16 MANUAL OF MICROBIOLOGICAL METHODS 



Hucker Modification 



Ammonium Oxalate Crystal Violet 



(See page 13) 



Gramas Modification of LugoVs Solution 



Iodine 1 g 



KI 2g 



Distilled water 300 ml 



Counterstain 



Safranin O (2.5 % solution in 95 % ethyl alcohol) 10 ml 



Distilled water 100 ml 



Staining schedule: 



1. Stain smears 1 min with ammonium oxalate crystal violet. This 

 formula has sometimes been found to give too intense staining, so that 

 certain gram-negative organisms (e.g., the gonococcus) do not properly 

 decolorize. If this trouble is encountered, it may be avoided by using 

 less crystal violet. 



2. Wash in tap water for not more than 2 sec. 



3. Immerse 1 min in iodine solution. 



4. Wash in tap water, and blot dry. 



5. Decolorize 30 sec with gentle agitation, in 95 per cent ethyl alcohol. 

 Blot dry. 



6. Counterstain 10 sec in the above safranin solution. 



7. Wash in tap water. 



8. Dry, and examine. 



Results: Gram-positive organisms, blue; gram-negative organisms, red. 



Burke and Kopeloff-Beerman Modifications 

 Alkaline Gentian Violet 



Solution A Solution B 



Gentian or crystal violet^ 1 g NaHCOs 1 g 



Distilled water 100 ml Distilled water 20 ml 



Burke's Iodine Solution 



Iodine, 1 g; KI, 2 g; distilled water, 100 ml 



Kopeloff and Beerman^s Iodine Solution 



Iodine 2 g 



Normal NaOH (40.01 g per liter) 10 ml 



After the iodine is dissolved, make up to 100 ml with distilled water. 



Burke^s Counterstain 



Safranin O (85% dye content), 2 g; distilled water, 100 ml 



^ The authors specify either crystal violet or methyl violet 6B. Probably any of the 

 gentian violets now sold under the commission certification are satisfactory, i.e., either 

 crystal violet or one of the bluer grades of methyl violet (e.g., methyl violet 2B). 



