142 METHODS FOR PREPARING, EXAMINING, AND MEASURING 



solubility, alters the extinction coefficient, gradually decolorizes the crystals, 

 and doubtless affects the water of crystallization. Alkalies and mineral 

 acids have likewise been avoided, because of their pernicious influences. 

 Hemoglobin, whether in crystalline form or in solution, especially when in 

 concentrated solution, undergoes rapid alteration; we therefore made our 

 studies as soon as possible after we obtained satisfactory crystals, usually 

 within a few hours. In none of our examinations have we used recrystal- 

 lized hemoglobin. Our specimens have been too small in quantity to permit 

 of satisfactory recrystallization, and, moreover, the disadvantages of 

 recrystallization, especially in so far as the methods of our investigation 

 are concerned, quite outweigh the advantages. The injurious effects of 

 recrystallization have been fully referred to in previous pages. 



At the inception of our research it seemed to us that the best results, 

 on the whole, were to be obtained by the use of fluid blood, either defibri- 

 nated or rendered incoagulable by oxalate, fluoride, or other anticoagulant, 

 so that in the case of bloods which do not crystallize readily the corpuscles 

 could be collected from the serum or plasma by centrifugalization, and thus 

 eliminate certain substances in these fluids which retard crystallization 

 and at the same time obtain a concentrated solution of hemoglobin. Since 

 it seemed impracticable to obtain defibrinated blood, owing to the circum- 

 stances under which our specimens were to be collected, and since one of 

 us (Reichert, page 128) had already found that the presence of an anti- 

 coagulant, such as neutral oxalate, was not only not injurious but actually 

 beneficial, we made use of oxalate of ammonium in all of our preparations 

 except in a very few instances, when for some special reason its absence 

 was desirable or necessary. The addition of oxalate, in the proportion of 

 1 to 5 per cent of the dried powder, it was found, very much favors crystal- 

 lization ; the larger the quantity up to the point of saturation the better the 

 effect, saturation not being a disadvantage beyond the appearance of 

 crystals of oxalate, which, however, are readily distinguishable from those 

 of hemoglobin. In fact, in several instances these crystals appeared to be 

 of advantage, because hemoglobin crystals formed on them, but not in 

 other parts of the preparations. When we had defibrinated blood or clots 

 to work with, oxalate was added at the proper time during our procedures 

 of preparation. 



Since the presence of foreign bodies may, as is well known, not only 

 augment or hinder crystallization, but also affect crystallization in other 

 and even more important ways, we made appropriate tests to determine 

 especially if the presence of the oxalate in any particular quantity affected 

 either the type of the crystals or the optical properties of hemoglobin. The 

 optical properties were not in any way appreciably affected. The habit 

 of crystallization, as in the case of \<di<rus, seemed to be affected in the 

 direction of causing the crystals to be shorter and thicker. The only im- 

 portant influence of the oxalate, apart from the accelerating effect, we 

 found in our experiments with the bloods of the horse and mule, in which 

 we discovered that by modifications in the quantity of oxalate we could 

 obtain a relative abundance of one or the other or of both kinds of oxy- 



