Peptides are being studied as novel antifungal molecules. This protocol can be used to study the antifungal efficacy against the human fungal pathogen Candida albicans. This technique provides quantitative data on antifungal activity while reducing experimental time and plastic waste compared to the other methods that require creating on agar.
In addition to testing the activity against Candida albicans, this protocol can be used to test the activity against other yeast-forming cells and to test the activity of small-molecule antifungal agents Begin by inoculating the desired Candida albicans strain into 10 milliliters of liquid yeast extract-peptone-dextrose, or YPD medium, in a culture tube. Grow the culture overnight at 30 degrees Celsius and 230 rpm on a rotary shaker. Subculture the overnight culture of C.albicans and grow to an optical density of approximately 1.0 to 1.2 at 600 nanometer wavelength.
Solubilize each peptide in sterile water and dilute it to twice the desired highest concentration, then add 40 microliters of the desired peptide stock solutions to the first well of each row in a round-bottomed 96-well plate. Next, add 20 microliters of sterile ultra-pure water to columns 2 to 12 of the rows containing the peptide. To serially dilute the peptide stock solutions across the plate up to column 10, transfer 20 microliters from column 1 to column 2 and mix it by pipetting up and down.
Repeat the dilution process up to column 10 which will contain 40 microliters of peptide solution at a 1-to-512 dilution of the column 1 concentration. Once done, remove 20 microliters of peptide solution from column 10 and discard it. Transfer the C.albicans subculture to a 15-milliliter centrifuge tube and centrifuge at 3, 900 g for 3 minutes at room temperature.
Remove the supernatant by pipetting or decanting. Resuspend the pellet with 1 milliliter of 2-millimolar sodium phosphate buffer and transfer the suspension to a 1.7-milliliter centrifuge tube. Pellet the cells by centrifuging and discard the supernatant.
Again, resuspend the pellet in 1 milliliter of 2-millimolar sodium phosphate buffer and wash 2 more times to leave the washed cells in 1 milliliter of sodium phosphate buffer. Determine the cell density of the washed cell suspension and dilute the suspension to 5 times 10 to the 5th cells per milliliter in a 2-millimolar sodium phosphate buffer. Take diluted C.albicans cell suspension and add 20 microliters to columns 1 to 10 and column 12 of each row, then add 20 microliters of 2-millimolar sodium phosphate buffer to column 11.
Cover plate 1 and place it into an incubator at 30 degrees Celsius for 30 minutes. Prepare a new 96-well culture plate to quantify viability by adding 100 microliters of YPD to all the wells of plate number 2, then add 100 microliters of 2-millimolar sodium phosphate buffer to all the wells of plate 2. To dilute the samples in plate 1, retrieve plate 1 from the incubator, and add 280 microliters of 1-millimolar sodium phosphate buffer in each well.
After mixing the contents well with a pipette, transfer 8 microliters from plate 1 to the corresponding well of plate 2. Once done, place the covered plate 2 on a microtiter plate shaker at 350 rpm for 17 hours at 30 degrees Celsius. Mix all the wells in plate 2 by pipetting up and down, ensuring all the cells are resuspended.
Obtain optical density readings at a 600 nanometer wavelength for each well in plate 2 using an absorbance plate reader. Plot the reduction in viability as a function of the peptide concentration to calculate the growth inhibition percentage and to determine the effect of the peptides on the C.albicans growth. After incubating the peptides with the C.albicans cells, the antifungal activity was quantified using the colony-forming units, or CFU counting method and the described protocol incorporating optical density measurements.
The data using the optical density readings were highly reproducible as indicated by the small standard deviations of the replicates. In performing this protocol, maintain the Candida albicans cells in the yeast form by incubating the cells at 30 degrees Celsius since optical density measurements are not reliable in the hyphal cells. The efficiency of this method has allowed researchers to perform high triple studies on peptide activity by testing larger numbers of peptides and peptide concentrations.
