38 A MANUAL OF BACTERIOLOGY 



/. Wash with 60 per cent alcohol until only a light brown 

 shade remains (as if the glass were smeared with dry blood). 



g. Rinse off alcohol with water. Dry and mount, or contrast 

 stain with eosin or Bismarck brown. If the bacteria are Gram 

 positive, they will appear a deep blue under the microscope. 



2. Ziehl-Neelson method of demonstrating B. tuberculosis and 

 other acid-fast organisms. 



a. Spread a thin film on the cover glass ; dry and fix as usual. 



b. Stain with hot carbol-fuchsin five to ten minutes (entire 

 film stained). 



tf. Decolorize with 25 per cent sulphuric acid or 30 per cent nitric 

 acid. (Removes stain from everything but acid-fast organisms.) 



d. Wash thoroughly with water. 



e. Counterstaiii the film with weak methylene blue. (Stains 

 non-acid-fast organisms, leucocytes, epithelial cells, etc.) 



f. Wash in water, dry, and mount. 



SECTION VIII 

 FUNDAMENTAL METHODS OF ISOLATION 



In nature single species of bacteria do not often grow alone. 

 Even in those cases where they do exist it is not always possible 

 to get them into artificial culture without contamination with 

 other species. Yet the student of bacteriology must have pure 

 cultures of a single species in order to learn anything definite 

 about them. This was one of the greatest handicaps from which 

 the earlier workers suffered. After the introduction of agar and 

 gelatin media the problem of isolating species was greatly simpli- 

 fied, since it was then possible to get colonies sufficiently sepa- 

 rated from each other, on plates, to obtain bacteria which were 

 the descendants of a single organism, and therefore of the same 

 species. For special methods of isolation in cases where this 

 procedure is not successful, the student is referred to Eyre, 

 Bacteriological Technique, Chapter XIII ; Muir and Ritchie, 

 Manual of Bacteriology, page 51 ; or Abel, Bacteriologisches 

 Taschenbuch. 



