Gram Staining 2023

Gram Staining


The Gram staining technique was invented in 1884 by Danish bacteriologist Hans Christian Gram.
Gram stain (Gram staining or Gram’s method) is a staining method used to divide bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria.


To describe standard procedure for detecting the Gram – positive and Gram Negative Bacteria by Gram- staining method.

2.0 SCOPE:

This SOP covers the procedure of Gram- staining in Microbiology Lab




Head Microbiology/Head QA


  1. Wear hand gloves, head cap and nose mask during handling of micro organisms.
  2. Prepare smear under LAF to avoid contamination.
  3. Use isolated colony for Gram staining.
  4. Gram staining:
    Take cleaned and dried glass slide and culture which is to be checked for Gram staining under LAF bench.
  5. Heat the nichrome wire loop in burner till it becomes red hot. Cool the nichrome wire loop.
  6. Put one drop of sterile saline water onto the slide with help of nichrome wire loop.
  7. Take isolated colony, which is to be checked for Gram staining with the help of sterile nichrome wire loop.
  8. Prepare a thin smear on the slide.
  9. Allow it to air dry and fix by gentle heat.
  10. Flood the smear with Gram’s crystal violet for 1 min.
  11. Wash with tap water.
  12. Flood the smear with Gram’s iodine. Allow it to remain for 1 min.
  13. Decolorize with Gram’s de-colouriser until the blue dye no longer flows from the smear.
  14. Wash with tap water.
  15. Counter stain with 0.5 % w/v safranin. Allow it to remain for 1 min.
  16. Wash with tap water.
  17. Allow the slide to air dry and examine under oil immersion objective of microscope at magnification sequence from 10X, 40X, &100XGram Staining


Gram-positive micro organisms: Bluish purple in colour.

Gram staining

Gram-negative micro organisms: Pinkish red in colour.

Note: The smear should be thin and the slide after staining should not show any foreign matter.


Q. C. : Quality Control
SOP : Standard Operating Procedure
QCM : Quality Control Microbiology
QA : Quality Assurance
LAF : Laminar Air Flow

8.0 RECORD(S):
       Gram staining Kit instruction.

Text Books of Microbiology from Prescott

Youe may also read the aticle about CCS Contamination Control Strategy Tricks.


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Isolation & Identification of Environment Isolates

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