SOP for Grams Staining test of microbial culture
To lay down a procedure for Gram’sstaining of culture in Microbiological Laboratory.
2.0 SCOPE:
This procedure is applicable for identification of gram’s nature of microbial culture in Microbiology Laboratory.
3.0 RESPONSIBILITIES:
Microbiologist : To perform the procedure of Gram’s staining as per SOP.
Head-QC : To review and implementation and ensure compliance of SOP.
Head-QA : To approve, implement and ensure compliance of SOP.
4.0 PROCEDURE:
4.1 Prepare a suspension of the microorganism in sterile saline solution.
4.2 Take a clean grease free microscopic slide & prepare a uniform smear of the suspension using wire loop.
4.3 Allow the smear to dry in air.
4.4 Heat fixes the slide.
4.5 Cool the slide & add few drops of crystal violet stain onto the smear & allow for 1 minute.
4.6 Drain off excess crystal violet stain & wash the slide with water.
4.7 Place Gram’s iodine onto the smear & allow for 1 minute.
4.8 Drain off excess iodine & wash the slide with water.
4.9 Decolorize the smear by adding drop wise Gram’s decolorize until no further violet color comes off.
4.10 Wash the slide with water.
4.11 Apply the counterstain like 0.5% Safranin solution for about 1 minute.
4.12 Drain off excess stain & wash the slide with water and let it dry.
4.13 Examine the slide first under objectives of the microscope for better magnification view the slide under oil immersion lens using cedar wood oil.
4.14 Determine the Gram nature of the organisms by viewing the Stained smear.
4.15 Gram’s positive organisms appear as violet to blue color while Gram’s negative organisms appear reddish or pinkish.
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