Nonstaining (KOH) method for determination of gram reactions of marine bacteria

Abstract

A rapid nonstaining (KOH) method for the determination of the Gram reactions of bacteria is described, and its application to marine isolates is discussed. To perform the test, place a drop of 3% aqueous KOH on a slide. If one prefers, 10 μl from an automatic pipette is adequate. In practice, as many as 8 to 10 tests per slide can be done conveniently. Using a sterile loop, transfer a visible amount of bacterial growth from an agar culture to the drop of KOH. Mix the cells and KOH thoroughly on the slide, constantly stirring over an area about 1.5 cm in diameter. If the bacterium-KOH suspension becomes markedly viscid or gels within 5 to 60 sec, the isolate is gram negative. If no gelling is observed, the isolate is gram positive. The best way to determine viscosity is to raise the loop about 1 cm from the slide. If an obvious stringiness is present, then the culture is gram negative. All gram-positive and gram-negative results obtained by gram staining were confirmed by the KOH method. Gram-variable bacteria produced equivocal results.