The overall goal of this method is to evaluate the activity of potential antifungal compounds, using a modification of the broth microdilution susceptibility test, used in clinical laboratories. This method is used to evaluate the fungal activity of natural or synthetic compounds, one of the first tests in the development of anti fungal therapies. We provide instructions for an initial screen for new antifungals using small volumes in a short time, especially, if the experiments are carried out with automation.
Demonstrating the procedure will be Daniel Zamith-Miranda, a post doctoral fellow in my laboratory. Begin this procedure by growing the fungal strains for the assay as described in the text protocol. On the following day, collect the cells by centrifuging the conical tubes at 1200 G, for five minutes at room temperature.
Discard the supernatant, add ten milliliters of PBS to each tube, and resuspend the cells. Centrifuge again at 1200 G for five minutes at room temperature. Repeat the PBS wash and centrifugation two more times.
After the third wash, resuspend the cells in five milliliters of double strength RPMI 1640 medium. Depending on the turbidity of the cell suspension, prepare one milliliter of a one to 100, or one to 1000, dilution of each strain, in a microcentrifuge tube. Aliquat ten microliters of this dilution, place it in a hemocytometer chamber, and count the total number of cells in the four corner quadrants under the microscope.
After calculating the density of each cell suspension, prepare stock cell suspensions in double strength RPMI 1640 medium. Prepare 4, 000 cells per milliliter for C.albicans strains, and 20, 000 cells per milliliter for C.neoformans strains. The lypholized peptides are stored in aliquats for one time use at minus 20 degrees Celsius, and only dissolved in deionized water before each experiment.
The peptide concentration should be twice the highest final concentration tested in the assay. Reference antifungals are also prepared. The antifungal assay is performed in triplicate and 96 well polystyrene microplates.
Using a two 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 RPMI 1640 medium to each well of columns one to three, plus column 11. The final concentration for C.albicans will be 2, 000 cells per milliliter. 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. Acquire an image of each plate using a digital camera.
In this representative assay, three antimicrobial peptides, or AMPs, with concentrations ranging from 100 micromolar to point seven eight micromolar, were evaluated for antifungal activity against C.neoformans. A growth control and a blank were included. Any well with clear media similar to the blank wells is considered a positive result, whereas any well with turbidity analogous to the negative growth control wells is considered negative.
Since the minimum inhibitory concentration, or MIC, is defined as the lowest antimicrobial compound concentration that completely inhibits visible fungal growth at the end of the incubation period, 25 micromolar is considered the MIC for AMP1. And, 12 point five micromolar, the MIC for AMP2. AMP3 will have to be reevaluated, as the fungus grew at all concentrations tested.
In the same manner, three distinct peptides tested against C.albicans, indicted 50 micromolar is the MIC for AMP1. Six micromolar is the MIC for AMP2, and 25 micromolar is the MIC for AMP3. An example of a poor result, likely from improper pipetting during the dilution step, is illustrated by columns five to seven of row C in this plate, where differences in C.neoformans growth are present between replicates.
Once mastered, this technique can be done in approximately two hours, plus the time of fungal growth, if it's performed properly. Remember to be aware of the physical chemical features of these tested molecules. Then use appropriate solvents as control in case they are needed, and to always use the cells in the same growth phase.
Following this procedure, other methods could be performed such as time kill assay, the use of viability dyes, and ST60 assays to answer additional questions, like whether the compound has a fungicidal or fungistatic effect. After it's development, this technique paved the way for researchers in the field of medical micrology to explore several sources of natural or synthetic compounds in the search for antifungal compounds. After watching this video, you should have a good understanding of how to start a fungal culture, prepare cells for the assay, perform the serial dilutions, and read the results after incubating the fungal strains with our tested compounds.
Don't forget, that working with fungal pathogens can be extremely hazard. And preparation such as wearing personal protection equipment, working inside a biological safety hood, and using appropriate safety practice should be taken while performing this procedure.
