.'^12 Microorganisms (chap. 20) 



13. Blot with filter paper. Do not dry. 



14. Flood with equal parts of aniline oil and xylene, rcflood until no 

 color washes out of sections. 



15. Xylene and mount. 



results: 



gram-positive bacteria — red 

 gram-negative bacteria — blue 

 fibrin — blue 

 other tissue elements — reds to purples 



Acid-Fast Staining 



Ziehl-Neelsen (modified from mali.ory, 1944) 

 fixation: any general fixative. 



solutions: 



Carbol-fuchsin: 



basic fuchsin, C.I. 42500, saturated in absolute 

 ethyl alcohol (approximately 6 gm./lOO ml. 



alcohol) 10.0 ml. 



carbolic acid, 5% (5 ml. melted carbolic acid/95 



ml. water) 90.0 ml. 



Store at room temperature. It is advisable to filter each time before 

 use. 



Acid alcohol: 



hydrochloric acid, concentrated 1.0 ml. 



70% ethyl alcohol 99.0 ml. 



Methylene blue solution: 



methylene blue chloride, C.I. 52015 0.5 gm. 



glacial acetic acid 0.5 ml. 



distilled water 100.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides to Avater; remo\e HgCU. 



2. Stain in carbol-fuchsin, 55 °C: 30 minutes. 



3. Wash in running water: 5 minutes. 



4. Destain in acid alcohol until sections are pale pink; check under 

 microscope. 



5. Wash in running water: 3-5 minutes. 



6. Dip one slide at a time into methylene blue, taking care that sec- 

 tions remain light blue; rinse briefly in distilled ^vater. 



