- the KOH test is a confirmation test of the Gram stain, especially with difficult bacterial species.
- The Gram stain is the basis for identification & classification in microbiology and is based on the fundamentally different cell walls of Gram- and Gram+ cells. Yet, the Gram stain is somewhat tricky, not very easy, must be used on fresh cells, must be used on a thin smear, and some species just stain poorly with it.
- A Japanese microbiologist who found the stain difficult decided there had to be an easier way. Instead of using two stains, it uses KOH in an attempt to dissolve the cells.
- Gram+ cells with their tough thick cell walls of peptidoglycan do not lyse.
- Gram- cells with their thinner more porous cell walls lyse. DNA comes out of the cells, and the bacterial smear becomes a viscous, stringy, sticky mess.
Procedure:
- Aseptically, place one or two large colonies on a slide.
- Add 1 drop of KOH 3% on top of the cells
- Let it sit for 1 minute
- After waiting for a minute, add your cool loop to the bottom of the mixture and slowly draw the loop up.
- If you see viscous strings and stuff, the cells are Gram(-) , if not they are gram (+)
KOH Test for Gram stain confirmation
Video Tutorial: How to do KoH Test
