EMBRYOLOGY 371 



graded alcohols, clear thoroughly, first in benzine (or benzol), and 

 then in oil of wintergrcen (Spalteholz). Embryos cleared by 

 Spalteholz's method may later be imbedded from oil of winter- 

 green by transferring to half wax, half oil of wintergreen, and then 

 pure wax. Tissues left in oil of wintergreen do not go brittle 

 even after a year or two (Sabin). 



Improved methods have recently been introduced by Franklin 

 P, Reagan {University of California Publications in Zoology, 

 192G). who injects with undiluted filtered India ink, fixes in 

 formalin of 4 per cent, to 10 per cent, with a trace of acetic acid 

 for twenty-four hours if the material is to be dissected in water. 

 The fixed tissue is somewhat clear, conveniently elastic. Carnoy's 

 fluid shrinks the tissue and renders it tough. After fixation in 

 formalin acetic, the embryo is washed for an hour in tap water, 

 bleached in a mixture of 3 parts of water to 1 of hydrogen peroxide, 

 which may produce a few bubbles in the tissue. The bubbles may 

 be liberated by punctures in the skin. The bleaching process 

 may last from a few minutes to a few hours. After bleaching, and 

 removal of bubbles, the material is washed for a short time in 

 water. It may then be returned to formalin if not wanted 

 immediately, 



Dehvdration is carried out in various strengths of alcohol as 

 usual, cleared in benzol, and then in pure benzyl benzoate, after 

 which they are remarkably clear if illuminated with transmitted 

 light. In direct light they exhibit a bluish opalescent sheen. 

 This sheen, if objected to, can be removed by adding a very 

 small quantity of oil of wintergreen to the benzol benzoate. For 

 storing in benzol benzoate, add a small quantity of Beechwood 

 creosote. Embryos done by Reagan's method are very beautiful. 

 810, Staining Embryological Sections.* Some years ago the 

 routine methods for embryological study were borax carmine 

 and picro-indigo-carmine, and a hsematoxylin and eosin. We 

 have yet to see preparations which surpass for beauty, really 

 good borax carmine and picro-indigo-carmine sections. It is 

 certainly true that some of the more recent polychromatic, 

 many process staining methods are much inferior to the carmine 

 ones. Nevertheless there has been a steady trend away from these 

 older techniques towards the use of certain methods which give 

 more information about the histology and cytology of develop- 

 ment. It is quite possible nowadays to combine good embryo- 

 logical results with a satisfactory cytological and histological 

 interpretation of the material being studied. 



For the study of the histology of the gonads, as an example, 

 the azan stain of Heidenhain, and Pasini, will be found excellent. 

 Clearer cytological results will be obtained with Masson's iron 



* §§ 810—815, suggested by Dr. J. P, Hill, 



