Our microplate method proved to be an easy to use and
effective tool in the identification of antibacterial principles
in P. galioides and we will continue to apply the method
in bioassay-guided fractionation of other Peruvian medicinal
plants identified in our initial screening. The method takes
the benefits of a known working assay, the NCCLS method,
and combines it with the ease of interpretation provided by a
color indicator, which can be used successfully with several
strains. Our protocol differs from Eloff’s procedure in that it
offers an approach to deal with extract solubility issues by
using an effective universal solvent, DMSO, for initial dissolution
of extracts. Dilute samples at various concentrations
can then be prepared using broth rather than water. Solubility
problems were overcome by sonication of the sample
in DMSO or in the solvent/broth test solution. This allows
a more accurate assessment of the sample’s biological activity.
For highly colored samples, extra wells of test sample
with no added indicator can be prepared as a reference
for color change comparison. With appropriate precautions
and careful attention to aseptic techniques, this assay can be
performed on the bench top and does not require a laminar
flow hood. Advantages over the agar disk-diffusion method
include increased sensitivity for small quantities of extract,
ability to distinguish between bacteriostatic and bactericidal
effects, and quantitative determination of minimal inhibitory
concentrations (MIC). The use of a colorimetric indicator
eliminates the need for a spectrophotometric plate reader and
avoids the ambiguity associated with visual comparison or
measurement of growth inhibition rings on agar plates. We
believe that this method will be of use to many researchers in
the field for the identification and evaluation of antibacterial
principles in medicinal plants.