Preparation of Gram Stain Reagents

Gram staining requires the use of different chemical reagents which can be purchased in ready-to-use forms from commercial suppliers or can be prepared at the laboratory by mixing different chemicals in an appropriate amount.

Gram staining technique requires simultaneous use of chemical reagents for a fixed period followed by washing; Primary stain (crystal violet), Mordant (iodine), Decolorizer (ethanol or acid-alcohol), and Counterstain (safranin or dilute carbol-fuchsin).

Stained slide is air-dried and observed under oil immersion (100x) using a bright field microscope. Gram-positive bacteria appear blue/purple while Gram-negative appear as pink/red.

Crystal violet

1. Dissolve 2.0 g certified crystal violet into 20.0 ml of 95% ethyl alcohol.
2. Dissolve 0.8 g ammonium oxalate into 80.0 ml distilled water.
3. Mix the two solutions together and allow them to stand overnight at room temperature (25°C).
4. Filter through coarse filter paper before use.
5. Store at room temperature (25°C).

Gram’s iodine

1. Grind 1.0 g iodine (crystalline) and 2.0 g potassium iodide in a mortar. Small additions of distilled water may be helpful in preparing the solution.
2. Add to 300.0 ml distilled water.
3. Store at room temperature (25°C) in a foil-covered bottle (to protect solution from light).

Decolorizer

Some workers prefer to use acetone by itself, ethanol 95% v/v, or ethanol-iodine as the decolorizing solution. A mixture of acetone-alcohol is recommended because it decolorizes more rapidly than ethanol 95% v/v, and is less likely to over-decolorize smears than acetone without alochol added.

Acetone-alcohol decolorizer

To make 1 litre :

• Acetone………..500 ml
• Ethanol or methanol, absolute* …………..475 ml
• Distilled water…………25 ml

*Technical grade is adequate.

1. Mix the distilled water with absolute ethanol (ethyl alcohol) or methanol (methyl alcohol). Transfer the solution to a screw-cap bottle of 1 litre capacity.
   Caution: Ethanol and methanol are highly flammable, therefore use well away from an open flame.
2. Measure the acetone, and add immediately to the alcohol solution. Mix well.
   Caution: Acetone is a highly flammable chemical that vaporizes rapidly, therefore use it well away from an open flame.
3. Label the bottle, and mark it Highly Flammable. Store in a safe place at room temperature. The reagent is stable indefinitely.
4. For use: Transfer a small amount of the reagent to a dispensing container that can be closed when not in use.

Counterstain

Safranin and dilute carbol-fuchsin are commonly used counterstain in Gram staining procedure, another being Neutral red (it stains gonococci and meningococci well).

Preparation of Safranin

1. Add 2.5 g certified safranin-O to 100.0 ml 95% ethyl alcohol.
2. Add 10.0 ml safranin and ethyl alcohol solution made in step 1 to 90.0 ml distilled water.
3. Store at room temperature (25°C).

Preparation of dilute carbol-fuchsin

(may be a more effective counterstain than safranin)

1. Dissolve 0.3 g basic fuchsin in 10.0 ml 95% ethyl alcohol.
2. Add 5.0 ml melted phenol crystals to 95.0 ml distilled water.
3. Add the 5% phenol solution to the fuchsin solution and let stand overnight.
4. Filter through coarse filter paper.
5. Store at room temperature (25°C) in a foil-covered bottle for up to 1 year.

References

1. Tripathi N, Sapra A. Gram Staining. [Updated 2023 Aug 14]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK562156/ 
2. Davies, J. A., Anderson, G. K., Beveridge, T. J., & Clark, H. C. (1983). Chemical mechanism of the Gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain. Journal of bacteriology, 156(2), 837–845. https://doi.org/10.1128/jb.156.2.837-845.1983