universidade de são paulo - Faculdade de Odontologia - Unesp

119
4 – 6 ºC during the experimental period. For preparation of the yeast inoculum,
two loopfuls of the stock culture were streaked onto YEPD medium and incubated
at 37 ºC for 48 h. Two loopfuls of this young culture were transferred to 20 ml of
yeast nitrogen base (YNB) medium with 50 mM glucose and incubated at 37 ºC
for 21 h. Cells of the resultant culture were harvested, washed twice with
phosphate-buffered saline (PBS, pH 7.2) at 5000 g for 5 min and resuspended in
YNB with 100 mM glucose. Candida suspensions were 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 10 . 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.
Crystal violet staining
Nine specimens from each group were evaluated by crystal violet staining
assay. 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 24 . Adherent yeast cells were counted in 10 different fields for each
specimen, using a light microscope (Olympus BX51, Japan) at 400 x
magnification and the mean values were calculated. Adherent yeast cells were
counted in a "blind" manner to avoid subjective bias. The results were expressed
as cells/mm 2 .