CHAPTER VIII. 



METHODS FOR PREPARING, EXAMINING, AND MEASURING 



CRYSTALS OF THE HEMOGLOBINS EMPLOYED 



IN THIS RESEARCH. 



METHODS FOR PREPARING CRYSTALS OF HEMOGLOBIN. 



The necessarily limited quantities of blood that have been furnished us 

 led, as a consequence, to the study of only such methods as are especially 

 applicable to very small supplies, such for instance as 1 to 5 c.c. of fluid 

 or clotted blood, although several of our processes may be used to advan- 

 tage in the preparation of very large quantities if a method be selected 

 that is suited to the species and to the condition of the blood. In only a 

 few instances were we unsuccessful in obtaining crystals, and when we 

 failed it was owing to an inadvertent selection of a wrong method or to 

 attendant conditions over which we had no control. Our difficulty was not 

 so much in the way of securing crystals as it was in the preparation of 

 specimens that were adapted to the peculiar requirements of our investiga- 

 tion. We found, as we gained experience with the bloods of different 

 species, that, while the blood of each species must be treated as an individ- 

 ual, we could nevertheless depend with some confidence upon the guidance 

 of certain generalizations in the selection of the best method to be pursued. 

 Thus, we found that usually the hemoglobins of Rodentia and Canidce 

 crystallize with great readiness, those of Marsupialia very readily, those of 

 Felidce readily, those of Ungulata not readily, those of Aves with difficulty, 

 etc. ; but there were so many unexpected exceptions that we were often 

 misled, and, as a consequence, obtained inferior results, as a number of 

 our photomicrographic reproductions show. 



Even in the case of species closely related, as, for instance, certain of 

 the rats, we found striking exceptions: The blood of the common albino 

 or white rat (Mus norvegicus var. albus)* and that of Mus decumanus Pall. 

 (Mus norvegicus Erxleben brown rat) crystallize with such readiness that 

 we found it desirable to use a restrainer to obtain crystals of desirable size 

 for study; on the other hand, the bloods of Mus rattus (black rat) and Mus 

 alexandrinus (alexandrine rat) crystallize much less readily, and hence 

 should be treated in an entirely different way. 



We absolutely avoided the use of alcohol, because, notwithstanding 

 the fact that it has proven one of the most widely used and most valuable 

 agents in the preparation of hemoglobin crystals, it so deleteriously affects 

 the hemoglobin molecule that even when present in dilute solution it lessens 



* Hatai (Biological Bulletin, Wistar Institute of Anatomy and Biology, Philadelphia, 1907, xn, 266) 

 states, upon morphological grounds, that the albino rats of Chicago and Philadelphia are a variety of Mus 

 norvegicus. 



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