528 APPENDIX C. 



Solutions : A. Medicinal methylene-blue (Griibler) in i per cent 

 watery solution, with .5 per cent sodium carbonate added. The solution 

 is heated for about twelve hours at 65 C., and then kept for about a 

 week at warm room temperature. 



B. Eosin "extra B.A." (Griibler) in 1:1000 watery solution. 



First Method. Dilute some of A and B, each with 25 vols. of 

 water. Then mix equal parts (say 2 c.c.) of the diluted stains. Stain 

 films (fixed in alcohol and ether or in alcohol) for half an hour or longer. 

 When the staining is sufficient the nuclei of the leucocytes, when examined 

 under the microscope, should have a ruby-red colour. Then decolorise 

 slightly, by washing in alcohol for two or three seconds, or in water for 

 about half an hour. Then allow to dry and mount in balsam. 



Second Method. Equal parts of A and B (say 500 c.c. of each) are 

 mixed, allowed to stand for 6-12 hours, the mixture being thoroughly 

 stirred from time to time. The mixture is then filtered, and the deposit 

 which is got on the filter is dried and powdered. A .15 per cent solu- 

 tion of this is made in methyl alcohol (Merck, "for analysis"). This 

 alcoholic solution fixes and stains at the same time. Place 3-4 drops on 

 the film for half a minute, then add 6-8 drops of distilled water, mix 

 with the stain, and allow to remain for five -minutes longer. (This in- 

 tensifies the staining, especially the red tint.) Then wash in water, allow 

 to dry, and mount in balsam, or simply examine in water. 



In the Romanowsky method, the chromatin of the parasites ought to 

 be brilliant red, the protoplasm blue. 



It is to be noted that with practically all the methods of staining, 

 better results are obtained when the blood films have been recently made 

 than when they have been kept for some time. 



