procedure for gram staining.

      1.0       Objective

     To lay down a procedure  for gram staining.

      2.0        Responsibility

      Microbiologist/QC-Executive.

      3.0        Accountability

      Head-QC executive

      4.0        Procedure

4.1              Prepare thin smear of 24 hours old culture suspension, dry in air and fix by gentle heat under aseptic conditions in laminar airflow.

4.2    Flood the smear with Gram’s crystal violet for one (1) minute.

4.3    Wash the smear with tap water.

 4.4        Flood the smear with Gram’s iodine for one (1) minute.

 4.5       Wash the smear with tap water and decolorize the smear with Gram’s decolorizer) until no further violet colour comes out from the smear.

4.6              Wash the smear with tap water.

4.7              Counter stain the smear with 0.5% safranin for about one (1) minute.

4.8              Wash the smear with tap water, dry it in air and observe under oil immersion objective of microscope.

4.9          Following table shall be considered for identification of Gram’s nature of the bacteria.

Bacteria.	Gram reaction
Gram positive bacteria	Violet color
Gram negative bacteria	Pink color