☛ When over-decolourized by either prolonged exposure to decolourizer or using acetone alone.

☛ When cell wall gets damaged by exposure to lysozyme or cell wall acting antibiotics such as Penicillin.

☛ Old cultures, where cell wall is weakened or action of autolytic enzymes

☛ Those bacteria that are phagocytosed. where cell wall is acted upon by lysosomal contents