1890-91.] AMPHIBIA BLOOD STUDIES. 225 



xylol, postponed, but did not prevent, fading. It seems that the essential 

 oils, even in small quantities, possess an oxidising power to which the 

 sulphindigotate of soda is subject. 



In order to get the best effects with this stain, the tissues are to be hard- 

 ened with reagents which preserve the haemoglobin and its- normal dis- 

 tribution in the corpuscles. Some of the ordinary hardening reagents do 

 not fix haemoglobin (Muller's Fluid and solutions of potassic bichromate), 

 others decompose it (w^eak solutions of chromic acid), while others again 

 cause the haemoglobin and the so-called stroma containing it to shrink 

 irregularly in the corpuscles. The very fact that a reagent removes or 

 decomposes the haemoglobin does not prevent its employment in the 

 study of the mode of formation of the pigment, but points to its useful- 

 ness in testing and controlling the results obtained by reagents which fix 

 the haemoglobin well. For instance, I have used chromic acid for the 

 purpose of removing the haemoglobin and fixing the antecedent. Even 

 in the list given below the haemoglobin-fixing property is not the same 

 in all, and again the reagent which fixes the haemoglobin in the red 

 corpuscles in pieces of the spleen may not have the same property as 

 regards cover-glass preparations of the blood. These facts should be 

 borne in mind in every research on red blood corpuscles. The method 

 which I adopted after a long series of experiments was as follows : — 



Small portions of the spleen oi Necturus were allowed to lie half-an- 

 hour in a saturated solution of corrosive sublimate, or five days in 

 Erlicki's Fluid, or twenty-four hours in a \ — ^% solution of chromic 

 acid, five hours in a saturated solution of picric acid or two to five hours 

 in i^ solution of osmic acid. They were afterwards washed in distilled 

 water and put in alcohol of 50% strength for two hours and then in 70^ 

 for twenty-four hours and finally in 95%- The 70^ alcohol was changed 

 several times, each at an interval of twenty-four hours in the case of the 

 chromic and picric preparations. The pieces were imbedded, either in 

 mucilage and sectioned on the freezing microtome, or by the chloroform 

 method in paraffin by which sections of about 5 — iO'.J- were made. The 

 latter were freed from paraffin with turpentine and passed through 

 absolute alcohol to water in the usual way. These, as well as those 

 prepared with the freezing microtome, were transferred to the Indigo- 

 carmine Fluid and treated in the manner described above. 



The great value of these preparations consists in the fact that haemo- 

 globin is stained grass-green or greenish-blue while other proteid elements 

 are colored red. This grass-green or greenish-blue is shown by a ^&^ 

 other elements, but these are so well known and so easily recognised that 

 no confusion can result. The numberof structures other than haematogenic 



