SOP on Identification of Microorganisms(Gram Staining Method)

SOP on Identification of Microorganisms
(Gram Staining Method) 

1.0 Purpose: This SOP describe a procedure for the identification of microorganisms

2.0 Scope: This SOP applies to the microbiology  department staff 

3.0 Responsibility: Quality Control Microbiologist to ensure that the procedure is followed.
4.0 Materials and Equipment: As required
5.0 Procedure:

5.1 In a clean grease free glass slide the smear of the organism is made, air-dried & heat fixed. The smear is first treated with basic dye methyl violet. Allow it to stand for 30 seconds. At this stage all the organisms take methyl violet and appear violet.
5.2 After washing, the smear is treated with iodine solution. Allow it to stand for 30 seconds. It forms a complex with methyl violet inside the cell.
5.3 After washing with water the smear is treated with absolute alcohol in order to decolorise the stained smear. During this process some bacteria loose the methyl violet complex and are gram negative, where is some bacteria retain the methyl violet colour are gram positive.
5.4 After washing the smear is treated with a red coloured dye, either safranine or diluted carbol fuschin. At this stage the organisms, which have lost the methyl violet colour complex, take up this red dye & appear red. The ones which did not loose methyl violet colour complex remain violet. Thus after the completion of all four steps in the staining procedure one gets either violet coloured organism or red stained organism in the smear.
Gram positive bacteria appears – violet
Gram negative bacteria appears – pink / red

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