1.0 PURPOSE:
To define the gram staining procedure to identify the purity of culture by checking the Gram characteristics and to differentiate unknown colony forming units found during microbiological testing.
2.0 SCOPE:
This Gram staining procedure is applicable during validation for out of specification results and whenever gram characteristics of the microorganism is required at (Company name).
3.0 DEFINITION:
4.0 RESPONSIBILITY:
4.1 Trainee/officer-QC is responsible for execution of procedure.
4.2 Head-QC is responsible for compliance of procedure.
5.0 PROCEDURE:
5.1 Use Gram stains kit that include all reagents required for Gram staining.
5.2 Take a clean, dry slide and label with respective sample name.
5.3 Pick the required bacterial colony make a thin smear over clean glass slide.
5.4 Fix the smear by gentle heating over flame avoid steaming.
5.5 Stain the Gram’s crystal violet solution for 1 minute.
5.6 Stain with Gram’s iodine solutions for 1 minute.
5.7 Drain off, rinse slide gently with purified water.
5.8 Decolorize with decolorizer until no further violet color comes off.
5.9 Rinse with purified water for 5 seconds.
5.10 Counter stain with safranin solution for 1 minute.
5.11 Rinse with purified water for 5 seconds. Allow to dry.
5.12 Flood the slide with a drop of immersion oil.
5.13 Examine under oil immersion lens of a microscope.
5.14 Gram positive bacteria retain crystal violet giving them purple color. Gram negative bacteria get decolorize with decolorizer and take up safranin stain (counter stain) giving them red color.