The KOH test is used to identify Gram-negative and Gram-positive bacteria as an alternative to the Gram stain. It works by dissolving the thin peptidoglycan cell wall of Gram-negative bacteria when exposed to 3% KOH, lysing the cell and releasing viscous DNA strands. Gram-positive bacteria are not lysed by KOH and do not release DNA. To perform the test, a bacterial sample is mixed with 3% KOH on a slide. Gram-negative bacteria will appear viscous and form mucoid strings within 15 seconds, while Gram-positive bacteria will not become viscous or form strings.

1. KOH test
Purpose
For identification of Gram (-) bacteria. This test
replaces the Gram stain. It is much quicker and
easier to perform than the traditional Gram stain. It is
just as accurate. It is a secondary test for
identification of Gram (+) vs. Gram (-) cultures. 3%
KOH dissolves cell walls of Gram (-) (thin layer of
peptidoglycan), but does not affectGram (+) cell
walls. Dissolution of Gram (-) cell wall lyses the cell
and spills its contents,including the DNA. DNA is
very viscous, and with a large enough cell mass, the
DNAstrands can be seen sticking to / dragging from
a loop when touched. Gram (+) cells are notlysed, no
free DNA, no viscosity will be observed.This test
requires a large amount of cells (visible clump).
Scrape samples of E. coli,Staphylococcus
epidermidis, and your unknown from a solid medium (agar slants).
Materials
 Microscope slide
 Toothpick
 3% KOH
Instructions
1. Place one drop of 3% KOH on a microscope slide
2. With a toothpick, transfer a generous amount of bacteria to the drop of KOH.
3. Stir
4. Raise and lower toothpick off slide
Expected results
 KOH + (Gram -); bacteria will be viscous and form a mucoid string in 15 sec.
 KOH - (Gram +); bacteria do not become viscous or form mucoid strings in 15 sec.
Notes
 Use 24-48h cultures
 It is a good idea to use + and - controls
 KOH should be fresh. Discard any bottles with a white precipitate.
 Coryneform bacteria can be Gram variable as cultures age.
Prepared by: Mr. Say Sinith

2. 3% KOH test
Prepared 3% KOH
reagent
Place one drop of 3% KOH on
slide
Pick colony from pure
culture
Mix and stir organism with
the reagent
3% KOH Positive 3% KOH Negative
Observed result when raise the loop off slide