APPENDIX. g 31 



40 mm.). Larger embryos should be opened by a ventral incision to allow the fixative to 

 penetrate; or portions may be removed and fixed as desired. Flemming's fluid is an excellent 

 fixing agent for small pieces of tissue. 



After Zenker's or Flemming's fluid, the specimen should be washed in running water 

 for a few hours and then hardened in graded alcohols to 80 per cent, and preserved in the 

 latter. After Bouin's fluid, the washing should be done in several changes of the weaker 

 alcohols instead of water. 



With some extra facilities and trouble, rabbit embryos also are available. Furthermore, 

 it is possible to obtain very early stages. The female rabbit should be allowed to become 

 pregnant and then be isolated until she gives birth to her young The date of birth should 

 be noted and 30 days later the male should be admitted and the exact time of coitus recorded. 

 At any time thereafter, the time depending upon the stage desired, the animal is killed and 

 the uterus and oviducts at once removed. Up to and including the third day after coitus, 

 the ova are in the oviducts. They may be found by injecting a weak solution of osmic acid 

 (0.1 per cent.) into the oviduct with a small syringe, collecting the liquid that escapes in a 

 series of watch glasses and examining it under the microscope. Or the oviducts may be 

 fixed, hardened and embedded in tola and cut into serial sections. 



During the fourth, fifth and sixth days the ova lie free in the cavity of the uterus. The 

 uterus should be opened carefully with fine scissors. The ova are small rounded bodies 

 with pearly luster. They can be examined in some "indifferent" medium, such as the 

 peritoneal fluid of the mother, or blood serum. After the sixth day the ova become attached 

 to the uterine mucosa. A little block of the wall of the uterus containing the ovum can be 

 cut out and fixed in Flemming's or Zenker's fluid. 



The way in which human embryos are usually obtained and the methods of treating them 

 are discussed on p. 159 et seq. 



II. Fixation. 



Embryonic tissues are soft and delicate and should be treated with reagents which cause 

 as little shrinkage or swelling as possible. The embryos should be fresh and should not be 

 handled more than is absolutely necessary before fixing. Large quantities of the fixative 

 should be used, at least ten times the volume of the object to be fixed. For ordinary histologi- 

 cal study, the fixative used should be of such a nature that the tissues will afterward take a 

 good differential stain. Bouin's, Zenker's and Orth's fluids are good, and prepare the 

 tissues for a brilliant hsematoxylin and eosin stain. Flemming's fluid (for small pieces of 

 tissue) gives an excellent fixation, and can be followed most satisfactorily by Heidenhain's 

 iron-haematoxylin. For special purposes, special fixatives and stains are necessary. The 

 fixatives which perhaps give the best results with certain structures are given in "Practical 

 Suggestions" in the chapters treating of those structures. The formulas and some general 

 directions are given below. 



1. Alcohol.— Strong alcohol (95 per cent.) may be used when no other fixative is at hand, 

 but always causes considerable shrinkage. It is sometimes convenient to use for fixing small 

 human embryos with the membranes intact (see "Practical Suggestions," p. 159). 



2. Bouiri 's fluid. — 



Picric acid, saturated aqueous solution . 75 parts. 



Formalin (commercial) 2 S parts. 



Acetic acid • • ■ -5 P arts - 



This fluid has good penetrating powers, fixes both nuclear and cytoplasmic structures 

 well, and causes no shrinkage. Embryos 35 to 40 mm. long are fixed in from 24 to 36 



