KOH Method for Gram “Staining”
Objective: To distinguish Gram-positive bacteria from Gram-negative bacteria.
Introduction: Gram character is a fundamental characteristic of bacterial cells. Along with cell
morphology, it is the first clue we have as to the identity of an unknown isolate. Gram character is
rooted in basic physiological differences between Gram-positive bacteria, which have very thick cell
walls, and Gram-negative bacteria, which have thinner cell walls and an additional protective or
barrier structure called the outer membrane. The traditional Gram stain takes advantage of differential
interactions of dyes with Gram-positive or Gram-negative cell walls. However, there are other, more
indirect ways of determining Gram reaction.
The thick peptidoglycan layer that makes up the cell wall of Gram-positive bacteria is much more
resistant to chemical digestion than that of Gram-negatives. Therefore, treatment with a dilute base
such as 3% potassium hydroxide (KOH) affects these two classes of bacteria differently. This forms
the basis for the KOH Gram “stain” – a method for determining Gram reaction based on chemical
digestion of the cell wall.
Basis of the Test: The thin-walled Gram-negatives will lyse in 3% KOH, releasing cell contents,
including DNA, which is very viscous. The thick-walled Gram-positives do not lyse and therefore do
not affect the viscosity of the KOH on the slide.
Note: Another indirect, non-microscopic approach for determining Gram reaction is to test for the
presence of L-alanine aminopeptidase, an enzyme that (almost) all Gram-negative bacteria express,
but that is not present in Gram-positive bacteria. This is done by placing a portion of an isolated
colony into a buffer that lyses Gram-negative bacteria and releases L-alanine aminopeptidase, which
is then detected colorimetrically using a test strip.
Procedure: Work singly or in groups.
a. As with “regular” Gram staining, use young cells (<24 hr old); ensure use of clean
b. Materials: microscope slides, toothpicks, 3% solution of potassium hydroxide (KOH)
c. Apply a 50µl portion of KOH to a microscope slide.
d. With an inoculum loop, add a visible portion of an isolated colony taken from a plate. Mix
with a toothpick.
e. Observe: watch for viscosity to develop. After 5 – 60 seconds, lift up the toothpick gently and
check for adherent, stringy “goo”.
f. Stringy, “gooey” cultures are Gram-negative. Those not forming a viscous, stringy goo are
Potential problems with the KOH method: Although this method is convenient, it has several
drawbacks: it is indirect and there is no morphology component – the cells are not looked at under the
scope; additionally, it must be performed on pure cultures – isolated colonies from a plate, while the
traditional Gram stain can be carried out on mixed cultures. Further, for any given Gram-negative
isolate, if you have too few cells, stringiness may not develop. If you have too many cells, stringiness
may not develop. Therefore, the results from this test can be variable. Still, in the hands of a skilled
technician, the KOH test does have some strengths – it is very convenient and does not require a