1890-yl.] AMPHIBIA BLOOD STUDIKS. 227 



when a quantity of solution B. (see p. 224) alone was mixed with a pure 

 solution of haemoglobin and the mixture treated with a saturated 

 solution of oxalic acid there resulted only a dirty brownish precipitate 

 from the decomposed haemoglobin. This proves that soluble haemo- 

 globin cannot yield any reliable reactions with the Indigo-carmine Fluid. 

 Acting on the v^iew that the haemoglobin in my preparation is a 

 fixed insoluble compound and therefore quite different from that obtained 

 for example, by mere crystallization from dog's blood, I modified Bayerl's 

 experiment. I took pure crystallized haemoglobin from dog's blood, 

 dissolved it in distilled water and mixed it with an equal volume of 

 a^^ar-agar solution* made liquid at 42'^C. Stirred rapidly and then 

 cooled by plunging the base of the containing vessel in pounded ice, tha 

 deep red agar-agar mixture becomes firm enough to cut with a knife. 

 I made pieces about one-eighth of an inch in thickness which I put in 

 various hardening fluids, as in the case of the spleen of the Necturus. 

 When the fixation was complete the excess of the reagent was removed 

 with alcohols 50/^, yo^/ and 90^ successively, sections of the pieces 

 were made on the freezing microtome anci stained with the Indigo- 

 carmine Fluid. The preparations made with chromic acid or Erlicki's 

 Fluid gave a grass-green reaction while those made with corrosive 

 sublimate gave a greenish blue, practically the same results as m 

 the case of the haemoglobin in the red corpuscles. The fact that the cor- 

 rosive sublimate preparations gave a greenish blue color with the Indigo- 

 carmine Fluid, while the other preparations gave a grass-green, would 

 lead one to suspect that there might be a difference in the chemical 

 composition of the reagent when absorbed in the two kinds of pre- 

 parations. If there is such a difference, it cannot be in the indigo 

 portion of the staining molecule, for blue and grass green sections with 

 the spectroscope, give alike the indigo absorption bands and no more. 



I used also in staining sections of the spleen alum-haematoxylin 

 solutions, in which ammonia alum is dissolved to saturation, and Czokor's 

 alum-cochineal. These two reagents are of great value, especially the 

 former, in connection with the studies on the haematoblasts in the Ainbly- 

 stonia larvae, the latter having been in various stages of their development 

 fixed in chromic acid (^°/o), Flemming's Fluid, corrosive sublimate, and 

 Erlicki's Fluid. Though the other reagents have their uses, the second 

 and third mentioned were the only ones to give good general results. 

 My preference is decidedly for Flemming's Fluid for larval or embryonic 

 tissues. Half an hour is long enough for this reagent to act, since with 



* Of the strength and characters recommended by Biondi. Arch, /ilr Mikr. Anat. Bd. 

 XXXI., p. 105. 



