SOP on Gram Staining

SOP on Gram Staining

1.0 Objective

To lay down the procedure for performing Gram Staining for differentiation of Gram-positive and Gram-negative microorganisms in the Microbiology Laboratory.

2.0 Scope

This SOP is applicable to Gram Staining performed in the Microbiology Laboratory for identification and characterization of microorganisms.

3.0 Responsibility

Microbiologist or above of Microbiology Laboratory:

• Preparation and implementation of this SOP.

• Performing Gram staining and recording observations.

Head – Microbiology Section / Nominee:

• Reviewing and checking the SOP.

• Ensuring proper execution of the procedure.

4.0 Accountability

Head – Quality Control / Nominee:

• Ensuring compliance with this SOP.

5.0 Procedure

5.1 Requirements

The following reagents and materials are required:

• Crystal Violet (Primary Stain)

• Gram’s Iodine (Mordant)

• 95% Alcohol (Decolorizing Agent)

• Safranin (Counter Stain)

• Clean glass slides

• Inoculating loop

• Bunsen burner

• Distilled water

• Blotting paper

• Microscope with oil immersion objective

5.2 Preparation of Smear

5.2.1 Using a sterile inoculating loop, prepare a thin smear by spreading a small amount of microbial colony or a loopful of broth culture on a clean glass slide.
5.2.2 Allow the smear to air dry completely at room temperature.

5.3 Heat Fixation

5.3.1 Fix the dried smear by passing the slide rapidly through the Bunsen flame three times with the smear side facing upwards.
5.3.2 Allow the slide to cool before staining.

5.4 Staining Procedure

5.4.1 Flood the smear with Crystal Violet and allow it to act for approximately 1 minute.
5.4.2 Rinse gently with water.
5.4.3 Cover the smear with Gram’s Iodine and allow it to act for approximately 1 minute.
5.4.4 Rinse gently with water.
5.4.5 Decolorize with 95% alcohol by applying it dropwise for 10–20 seconds (for thin smears) or until the runoff becomes colorless.
5.4.6 Immediately rinse with water to stop the decolorization process.
5.4.7 Counterstain with Safranin for 20–30 seconds.
5.4.8 Rinse with water and gently blot dry using blotting paper.

5.5 Microscopic Examination

5.5.1 Apply a drop of immersion oil on the stained smear.
5.5.2 Examine the slide under the oil immersion objective (100X).
5.5.3 Observe cell morphology, arrangement, and staining characteristics.

6.0 Interpretation of Results

6.1 Gram-positive organisms retain Crystal Violet and appear purple or violet under the microscope.
6.2 Gram-negative organisms lose the primary stain during decolorization and take up Safranin, appearing pink or red in color.

7.0 Precautions

7.1 Use fresh (18–24 hour) cultures for accurate results.
7.2 Ensure smear is thin to avoid improper decolorization.
7.3 Avoid over-decolorization or under-decolorization.
7.4 Handle slides carefully to prevent contamination and breakage.
7.5 Ensure proper heat fixation to prevent washing off of the smear.

8.0 Documentation

8.1 Record the following details in the laboratory record:

• Name of organism/sample

• Date of staining

• Observations (Gram reaction, morphology, arrangement)

• Analyst signature

• Reviewer signature

Environmental and personnel monitoring – viable particle EUGMP- Annex-1