Gram Staining protocol-gram staining principle, procedure,result,precautions

Gram Staining protocol-gram staining principle, procedure,result,precautions



In gram staining protocol, crystal violet,the primary stain,causes both gram positive and gram negative organisms to become purple after 20 seconds of staining.When Gram’s Iodine, the mordant, is applied to the cells for 1 minute,the color of gram positive and gram negative bacteria remains the same;purple color.The function of the mordant here is to combine with crystal violet to form a relatively insoluble compound in the gram positive bacteria remain purple.In the final step a counter-stain, safranin ,adds a pink color to the de-colorized gram negative bacteria without affecting the color of the purple gram positive bacteria.

This techniques seems quite simple,performing it with a high degree of reliability is a goal that requires some practice and experience.Here are some suggestions that can be helpful;

  • First, don’t make your smear too thick. Only a very small amount of culture is needed; a visual detection of the culture on an inoculation loop already indicates that too much is taken
  • Second, when working with unknowns keep in mind that cultures of gram positive bacteria tends to de-colorized more rapidly than young ones.
  • Leaving the decolorizer on for longer than 8 seconds will cause excess decolorization in the gram-positive cells, and proper staining will not occur.
  • While working with Bacillus,causing them to appear gram positive, another point to remember that, one should use cultures that are totally fresh.Because Bacillus shows gram variability over aging of cultures i.e. sometimes it may be gram positive and sometimes gram negative in nature.



A. Gram staining reagents

  • Grams iodine solution
  • Acetone
  • Distilled water drop
  • 95% ethyl alcohol
  • Safranin

B. Others Materials

  • Inoculation loop
  • Glass rods
  • Blotting paper
  • Burner
  • Microscope

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