sop for Gram Staining of Bacteria in Microbiology Laboratory

sop for Gram Staining of Bacteria in Microbiology Laboratory


1.1 To lay down the Procedure for Gram Staining of Bacteria.


2.1 This procedure applicable for Gram Staining of Bacteria in Microbiology Laboratory


3.1 Microbiologist QC


4.1 QC Manager


5.1 Requirement
5.1.1 Crystal Violet (The Primary Stain)
5.1.2 Iodine Solution (The Mordant)
5.1.3 Decolorizer (Such as ethanol)
5.1.4 Safranin (The Counter stain)
5.2.1 Gram-staining is a four part procedure which uses certain dyes to make a bacterial cell stand out against its background. The specimen

should be mounted and heat fixed on a slide before you proceed to stain it the reagents you will need to successfully perform this operation are:
1. Crystal Violet (The Primary Stain)
2. Iodine Solution (The Mordant)
3. Decolorizer (Such as ethanol)
4. Safranin (The Counter stain)

5.3.1 Take a clean glass slide.
5.3.2 Deposit a drop of purified water on the glass slide.
5.3.3 Flame the loop to red hot under biosafety, allows it to cool and take a small portion of the colony to stain.
5.3.4 Put this colony on to the previously deposited drop of purified water and mix. Spread the culture with an inoculation loop to an even thin film or smear. Note that only a very small amount of culture is needed.
5.3.5 The smear should be thin enough to dry completely within a few seconds.
5.3.6 Fix the smear by heating lightly on the opposite side of the smear, by passing quickly over the flame two or three times.

5.4.1 Take a clean glass slide.
5.4.2 Flame the loop to red hot and cool in air.
5.4.3 Spread on the surface of the slide properly.
5.4.4 Fix the smear by heating lightly opposite side of the smear, by passing quickly over the flame two or three times.
5.5.1 The staining is carried out with the help of Gram Staining reagent. We can use Gram staining kit in ready to use form, from some commercial supplier such as HIMEDIA.
5.5.2 Keep the slide on the stand and flood it with Gram’s primary stain Crystal Violet for one minute.
5.5.3 Wash with water and drain the excess water.
5.5.4 Flood the smear with iodine solution and kept for 1 minute.
5.5.5 Wash the slide with water and decolorizes with Gram’s Decolorizer (Ethyl alcohol 95%.)Until no further violet colour comes off.
5.5.6 Wash with water and counter stain with Safranin. Allow to stand for about 1Minute.
5.5.7 Dry the smear with filter paper or allow it to dry at room temperature.
5.5.8 See the slide under Microscope with the use of immersion oil on the smear and observe in the Microscope using oil immersion lens (100X).
5.5.9 Absorb gauze in the cleaning solution and apply to the Oil immersion lens till all oil is remove and lens is clean.

The Gram positives Bacteria will take a color between Blue to Violet and the Gram negatives will take up the color of the counter

stain i.e. pink color. For example – E. coli (Gram negative short rods) S.aureus (Gram positive cocci in cluster).

6.0 Abbreviation

Abbreviation used Extended Form
SOP Standard Operating Procedure
S.NO. Serial No.

sop for calibration and validation of micro autoclave

sop for Sterility failure investigation

cleaning and operation of discard autoclave

sop for operation of fogger machine

sop for Biological assay of lactic acid bacillus

sop for preparation of culture inoculum

STP for sterility testing of sterile gloves

sop for Operation and calibration of active air sampler

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

entry & exit procedure in microbiology laboratory

Growth Promotion Test In Microbiology Laboratory

Operation of B.O.D in Microbiology Laboratory

Operation of Horizontal Laminar Air Flow in the microbiology laboratory

Operation and cleaning of Pass Box.

Operation and cleaning of air sampler

Cleaning and Sterilization of Glassware

Analysis of water for microbial load in microbiology lab

Operation and temperature monitoring of Refrigerator

Fumigation of Microbiology Laboratory.

Entry & Exit procedure In Sterility Area

SOP for Microbial analysis of Raw Material Finished Products

SOP for Operation & Calibration of pH meter in Micro Department

SOP for Operation & Calibration of pH meter in Micro Department

SOP Operation and calibration of Hot Air Oven In Microbiology

SOP for operation cleaning & calibration of Digital colony counter

SOP for Operation And Cleaning of Microscope

sop for Media Preparation and Consumption

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

sop for entry & exit procedure In Microbial limit test and Biosafety

sop for Gram Staining of Bacteria in Microbiology Laboratory

sop for Monitoring of Compressed Air/gases for microbiological analysis

sop for BET (Bacterial Endotoxin) test in Microbiology

sop for receipt storage and Determining the population of Biological indicators

sop for qualification of analyst microbiologist

sop for Bioburden test of Packing materials in Microbiology Laboratory

sop for microbiological assay of erythromycin antibiotic

sop for liquid particle counter

sop for operation and calibration of digital zone reader

sop for monitoring of ultraviolet efficiency LAF and pass box

microbiological assay of cyanocobalamin or vitamin B12

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

Sop for Operation of Airborne Particle Counter

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

Sop for UV Efficacy Test

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

preparation of settle plates

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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