In this research, we present a protocol for the examination of oral Candida infection in patients with primary Sjogren's syndrome, which can be used for timely treatment to avoid infection related complications. We provide a series of systematic, simple and feasible methods for detecting oral Candida infections, identifying Candida strains and testing the antifungal susceptibility of commonly used drugs in patients with primary Sjogren's syndrome. Maybe are focused on the role of oral candidate infection in the onset and the progression of primary Sjogren's syndrome pathologists in our future studies.
To begin, select the participants based on the framed inclusion criteria. For saliva collection, instruct the patient to hold the tube with a funnel and let the saliva flow gradually into the tube along the lower lip for 15 minutes. At the end, ask the participant to spit all the remaining saliva into the tube.
In case of low unstimulated saliva flow, swab the oral mucosa in infection suspected areas at least 10 times, and let the patient rinse their mouth with five milliliters of PBS for one minute. Then collect the mouthwash in a 50 milliliter sterile tube and put the swab into it. Add one milliliter of PBS to the collected saliva and vortex the tube for one minute.
To begin, obtain the patient's saliva sample in PBS. Dissolve 7 grams of SD agar powder in 100 milliliters of double distilled water. Using an autoclave sterilize the medium at 121 degrees Celsius for 15 minutes.
Pour around 10 milliliters of the medium into a 10 centimeter microbiological culture dish and let it cool. Then streak 200 microliters of the saliva sample on the surface of the agar and incubate for 48 hours at 37 degrees Celsius. To prepare Candida diagnostic agar medium, mix 4.77 grams of the powder with 100 milliliters of double distilled water.
Heat the mixture to 100 degrees Celsius until it reaches a slight boil. Then dispense around 10 milliliters of media into each 10 centimeter microbiological culture dish and let it solidify. Next, transfer the Candida colonies into 500 microliters of SD medium and suspend them thoroughly using a pipette.
Plate 50 microliters of the suspension onto the diagnostic agar and incubate the plate at 37 degrees Celsius for 48 hours. Finally, identify the strains from the mature colonies using the specific colony color and pattern. The Candida strain identification assay using the Candida diagnostic agar showed that primary Sjogren's syndrome patients had oral infections of Candida albicans, Candida glabrata, and Candida albicans plus Candida krusei.
To begin, obtain the patient's saliva sample in PBS. Using a precision molded loop, smear the sample on a glass slide and let it dry at room temperature. Meanwhile, dissolve 10 grams of potassium hydroxide in 100 milliliters of double distilled water.
Then heat the slide with an alcohol lamp and stain it with 10%potassium hydroxide. Cover the sample with a cover slip and press it slightly to make it transparent. Using a microscope, visualize the hyphae and yeast cells.
For CFW staining assay, stain the smeared slide with a drop of CFW. Cover it with a cover slip and place it aside for a minute. Visualize the hyphae using a fluorescence microscope and repeat three times.
Potassium hydroxide smear test of primary Sjogren's syndrome patients oral samples indicated the presence of Candida hyphae and CFW staining confirmed the existence of hyphae and fungal spores.
