|String production by E. coli mixed in a 3% solution of
KOH (a typical result for Gram-negative bacteria).
Most microbiologists use Gram staining to separate bacteria into two groups, based on their Gram stain reaction: Gram-positive and
Gram-negative. But, some Gram-positive bacteria decolorize more readily than others, often resulting in being perceived incorrectly as
Gram-negative. An alternative to Gram staining is the KOH test.
Like the Gram stain reaction, the potassium hydroxide test is based on differences in
the bacterial wall composition. The cell wall of Gram-negative bacteria is easily
disrupted when exposed to dilute alkali solutions while the tough thick peptidoglycan
wall of Gram-positives do not lyse. When the cell walls are disrupted, the suspension
in KOH becomes viscous due to the release of relatively unfragmented threads of
Prepare a 3% KOH solution.
Use a 24-48 h bacterial culture from solid media.
Gram-negative (E. coli) and Gram-positive (Staphylococcus aureus) controls are
Place 1 drop of KOH solution on a slide. Using the loop, harvest one large colony (or
several small colonies) and mix the culture with the solution. Homogenize and wait
for about one minute.
Immerse the loop in the mixture and slowly draw it up 1-2 cm. The KOH solution
characteristically became very viscous and mucoid with Gram-negative bacteria. A
string of the mixture would follow the loop when it was raised. Gram-positive bacteria
suspended in the KOH solution generally display no reaction (no string formation).
False negative reaction may appear when using cultures older than 5 days.
The KOH test may be used on Gram-negative staining Clostridia to demonstrate the
Gram-positive structure of their cell wall.
1. S. Halebian, B. Harris, S.M. Finegold, and R.D. Rolfe: Rapid method that aids in
distinguishing Gram-positive from Gram-negative anaerobic bacteria. J Clin Microbiol. 1981 March; 13(3): 444–448.
2. George M. Carlone, Manuel J. Valadez, and M. John Pickett: Methods for Distinguishing Gram-Positive from Gram-Negative
Bacteria. Journal of Clinical Microbiology, Dec. 1983, p. 1157-1159.
(c) Costin Stoica