MICHIGAN ACADEMY OF SCIENCE. Ill 



methylene blue stainiiisr. Even when the sections were subjected to the 

 action of the stain for twenty-four hours with several changes of the solution 

 no chromatin staining resulted. The temperature of the staining solution 

 was now raised by placing the solution containing the preparation in the 

 hot-room at 35° C* When removed at the end of one hour and examined 

 in water the nuclei of the cells showed a red chromatin stain, the protoplasm 

 being blue. The attempt was made to dehydrate in absolute alcohol but a mo-^ 

 mentary dip changed the red in the chromatin to lilue. After drying in the air, 

 even in 3 micron sections, the structure of the tissue worked with was de- 

 stroyed owing to the shrinkage. After placing the sections in 2%tannin solution 

 it was found that they could be hurriedly passed through absolute alcohol, 

 to xylol and balsam. The best results have been obtained by placing the 

 solution containing 3 micron sections in the hot-room at 35° for one hour and 

 twenty minutes, renewing the solution at the end of forty and eighty minutes 

 respectively. They are then washed in water — if quite blue wash in 

 1-1000 acetic [acid imtil pink — and placed in a 2% tannin solution 

 for ten to forty minutes. The tannin not only makes dehydration 

 in alcohol possible but also differentiates, taking out the excess of blue. 

 The tannin solution is washed off with water, the excess of water removed 

 by blotting, and the ])re])aration hurriedly passed through absolute alcohol 

 to xylol and mounted in balsam. Sections of spleen, liver, and lymi)h- 

 gland containing Leishmania infantum have been successfully stained In- 

 this method, the large and the small chromatin masses staining violet, the 

 protoplasmic body of the parasite a robin's egg blue. Sections of lung of 

 an owl infected with //. ziemanni stained by this method show the chro- 

 matin of the parasite stained a violet red, the protoplasm blue. 



It would seem then, at least in the case of some polychrome solutions, 

 that it is necessary to raise the temperature of the staining fluid in order to 

 get chromatin staining, and that insolubilization in some fluid such as tannin 

 is necessary before dehydration in alcohol is possible. 



I wish to express my indebtedness to Dr. F. G. No\y for the suggestions 

 which led to the development of this method and to Dr. G. Carl Huber 

 for the loan of apparatus used in cutting sections. 



Hygienic Laboratory, U. of M., Ann Arbor, March 31, 1909. 



*NoTE. — The Borrel staining glass was found to be well adapted for tliis procedure. The section 

 was fixed on a slide near one end and placed in the solution in the cylinder which was then placed in 

 the hot room. The solution may be renewed by washing the stain out of the glass under the tap. 



iGrassiand Feletti. Centrabl. f. Bakt., 1891. X. p. 517. 



2Romanowsky. St. Petersburg. 1891, Zur Frage der Parasitologie and Therapie der Malaria. 



3Nocht. Centrabl. f. Bakt., 1898, 24 p. 839; 1899, 25, p. 764. 



^Renter. Munch. Med. Wochensch., 1901, 48. p. 1261. Centralbat. Bakt., 1901, 30, p. 248. 



sLeishman. Brit. Med. Jour., 1901, 1, p. 635. Brit. Med. Jour., 1901, 2, p. 757. 



cWright. Jour. Med. Res.. 1902, 7, p. 138. 



^Giernsa. Centrabl. f. Bakt.. 1902. 31. p. 429; 1902. 32, p. 307. 



SMarino. Ann. de L' Inst. Pasteur, 1904. 18, p. 761. 



9Unna. Monatsh. f. Prakt. Dermatol., 1904, 38, p. 119. 

 '"Christophers. Sc. Mem. by Officers of the Med. and sanitary Dept. of Gov. of India, 1904. 

 iiLeishman. Jour. Hyg., 1904, 4. p. 434. 

 i^Nicolle. .\rchiyes de L' Inst. Pasteur de Tunis, 1908, 1, p. 17. 



