E. coli from an overnight culture o

E. coli from an overnight culture on Nutrient Agar plates were
resuspended in Ringer’s solution to a turbidity of McFarland
0.5 and from this suspension serially diluted 1/10 in Ringer’s
solution. The MUG substrate from the FluorAce kit (Bio-Rad,
Hemel Hempstead, UK) was prepared as described in the
manual and dispensed into black 96-well plates (Greiner Bio-
One, Frickenhausen, Germany). The E. coli dilutions were
assayed with the MUG substrate. The commercially available
miniaturised method, FluorAce kit, for the quantification of b-
D-glucuronidase activity, was modified as described by Garcia-
Armisen et al. (2005). The plates were freeze dried over night,
sealed and stored at 20 C until use. E. coli suspensions
(100 ml) and controls (b-D-glucuronidase from kit, Ringer’s)
were added to the freeze dried MUG substrate and plates
incubated for 24 h at 37 C. Stopping Buffer (100 ml) was added
and the fluorescence measured with a FLUOstar OPTIMA plate
reader (BMG Labtech, Offenburg, Germany)