sop for Gram Staining of Bacteria in Microbiology Laboratory

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sop for Gram Staining of Bacteria in Microbiology Laboratory

1.0 OBJECTIVE

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

2.0 SCOPE

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

3.0 RESPONSIBILITY

3.1 Microbiologist QC

4.0 ACCOUNTABILITY

4.1 QC Manager

5.0 PROCEDURE

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 PROCEDURE
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 PREPARATION OF THE SMEAR FROM SOLID GROWTH SUCH AS SLANT OR PLATE.

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 PREPARATION OF THE SMEAR FROM LIQUID GROWTH
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 STAINING OF THE SMEAR
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.

5.6 INTERPRETATION OF THE RESULTS
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