2590 Journal of Applied Microscopy 



warm normal salt solution until the yolk lies just below the surface. The embryo 

 will usually lie uppermost. If it does not, it will revolve to this position, if the 

 chalazare be cut. 



With fine scissors rapidly cut completely around the embryo and so much of 

 the embryonic areas as it is wished to retain. Usually this cut is just outside 

 the area vasculosa. This is the most critical part of the entire operation, for as 

 soon as the vitelline membrane is punctured the yolk contents begin to flow out 

 and mix with the albumen and the embryo tends to revolve in the opposite direc- 

 tion. This latter is best prevented by placing the thumb or finger of the left 

 hand lightly upon the yolk before beginning to cut, thus holding it firmly in place. 



Having cut completely around the embryo, remove it, together with some of 

 the underlying yolk contents, to another dish of warm salt solution. This is best 

 done with a lifter or a small horn spoon. The embryo may now be separated 

 from the adhering yolk and from the vitelline membrane, either by gentle jets 

 from a pipette or by shaking gently. 



Now float the embryo upon a flat glass plate (a two by three inch slide is 

 convenient) and remove it from the salt solution. The embryo should be spread 

 out on the glass perfectly fiat without folds or wrinkles. 



Prepare previously a square of unglazed paper (thin filter paper, ordinary 

 toilet paper, or thin tissue meets the requirements) one to two inches square, ac- 

 cording to size of embryo. In the center of this cut an oval hole larger than 

 the embryo and area pellucida. Put this dry paper over the embryo as it lies on 

 the glass plate. The embryonic area will adhere to the paper while the embryo 

 is suspended without any distortion or strain in the hole. 



With a pipette put a few drops of fixative on the area of paper to which the 

 embryonic area is attached and let stand for one or two minutes only. In this 

 time the embryonic area will become fixed to the paper, but not to the glass. 



Slowly immerse the plate in the fixative when the paper with embryo attached 

 will float off on the surface of the fixative. If filter paper is used it will sink at 

 once, but if non-absorbent paper is used it will continue to float on the surface, 

 and by reversing the slide before immersing the chick may be thus held suspended 

 in the fixative, with no danger of flattening from pressure against bottom of 

 the dish. 



After fixation and washing the embryo may be removed from the paper by 

 jets from a pipette or with a thin-bladed lifter. Since the paper serves as a very 

 convenient means of handling the embryo, it is advisable not to remove it until 

 just before clearing. It cannot well be done after clearing, because of the 

 brittleness acquired in this process. 



This method gives preparations which are neither distorted, torn, nor wrinkled, 

 and the loss from accidents is very small, since the paper serves as a protective 

 frame for the embryo. I fi.nd that it is not convenient to use for chicks under 

 20 hours, but is available for all older stages. 



In making whole mounts, the edge of the embryonic area is often ragged. 

 This may be trimmed to a smooth, circular periphery by placing on glass or wax 

 and cutting with a cork borer of the proper size. F. C. Waite. 



Medical Department, Western Reserve University. 



