1 bjective
To lay down the Procedure for Gram staining.
2 Scope
This procedure is intended as a standard test for identification of gram
negative and gram-positive bacteria.
This staining procedure differentiates bacteria into Gram positive and
Gram negative based on their ability to retain the primary dye (Crystal
violet) or lose the primary dye and accept the colour of the counterstain
(Safranin). Gram positive bacteria produce a Blue/Purple colour with
this staining procedure While Gram negative bacteria produce a
Red/Pink in colour.
Staining is an auxiliary technique used in microscopy to enhance
contrast in the microscopic image.
Gram staining: Gram staining is a method of staining used to
distinguish and classify bacterial species into large two groups (Gram
positive and Gram negative).
3 Responsibility
It is the responsibility of the Quality Officer to ensure the SOP is
followed.
4 Abbreviation and Definitions
NA
5 Procedure
6.1 Materials and Requirements
6.1.1 Bacterial cultures
6.1.2 Clean glass slides
6.1.3 Bunsen burner
6.1.4 Microscope (Leica)
6.1.5 Immersion oil
6.1.6 Wash bottle filled with distilled water
6.1.7 Crystal Violet-the primary dye (HiMedia-S012)
6.1.8 Iodine-the complex agent (HiMedia-S013)
6.1.9 95% ethanol/acetone-decolourizer (HiMedia-S032)
6.1.10 Safranin-counterstain (HiMedia-S027)
6.1.11 Timer (stop watch)
6.2 Procedure
6.2.1 Preparation of a Slide smear
6.2.1.1 Use inoculation loop to transfer a drop of suspended culture to the
microscope slide.
6.2.1.2 if the colony is present in a Petri dish or a slant culture tube, a drop or
a few loopfuls of water are added to facilitate a minimal amount of
colony transfer to the examination slide.
6.2.1.3 Air dry and heat fix the slide by waving it a few times using Bunsen
burner. Allow the slide to cool.
6.2.2 Staining
6.2.2.1 Place the slide on the staining rack.
6.2.2.2 Flood the slides smears with crystal violet and let stand for 1 minute.
6.2.2.3 Wash the smear in a gentle and indirect stream of water.
6.2.2.4 Flood the smear with Gram's iodine and let stand for 1 minute.
6.2.2.5 Rinse with water.
6.2.2.6 Decolorize with 95% ethanol for 15 to 30 seconds till the violet colour
comes off the slide.
6.2.2.7 Rinse with water.
6.2.2.8 Counterstain with safranin for about 20 to 30 seconds.
6.2.2.9 Rinse with water.
6.2.2.10 Allow the slide to air dry or blot dry between sheets of clean bibulous
paper, place a drop of oil on the slide and examine under oil immersion
Lens.
6.2.3 Microscopic observation
6.2.3.1 Gram positive organisms stain blue to purple; gram negative organisms
stain pink to red.
7 Appendices
NA
8 References
Gram Staining Kit (HiMedia – K001)
9 Review History
Date Version/Revision Reason of Review
No.
10/07/2021 PELL/MIC/002_1.0 Initial Release
27/08/2021 PELL/MIC/002_2.0 Structure and format change
10/06/2023 PELL/MIC/002_2.1 Media Catalogue Addition
27/08/2025 PELL/MIC/002_2.2 Periodic Review
Flow charts:
Smear
preparation
Crystal violet (1 Minute)
Water Rinse
Gram’s Iodine (1
Minute)
Decolorize (30
Seconds)
Counterstain (30
Seconds)
Water Rinse
Blot dry
