In gram staining, 95% ethanol is used to destain the gram-negative bacteria but gram-positive bacteria do not get destain because peptidoglycan layer in gram-positive cell wall does not allow crystal violet- iodine complex to come out of the cell wall.
Gram-negative bacteria do not have peptidoglycan cell wall and so crystal violet comes out of the cell wall by using 95% ethanol because ethanol dissolves the lipopolysaccharide membrane of gram-negative bacteria and allow the dye to leak out.
Therefore if I forgot to apply 95% ethanol than crystal violet dye will not come out of the lipopolysaccharide cell wall of gram-negative bacteria and it will appear violet under the microscope.
