Follow
1.0 OBJECTIVE : To lay down the procedure for Gram Staining of bacterial cells.
2.0 SCOPE : This SOP is applicable for Microbiological Laboratory of Quality Control Department of (Company Name).
3.0 RESPONSIBILITY
3.1 Each individual in the laboratory is responsible for carrying out the procedure.
3.2 Overall responsibility for training, implementation & follow-up with the QC Manager or his nominee.
4.0 ACCOUNTABILITY
4.1 Head of Quality.
5.0 PROCEDURE
5.1 Precaution
5.1.1 Staining should be carried out in the washing area near the tap.
5.1.2 Smear should be prepared under LAF bench.
5.1.3 Extra precautions should be taken for avoiding spillage of stains as the stains are very difficult to remove.
5.1.4 Do not overheat the slide during fixing as this can lead to dehydration of the cells.
5.1.5 Hand gloves should be used.
5.2 Preparation of smear
5.2.1 Take a loop full of microbial culture under test and make a suspension in 2 ml of 0.85% of sterile sodium chloride solution (Normal saline).
5.2.2 Transfer a loop full of the suspension on a clean dry grease-free glass slide.
5.2.3 Spread the suspension on the slide with the same loop to form an even smear of approximate area of 1 cm2.
5.2.4 Heat fix the smear by passing it two to three times through a flame.
5.2.5 Keep the slide for approximately 10 minutes for complete drying.
5.3 Staining
5.3.1 Cover the entire smear with Gram’s Crystal Violet and leave the slide for 1 min.
5.3.2 Wash the slide under continuous flow of water by holding the slide in slanting position. Do not allow the water to directly fall on the smear.
5.3.3 Cover the entire smear with Gram’s Iodine and leave the slide for 1 min.
5.3.4 Wash the slide under a slow and steady flow of water by holding the slide in slanting position. Do not allow the water to directly fall on the smear.
5.3.5 Decolorize with Gram’s Decolourizer by holding the slide in slanting position till further violet color stops coming.
5.3.6 Wash the slide again under a slow and steady flow of water by holding the slide in slanting position to ensure complete removal of the decolorizer.
5.3.7 Counter stain with 0.5% Safranin solution by covering the entire smear and leave the slide for 30 seconds.
5.3.8 Wash the slide under a slow and steady flow of water by holding the slide in slanting position. Do not allow the water to directly fall on the smear.
5.3.9 Keep the slide in slanting position on a tissue paper so that the access water is drained off and air dry the slide.
5.3.10 Add one drop of immersion oil on the stained smear and observe the slide under microscope using oil immersion lens.
5.3.11 Make decision on the colour of the cells. If the cell retain colour of crystal violet (violet) the organism is Gram positive and if the cell retain colour of the safranin (pink to red) the organism is Gram negative.
6.0 REFERENCES
6.1 United States Pharmacopeia <1113>.
