Gram staining procedure explained in Hindi | gram staining principle | gram staining steps

Learn that gram staining differentiates gram-positive bacteria (blue/violet color) from gram-negative bacteria (pink/red color) based on their cell wall peptidoglycan layer thickness differences.

Place bacterial culture on a glass slide and apply heat fixation to kill and adhere bacteria to the slide surface.

Add crystal violet (CV) stain to the fixed bacteria, which imparts a blue-violet color to both gram-positive and gram-negative cells.

Apply iodine as a mordant to form crystal violet-iodine (CVI) complex, which stabilizes and retains the crystal violet stain in bacterial cells.

Treat slides with alcohol or acetone to wash away the CVI complex; gram-negative bacteria lose the stain due to thin peptidoglycan layer while gram-positive bacteria retain it due to thick peptidoglycan layer.

Add safranine (pink-red secondary stain) which is absorbed only by decolorized gram-negative bacteria; gram-positive bacteria retain their blue-violet color.

Examine the stained slide under microscope to identify gram-positive bacteria (blue-violet) and gram-negative bacteria (pink-red) based on their color differentiation.