APPENDIX 373 



STAINS 



Methylen blue for differentiating living from dead 

 yeast cells: 



Methylen blue 0.1 gm. 



Distilled water. . . 1000.0 c.c. 



Aqueous-alcoholic stains, fuchsin, methylen blue and 

 gentian violet: 



1. A saturated alcoholic solution of a stain is prepared 

 by shaking frequently about 10 gms. of the stain with 

 100 c.c. of absolute alcohol. If the stain dissolves quickly, 

 add more dry stain. The alcoholic solution should be 

 slightly supersaturated. 



2. Allow the undissolved stain to settle over night. 



3. Decant. 



4. Dilute 1 part of the alcoholic solution with 9 parts 

 of distilled water. 



Note 1. If 95% alcohol is used instead of absolute alcohol to 

 dissolve the stain, the dilution should be made 1 : 7. 



Note 2. These aqueous solutions may not keep longer than about 

 a month, while the saturated alcoholic solutions keep indefinitely. 



Note 3. The vegetative forms of bacteria stain more or less 

 readily with all aqueous-alcoholic stains but not with saturated alcoholic 

 stains. Acid-fast bacteria, e.g., Bact. tuberculosis, are the exception 

 to the former. 



Anilin- water gentian violet: 



1. Shake 5 c.c. of anilin oil vigorously with 100 c.c. of 

 distilled water in a stoppered bottle for several minutes. 



2. Filter through a wet filter immediately before use. 



3. Add 1 part of saturated alcoholic solution of gentian 

 violet to 9 parts of the freshly prepared anilin-water and 

 filter immediately before use. 



Note. Anilin-water stains do not keep longer than about a week. 

 The stock solutions will keep indefinitely if kept separate. 



