universidade de são paulo - Faculdade de Odontologia - Unesp

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For group G 3 , whole human unstimulated saliva was collected from one
healthy adult volunteer. The saliva was expectorated into sterile 50 ml Falcon
tubes on ice, pooled and clarified by centrifugation at 10000 rpm for 5 min at 4 ºC
[16-17, 25, 30-31].
For all experimental groups, the supernatant of saliva was sterilized using
membrane filter with a 0.22 μm pore size [20, 24]. The resulting saliva was
immediately stored at -70 ºC until use. The study was approved by the Ethics
Committee of Araraquara Dental School, and all subjects volunteered to
participate and signed an informed consent form.
In vitro salivary pellicle formation
The specimens of the three experimental groups were incubated in 12-well
microtiter plates with 3 ml of the saliva preparation at room temperature for 30
minutes prior to the adherence assay [29].
Preparation of Candida suspension and adherence assay
The stock culture of Candida albicans strain ATCC 90028 was maintained
in YEPD medium (1% yeast extract, 2% peptone, 2% dextrose, 2% agar) stored at
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), centrifuged at 4000 g for 5 min and
resuspended in YNB with 100 mM glucose. Candida suspensions were