Question

You view your Gram stain of the Gram Standard and see only purple cells. What may have gone wrong?

Select one:

a. used iodine as the mordant

b. Decolorized with 95% ethanol.

c. Used crystal violet for the counterstain

d. did not heat fix the smear

Answer 1

Answer: b-decolourized with 95% ethanol

gram stain is based on the ability of the bacterial cell walls to retain crystal violet dye during decolorizing treatment with a decolourizing agent. Crystal violet is used to stain cell walls of gram+ve beacteria have high peptidoglycon and low lipid content than gram-ve. All bacteria stain violet.

Iodine is added as a fixative so that dye cannot be easily removed.

Then it is treated with decolourizing agent, a mixture of ethanol and acetone.

70% alcohol is used to decolourize. But high concentration of 95% alcohol is not effective. Either it will evaporate or it will pass through cell wall of the bacteria very fast in all directions and starts coagulating the protein at must faster rate. So all the bacteria remain purple as the gram-ve bacteria are not decolourized.