J. C. Johnson 
277 
placed in Petri dishes arranged as moist chambers and kept in the dark. 
Having had to make so many preparations I found this method rather 
inconvenient, besides not gauging the dilution of the blood definitely, 
and abandoned it for the collection of the blood in tubes of small bore. 
A certain amount of the methylene-blue-citrate mixture was placed in 
the tube and the blood was allowed to run in until it reached a mark 
previously made on the tube. Several specimens were thus taken from 
each animal containing blood diluted from one-sixth to several times its 
volume with the staining solution. Great care was taken that no clot 
should form on the skin of the animat, and as the blood entered the tube 
the latter was gently shaken to distribute the corpuscles. The tubes 
were f)rotected from light. In every case at least one specimen of the 
blood was run into tubes containing Hayem’s solution for microscopical 
comparison with blood mixed with methylene blue. 
In the normal animals I fouud the quantity of stain recommended 
by Braddon (from one-fifth to one-third volume of blood) to be inade¬ 
quate in preventing coagulation. Thus, one-fourth dilution of rabbit’s 
blood produced a distinct but delayed clot while the same dilution of 
the more hydraemic blood of a dog with acute piroplasmosis produced 
none. 
Examination. Four methods of examination were used in observing 
the condition of the erythrocytes:— 
1. A drop of the mixture from the tubes was transferred to a slide, 
covered, and evaporation prevented by means of vaseline around the 
edge of the cover-slip. 
2. Cover-glass films were made from the tube specimens, fixed wet 
over formalin, or with a saturated aqueous solution of mercuric chloride, 
and finally mounted in Canada balsam. 
3. Films of the fresh blood were made at the time of collection, and 
treated with either Giemsa’s or Leishrnan’s stain. 
4. For correlation with the above, unstained films were observed of 
the blood mixed with Hayem’s solution. 
Specimens that had clotted were, of course, useless for microscopical 
observation, and in addition to these a great many films had to be 
rejected owing to the osmotic changes in the red corpuscles. Many 
varieties of di'opsical, crenate, buckled and gastrula-shaped erythrocytes 
were found in preparations in which isotonicity had not been pre¬ 
served. 
The logical objection to the method of blood-collection as carried out 
above lies in the uncertainty of knowing whether the right amount of 
