48 LABORATORY MANUAL OF MICROBIOLOGY 



If used in staining root-nodule bacteria dilute the carbol 

 fuchsin 1 part of stain to 9 parts of distilled water. 



Gram Stain 



This is one of the most important methods of staining employed 

 in a study of bacteria. It is commonly used to distinguish 

 certain organisms from others and also to show the general 

 morphology. It has been found that when stained with gentian 

 or crystal violet, and treated with an iodine solution, some 

 organisms are easily decolorized while others are much more 

 difficult to decolorize.. The method follows. Prepare a thin 

 smear of the culture on a slide. Dry in the air and fix with heat. 

 Stain 1 minute with the crystal violet dye and then treat with the 

 iodine solution for 1 minute. 



Crystal Violet 



Saturated alcoholic solution of crystal violet 10.0 cc. 



Ammonium oxalate, 1 per cent aqueous solution 40.0 cc. 



Lugol's Iodine Solution 



Iodine 1.0 gm. 



Potassium iodide 2.0 gm. 



Water 300.0 cc. 



Decolorize for 30 seconds in 95 per cent alcohol. Wash 

 between each step. Counter stain for 10 seconds with safranin. 

 Wash and dry. 



Safranin 



Saturated alcoholic solution of safranin 10.0 cc. 



Distilled water 100.0 cc. 



If gram-positive, the cells should retain the crystal violet 

 stain and thus appear under the microscope as a deep violet. 

 If gram-negative, the cells are decolorized by the alcohol and 

 thus show a pale pink color. 



Carbol Thionin (Nicolle's) 



Prepare a stock saturated alcohoUc solution of thionin in 50 

 per cent alcohol. 



Take 10 cubic centimeters of this saturated solution of thionin 

 and 100 cubic centimeters of a 2 per cent carbohc acid solution. 



