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).

• • Purified Water.

• • 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.

• • Wash the slide with purified water and blot dry with Whatman filter paper.

• • 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.