SOP for Gram Staining

It describes the (Standard Operating procedure) SOP for Gram Staining

 SOP for Gram Staining

I: Purpose and Scope :

  • The purpose of this SOP is to lay down the procedure for Gram Staining of Microbial Culture
  • This Standard operating procedure shall be applicable for Gram staining of Microbiological cultures in the Microbiology section of Quality Control Department

II: Responsibilities:

  •  All Microbiologist, Quality control personnel shall be responsible to follow and implement this SOP.

III: Introduction and Procedure:

Principle of Gram Staining :

Gram’s staining is usually done to study the microscopic cell character of bacteria. This technique is useful in distinguishing the bacteria as `Gram Positive’ and `Gram Negative’. When the bacterial cells resist decolourization with alcohol or acetone after being stained with a triphenyl methane dye, e.g. crystal violet and subsequent treatment with iodine (which acts as a mordant) they are called as `Gram Positive’.  They are therefore stained dark purple or violet.  ‘Gram Negative bacteria’ are decolourized with alcohol or acetone and are then stained light pink with the counter stains like basic fuschin, Safranine, neutral red.

General Procedure for Gram staining : 

  • Prepare a suspension of an intended microbial colony or culture.
  • By using sterile loop spread this solution to make the smear on a clean, grease free glass slide.
  • Heat fix the smear by heating the slide. Allow to cool the slide.
  • Put 2 to 3 drops of the primary stain i.e. crystal violet on the smear, Hold it for one minute.
  • Washout the stain under gentle flow of tap water.
  • Flood the smear with Gram’s iodine; allow it to remain for one minute.
  • Gently rinse the smear with tap water.
  • Decolourize the smear with Gram’s decolourizer until the blue dye no longer flows from the smear. Gently wash the slide with running tap water.
  • Put the counter stain i.e. Safranin on the smear. Allow it to remain for one minute. Gently wash the slide with running tap water.
  • Allow the slide to air dry or using blotting paper carefully soak off the moisture. Put a drop of cedar wood oil on the smear and examine it under oil immersion objective of Microscope.
  • Gram positive organisms will get stained by crystal violet so remains as violet coloured and Gram negative organisms will get stained by counter stain safranin so appears as Pink-red colour.
  • Record the Gram staining results in Annexure-I.
  • Perform the Gram staining of cultures after sub-culturing or during identification of Micro-organisms.

Annexure I: Gram Staining Record

Annexure- I

Gram Staining Record


Date of Staining :  ______________                   

Sr. No. Name of Organism


Strain No. Observation Done By. Checked By





















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