METHODS OF INVESTIGATION. 19 



1 per cent hydrochloric acid. After this primary procedure, during which the 

 ferrocyanide was precipitated, the embryo was transferred to Bouin's fluid (satu- 

 rated aqueous picric acid, 75; formaldehyde 40 per cent, 20; glacial acetic acid, 5). 

 The specimens were allowed to fix over night and were then dehydrated in graded 

 alcohols. From 30 per cent alcohol, use was made of 4 per cent changes up to 60 

 per cent; and from this point to absolute the changes were by 2 per cent gradations. 



In addition to the technique outlined above, Carnoy's solution and 10 per cent 

 formol were employed. The Carnoy fluid, containing acid (absolute alcohol, 60; 

 chloroform, 30; glacial acetic acid, 10; hydrochloric acid, 1) proved to be of particular 

 service in the study of specimens cleared by the Spalteholz method; histologically, 

 however, it has not been as valuable as Bourn's fluid. 



Besides the ferrocyanide solution, two other injection masses were constantly 

 employed. Solutions of silver nitrate in concentrations of 0.5 per cent were injected 

 into the central canal of the spinal cord and into the cerebral ventricles. This 

 method, with reduction of the silver salt in the sunlight, gives very pleasing prepara- 

 tions. It is, however, subject to obvious limitations. The intraspinous toxicity 

 of the silver, together with its action as a precipitant of albuminous substances, 

 renders its use unsatisfactory in replacement experiments. Furthermore, it reacts 

 apparently with any protein tissue, irrespective of the true function of that tissue 

 (as, for example, its coagulation of the lining ependyma of the ventricles). 



India ink, the other substance employed, is of extreme value in anatomical 

 studies. Because of the suspension of carbon granules it possesses the disadvan- 

 tages already commented upon for the study of any true pathway of fluid. It has 

 been of service, however, in the present work in showing marked differences in 

 spread from that of true solutions and in furnishing information in regard to fluid 

 passage through a membrane. 



This investigation has been carried out on the basic idea of correlating the 

 physiological spread of the embryonic cerebro-spinal fluid with the gradual trans- 

 formation of the perimedullary mesenchyme into the three fully formed meninges. 

 This has necessitated a histological study of the embryo. Pigs for the most part 

 were the animals used, but the findings have all been verified by a study of the same 

 regions in the human embryos in possession of the Carnegie Institution of Washing- 

 ton. In addition, certain structural characters have likewise been identified in 

 sections of chick, rabbit, and cat embryos. 



It was early apparent that the material to be of value must be free from any 

 great shrinkage about the central nervous system. Comparative freedom from 

 this artifact was obtained by fixing the embryo alive in Bouin's fluid and dehydrat- 

 ing by 2 and 4 per cent gradations of alcohol. The material was chiefly cut in 

 paraffin after being embedded by means of xylol. 



The methods of investigation outlined in the foregoing paragraphs have been 

 followed throughout the major portion of the work. In many minor instances 

 other procedures not commented upon have been employed; these will be detailed 

 in appropriate subdivisions of this paper. 



