110 ELEVENTH REPORT. 



THE APPLICATION OF THE R0MAN0W8KY METHOD OF STAINING 



TO SECTIONS. 



Frank A. McJunkin, M. D. 



Grassi and Feletti \ in 1891, succeeded in staining the chromatin of the 

 malarial parasite by the prolonged application of a methylene blue sohition, 

 one drop of the saturated alcoholic solution to the watch-glass of distilled 

 water. Romanowsky ^ later in the same A'ear recommended a methylene 

 blue-eosine solution for staining films of malarial blood, which colored the 

 chromatin of the parasite red, the protoplasmic portions blue. This specific 

 differential staining of chromatin is common to the many polychrome methy- 

 lene blue solutions subsequently recommended. Nocht ^, Renter *, Leish- 

 man \ Wright °, Giemsa ', and i\Iarino * developed modifications, recommended 

 for the staining of blood films and smears containing bacteria. 



Unna*, early in 1904, reported the results of sections stained with poly- 

 chrome methylene blue, and various combinations of methylene azure 

 (Giemsa). The gross appearence of all sections was blue or blue-violet 

 with blue or blue-violet nuclei. The tissue with Avhich he worked contained 

 no protozoa. Christo])hers ' " stained sections of organs from cases of "trop- 

 ical, spleno-megaly," containing the " Leishman-Donovan bodies" with a 

 polychrome solution. Leishman ^ ' later this same year published a method 

 for staining chromatin in sections. He was not able to get good results by 

 the method of Christophers owing to the shrinkage of the tissue lirought 

 about by the drying to which it was subjected. Previous to this time he 

 had failed to get chromatin staining in sections with his polychrome stain 

 and thought that a chemical change was produced in the tissue by the harden- 

 ing and embedding which made it impossible. He cuts 5 micron sections 

 and after removal of paraffin with xylol, and passage through alcohol places 

 on them fresh blood serum for five minutes, ]:)lots off the excess and allows 

 the remainder to dry on the preparation. He then places on the section 

 a solution composed of two parts of "Leishman stain" and three parts of 

 distilled water for one and a half hour, renewing the solution once or twice 

 during this time. The section is now differentiated in 1-1500 acetic acid 

 and a 1-7000 sodium hydroxide, dehydrated in absolute alcohol, passed 

 through xylol and mounted in balsam. He remarks that this method 

 induces chromatin staining which resists the decorization of absolute alcohol, 

 although he qualifies this statement by remarking that the red chromatin 

 of the cell nuclei is often changed to blue b}' the action of the alcohol. He 

 worked with sections containing Tr. brucei, the nucleus and micronucleus 

 being stained red, while the protoplasm was at times blue. Nicolle ^ '^ in 

 working with Leishmania infantum stained sections with a Giemsa solution 

 followed by 2% tannin, absolute alcohol, xylol, Ijalsam. His j^lates show 

 the two nuclear bodies chromatin-stained while the protoplasm is unstained. 



The method Avhich I shall now describe was developed while attempting 

 to stain Leishmania infantum in sections of si-)leen, liver, etc. Blood films 

 and smears containing ])rotozoa are ordinarily placed in the jiolychrome- 

 eosine solution which is in use in this laboratory for twenty to forty minutes 

 with one change of solution, chromatin and differential staining l>eing well 

 brought out in this time. Sections Avhen treated in this way show only 



