224 TRANSACTIONS OF THE CANADIAN INSTITUTE. [VoL. II. 



antecedent if such existed. I need hardly go over the list of wearisome 

 experiments which I made for this purpose. Many, but not all, of these 

 were resultless. Of the dyes at my disposal belonging to the aromatic 

 group of organic compounds, Eosin is the only one which I found useful. 

 As will be shown below it reacts with haemoglobin and, in conjunction 

 with alum-haematoxylin or alum-cochineal, it is a reagent for the 

 antecedent of the pigment. Taken of course alone, without employing 

 any other reagent for control purposes, it gives results far from satisfactory 

 and it is also very misleading. Another reagent, the employment of 

 which has been of great value to me, is the staining fluid of Shakespeare 

 and Norris,* and which I shall name throughout this paper, for the sake of 

 brevity, the Indigo-carmine Mixture or Fluid. 



This fluid is made according to a formula which I have modified from 

 that given by Rayerl, and consists of a mixture of equal volumes of the 

 following solutions : — 



A — Carmine, 2 grms ; Borax, 8 grms ; Distilled Water, lOO c.c. 

 B. — Sulphindigotate of Soda (Indigo-carmine), 8 grams ; Borax, 

 8 grms ; Distilled Water, lOO c.c. 



In preparing each of these solutions, the borax is ground up in a mortar 

 with the dye, the water poured on, and the whole allowed to stand for 

 from five to seven hours before filtering. Owing to the fact that much of 

 the Indigo-carmine in the market is impure, and consequently alters its 

 composition in solution in a couple of weeks, it is not advisable to prepare 

 more than 25 — 50 cc. of solution B at a time. I have obtained 

 quantities of the reagent which retained in solution for three months its 

 normal staining properties. As A, when kept for a year or more, readily 

 shows undiminished staining power, a larger quantity may be prepared 

 as " stock " solution. 



The section to be stained is left in the fluid for fifteen minutes, then 

 plunged in a saturated solution of oxalic acid for ten minutes, washed in 

 distilled water, deh\'drated with absolute alcohol, cleared in pure xylol, 

 and mounted in benzol balsam. Preparations made in this way two 

 years ago still retain undiminished their original stain. When I first 

 employed the fluid, four years ago, I used clove-oil for clearing, and 

 found that my preparations faded, or contained a dirty precipitate after 

 three or four weeks. The removal of the clove-oil after clearing with 



* I have not seen the paper of Shakespeare and Norris describing the stain or its properties 

 and capacities and my attention was first directed to it by Bayerl's work on the formation of 

 blood corpuscles on the margins of ossifying zones in bones : Arch, fur Mikr. Anat. Bd. 

 xxni. p. 30. 



