universidade de são paulo - Faculdade de Odontologia - Unesp

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standardized to a concentration of 1 x 10 7 cells/ml, spectrophotometrically. Three
ml of the standardized C. albicans cell suspension was added to each well
containing the specimen. The cells were left to adhere for 90 min at 37 ºC in a
shaker at 75 rpm [20]. The non-adherent cells were removed from the specimen
by gently washing with 3 ml PBS twice. The negative controls were acrylic
specimens to which no cells were added. All experiments were performed in
triplicate on three independent occasions.
XTT reduction assay
Nine specimens from each group were evaluated by XTT reduction assay,
as described elsewhere [22]. Briefly, XTT (Sigma, MO, USA) was prepared in
ultrapure water (1mg/ml), filter sterilized and stored at -70 ºC until used.
Menadione (Sigma, MO, USA) solution was prepared in acetone at 0.4 mM
immediately before each assay. After washing, the specimens were transferred to
new wells with XTT solution in the following proportion: 158 μl PBS with 200
mM glucose, 40 μl XTT and 2 μl menadione. The plates were incubated for 3 h in
the dark at 37 ºC. The whole content of each well was centrifuged at 5000 g for 2
minutes and the colorimetric change of the supernatant was measured using a
microtiter plate reader (Thermo Plate – TP Reader) at 492 nm.
Crystal violet staining
Nine specimens from each group were evaluated by cell counting after
crystal violet staining. After the non-adherent cells were removed by washing, the
specimens were fixed in 80% ethanol, stained with crystal violet for 1 minute and
washed with PBS [36]. Adherent yeast cells were counted in 10 different fields for