Frog embryos 



PARAFFIN SECTIONS 



135 



of the rubber paraffins with a melting 

 point of from 50° to 55°C. be employed. 



In any case we now have amphibian 

 embryos and eggs accumulated in the 

 embedding oven in whatever medium has 

 been decided to use. It is usually necessary 

 in cutting sections of this type that the 

 orientation of the embryo in relation to 

 the knife should be known; this is difficult 

 to estabhsh by ordinary means when one 

 is deahng with a more or less spherical 

 embryo. The method preferred by the 

 writer for indicating one of the planes is 

 to embed at the same time and alongside 

 the spherical embryo a little rectangular 

 block of liver or some other soft tissue. It 

 is easy to dehydrate clear and impregnate 

 with wax a piece of liver and keep this 

 permanently in the embedding oven in 

 paraffin. When one is ready to embed the 

 eggs a small strip (in the case of the frog 

 embryo about 3 mm. X 1 mm. X 1 mm.) 

 is cut from this slab of hver, and is first 

 of all laid in the paper box to which one 

 has added the wax in the manner already 

 described. The egg is then transferred to 

 the same box and is most carefuUj^ ori- 

 entated with regard to the strip of liver, 

 so that if the liver be cut exactly at right 

 angles, the egg will be cut in the desired 

 plane. A strip of hver this size does not 

 greatly increase the total area of this 

 block, nor does it in any way interfere 

 with whatever staining technique is em- 

 ployed for the sections. An identical pro- 

 ' cedure is followed, whether one is dealing 

 with eggs fixed by the technique of Gregg 

 and Puckett, or fixed and prestained by 

 the technique of Smith. 



After the block has been hardened under 

 the surface of water (Smith specifies 70 % 

 alcohol for the purpose, but it does not 

 appear to matter) it is removed, allowed 

 to attain room temperature, and the sides 

 trimmed away until the strip of liver is 

 clearly seen. The block is then attached 

 to the holder, mounted in the block holder 

 of a microtome, and a ribbon is prepared 

 in the usual manner. The only difficulty 

 that is hkely to arise is that, after the 

 ribbons have been flattened and are dry- 

 ing, the entire yolky center of the embryo 

 may rise in a dome. This event usually 

 indicates that the vitelline membrane has 



remained on the egg and there is nothing 

 whatever tliat can l)e done about it. Such 

 sections always become detached in the 

 course of staining and are in any case 

 worthless, for if they are varnished in place 

 they will still be so domed as to render 

 microscopic examination almost imi)ossi- 

 ble. If, however, several successive batches 

 of sections, in which one is quite certain 

 that the vitelline membrane has been re- 

 moved from the embryo, behave in this 

 manner, it is sometimes possible to stop 

 the trouble by drilling a little liole in the 

 block until one just comes to the egg itself, 

 and then soaking the block in glycerol- 

 alcohol. Blocks so treated will, when cut, 

 usually be found to lie flat on the slide. 

 If this dexice fails it is strongly recom- 

 mended that 70% alcohol be substituted 

 for water used to flatten the sections (any 

 of the customary adhesives may be mixed 

 with alcohol of this strength just as readily 

 as with water) and that the technique of 

 using a wet blotter and a rubber roller 

 be used, as described in the body of this 

 chapter. It must be emphasized that these 

 defects are uncommon in materials fixed 

 in the manner described; they are men- 

 tioned only because they occur so fre- 

 quently in the handling of amphibian 

 embryos fixed and prepared by other 

 methods. 



After the ribbons have been flattened 

 and dried, they are then put through the 

 ordinary series of reagents until they are 

 ready to be stained. The technique differs 

 very greatly according to whether one is 

 dealing with the technique of Gregg and 

 Puckett or that of Smith. In the technique 

 of the latter the slides are removed from 

 absolute alcohol and flooded with the 

 Lyons blue and picric acid mixture of 

 Smith (Chapter 20 DS 12.221) for a 

 period of about one minute. They are then 

 returned to absolute alcohol until no 

 color comes away, and cleared in xylene 

 before being mounted in a resinous me- 

 dium. The writer prefers this technique to 

 any other because of the gross swelling 

 which occurs in yolk when exposed to 

 aqueous solutions of stains. The rising up 

 of the center of the section, commented 

 on in the last paragraph, is not confined to 

 the time when the sections are flattening. 



