A modified microtiter-plate test for quantification of staphylococcal biofilm formation

The tube test and the microtiter-plate test are the most frequently used te chniques for quantifying biofilm formation, an important indicator for the pathogenicity of staphylococci. The purpose of the present study was to dev elop a modified microtiter-plate technique for quantification of biofilm fo rmation. This technique involves fixing the bacterial film with methanol, s taining with crystal violet, releasing the bound dye with 33% glacial aceti c acid, and measuring the optical density (OD) of the solution at 570 nm by using an enzyme immunosorbent assay reader. Biofilm formation of 30 Staphy lococcus strains was estimated by the tube test, the standard microtiter-pl ate test and the modified microtiter-plate test. The modified microtiter-pl ate test, as a quantitative assay, is superior to the tube test in terms of objectivity and accuracy. It is also superior to the standard microtiter-p late test because it enables indirect measuring of bacteria attached both t o the bottom and to the walls of the wells, while in the standard test only the dye bound to the bacteria adhered to the bottom of the wells is spectr ophotometrically registered. Highly significant differences between OD valu es obtained by the standard microtiter-plate test and those obtained by the modified test suggest that large number of bacteria were attached to the w alls of the wells. Therefore, the modification of the standard microtiter-p late test by introduction of an additional step of decolorization by acetic acid seems to be a useful improvement of the technique. (C) 2000 Elsevier Science B.V. All rights reserved.