278 



METHODS AND FORMULAS 



DS 11.10 



or the end of the pipet, to make sure that 

 adherence is perfect. The whole should be 

 left for a moment or two before being very 

 gently shaken from side to side to make 

 quite certain that the embryo is not stick- 

 ing to the watch glass. If it is sticking, the 

 end of the pipet containing the fixative 

 should be slid under the edge of the paper 

 and a very gentle jet of fixative used to 

 free the embryo. As soon as the embryo is 

 floating freely in fixative the Syracuse 

 watch glass may be filled with fixative and 

 placed on one side while the same cycle of 

 events is repeated with the next embryo. 

 After about 10 minutes in the fixative, the 

 paper may be picked up by one corner 

 and moved from reagent to reagent with- 

 out the shghtest risk of the embryo be- 

 coming either detached or damaged. The 

 paper must not be picked up with a pair 

 of metal forceps unless these have been 

 waxed, or the mercuric chloride in the 

 fixative will damage the metal. It is the 

 writer's custom to leave the embryos in 

 the watch glass for about 30 minutes be- 

 fore picking them out and transferring 

 them to a large jar of the fixative which is 

 l^referably kept in a dark cupboard. The 

 total time of fixation is not important, but 

 it 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 

 then be stored in 70% alcohol. 



When one is ready to stain a batch of 

 embryos it is only necessary to transfer 

 them from alcohol back to distilled water, 

 leaving them there until they are thor- 

 oughly rehydrated, and then to transfer 

 them to a reasonably large volume of 

 Carazzi's hematoxylin (DS 11.122 Ca- 

 razzi 1911) in which they may remain 

 overnight. It is a mistake to stain them 

 initially for too short a period, for the 

 result will be that the outer surface of the 

 embryo becomes adequately stained while 

 the inner structures do not. This defect, 

 however, is very difficult to detect until 

 the embryo is finally cleared for mounting. 

 When the embryos are removed from the 

 stain, at which time they should appear a 

 deep purple, they should be transferred 

 to a large fingerl)owl of distilled water 

 and rocked gently backward and forward 



until most of the stain has been removed 

 from the papers to which they are at- 

 tached. Each embryo should now be taken 

 separately and placed in 0.1% hydro- 

 chloric acid in 70% alcohol. The color will 

 immediately start to change from a deep 

 purple to a pale bluish-pink. They should 

 remain in this solution until, on examina- 

 tion under a low power of the microscope, 

 all the required internal structures appear 

 clearly differentiated. Most people differ- 

 entiate too little, forgetting that the pale 

 pink of the embryo will be changed back 

 to a deep blue by subsequent treatment 

 and that the apparent color will also in- 

 crease in density when the embryo is 

 cleared. No specific directions for the ex- 

 tent of the differentiation can be given 

 beyond the general ad\'ice to differentiate 

 far more than you anticipate to be neces- 

 sary. After the embryos have been suffi- 

 ciently differentiated each one should be 

 placed in alkaline tap water, either as it 

 occurs in nature or as it is rendered alka- 

 line with the addition of sodium bicar- 

 bonate. Here it should remain until all the 

 acid has been neutralized and the embryo 

 itself has changed from a pink back to a 

 blue coloration. It may then be dehy- 

 drated in the ordinary manner through 

 successive alcohols, and it is the author's 

 custom to remove it from its paper only 

 w^hen it is in the last alcohol and before 

 it is placed in the clearing reagent. Some 

 persons place it in the clearing reagent 

 attached to its paper and remove it only 

 before mounting. Any clearing reagent 

 may be tried at the choice of the operator. 

 The author's preference for chicken em- 

 bryos is terpineol which has the advantage 

 of not rendering these delicate structures 

 as brittle as do many other reagents. The 

 mountant may be Canada balsam or any 

 of its synthetic substitutes. 



Preparation of a series of demonstra- 

 tion slides, each having six typical 

 transverse sections of a 72-hour 

 chicken embryo, using the acid 

 alum hematoxylin stain 

 of Ehrlich 1896 



The last example described in some de- 

 tail the manner in which a cliicken embryo 



