PREPARATION OF OXYHsEMOGLOBIN. 195 



A. Schmidt 1 made the same observation in reference to the blood near 

 the positive pole, when this liquid is subjected to the action of a 

 constant current. 



Without undergoing any other treatment, the blood of asphyxiated 

 animals often crystallises. Blood which has been deprived of its gases, 

 by boiling in vacuo, often crystallises. Indeed, by this method, Preyer 2 

 succeeded in the very difficult task of crystallising sheep's blood. 



Methods of preparing considerable quantities of oxyheemo- 

 globin. Certain of the methods already recommended for the prepara- 

 tion of haemoglobin, when small quantities only are needed for the 

 purposes of microscopic investigation, might be employed in the pre- 

 paration of larger quantities. Other processes, however, are to be 

 preferred, and of these some which are specially to be recommended 

 are given below. Of these processes, the first, or Hiifner's modification 

 of it, should, by preference, be employed, especially if a preparation, as 

 free as possible from products of decomposition, be desired. 3 



Hoppc-Seylcrs method.^ Defibrinated blood is mixed with ten times 

 its volume of a solution of sodium chloride 5 (made by diluting one 

 volume of a saturated solution of NaCl with nine volumes of water), 

 and the mixture is poured into shallow basins, which are set aside in a 

 cool place, so as to allow the greater part of the blood corpuscles to 

 settle. 6 The supernatant liquid is decanted, and the magma of corpuscles, 

 mixed with a small quantity of water, is poured into a stoppered 

 separating funnel. The contents of this funnel are treated with an 

 equal volume of ether. After repeated, but not too violent, agitation, 

 the deep red aqueous solution is separated from the supernatant ether, 

 and filtered as quickly as possible. The clear red filtrate cooled to C. 

 is then mixed with one-fourth of its volume of absolute alcohol, likewise 

 cooled to C. The mixture is then maintained for a couple of days 

 (and, if crystallisation has not occurred, even longer), at a temperature of 

 5 C. to 10 C. In a period varying between twenty-four and 

 forty-eight hours crystallisation has usually occurred, and, unless the 

 solution of haemoglobin was too dilute, the whole of the liquid has set into 

 a mass of crystals. The crystals are now collected on a filter (the process 

 of filtration being carried on at as low a temperature as possible, in any 

 case below C.) and washed several times with a previously cooled 

 mixture, composed of one volume of absolute alcohol and four volumes 

 of distilled water. The filter with its contents is now placed between 

 sheets of filtering paper, and as much as possible of the adhering mother- 

 liquor is removed by gentle pressure. The oxyhsemoglobin thus 



1 "Zuv Krystallisation des Blutes," Virchow's Archiv, 1864, Bd. xxix. S. 29. 



2 "Die Blutkrystalle," S. 19 and 20. 



3 The blood of the dog, and especially of the horse, are to be preferred for the preparation 

 of large quantities of oxyhiemoglobin. As the success of the various operations depends 

 upon their being conducted at a low temperature, the preparation of haemoglobin for purposes 

 of research should only be attempted in the depth of winter. 



4 " Beitrage zur Kenntniss des Blutes des Menschen und der Wirbelthiere," Med. 

 Chem. Untersuch., Berlin, 1866, S. 170, 180-185; "Handbuch d. physiologisch. cheni. 

 Analyse," Berlin, 1893, Aufl. 6, S. 274. 



5 In the preparation of haemoglobin from the blood of birds, amphibia, and fish, sodium 

 sulphate is to be employed in the place of sodium chloride. In the case of mammalian 

 blood, it presents no advantages over sodium chloride. 



6 Instead of allowing the corpuscles to separate, as described, it is preferable to employ a 

 centrifugal machine. The separation of the corpuscles from the mixture of serum and salt, 

 solution is not only very much more rapid, but also much more complete, and therefore the 

 obtaining of pure oxyluiemoglobm is facilitated. 



