Answer:
Gram staining procedure is used to differentiate between the gram-positive and gram-negative bacteria. various reagents used in the gram staining are crystal-violet, iodine, ethanol, and safranin.
1. First, the primary strain that is crystal violet is used to stain the bacterial cells present present on the slide.
2. Then the iodine solution is added over the bacterial culture and held for 1 minute. Iodine makes a complex with the crystal violet in the cell wall of bacteria.
3. Then acetone is used as a decolorizer that decolorizes the gram-negative bacteria by dissolving its lipopolysaccharide cell wall but do not decolorize gram-positive bacteria because its cell wall is made up of peptidoglycan layer.
4. The last reagent is safranin which is used to stain decolorized gram-negative cells pink. Therefore the correct order of staining is:
Crystal violet→Iodine→acetone→sefranine
