universidade de são paulo - Faculdade de Odontologia - Unesp

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cells/ml, spectrophotometrically. Three ml of the standardized C. glabrata cell
suspension was added to each well containing the specimen. The cells were left to
adhere for 90 min at 37 ºC [23]. The non-adherent cells were removed from the
specimen by gently washing with 3 ml PBS twice. For all experimental
conditions, the negative controls were acrylic specimens to which no cells were
added. All experiments were performed in triplicate on three independent
occasions.
Preconditioning with saliva
To investigate the effect of the saliva on Candida glabrata adhesion to the
denture acrylic resin, half of specimens from each group (n=9) were incubated
into the 12-well microtiter plates and coated with 3 ml of prepared saliva for 30
min at room temperature prior to the adhesion assay.
Measurement of adherent C. glabrata
Crystal violet staining
After the non-adherent cells were removed by washing, all specimens were
fixed in 80% ethanol, stained with crystal violet for 1 minute and washed with
PBS [37]. Adherent yeast cells were counted in 10 different fields for each
specimen, using a light microscope (Olympus BX51, Tokyo, 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 .