THE ROMANOWSKY STAINS 115 



the fluid is cold, 1-1000 solution of extra B. A. eosiii 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 Leishman'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, 

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

 alcohol (Kahll.aum, 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 

 spirochiete 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 

 solutions from Griibler ready for use ; the powder for Leishman's stain 

 may be obtained from the same source, and the solution made up by 

 the worker himself. Cabot states that Wright's stain can be obtained 

 from the Harvard Co-operative Society, Boylston Street, Boston, 

 U.S.A. 



Neisser's Stain. (a) The following is the original method introduced 

 by Neisser as an aid to the diagnosis of the diphtheria bacillus. Two 

 solutions are used as follows : (a) I grm. methylene-blue (Griibler) is 

 dissolved in 20 c.c. of 96 per cent, alcohol, and to the solution are added 

 950 c.c. of distilled water and 50 c.c. of glacial acetic acid ; (ft) 2 grins. 

 Bismarck-brown (vesuvin) dissolved in a litre of distilled water. Films 

 are stained in (a) for 1-3 seconds or a little longer, washed in water, 

 stained for 3-5 seconds in (6), dried, and mounted. The protoplasm of 

 the diphtheria bacillus is stained a faint brown colour, the granules a blue 

 colour. Neisser considers that this reaction is characteristic of the 

 organism, provided that culture..* on Loffler's serum are used and examined 

 after 9-24 hours' incubation at 34 to 35 C. Satisfactory results are not 

 always obtained in the case of films pn-pan-d from membrane, etc., but 

 there is no doubt that here also the method is one of considerable 

 value. 



