METHODS OF STAINING OF BACTERIA 51 



By the use of nigrosin it is possible to examine organisms 

 unstained. There are many points in favor of this method, e.g., 

 the organisms do not shrink or change their form. Nigrosin may 

 be used in demonstrating the motihty of bacteria. For this 

 purpose add a very small amount of nigrosin to a hanging drop. 



Congo Red for Negative Mounts 

 (For differentiating living and dead bacteria) 



1. Place a drop of 2 per cent aqueous Congo red solution 

 (free from bacteria) on a clean glass slide. 



2. Mix with it a loopful of the bacterial culture. 



3. Allow it to dry thoroughly in air 10 minutes or more. 



4. Flood with acid alcohol (1 or 2 per cent HCl). This 

 changes the color to blue and fixes the film. 



5. Dry without washing and examine in oil, with or without 

 cover glass. Living cells appear unstained-white against blue. 



Unless preserved with oil or balsam the preparations fade 

 rapidly. 



This method of preparing negative mounts is recommended 

 for root-nodule bacteria. The active living cells are negative 

 while the dead cells are more or less positive. 



Erythrosin for Direct Staining of Soil Bacteria 



1. Prepare a suspension of soil (about 1 : 10) in a 0.015 per cent 

 solution of gelatin. 



2. Smear a thin film on a slide and dry. 



3. Immerse in 40 per cent acetic acid for 1 to 3 minutes, wash 

 and dry on a water bath. 



4. While on the water bath, stain for 1 minute with 1 per cent 

 aqueous solution of erythrosin Y or rose bengal. 



Barlow Stain for Root Nodule Bacteria 

 (See p. 109) 



