Determination of MIC was performed using the broth microdilution
method, following the norm M38-A of the National Committee
for Clinical Laboratory Standard (NCCLS, 2002). TEO was diluted
in a sterile solution of Tween 80 at 0.001% (Vetec, Rio de Janeiro,
Brazil) and evaluated in final concentrations that ranged from
6.25 to 4000 lg/mL. At each different TEO concentration, 100 lL
of a suspension containing 4 105 CFU/mL of A. flavus were added
to synthetic medium RPMI-1640 (0.1 mL). The microplates were
incubated at 35 C for 72 h. MIC was determined according to the
lowest concentration of TEO that was capable of inhibiting the
visual growth of A. flavus. The positive control was performed in
the medium containing only the conidia suspension. For MFC
determination of the essential oil, samples were taken from the
wells that did not show any visible fungal growth and
re-inoculated in plates containing Sabourand Agar. These plates
were incubated at 25 C for 72 h. The lowest concentration of
TEO that was capable of inhibiting fungal growth was considered
to be the MFC. At the other experiments, the methodology
proposed by Shukla, Singh, Prakash, and Dubey (2012) was utilised
with some modifications, being utilised three concentrations
below and one above the MIC and MFC.
2