoff found that this medium could sustain a nearly constant rate of res- 

 piration of all embryonic tissues tested, without providing any pH change.) 



^- Ringer-al bumen-Agar medium : This medium is made up from 2 components, as 

 follows. 



a-. Ringer-albumen compon ent: 



(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. 



* The author is indebted to Dr. N. T. Spratt, Jr. for these procedures, and for helpful suggestions in the organization of this part 

 of the exercise on the chick embryo. 



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