An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds

The antifungal assay is performed in triplicate in 96-well polystyrene microplates using a 2-fold serial dilution of each peptide and control antifungal and a final volume of 50 microliters per well.

The most crucial step in the protocol is the serial dilutions. So be very careful during the next few steps to avoid any error that will compromise your whole analysis.

Using a pipette, add 100 microliters of the antifungal peptide in the double-strength concentration of the highest desired final concentration in columns one through three in row A. Using a multichannel pipette, add 50 microliters of sterile water to the other wells in rows B to H. Remove 50 microliters of the wells with the highest concentration, transfer to the corresponding wells of the next concentration, and homogenize by pipetting up and down several times.

Repeat the serial dilution until the wells with the lowest concentration are reached, and discard 50 microliters from these wells. Leave columns 11 and 12 of rows A, B, and C with only water for blank controls or negative growth controls. Add 50 microliters of the double-strength-adjusted inoculum in RPMI1640 medium to each well of columns 1 to 3 plus column 11.

The final concentration for C. albicans will be 2,000 cells per milliliter, and the final concentration for C. neoformans will be 10,000 cells per milliliter. Prepare negative growth controls by mixing 50 microliters of water and 50 microliters of adjusted inoculum without the antifungal. Prepare blank controls by combining 50 microliters of water and 50 microliters of medium without cells.

Load the controls into the appropriate wells. Observe all wells using an inverted optical microscope and then incubate the plates at 37 degrees with shaking at 200 RPM. After the incubation period, observe all wells again to verify changes in morphology, as well as to check for signs of contamination.