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

1. Solutions and Media

1. Prepare 2X Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate-buffered saline (PBS), Sabouraud dextrose broth, and Sabouraud dextrose agar as per Table 1.

2. Fungal Inoculum Growth Conditions

1. Store all fungal strains as frozen stocks in 35% glycerol at -80 °C, until needed.
2. Perform the following steps before each experiment.
   1. For Candida albicans strains:
      1. Thaw a stock vial and transfer 200 µL to 10 mL of Sabouraud dextrose broth in a sterile 50 mL polypropylene tube with a cap and culture overnight at 30 °C with agitation (200 rpm). Remember to incline the tube and leave the cap slightly open for better aeration of the culture.
   2. For Cryptococcus neoformans strains:
      1. Scrape the frozen surface of a stock vial. Plate the cells onto Sabouraud dextrose agar plates and incubate for 48 h at 30 °C. After visible growth of isolated colonies, keep the plates in the refrigerator (4 °C) sealed with paraffin film for up to 15 days.
      2. Collect a medium-sized isolated colony from the plate using a sterile toothpick or sterile inoculating loop and inoculate 10 mL of Sabouraud dextrose broth in a sterile 50 mL conical tube. Incubate for approximately 24 h at 30 °C under agitation (200 rpm).
      3. Do not exceed the 24 h incubation. Remember to incline the tube and leave the cap slightly open for better aeration. It is always good to standardize the growth stage of the cells before each test, since this is an important factor affecting antimicrobial resistance.
         NOTE: Both fungi were grown at 30 °C for rapid propagation in our experiments, but the temperature can be changed to reflect the aims of the study, for example clinical treatment (37 °C). Most importantly, this initial incubation time should be established beforehand for each fungal isolate and maintained throughout all the tests to ensure reproducibility.
3. After fungal growth, collect the cells by centrifuging the conical tubes at 1,200 x g for 5 min at room temperature. Discard the supernatant and add 10 mL of PBS.
4. Resuspend the cells and centrifuge again at 1,200 x g for 5 min at room temperature.
5. Repeat the PBS wash and centrifugation twice more. After the third wash, resuspend the cells in 5 mL (according to the pellet size) of 2X RPMI-1640 medium.
6. Prepare 1 mL of a 1:100 or 1:1,000 dilution in a microcentrifuge tube (depending on the turbidity of the cell suspension).
7. Aliquot 10 µL of this dilution, place it in a hemocytometer chamber and count the total number of cells in the four corner quadrants under the microscope. Calculate the concentration by the formula: (total cell number/4) x dilution factor x 10⁴ (chamber dilution constant).
   1. Consider a 100% viability if viability counts and growth conditions have been systematically correlated for the isolate being studied.
      NOTE: The standardization and quality control of viability counts can be done by back-plating in accordance to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) antifungal Minimum Inhibitory Concentration (MIC) method for yeasts.
   2. If the growth/viability correlation has not been established for the isolate under study, measure the fungal viability by counting live cells in a hemocytometer with the aid of dyes that selectively color dead cells, such as phloxine B11, trypan blue, or Janus Green. Use the fungal cells for this protocol only if the viability of the population at this point is 90% or above.
      ​NOTE: Dead/alive dyes may not work well with the fungus of choice. Please test them before use. For example, the trypan blue dye does not work well with C. neoformans.
8. After that, prepare the cell suspensions in 2X RPMI-1640 medium (2X adjusted inoculum in RPMI-1640 medium). For 96-well plates consider a volume of 5 mL for each plate.
   1. For all C. albicans strains, prepare a stock cell suspension of 4 x 10³ cells/mL. This concentration is 2X of the final cell concentration in each well (2 x 10³ cells/mL).
   2. For C. neoformans strains, prepare a stock culture of cells 2 x 10⁴ cells/mL. This concentration is twice the final cell concentration in each well (1 x 10⁴ cells/mL).
   3. For other fungi, test with a known antifungal which concentration will be ideal for the particular study in relation to the incubation time. Take metabolism and duplication time of the fungus into consideration.

3. Peptides (Unknown Agent)

1. Store the lyophilized peptides at -20 °C, and dissolve them in deionized water before each experiment. The maximum storage time once dissolved in water will depend on the nature of each peptide.
2. Prepare aliquots of twice the highest final concentration tested in the assay (2X). Ideally, prepare a small number of aliquots with enough peptide for one time use to avoid freeze-thaw cycles.
   NOTE: The choice of concentration should be based on literature and the properties of the peptide. It is recommended to start the serial dilutions with approximately 100 µM of the peptide, and then decrease or increase this concentration range depending on the obtained results.

4. Reference Antifungals (Positive Controls)

1. For those diluted in water: prepare a 2X solution of the highest concentration of the analysis (see Section 5: Antifungal Assay for the dilution steps).
   1. For C. albicans strains, prepare a solution of 128 µg/mL of fluconazole or 128 µg/mL of caspofungin. The plate concentration for both will be 64 µg/mL.
   2. For C. neoformans strains, prepare a solution of 32 µg/mL of amphotericin B (water-soluble solution). The plate concentration will be 16 µg/mL.
      ​NOTE: Normally amphotericin B is diluted in dimethyl sulfoxide (DMSO), since this anti-fungal is poorly soluble in water. However, there are water-soluble amphotericin B preparations commercially available.
2. For antifungals diluted in an organic solvent: prepare a 100X stock solution in DMSO, then dilute it to 10X in water for use.
   NOTE: Thus, in the well, the final concentration of DMSO will not exceed 1%. A well where the fungus will grow in media containing 1% DMSO as control is required given that some fungi do not tolerate well this concentration of the solvent. Remember that DMSO is photosensitive, so cover the plate with foil or place it in a dark chamber for the duration of the incubation period.

5. Antifungal Assay

NOTE: In vitro antifungal assays are performed based on the broth microdilution susceptibility test from Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with some modifications.

1. Prepare a two-fold serial dilution of each peptide and control antifungal in 96-well polystyrene microplates to a final volume of 50 µL.
   1. Using a pipette add 100 µL of the antifungal/peptide in the 2X concentration of the highest desired final concentration in columns 1-3, in row A.
   2. Using a multichannel pipette, add 50 µL of sterile water into the other wells, in rows B to H.
   3. Remove 50 µL of the wells with the highest concentration (row A), transfer to the next well of the next concentration (row B) and homogenize.
   4. Repeat the above steps until the well with the lowest concentration and discard 50 µL of this well (row H). Leave Columns 11 and 12 (rows A, B, C) with only water for either blank control or negative/growth control.
   5. Add 50 µL of the 2X adjusted inoculum in RPMI-1640 medium to each well (columns 1-3 plus column 11 (negative/growth control)); the final concentration for C. albicans will be 2 x 10³ cells/mL, and the final concentration for C. neoformans strains will be 10⁴ cells/mL.
   6. Prepare blank controls (50 µL water + 50 µL 2X RPMI-1640 medium without cells) and negative/growth control (50 µL water + 50 µL adjusted inoculum 2X RPMI-1640 medium without antifungal), and load into the well plate as described above.
2. Repeat the procedure for each compound to be tested and the chosen antifungal control (columns 4-10).
   NOTE: The serial dilutions can be done vertically as the experiment below, or horizontally in the plate (i.e., if the number of dilutions of the given compound/extract that needs to be tested is eight or more).
   1. If the compounds, reference drugs, or any of its components are photosensitive, perform the assay in reduced light, cover the plates with foil, or place in a dark chamber during incubations.
3. Consider the following optional recommendations.
   1. Seal the plate with a clear cover plate that permits gas exchange, as this helps reduce evaporation.
   2. Place the plate in a humid chamber; it will also help reduce evaporation.
4. Incubate the plates at 37 °C for 24 h or 48 h for all C. albicans strains, and for 48 h with 200 rpm shaking for C. neoformans. The lower final volume (100 µL) ensures that no spillover occurs.
   NOTE: No significant difference was observed between MIC readings at 24 h and 48 h for C. albicans strains. Nonetheless, readings at 48 h are easier to visualize.
5. Observe all wells, with the assistance of an inverted optical microscope, before the incubation period and after to verify changes in morphology as well as to check for signs of contamination.
   NOTE: Readings can be done visually and photographed at the end of the experiment. Before each reading, homogenize slightly the plate. The reads can also be performed by measuring its OD at 600 nm, if cell clumps or filamentation are not observed.
6. Perform the experiments at least three times on separate dates.

Table 1. Media and reagent preparation.