SOP of Gram Staining

SOP of Gram Staining

Objective:

To lay down the procedure for Gram Staining This SOP is applicable for Gram Staining in Microbiology Laboratory of (Pharmaceutical Company Name).

• Responsibility
  • Microbiologist or above of Microbiology Laboratory: Preparation of SOP.
  • Head – Microbiology section / Nominee: Checking of the SOP.

• Accountability
  • Head – Quality Control / Nominee: Compliance of SOP.

• Abbreviations and Definitions

SOP                             : Standard operating procedure

No.                              : Number

QC                              : Quality Control

QA                              : Quality Assurance

• Procedure
  • Requirements: Crystal violet (primary stain), Gram’s iodine (mordant), 95% alcohol (decolorizing agent), Safranin (counter stain)
  • 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.
  • Air dry, and fix the smear by passing the slide rapidly through the Bunsen flame for three times.
  • Stain with crystal violet for about 1 min.
  • Rinse with water.
  • Cover the film with gram’s iodine and allow it to act for about 1 min.
  • Rinse with water.
  • Decolorize with 95% alcohol. For a thin smear, 10-20 seconds is long enough; after the proper time interval, alcohol drippings from the slide are no longer colored.
  • Rinse with water. Counter stain with Safranin for about 20-30 seconds.
  • Rinse with water and blot dry.
  • Examine under the oil-immersion objective.
  • Result: Gram-positive cells stain purple while. Gram-negative cells stain pink colour.

• Forms and Records (Annexures)
  • Not applicable

• Distribution
  • Master copy       –       Quality Assurance
  • Controlled copies   –       Quality Assurance, Production, Quality Control, Stores, Engineering and Human Resource
• History:

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