2178 Journal of Applied Microscopy 



To this mixture is then added, 



Saturated aqueous solution of methyl green - 65 

 Distilled water ------ 50 



Alcohol -------- 12 



This mixture must then stand 1 to "2 weeks before use. It gives good stain- 

 ing after dry heat or alcohol and ether fixation. Stain 10 to 15 minutes, wash 

 in water, dry in the air and mount. Nuclei are stained green, hemoglobin 

 orange, acidophile granules yellowish red, neutrophile granules violet red. 



Neiiser (1894) used a modified triple stain for staining his " perinuclear 

 basophile granules": 



Saturated watery solution of acid fuchsin - 50 c. c. 



" " " " orange G - - 70 c. c. 



" " " " methyl green - 80 c. c. 



Distilled water . - . - - 150 c. c. 



Absolute alcohol 80 c. c. 



Glycerin - - - - - - - 20 c. c. 



Dry preparations stained with this compound show in certain diseases (gout, 

 leukaemia, etc.,) a grouping of dark blue stained granules around the nuclei of 

 the mononuclear leucocytes and over and around the nuclei of the polymorpho- 

 nuclear leucocytes. 



Prince (1898) substituted toluidin blue for methyl green and eosin for orange 

 G in Ehrlich's tricolor stain. 



Saturated solution of toluidin blue (Griibler) - 24 

 " " " acid fuchsin - - - 1 



2 per cent, eosin ------ 2 



The solutions should be made in distilled water and should be mixed in the order 

 given and then be thoroughly agitated for a few moments to prevent precipita- 

 tion of the toluidin blue by the acid dye. Only the supernatant fluid is to be 

 used for staining. This mixture is ready for immediate use after agitation. 

 Stain from 30 to 60 seconds with fresh stain. It possesses good staining qual- 

 ities at the end of 10 to 12 weeks, but it then requires 5 to 7 minutes' action to 

 produce good results. Preparations should be fixed by dry heat 120°C. for at 

 least 20 minutes. 



Ehrlich has recommended several modifications of his original formula, the 

 latest (1S98) is as follows: 



Saturated watery solution of orange G - 13-14 c. c. 



" " " " acid fuchsin 6-7 c. c. 



Distilled water - - - - - 15 c. c. 



Alcohol - - - - - - 10 c. c. 



Saturated watery solution of methyl green 12.5 c. c. 



Alcohol - - - - - - - 10 c. c. 



Glycerin ------- lu c. c. 



These are to be measured in the order given in the same measuring glass, and 

 on the addition of the methyl green the solution is thoroughly shaken. The 

 solution can be used immediately and remains good for a longtime. Only slight 

 fixation is necessary. Stain not more than 5 minutes. Nuclei stain greenish, 

 red corpuscles orange, acidophile granules copper color, neutrophile granules 

 violet. Ernest L. Walker. 



Massachusetts State Board of Health. 



