CRYSTALS OF HEMOGLOBINS EMPLOYED IN THIS RESEARCH. 143 



hemoglobins that are normally present in these bloods. In no instance did 

 we find any evidence of any influence on the type of crystals that is peculiar 

 to the species. We did not make any investigations of the possible influ- 

 ences of diseased conditions upon the form of crystallization, because, in 

 the first place, of an insufficiency in our supplies, and, secondly, because 

 Dr. S. Weir Mitchell and others have found, as far as their studies have 

 gone, that disease is without influence on the type of crystals. Assuming, 

 however, that the presence of ammonium oxalate might have some undis- 

 covered effect upon the morphological or optical properties of the crystals, 

 we, with the few exceptions indicated, always introduced the oxalate, and 

 as nearly as possible in the same proportion. 



Most of our specimens were in various stages of putrefaction. For- 

 tunately, hemoglobin, in comparison with other proteins, is remarkably 

 resistant to putrefaction, so that even when the plasma proteins are in an 

 advanced state of decomposition the hemoglobin may have merely suffered 

 a partial alteration to reduced hemoglobin and metoxyhemoglobin. Upon 

 exposure of the blood to the air, especially shaking with the air or with an 

 atmosphere of pure oxygen, a rapid restoration of oxyhemoglobin is readily 

 brought about, and unless the blood is excessively putrid the exposure of 

 the drops of the prepared blood upon the slides antecedent to the covering 

 with cover-glasses is sufficient to yield good preparations of oxyhemoglobin. 



Each of our processes is characterized by three major procedures, which 

 are accompanied by such accessory procedures as conditions indicated: 

 (1) the addition of oxalate; (2) laking with ethyl ether (Squibb's); (3) 

 centrifugalization ; (4) occasional accessory procedures, such as variations 

 in temperature, the addition of asbestos wool, alumina, etc., to aid in the 

 separation of the stromata or the nuclei of erythrocytes, keeping the prep- 

 arations in a moist chamber, etc. Oxalate was added (a) to prevent coagu- 

 lation, (b) to increase crystallizability, or (c) to obtain one or another form 

 of oxyhemoglobin present in the same blood. In the process of laking, the 

 ether was usually added in three or four portions, the mixture being shaken 

 vigorously after each addition, and sufficient ether being added to cause 

 marked pressure within the test-tube when the opening of the tube is closed 

 by the finger. When the blood is very putrid no more ether should be used 

 than is absolutely necessary to cause complete laking, otherwise the hemo- 

 globin is likely to be thrown down in the form of an insoluble precipitate. 

 Caution must also be practised when working with bloods, oxalated or not, 

 which contain nucleated erythrocytes. An excess of ether is likely to cause 

 coagulation, and especially so the larger the quantity of oxalate present. 

 One of us (Reichert, Journal of Experimental Medicine, April, 1905) has 

 found that even oxalated defibrinated blood may be converted into a gelat- 

 inous mass by the addition of ether. Centrifugalization was practised, 

 (a) to collect the corpuscles, and thus get rid of substances which hinder 

 crystallization, and at the same time to secure a concentrated solution of 

 hemoglobin; and (b) to clear the laked preparation of the stromata and 

 other bodies in suspension. Occasionally our specimens were too small to 

 centrifugalize. 



