116 MICROSCOPIC METHODS 



be restored by washing the film with acetic acid, 1-1500. The film is 

 dried between filter- paper and mounted. 



For staining sections a little modification is necessary. A paraffin 

 section is taken into distilled water as usual, the excess of water is drained 

 off, and a mixture of one part of stain and two parts of distilled water 

 is placed on it. The stain is allowed to act for five to ten minutes till the 

 tissue appears a deep Oxford blue ; it is then decolorised with 1-1500 

 acetic acid the effect being watched under a low-power lens. The blue 

 begins to come out, and the process is allowed to go on till only the 

 nuclei remain blue. The section is then washed with distilled water, 

 rapidly dehydrated with alcohol, cleared, and mounted. If, as some- 

 times happens, the eosin tint be too well marked, it can be lightened 

 by the action of 1-7000 solution of caustic soda, this being washed off 

 whenever the desired colour has been attained. 



In certain cases, e.g., for the staining of old films or of trypanosomes 

 or Leishmanise in sections, Leishman recommends an initial treat- 

 ment of the preparation with serum. This modification is described in 

 Appendix E. 



3. J. H. Wright's Stain. In this modification 1 per cent, methylene- 

 blue (BX or Ehrlich's rectified) and \ per cent, sodium carbonate (both 

 in water) are mixed and placed in a Koch's steriliser for an hour. When 

 the fluid is cold, 1-1000 solution of extra B. A. eosin is added till the mix- 

 ture becomes purplish and a finely granular black precipitate appears in 

 suspension (about 500 c.c. eosin to 100 c.c. methylene-blue solution are 

 required) ; the precipitate is filtered off and dried without being washed. 

 A saturated solution of this is made in the pure methyl alcohol ; this is 

 filtered and diluted by adding to 80 c.c. of the saturated solution 20 c.c. 

 of methyl alcohol. The application of the stain is almost the same as 

 with Irishman's. A few drops are placed on the preparation for a 

 minute for fixation ; water is then dropped on till a green iridescent 

 scum appears on the top of the fluid, and staining goes on for about 

 two minutes ; the stain is then washed off with distilled water, and 

 a little is allowed to remain on the film till differentiation is com- 

 plete ; the preparation is carefully dried with filter-paper, and mounted. 



4. Giemsa's Stain. Giemsa believes that the reddish-blue hue 

 characteristic of the Romanowsky stain is due to the formation of 

 methyl-azure, and he has prepared this by a method of his own under 

 the name " Azur I." From this, by the addition of equal parts of 

 medicinal methylene-blue, he prepares what he calls "Azur II.," and 

 from this again by the addition of eosin he prepares "Azur II. -eosin." 

 The latest formula for the finished stain is as follows : Azur II. -eosin, 

 3 gr. ; Azur II., 8 gr. ; glycerin (Merck, chemically pure), 250 gr. ; methyl 

 alcohol (Kahlbaum, I.), 250 gr. This stain has been extensively used 

 for demonstrating the spirochsete pallida, but it can be used for any 

 other purpose to which the Romanowsky stains are applicable. For the 

 spirochsete the following are Giemsa's directions : 



(1) Fix films in absolute alcohol for fifteen to twenty minutes, dry 

 with filter-paper. (2) Dilute stain with distilled water one drop of 

 stain to 1 c.c. water (the mixture being well shaken). (Sometimes the 

 water is made alkaline by the addition of one drop of 1 per cent, potassium 

 carbonate to 10 c.c. water.) (3) Stain for fifteen minutes. (4) Wash in 

 brisk stream of distilled water. (5) Drain with filter-paper, dry, and 

 mount in Canada balsam. 



With regard to the Jenner and Giemsa stains it is best to obtain the 



