432 EXPLANTING AND CULTIVATING 



Procedure for culturing: 



1. Transfer the embryo, or parts, to be cultivated by means of a wide-mouthed 

 pipette to the surface of a culture medium, orient as desired, and flatten it out 

 by sucking away all excess superficial medium with a fine pipette. The embryo 

 (or part) should float on a few drops (2 cc. ) of culture medium. 



2. Cover the Petri dish and return it carefully to the incubator. 



Variations in the procedure: 



1. Glucose medium: Some interesting effects can be seen if 100 to 800 mg. percent 

 of glucose is added to the Ringer-Agar medium as a substitute for the albumen. 

 Such a medium should be buffered to pH 7. 8 to 8. 3. (Sterilize the medium before 

 adding the sterile buffer. ) Spratt (1948, p. 64) gives the following formula for 

 his "minimal medium": 



a. Add 0. 10 to 0. 15 grams powdered Agar (U. S. P. XI) to 36 cc. of chick 

 Ringer's, shake, add 0. 34 grams of glucose, and 0. 001 gram percent of 

 phenol red. This n:iixture is autoclaved in a small Erlenmeyer flask for 8 to 

 10 minutes. 



b. Cool the mixture to 40°C. 



c. Add 2 cc. of 0. 290 grams percent of Na^HPO^- IZH^O, and 0. 052 grams per- 

 cent of KH2PO^ (previously mixed). 



d. Add 2 cc. of 1. 10 grams percent of NaHC03. 



e. Saturate the mixture with COt. 



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, maltose. 



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



3. Medium with amino acids added : See Spratt (1948) for list of amino acids which 

 can be tested for nutrient value. 



4. Cultivation of chick structures on plasma-embryonic extract: 



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

 of chick Ringer's and embryonic extracts. It is suggested that the student follow 

 this procedure to compare its benefits with those described above. 



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

 an equivalent volume of sterile Tyrode's solution, or sterile chick Ringer's. 

 The embryos must be finely chopped. 



b. Centrifuge the embryonic mash at 2500 r. p. m. for 15 minutes. 



c. With sterile pipette, remove the supernatent fluid and, without dilution, place 

 in culture dishes. About 1 to 2 cc. per dish. 



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



(1) Depression slides which are to be covered with coverslip, ringed with 



vaseline. 

 (2) 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. 



e. 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 coverslip (hanging drop) method of culturing, add the cul- 

 ture medium (within the paraffin ring), transfer the anlage to the medium, 

 ring the margin of a depression slide with white vaseline, and invert the de- 

 pression slide and bring it down over the coverslip so that it covers the ex- 

 plant. Gently press the depression slide into place, and transfer (without 

 righting the depression slide) the whole to the incubator at 38 C. After the 

 explant has been in the incubator for 6 to 8 hours, the depression slide and 

 coverslip may be quickly inverted, whereupon the culture becomes a hanging 



