Hematoxylin-stained, 33-hour Chick 187 



should be applied from an eye-dropper type of pipette in the following 

 manner. A few drops are first placed on the center of embryo, so that a 

 thin film of fixative is spread over it. After a moment or two a little more 

 may be added with a circular motion on the paper surrounding the em- 

 bryo. Again the paper should be pressed on the periphery of the blasto- 

 derm with a needle or the end of the pipette to make sure that the ad- 

 herence is perfect, and the whole should be left for a moment or two be- 

 fore being shaken gently from side to side to make quite certain that 

 the embryo is not sticking to the watch glass. If it is sticking, the end 

 of the pipette containing the fixative should be slid under the edge of the 

 paper, and a very gentle jet or fixative used to free the embryo. As soon 

 as the embryo is floating freely in fixative, the Syracuse watch glass may 

 be filled up with fixative and placed to one side, while the same cycle 

 of events is repeated with the next embryo. After about 10 min in the 

 fixative, the paper may be picked up by one corner and moved from 

 reagent to reagent without the slightest risk of the embryo becoming 

 either detached or damaged. Care, of course, must be taken not to 

 pick up the paper with metal forceps unless the instrument has been 

 waxed first because the mercuric chloride in the fixative will damage the 

 metal. It is the author's custom to leave the embryos in the watch glasses 

 for about 30 min before picking them out and transferring them to a 

 large jar of the fixative, preferably kept in a dark cupboard. The total 

 time of fixation is not important but should be not less than one day nor 

 more than one week. When the embryos are removed from the fixative, 

 they should be washed in running water overnight and can then be stored 

 in 70 per cent alcohol for an indefinite period. 



When a batch of embryos is ready to stain, it is necessary only to take 

 them from 70 per cent alcohol back to distilled water until they are 

 thoroughly rehydrated and then transfer them to a reasonably large vol- 

 ume of Delafield's hematoxylin, where they may remain overnight. The 

 gravest mistake that can be made in this type of staining is to stain initially 

 for too short a period. The result is that the outer surface of the embryo 

 becomes adequately stained while the inner structures do not, but this 

 defect is difficult to detect until the embryo is finally cleared for mount- 

 ing. When the embryos are removed from the stain, at which time they 

 should appear a deep purple, they should be transferred to a large finger 

 bowl of distilled water, where they are rocked gently backward and 

 forward until most of the stain has been removed from the paper to 

 which they are attached. Each embryo should then be taken separately 

 and placed in 0.1 per cent hydrochloric acid in 70 per cent alcohol. The 

 color will immediately start to change from a deep purple to a pale 

 bluish pink, and the embryos should remain in this solution until, on 

 examination under a low power of the microscope, all the required in- 



