SOP on Gram Staining
Objective
To lay down the procedure for Gram Staining.
Scope
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:
Date | Revision Number | Reason for Revision |
00 | NEW SOP |
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