WRIGHT'S STAIN OF EOSINATE OF METHYLENE BLUE U3 



Staining the Polar Bodies or Babes-Ernst Granules. Neisser's 

 Method. 1. Prepare and fix cover-glass as usual. 



2. Stain with the following solution for one to three seconds : 



Methylene blue . 1 gram 



Absolute alcohol * . . . 20 c.c. 



Glacial acetic acid ............. 50 c.c. 



Distilled water . . . ; .'. T * "... . . . . . . 1000 c.c. 



3. Wash in water. 



4. Counter-stain in a 2 per cent, watery solution of Bismarck- 

 brown. 



5. Wash in water, dry, and mount in Canada balsam. Result of 

 the stain polar bodies blue, other parts of bacterium light brown. 



Piorkowski's Method. 1. Prepare cover-glass as usual and stain 

 for one-half to one minute in Loeffler's alkaline methylene blue. 



2. Decolorize in alcohol containing 3 per cent, hydrochloric acid 

 for five seconds. 



3. Wash rapidly in water. 



4. Counter-stain in a 1 per cent, watery solution of eosin, very 

 rapidly. 



5. Dry between filter paper and mount. Result of the procedure 

 polar bodies blue, rest of bacterium eosin pink or yellow. 



Wright Stain of Eosinate of Methylene Blue. This stain is an ex- 

 ceedingly useful one, and it has a wide field of application, not so 

 much for staining bacteria from pure cultures, as for bacteria in pus, 

 and particularly for protozoa, such as malarial plasmodia, trypan- 

 osomes, piroplasmata, and for blood films in general. It is a modi- 

 fication of the stains of Romanowsky and Leishman. The dye can 

 be bought ready made or can be easily prepared as follows : 



1. Prepare in a flask a \ per cent, of sodium bicarbonate in water. 

 Add, after complete solution, 1 per cent, of methylene-blue in sub- 

 stance (either one of the following three preparations of Gruebler's 

 may be used: methylene-blue BX, Koch's or Ehrlich's rectified). 



2. Place the sodium bicarbonate methylene-blue solution in the 

 steam sterilizer, where it is left for one hour after the steam is up. 

 Then remove and allow to cool. 



3. Prepare a -fa per cent, watery solution of eosin (yellowish eosin 

 of Gruebler). 



4. Place the methylene-blue solution in a large flat dish and add the 

 YQ- per cent, eosin solution gradually, stirring constantly with a glass 

 rod. Keep this up until the mixture assumes a purplish color and 

 until a scum with a yellowish metallic lustre forms on the surface 

 and a finely granular black precipitate appears in the suspension. 

 This will generally require about 500 c.c. of the -fa per cent, eosin 

 solution to 100 c.c. of steamed alkaline methylene-blue solution. 

 After the precipitate has formed the fluid is run through a dense 

 paper filter and the precipitate collected. The fluid which runs 

 through the filter is of no further use, and is not kept. The pre- 



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