kj,6 EXPERIMENTAL CHICK EMBRYOLOGY 



Varlationa In the procedure : 



(1) Glucoae medium : Some interesting effects can be seen if 100 to 800 mg. per- 

 cent of glucose la added to the Binger-Agar medium as a substitute for the 

 albumen. Such a medium should be buffered to pH 7-8 to 8. 5* (Sterilize the 

 medium before adding the sterile buffer.) Spratt (19'+8, p. 6h) gives the 

 following formula for his "minimal medium": 



a. Add 0.10 to 0.15 grams powdered Agar (U.S. P. XI) to 56 cc. of chick 

 Singer's, shake, add 0.5'<- grama of glucose, and 0.001 gram percent of 

 phenol red. This mixture is autoclaved in a small Erlenmeyer flask for 

 8 to 10 minutes. 



b. Cool the mixture to Uo°C. 



c. Add 2 cc. of 0.290 grams percent of Nag HPOjj. -12320, and 0.052 grams per- 

 cent of KE^POjj. (previously mixed). 



d. Add 2 cc. of 1.10 grams percent of NaffiOj. 



e. Saturate the mixture with CC^. 



f. Immediately place 2 cc. of the prepared medium in each watch crystal. 



(2) Medium with other sugars : 



a. Sugars which serve as exogenous energy supply: mannose, fructose, mal- 

 tose. 



b. Sugars which do not serve as an exogenous energy source: lactose, suc- 

 rose. 



(5) Medium with amino acids added : See Spratt (19^) for list of amino acids 

 which can be tested for nutrient value. 



{k) Cultivation of chick structures on plasma-embryonic extract : 



The classic method of explant culturlng has been on plasma clot or on a 

 mixture of chick Elnger's and embryonic extracts. It is suggested that the 

 student follow this procedure to compare its benefits with those described 

 above . 



1. Place whole chick embryos (5 to 8 days) in a sterile beaker and cover 

 with an equivalent volume of sterile lyrode's solution, or sterile 

 chick Ringer's. The embryos must be finely chopped. 



2. Centrifuge the embryonic mash at 25OO r.p.m. for I5 minutes. 



5. '<^lth sterile pipette, remove the supematent fluid and, without dilu- 

 tion, place in culture dishes. About 1 to 2 cc. per dish. 

 h. The culture dishes may be of several types, all dry sterilized. 



a. Depression slides which are to be covered with coversllp, ringed 

 with vaseline. 



b. Coverslips ringed with paraffin (to limit the spread of the culture 

 medium) inverted over a depression slide (hanging drop method) 

 which acts as a moist chamber. 



5. With sterile pipette transfer the excised anlage* to the culture medium, 

 seal, and Incubate for from 1 to 8 days. 



In the case of the coversllp (hanging drop) method of culturlng, 

 add the culture medium (within the paraffin ring), transfer the anlage 

 to the medium, ring the margin of a depression slide with white vaseline, 

 and invert the depression slide and bring it down over the coversllp so 

 that it covers the explant. Gently press the depression slide into 

 place, and transfer (without righting the depression slide) the whole to 

 the Incubator at 58'-'C. After the explant has been in the incubator for 

 6 to 8 hours, the depression slide and coveralip may be quickly inverted, 

 whereupon the culture becomes a hanging drop. During the preliminary 

 Interval the cells of the explant will have had an opportunity to become 

 adherent to the coversllp, and the whole explant will be visible under 

 the microscope through the thickness of the coversllp. 



If the explant Is transferred to fresh, sterile medium every 3 to 'i- 

 days. It may be carried for longer periods (e.g., 18 days). 



