6/14/23, 3:22 PM BAM R32: Gram Stain | FDA

BAM R32: Gram Stain

 
January 2001

(commercial staining solutions are satisfactory)

Hucker's crystal violet

Solution A
Crystal violet (90% dye content) 2g

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 1g

Potassium iodide (KI) 2g

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

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6/14/23, 3:22 PM BAM R32: Gram Stain | FDA

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.

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