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
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