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

Gram stain reagent
  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).


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.


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.


  1. Tripathi N, Sapra A. Gram Staining. [Updated 2023 Aug 14]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: 
  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.

Nisha Rijal

I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

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