3.

Gram staining
1. smear with crystal violet
2. treat with iodine (mordant)
3. the smear is decolourized by washing with ethanol
4. smear is counterstained with a basic dye different
in colour from crystal violet. Safranin is used.
Gram negative: pink
Gram positive: dark purple
Mechanism of gram staining:
difference in gram positive and negative is due to
their physical cell walls
peptidoglycan is not stained; act as a permeability
barrier preventing loss of crystal violet.
Ethanol is taught to shrink pores of the thick pep.
Hence the dye complex is retained.
Gram negative pep is very thin, not highly cross-
linked, has larger pores. Hence alcohol readily
remove the crystal violet.
Effect of lysozyme on the cell wall Cell wall :
protect bacteria against osmotic lysis

Lysozyme : enzyme which attacks pep

by hydrolysis the bond that connects N-
M acid with N-G. Hence weakening the
cell wall
Formation of spheroplasts- gram
negative bac. will have some residual
wall

Formation of protoplasts- gram

positive bac. continue to grow
without a cell wall