308 Microorganisms (chap. 20) 



solutions: 



Mayer's hematoxylin, see page 125. 

 Gentian violet: 



crystal violet, C.I. 42555 (gentian violet) 1.0 gm. 



distilled water 100.0 ml. 



Sodium bicarbonate: 



sodium bicarbonate 5.0 gm. 



distilled water 100.0 ml. 



Gram's iodine, see page 410. 

 Basic fuchsin: 



basic fuchsin, C.I. 42500 0.25 gm. 



distilled water 100.0 ml. 



Dilute 0.1 ml. to 100 ml. distilled water for use. 



Acetone-picric acid solution: 



picric acid O.I gm, 



acetone 100.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides to water. 



2. Stain in Mayer's hematoxylin: 2-3 minutes. 



3. Wash in rimning water: 5 minutes. 



4. Mix 26 drops of gentian violet and 5 drops of soditim bicarbon- 

 ate solution. Flood slide, agitate occasionally: 2 minutes. 



5. Rinse in water. 



6. Treat with Gram's iodine: 1 miniue. 



7. Rinse in water and blot. 



8. Decolorize ^vith 1 part ether and 3 parts acetone, flooding slide 

 until no more blue color comes off. Blot. 



9. Stain in basic fuchsin: 5 miniues. 



10. Rinse quickly in water, blot btu do not dry. 



11. Dip briefly in acetone, and iminediately differentiate in acetone- 

 picric acid until sections ttirn yellowish pink. Overdifferentiation 

 may take out gxam negative stain. 



12. Rinse quickly in acetone, transfer to acetone-xylene (equal parts), 

 then to xylene and motmt. 



results: 



gram-positive organisms — deep \iolet or black 

 gram-negative organisms — bright red 

 cell nuclei — reddish bro^vn 



