96 LABORATORY MANUAL OF MICROBIOLOGY 



of contamination which may be present in the dye solutions it is 

 always well to mount a drop of the uninoculated dye on the same 

 slide. 



Examine: Cultures of bacteria from hay infusion, B. suhtilis 

 or B. mesentericiis. 



1. Congo Red for Negative Mounts : 



(For differentiating living and dead bacteria.) 

 (a) Place a drop of 2-per cent aqueous Congo red solution (free 

 of bacteria) on a clean glass slide. 



(6) Mix with it a loopful of the bacterial culture. 



(c) Allow it to dry thoroughly in air 10 minutes or more. 



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

 changes the color to blue and fixes the film. 



(e) 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. 



2. Nigrosin for Negative Mounts : 



(a) Place a loopful of culture on a clean glass slide, spread, and 

 allow it to air dry. 



(b) Spread thinly over the smear a loopful of saturated aqueous 

 solution of nigrosin B. Spread either with the wire loop or with 

 a glass slide (as for blood smears). Dry and examine in oil, or 

 mount in Canada balsam. 



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. 



B. Examination of Yeasts and Yeast Spores in Erythrosin or Rose 

 Bengal Preparations 



In a drop of sterile water on a glass slide, mix a small amount of 

 the yeast culture. 



Add a large loop of Erythrosin or Rose Bengal (0.5 gram in 100 

 cubic centimeters of water). The dead cells stain a deep pink 

 while the living cells remain colorless. 



To prevent evaporation make a ring of vaseline around the 

 edge of the drop culture and place cover slip over the top. 



