x HEMOGLOBIN-CRYSTALS 475 



time. To prepare oxyhsemoglobin crystals proceed thus : Free the 

 red blood-corpuscles as thoroughly as possible from all the albumins 

 of the blood-plasma or blood-semm by decantation, or, still better, by 

 means of the centrifuge, suspending the corpuscles in isotonic salt-solu- 

 tion and centrifugalising them repeatedly. Now lake the corpuscles 

 by adding distilled water, or by repeated freezing and thawing, or 

 by the addition of a little ether, which is then allowed to evaporate. 

 Schuurmans-Stekhoven 1 disintegrates the corpuscles without the 

 addition of any chemicals by shaking them vigorously with particles 

 of asbestos. On cooling laked blood, crystals separate out with 

 the more readily crystallisable species of blood. Crystals in small 

 amounts may be easily obtained by laking defibrinated rat's blood 

 and then allowing the water to partially evaporate ; Ewald 2 allows 

 evaporation to take place after having covered laked blood with a micro- 

 scopic cover-glass ; with the blood of the squirrel good results are obtained 

 by simply mixing the blood with Canada balsam and then adjusting 

 the cover -glass. 3 To prepare oxy- haemoglobin crystals in larger 

 quantities it is necessary to add alcohol to the laked blood according 

 to the directions given by Hoppe-Seyler : 4 cool the laked blood to 

 zero centigrade, add one quarter the volume of alcohol also cooled to 

 zero, and keep the mixture for several days at - 5 to - 10. The 

 crystals are then dissolved in a little water warmed up to 35 \ the 

 insoluble residue, consisting of the stromata of the red corpuscles, is 

 filtered off, and the filtrate is again exposed to the cold after the 

 addition of alcohol. If this procedure is then repeated several times 

 more, a very pure, ash-free preparation is obtained. Zinnofsky 5 lakes 

 the blood directly by the addition of ether, without having previously 

 removed the albumins of the plasma ; he then dissolves the stromata 

 by the addition of very dilute ammonia, and removes them by very 

 careful neutralisation with hydrochloric acid. Jaquet 6 adds to the 

 blood of hens one-third its volume of ether at 35, as otherwise the 

 nucleated red corpuscles set into a jelly. Schuurmans-Stekhoven 

 places the laked blood into a dialysing tube and then suspends the 

 latter in 45 per cent alcohol ; as soon as crystallisation commences, 



1 Schuurmans-Stekhoven, Maly's Jahresb. f. Tierchemie, 31. 212 (1901). 



2 A. Ewald, Zeitschr.f. Biol. 22. 459 (1886). 



3 Professor Francis Gotch tells me that he first heard of this method from a student 

 who worked in Krukenberg's laboratory, and that the method was discovered by a pupil 

 of Krukenberg's. The author. 



4 F. Hoppe-Seyler, Med.-chem. Untersuckungen, p. 169 (1867) ; Handbuch der 

 physiol.-chem. Analyse. 



5 0. Zinnofsky, Zeitschr. f. physiol. Chem. 10. 16 (1885). 



6 A. Jaquet, ibid. 14. 289 (1889). 



