Medicinal Plants Classification Biosynthesis and ... - Index of

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Philippe N. Okusa, Caroline Stevigny and Pierre Duez
The agar diffusion technique can also be used to study the effect of plant extracts on
antibiotics resistance. In this purpose, inactive plant extracts are dissolved and incorporated
in molten agar medium and cast in Petri dish (Okusa et al., 2007); suitable controls are
prepared with medium without plant extracts. The susceptibility of antibiotics is then
evaluated by a classical disk diffusion method. Another possible approach is the
incorporation of antibiotic in the agar layer overlaid by disks containing tested plant extracts
(Shahverdi et al., 2004).
2.3. Liquid-and Agar-Dilution Methods
Liquid broth dilution methods, including microdilution methods, are widely used to
quantitatively measure the in vitro activity of an antimicrobial agent against a given bacterial
strain. A series of serial dilutions of the tested agent in broth tubes or multiwell plates are
inoculated with a standardized suspension of the test microorganism. After overnight
incubation, the minimum inhibitory concentration (MIC) is visually or spectrometrically
estimated by determination of turbidity (NCCLS, 2003) or of reaction products from redoxindicators
such as MTT or resazurin (Gabrielson et al., 2002). Test samples may react with
the chromogenic reagent or, if not fully soluble, may interfere with turbidity readings,
emphasizing the need for suitable controls, i.e. extract dissolved in blank medium without
micro-organisms. The liquid-dilution method also allows determination whether a compound
or extract has a cidal or static action at a particular concentration. The minimal bactericidal or
fungicidal concentration (MBC or MFC) is determined by plating-out samples of completely
inhibited dilution cultures on agar medium and visually assessing growth (static, MIC) or nogrowth
(cidal, MBC) after incubation (NCCLS, 2002, 2003).
In agar-dilution methods, various concentrations of antibacterial substance are mixed
with nutrient agar and caste. Casted agar plates are then inoculated and incubated. The lowest
concentration of antimicrobial compound resulting in no visible growth is considered as the
MIC value.
Liquid-and agar-dilution methods can also be used to study the effect of plant extracts on
antibiotics resistance.
2.4. Synergy between Plant Extracts and Antibiotics
Antimicrobial combinations are considered to be synergistic if the effect of combination
is greater than the effect of either agent alone or greater than the sum of the effects of
individual agents. Antagonism results if the combination provides an effect less than the
effect of either agent alone or less than the sum of the effects of the individual agents.
Indifference results if the combination provides an effect equal to the effect of either agent
alone. The distinction between synergy, antagonism and indifference classically relies on the
determination of Fractional Inhibitory Concentrations (FIC= MIC of a drug given in
combination/MIC of the same drug alone) and FIC index (for a mixture of drugs A and B,
FIC index = FICA + FICB). The FIC index is classically evaluated as follows: synergy (FIC