This article describes a method for evaluating the antifungal activity of novel antimicrobial peptides (AMPs) against pathogenic fungi using a microtiter plate assay. The protocol involves serial dilutions of AMPs and observing fungal growth under controlled conditions.
Take a microtiter plate, and add serial dilutions of novel antimicrobial peptides or AMPs — the test compounds.
Designate a subset of wells as growth controls, and add sterile water without the AMPs.
Add a pathogenic fungus suspended in growth media into selected wells.
Add only the media, devoid of the fungus, in a subset of wells designated as blank controls.
Seal the plate and incubate under humid conditions conducive to fungal growth.
Post-incubation, observe the wells under a microscope.
The growth controls appear turbid due to fungal growth in the absence of AMPs. Turbidity across all dilutions of an AMP indicates its ineffectiveness against the fungus.
The blank controls, devoid of fungal growth, appear clear. Compare test wells with the blank controls to confirm the absence of growth, indicating the antifungal activity of AMPs.
The lowest concentration of an AMP that inhibits fungal growth is termed the minimum inhibitory concentration, or MIC.
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.
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