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Introduction of  Hucker Modification Gram stain

The Hucker modification Gram stain test was originally developed by Christian Gram in 1884. The modification currently used for general bacteriology was developed by Hucker in 1921. Gram-stain can be used effectively to divide all bacterial species into two large groups: those that take up the basic dye, crystal violet (Gram-positive), and those that allow the crystal dye to wash out easily with the Decolorizer alcohol or acetone (Gram-negative)

Hucker Modification Gram Stain and Reagent Preparation

Crystal violet solution (Hucker’s crystal violet)

Crystal violet stock solution

Crystal violet (90% to 95% dye content)       40 g

Ethanol, 95%                                                  400 ml

Preparation

1. Dissolve and mix in a glass bottle.
2. Label with a 1-year expiration date, and stored at room temperature.

Ammonium oxalate solution (1%)

Ammonium oxalate (reagent grade)               16 g

Distilled water                                                1600 ml

Preparation

1. Dissolve and mix in a brown glass bottle,.
2. Label with a 1-year expiration date, and stored at room temperature.

Crystal violet working solution

Crystal violet stock solution                           40 ml

Ammonium oxalate solution (1%)                 160 ml

Preparation

1.  Filter crystal violet stock solution into a glass.
2. Allow to filter completely, and then filter ammonium oxalate solution.
3. Label with the earliest expiration date of stock solutions.

Gram’s iodine

Stock Lugol’s iodine solution

Iodine crystals (reagent grade)                       25 g

Potassium iodide (reagent grade)                   50 g

Distilled water                                                500 ml

Preparation

1.  Mix and let stand until dissolved in a brown glass bottle
2. Label with a 6- month expiration date, and store at room temperature.

Sodium bicarbonate, 5% (w/v)

Sodium bicarbonate (reagent grade)                 50 g

Distilled water                                                1000 ml

Preparation

1. Dissolve in a glass bottle
2. Label with a 1-year expiration date, and stored at room temperature.

Gram’s iodine

Stock Lugol’s iodine solution                       60 ml

Distilled water                                                220 ml

Sodium bicarbonate (5%)                               60 ml

Preparation

1.  Mix in a brown glass bottle.
2. Label with a 6-month expiration date, and stored at room temperature.

Acetone-Alcohol Decolorizer

Acetone                                                           500 ml

Ethanol (absolute)                                           475ml

Distilled Water                                                25 ml

Preparation

1. Add to 25 ml D/W, 475 ml of absolute alcohol.
2. Mix and transfer into a clean bottle.
3. Then add immediately, 500 ml acetone to the bottle and mix well.

Safranine (Counter Stain)

Safranine                                                                     10.0 gm

Distilled Water                                                             1000 ml

Preparation

1. Weigh 10 gm of Safranine in a clean piece of paper, and transfer to a clean bottle.
2. Then add 1-liter D/W to the bottle and mix well until Safranine dissolves completely.

Gram Staining Procedure of Hucker Modification Gram stain

The following steps are involved in Gram stain:

1. Prepare and dry a thin film of the material to be examined.
2. Heat fix the material on the slide and allow it to cool before staining.
3. Flood the slide with a crystal violet stain and allow it to remain without drying for 30 to 60 seconds.
4.  Rinse the slide with tap water, shaking off excess.
5. Flood the slide with iodine solution and allow it to remain on the surface without drying for twice as long as the crystal violet is in contact with the slide surface.
6.  Rinse the slide with tap water, shaking off excess.
7. Flood the slide with acetone alcohol decolorizer for 10 seconds and rinse immediately with tap water until no further colors flow from the slide with the decolorizer. Thicker smear requires more aggressive decolorizing.
8. Flood the slide with counterstain (Safranine) for 30 seconds and wash off with tap water.
9. Blot the slide between two clean sheets of blotting paper and examine microscopically under oil immersion at 100X.

Further Readings

1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
2. Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
3. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
4. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
6.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.