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Gram’s Staining Procedure of Microorganisms

Standard Operating Procedure (SOP) for Gram’s Staining of Microorganisms.

Gram stain or Gram staining, also called Gram’s method, is a method of staining used to distinguish and classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique

Procedure for Gram’s Staining

1.0   Purpose:

    • To lay down the procedure for Gram’s Staining of Microorganisms..

2.0   Scope:

    • This Standard Operating Procedure is applicable at microbiology section of Quality Control department.

3.0   Abbreviations and Definition of Terms :

    • RTD :  Resistance Temperature Detectors
    • Mordant : A substance used to set the dies.
    • Decolorizing Agent : A substance to decolorize the excess colour of dies.

4.0   Procedure for Gram’s Staining:

  • Procedure 1 (Gram’s Staining)

    • Prepare smears of organisms by spreading cell mass from a colony or by taking a loopful from the broth as a thin film on the slide.
    • Dry in air and fix the smear by passing the slide rapidly through the Bunsen flame three times.
    • Staining with Gram’s Crystal Violet as primary stain and allow to stand for about one minute.
    • Gently rinse the slide with purified water.
    • Put one to two drops of Gram’s Iodine on the smear and allow it to act for about 1 minute. Gram’s iodine acts as a mordant.

Also Read: Microbiology Media Management

    • Gently rinse the slide with purified water.
    • Cover the slide with 95% Alcohol for 10-20 seconds. Alcohol acts as a decolorizing agent.
    • Gently rinse the slide with purified water.
    • Counter stain with Safranin for about 20-30 seconds.
    • Gently rinse the slide with purified water and blot dry.
    • Examine under Microscope through the oil-immersion objective.
    • During examination identify the cells as Gram negative or Gram positive.
    • Gram positive cells retains the Gram’s staining and looks purple, while gram negative cells do not retain Gram’s stain and looks pink in colour.

  • Procedure 2 (Gram’s Staining)

    • Precautions:

    • Wear gloves and face mask before performing the Gram’s staining.
    • Proper disposal procedures to be followed after the completion of the testing.
    • Sterilize all biohazard waste before disposal.

    • Requirement:

    • Gram’s Staining Kit (Reagents: Grams Crystal Violet, Gram’s Iodine, Gram’s Decolorizer / 95% Ethanol, Safranin).
    • Glass slide.
    • Cover slip.
    • Wash bottle.
    • Inoculation loop.
    • Bunsen burner / Spirit lamp.
    • 5% Aqueous Solution of Malachite Green.
    • Safranin (Counter Stain).
    • Lacto Phenol Staining Kit.
    • Spore Staining Kit.

    • Gram’s Staining:

    • Gram’s Staining is used to differentiate bacterial species into Gram Positive and Gram Negative.
    • Take a small drop of purified water on a clean and dry glass slide.
    • Take out a small portion of young bacterial colony from agar plate or broth medium with the help of inoculation loop and
    • Prepare a thin and uniform smear on the glass slide.
    • Allow to air dry and fix the smear by heating gently.
    • Staining with Gram’s Crystal Violet for 1 min.
    • Rinse with purified water.
    • Cover the slide with Gram’s Iodine for 1 min after staining.
    • Rinse with purified water.
    • Decolourize with Gram’s Decolourizer or 95% Ethanol for 30 seconds.
    • Rinse with purified water.
    • Counter stain with Safranin for 1 min.
    • Rinse with purified water.
    • Allow the slide to air dry and examine under Microscope; 40X followed by 100X using immersion oil.

    • Interpretation of Results:

    • Gram Positive cells are stained purple colour.
    • Gram Negative cells are stained pink colour.

    • Lacto Phenol Staining:

    • Use the Lacto Phenol Staining for identification of fungal species.
    • Take 2-3 drops of Lacto Phenol Cotton Blue Stain on a clean and dry glass slide.
    • Take out a small portion of young fungal culture along with the spores from agar plate with the help of inoculation loop / needle.
    • Transfer it on the Lacto Phenol Cotton Blue Stain.
    • Place a clean and dry cover slip on the culture and ensure that no air bubble is formed under cover slip.
    • Examine the slide under Microscope; 40X followed by 100X using immersion oil for morphological characteristics of fungi.

    • Spore Staining:

    • Use the Spore Staining for identification of Spore forming microorganism.
    • Prepare smear of the isolated pure culture.
    • Allow to air dry and fix the smear by heating gently.
    • Place the slide on a small beaker, containing boiling water,
    • Keep on the hot plate.
    • Cover the smear with a small piece of filter paper.
    • Keep the filter paper saturated with a 5% aqueous solution of Malachite Green for 30 minutes.
    • Wash the slide gently with water.
    • Counter stain with Safranin for 30 seconds.
    • Examine the slide under Microscope; 40X followed by 100X using immersion oil.
    • The spores are stained green where as the vegetative cells are stained pink.
    • The spores are stained green where as the vegetative cells are stained pink.
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Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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