sop for gram staining


sop for gram staining


1.1 To describe a procedure to differentiate the bacteria into Gram-positive and Gram negative by Gram staining
2.1 This procedure applicable for Gram’s Staining for microbes in Microbiology Laboratory
3.1 Officer Microbiologist
3.2 Executive Microbiologist

4.1 Head – QC

5.1 Requirements: Crystal violet (primary stain), Gram’s iodine (mordant), 95% alcohol (decolorizing agent), Safranin (counter stain).

5.2 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 three times.

5.3 Stain with crystal violet for about 1 min. Rinse with water
5.4 Cover the film with gram’s iodine and allow it to act for about 1 min.
5.5 Rinse with water.

5.6 Decolorize with 95% alcohol. For a thin smear, 10-20 seconds is long enough; after the proper
t time interval, alcohol drippings from the slide are no longer colored.
5.7 Rinse with water.

5.8 Counter stain with Safranin for about 20-30 seconds.
5.9 Rinse with water and air dry.
5.10 Examine under the oil-immersion objective.
5.11 Result: Gram positive cells stain purple while gram negative cells stain pink.
6.1 Nil

7.1 Indian Pharmacopeia
8.1 Nil


9.1 QC : Quality Control
9.2 QCM. : Quality Control Microbiology

9.3 SOP : Standard Operating Procedure
9.4 QA : Quality Assurance
9.5 % : Percent


Revision Reason for Revision Effective Date



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cleaning and operation of discard autoclave

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STP for sterility testing of sterile gloves

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sop for transfer of material for testing and sampling in sterile area

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sop for Receipt Storage and Usage of Culture Media

sop for Cleaning Sanitization And Disinfection In Microbiology

sop for Environmental monitoring of all the Classified area

sop for Handling and Sub culturing of Microbial cultures

sop for Media Growth Promotion Test and various Microbiological test

sop for BOD incubator operation and cleaning

sop sampling of water for microbiological analysis

sop for Disinfectant Efficacy Test

sop for for cleaning and operation of vortex mixture

sop for Temperature & Relative Humidity Monitoring

sop for Operation and Calibration of Heating Block

sop for Sterility Testing of Microbiology

sop for Disposal of Culture Media

sop for Drain point of Microbiology

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sop for Gram Staining of Bacteria in Microbiology Laboratory

sop for Monitoring of Compressed Air/gases for microbiological analysis

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sop for microbiological assay of erythromycin antibiotic

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sop for monitoring of ultraviolet efficiency LAF and pass box

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gowning procedure for microbiological testing area

swab testing of various surfaces for bioburden determination

sop for endotoxin challenge test

Hold time study protocol for sterilized media

sop for personnel Qualification protocol for aseptic area

sop for sampling and testing of drain water

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sop for Inoculum Preparation

sop for Validation protocol of steam sterilizer autoclave

sop for pathogen detection from drain point

Sop for Analysis of Raw water Purified water water for injection and pure steam water

sop for preservatives efficacy test

sop for collection and preservation of in house isolated microorganisms

sop for Operation Calibration and Maintenance of Micropipette

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sop for gram staining

Sop for swab testing

sop for microbiological testing of water


sop for depyrogenation of apparatus

sop for media preparation

sop for fertility test growth promotion test of media

sop for Operation and cleaning of moist heat sterilizer

sop for monitoring by active air sampler

sop for swab sampling and testing for clean rooms in production area

sop for monitoring in microbiology laboratory

sop for Fumigation of aseptic area and microbiology lab

sop for monitoring of personnel in aseptic area

sop for maintenance of cultures

sop for Operation and cleaning of laminar bench

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sop for monitoring of pure steam

sop for entry and exit procedure to m.l.t and b.e.t room

sop for storage of and use of media

sop for disposal of microbiological media and cleaning of microbiological glassware







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