EXPLANTING AND CULTIVATING 



433 



drop. During the preliminary interval the cells of the explant will have had 

 an opportunity to become adherent to the coverslip, and the whole explant 

 will be visible under the microscope through the thickness of the coverslip. 



If the explant is transferred to fresh, sterile medium every 3 to 4 days, it 

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



For such in vitro studies it is suggested that the whole or parts of the eye of 

 embryos ranging from 1 to 7 days be used (Dorris, 1938). The eyes may be 

 carefully dissected out in sterile chick Ringer's using fine glass needles. 

 When whole eyes are transplanted, remove as much of the adherent mesen- 

 chyme as possible, leaving only the ectoderm immediately covering the optic 

 cup. When parts of the optic cup are explanted, it is first necessary to punc- 

 ture the border of the cup with a glass needle, impale the lens on the needle, 

 and pull it out. With the cup in position, the needle can be inserted between 

 the two layers at the chorioid fissures, thus isolating the retina which can be 

 removed as a firm cup-shaped structure. In the older eyes the lens can be 

 removed by dissecting away the overlying ectoderm, trimming the edge of 

 the cup with fine scissors, and then separating the two layers with fine glass 

 needles. The cup, or parts of either layer, can then be cut into smaller 

 pieces for explantation. 



This type of study provides information relative to the self-differentiating 

 capacities of whole eyes, and (in explantation of pieces) to the capacity for 

 independent differentiation of the parts of the eye. 



DISCUSSION: 



The technique of tissue culture was first used by Dr. Ross Harrison in 1907. Most of 

 the work that has been done since with this technique has been with isolated cells of the 

 relatively older (4 to 8 days) chick embryos. Only recently has Spratt (1947, 1948) re- 

 vitalized interest in this technique by finding that the entire blastoderm of the early (18 

 to 24 hour incubated embryo) stages could be cultured in vitro, either in toto or in 

 transected parts. 



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Camera lucida drawings of the anterior portion of a 

 living 4-somite blastoderm explanted to a non- 

 nutrient saline-agar medium: a, at the time of ex- 

 plantation; b, after 20 - hours' cultivation. Note 

 the failure of development and the loss of organiza- 

 tion which had been attained at the time of embryo 

 was removed from its normal food supply - the yolk 

 and albumen. Note also that presence of part of the 

 opaque area has not prevented the characteristic 

 "degenerative" changes. 



From Spratt 1948: Jour. Exp. Zool. 107:39 



Camera lucida drawings of a living ex- 

 plant to a yolk-albumen extract saline- 

 agar medium, a, at the time of explan- 

 tation. b, after 55 hours' incubation. 

 Note the remarkably normal morpho- 

 genesis, differentiation, and increase in 

 size of the explant. The heart was beating 

 rhythmically when the drawing was made. 



From Spratt 1947: 

 Jour. Exp. Zool. 106:345 



