aliquots of at most 3μl sequentially and let the plates dry in
between additions, to avoid excessive spreading of the extract
on the plate.- If far more than 3μl of extract has to be used and the sample
cannot be reconcentrated (e.g., when using directly culture
supernatant instead of ethyl acetate extract), a hole can be
made on the agar plate using the top of a glass Pasteur pipette
(diameter approximately 7 mm) and then resealing the bottom
with 0.5 ml of melted DNAgar. The extract can then be placed
in the resulting well, avoiding spilling. In this case, the
butyrolactone extract in methanol must be diluted adding TE
buffer (10 mM Tris, 1 mM EDTA) pH 7.0 with at least
threefold volume of the extract used. - For the kanamycin bioassay, the intensity of the halo can be
increased by using a higher concentration of inoculum of
LW18 or LW94 strain. - Hsiao et al. [13] determined the minimum concentration of
several different GBLs required to induce the kanamycin resis-
tance phenotype. For SCB1, the minimum concentration was
of 0.025μg/μl. Using the halo diameter for quantification is
discouraged, as this shows a sigmoidal response to the amount
of GBLs [13].
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