2230 Journal of Applied Microscopy 



stain have employed various reagents, as soda, alcohol, acetic acid, etc., for dis- 

 solving the eosin-methylen blue precipitate. Secondly, there has been the pro- 

 duction by various treatment of a polychrome condition of the methylen blue, 

 whereby there is secured a greater differentiation in staining. And finally sev- 

 eral hematologists have succeeded in isolating the active staining ingredient of 

 the compound dye and applying it as a separate stain. 



Nocht (1898) modified the Romanowsky stain as follows: To 2 or 3 

 drops of a 1 per cent, eosin solution diluted with 1 to 2 c. c. of water is added 

 drop by drop a cool solution, consisting of 1 per cent, methylen blue and one- 

 half per cent, soda which has stood several days in a thermostat at 50° to 60°C. 

 until the eosin solution becomes more and more blue-red, and finally so dark 

 that the original eosin solution can scarcely be recognized. On this mixture 

 the blood preparations swim 5 to 10 minutes. 



Z(?7'^r<:z/r (1900) recommends the following eosin-methylen blue compound 

 for staining malarial blood : 



Solution No. 1. Silver oxid methylen blue (Borrel blue) is mixed with 150 

 c. c. of distilled water in a flask. Enough of the crystals are used to form a 

 nearly saturated solution. When the dye is dissolved the flask is filled with a 

 solution of soda and shaken, which causes a black precipitate of silver oxid. 

 This is carefully washed and a saturated aqueous solution of medicinal methylen 

 blue is poured over it and allowed to stand eight days, shaking occasionally. 



Solution No. 2. An aqueous solution of eosin 1 to 1000. 



Solution No. 3. A 5 per cent aqueous solution of tannin. 



The stain is prepared just before using as follows : 



Solution No. 1 - - - - - 1 c. c. 

 Solution No. 2 ... - 4 c. c. 



Distilled water - - - - - 6 c. c. 



Each should be filtered before mixing. 



Blood smears are fixed in alcohol 2(1 minutes and stained 5 to 10 minutes. 

 When stained the preparation is treated with the tannin (Solution No. 3) for a 

 minute. Nuclei are stained deep violet, red corpuscles pink, protoplasm of 

 malaria parasites a pale blue, chromatin a violet-red. 



Laurent (1900) studied the chemistry of the neutral stains and determined 

 from their molecular weights the proper proportions of the two dyes to mix for a 

 neutral stain. He used 1 per cent, solutions and to 1000 c. c. of the eosin solu- 

 tion was added 882.3 c. c. of the methylen blue solution. . The mixture is allowed 

 to stand at least 48 hours, when almost all of the neutral dye will have fallen as 

 a precipitate. The mixture stands as a suspension of neutral dye in a watery 

 fluid. This suspension remains good for 6 months, and will keep indefinitely if 

 the solution is kept practically sterile after the fall of the precipitate by storing, 

 after thorough shaking, in small, well-corked flasks. 



For staining one takes 1 part of this well-shaken mixture to 24 parts of water 

 in a reagent glass and brings it to a boil as quickly as possible over the flame of 

 a Bunsen burner. As soon as the solution comes to a boil the reagent glass is 

 cooled somewhat in water, and the preparation to be stained is placed in this 

 still warm clear fluid. A too hot solution spoils the preparation. The staining 



