Question

.  When performing the Gram stain procedure, why is it essential to limit the decolorizing step to less
     than 10 seconds?   

Answer 1

Decolorization - The step is pouring few drops of decolourizer to smear. Ex acetone alcohol (10 sec) or iodine acetone.Then slide is immediately rinsed with water .

This decolorizer removes the primary stain from the Gram negative bacteria. While Gram positive bacteria retains primary stain.

It is important to note the time of decolorizer .If decolorizer poured for more time. then even Gram positive bacteria (G+) loose colour. This is called Over decolourization.

If decolorizer poured for less time. Then Gram negative bacteria (G-)doesn’t loose colour . This is called under decolourization.