MS 31.0 



METAL STAINS 



547 



the shell, and then remove the shell, piece 

 by piece, from one end of the egg, until a, 

 hole is left of sufficient size for the yolk 

 and attached embryo to be slid from it 

 into the normal saline. Before going fur- 

 ther, the embrj'^o is examined and a piece 

 of filter paper taken of a size which will 

 approximately fit one of the Syracuse 

 watch glasses. A hole is then cut in this 

 filter paper of a size that will leave about 

 a J.^-inch gap around the embryo itself. 



The next stage is to take a pair of large 

 scissors and make as few cuts as possible 

 around the outside of the extra-embryonic 

 area, so as to isolate the entire blastoderm 

 containing the embryo. The fewer the 

 cuts which are made, and the larger the 

 scissors used, the less will be the leakage 

 of yolk into the surrounding normal saline. 

 It would be ideal if it were possible to re- 

 move the embryo with only four cuts, but 

 this requires a larger pair of scissors than 

 is possessed by most technicians. What- 

 ever method is adopted, the utmost care 

 should be taken to get as little yolk as 

 possible distributed through the normal 

 sahne. A common cause of failure in this 

 metal-staining technique is the carrying 

 over of a considerable quantity of yolk 

 to the final dish of sahne. As soon, there- 

 fore, as the embryo has been separated 

 from the yolk, it is taken with a pair of 

 forceps and pulled gently backward and 

 forward through the saline in the first 

 fingerbowl until it ajDpears to be yolk-free. 

 It is then picked up, preferably in a spoon, 

 transferred to the second fingerbowl of 

 clean saline, and again washed. Transfer- 

 ence to the third fingerbowl, where a con- 

 tinual rinsing of the embryo backward and 

 forward should fail to disclose the slightest 

 milky trace of yolk coming from it, com- 

 j)letes the washing. If, however, milky 

 trails of yolk are still observed coming 

 from the embryo, it must be washed in a 

 fourth batch of saline. Next take a chem- 

 ically clean Syracuse watch glass, and 

 transfer the embryo so that it lies in the 

 watch glass with its ventral surface upper- 

 most. This can most readily be established 

 by observing the entry of the vitelline 

 veins and arteries into the embryo. The 

 saline carried over with the embryo is 

 now removed with an eye dropper. 



The piece of filter paper with the hole 

 in it is now dipped into clean saline and 

 dropped on top of the embryo in such a 

 manner that the embryo is centered in the 

 hole. Next take the tip of a pair of fine 

 forceps and make, as it were, a series of 

 dots with these forceps on top of the filter 

 paper in the region where the extra- 

 embryonic membranes lie under it. Each 

 time the tip of the forceps is pressed down 

 on the filter paper, it causes the adhesion 

 of the membrane to the paper. Fifty or 60 

 such dots, evenly spaced around the 

 extra-embryonic membrane, will be none 

 too many to insure proper adhesion. Now 

 place a few drops of alcohol on the filter 

 paper without getting any on the embryo 

 itself. The purpose of this is to insure the 

 adhesion of the embryo to the filter paper 

 for ease in after-handling. Next place a 

 few drops on top of the eml)ryo itself, wait 

 a moment or two, and then very carefully 

 and slowly fill the watch glass with al- 

 cohol. Leave it for three or four minutes 

 and then, pressing downward on the filter 

 paper with the end of the pipet, move the 

 whole filter paper backward and forward 

 to make sure that the embryo is adhering 

 to the filter paper and not to the watch 

 glass. If the filter paper moves without 

 the embryo, thus indicating that the ad- 

 hesion of the embryo is to the watch glass, 

 it is almost certain that the embryo has 

 been placed upside down in the watch 

 glass (that is, with its ventral surface 

 against the glass) and there is nothing 

 which can be done about it save to start 

 with a new embryo. If, however, the 

 embryo is attached to the filter paper, it 

 may be picked up with a pair of forceps 

 and removed to a stoppered, four-ounce 

 jar of alcohol. This jar must be chemically 

 clean. Tip the jar upside down from time 

 to time to make sure that the water 

 coming out of the embryo does not dilute 

 the alcohol on the bottom. The embryo 

 should remain in alcohol for at least 2-1 

 hours. 



Make up the solutions which will be re- 

 quired. These are: 1.5% silver nitrate, of 

 which 100 milliliters should be placed in a 

 wide-mouthed, chemically clean, stop- 

 j)ere(l l)ottle; and the developer (AMS21.1 

 Cajal 1910, Chapter 24) which is pre- 



