SOP on Gram Staining

SOP on Gram Staining


To lay down the procedure for Gram Staining.


This SOP is applicable for Gram Staining in Microbiology Laboratory of (Pharmaceutical Company Name).


Microbiologist or above of Microbiology Laboratory: Preparation of SOP.

Head – Microbiology section / Nominee: Checking of the SOP.


Head – Quality Control / Nominee: Compliance of SOP.

Abbreviations and Definitions

SOP                             : Standard operating procedure

No.                              : Number

QC                              : Quality Control

QA                              : Quality Assurance


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


Master copy    –       Quality Assurance

Controlled copies   – Quality Assurance, Production, Quality Control, Stores, Engineering and Human Resource


Date Revision Number
Reason for Revision
00   NEW SOP

For More Pharma Updates Visit –

About Pharmaceutical Guidanace

Ms. Abha Maurya is the Author and founder of pharmaceutical guidance, he is a pharmaceutical Professional from India having more than 18 years of rich experience in pharmaceutical field. During his career, he work in quality assurance department with multinational company’s i.e Zydus Cadila Ltd, Unichem Laboratories Ltd, Indoco remedies Ltd, Panacea Biotec Ltd, Nectar life Science Ltd. During his experience, he face may regulatory Audit i.e. USFDA, MHRA, ANVISA, MCC, TGA, EU –GMP, WHO –Geneva, ISO 9001-2008 and many ROW Regularities Audit i.e.Uganda,Kenya, Tanzania, Zimbabwe. He is currently leading a regulatory pharmaceutical company as a head Quality. You can join him by Email, Facebook, Google+, Twitter and YouTube

Check Also


HANDLING OF ISOLATES This procedure elaborates the systematic approach for the isolation and identification of …