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U.S. Department of Health and Human Services

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BAM R32: Gram Stain

January 2001

Bacteriological Analytical Manual

R32
Gram Stain
(commercial staining solutions are satisfactory)

 

Hucker's crystal violet

 

Solution A

 

Crystal violet (90% dye content) 2 g
Ethanol, 95% 20 ml

 

 

Solution B

 

Ammonium oxalate 0.8 g
Distilled water 80 ml

 

Mix solutions A and B. Store 24 h and filter through coarse filter paper.

 

Gram's iodine

 

Iodine 1 g
Potassium iodide (KI) 2 g
Distilled water 300 ml

 

Place KI in mortar, add iodine, and grind with pestle for 5-10 s. Add 1 ml water and grind; then add 5 ml of water and grind, then 10 ml and grind. Pour this solution into reagent bottle. Rinse mortar and pestle with amount of water needed to bring total volume to 300 ml.

 

Hucker's counterstain (stock solution)

 

Safranin O (certified) 2.5
Ethanol, 95% 100 ml

 

Working solution: Add 10 ml stock solution to 90 ml distilled water.

 

Staining Procedure (Gram stain)

 

Fix air-dried films of food sample in moderate heat. Stain films 1 min with crystal violet-ammonium oxalate solution. Wash briefly in tap water and drain. Apply Gram's iodine for 1 min. Wash in tap water and drain. Decolorize with 95% ethanol until blue color is no longer released (about 30 s). Alternatively, flood slides with ethanol, pour off immediately, and reflood with ethanol for 10 g. Wash briefly with water, drain, and apply Hucker's counterstain (safranin solution) for 10-30 g. Wash briefly with water, drain, blot or air-dry, and examine.

 


Original Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998.