Gram stain

This is used most widely in bacteriology and was developed by Christian Gram (1884) to stain bacteria. The reagents used are:

• Crystal violet( primary dye)
• Gram’s iodine(mordant)
• Acetone/alcohol(decolorizer)
• Safranin (counter stain)

Principle of Gram staining:

  The cell wall of gram positive bacteria is thick(less permeable) with more peptidoglycan.  When stained with primary stain(crystal violet) followed by iodine, a CV-I  crystal complex is formed which gets trapped in the cell wall and the decolorizer (acetone or alcohol) cannot wash this stain. So the gram +ve bacteria appear violet/purple in color.

However, the cell wall of gram negative bacteria is thin(permeable) with high lipid content and less peptidoglycan. During staining process the primary stain with iodine can enter the cell wall, but after the application of decolorizer (alcohol/acetone), the cell wall gets shrink(high lipid) creating large pores and wash away the primary stain. At last the counter stain safranin(pink) is applied which gives the gram –ve bacteria a pink/red color.

Gram staining (method/steps)

• Heat fix smear of specimen with crystal violet stain (1 minute)
• Pour off crystal violet. Add dil.solution of Gram’s iodine (1 minute)
• Wash with water.
• Decolourization with organic solvent(alcohol/acetone)(10-30 seconds)
• Wash with water
• Counter stain with safranin (20-30 seconds)

Results:

Note: (only to assist memory)

All cocci are gram+ve except Nisseria(gram-ve)

All bacilli are  gram-ve except Bacillus, Clostridium, Corynebacterium)