326 Microorganisms (chap. 20) 



Methylene blue solution: 



Dissolve methylene blue, C.I. 52015 21.0 gm. 



in 95% ethyl alcohol 300.0 ml. 



Dissolve potassium hydroxide 0.1 gm. 



in distilled water lOOO.O ml. 



Mix the two solutions and let stand 24 hours. 

 Staining solution: 



Mix buffer working solution with methylene blue solution. 



procedure: 



1. Deparaffinize and hydrate slides as far as 50% alcohol. 



2. Stain in methylene blue: 2-3 minutes. 



3. Wash in running water: 30 seconds. 



4. Counterstain in 1% safranin (1 gm./lOO ml. water): 1-2 minutes. 



5. Dip briefly in 95% alcohol. 



6. Dehydrate in 2 changes absolute alcohol, clear, and mount. 



results: 



rickettsiae — light blue 



comments: 



Burrows {1954) recommends this as one of the best methods for 

 rickettsia. 



Ordway-Machiavello Method (gradwohl, 1956) 

 fixation: Regaud recommended. 



solution: 



Staining solution: 



Poirrier's blue (C.C), 1% aqueous 10.0 ml. 



eosin bluish, C.I. 45400, 0.45% aqueous 15.0 ml. 



Mix just before use. Add slowly with constant shaking: 



distilled water 25.0 ml. 



Use within 24 hours. 



procedure: 



1. Deparaffinize and hydrate slides to water. 



2. Stain: 6-8 minutes. 



3. Decolorize in 95% ethyl alcohol until slides appear pale bluish 

 pink. 



4. Dehydrate in absolute alcohol: 1 minute. 



5. Clear and mount. 



