Source: de-Souza-Silva, C. M., et al. Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds. J. Vis. Exp. (2018).
This video demonstrates an in vitro assay to screen antifungal compounds using a broth microdilution assay. Test compounds are serially diluted in a microtiter plate and incubated with a fungal inoculum. Post-incubation, the plate is examined for wells without visible fungal growth to assess the antifungal potential of the test compounds and their minimum inhibitory concentration (MIC).
1. Solutions and Media
2. Fungal Inoculum Growth Conditions
3. Peptides (Unknown Agent)
4. Reference Antifungals (Positive Controls)
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.
Table 1. Media and reagent preparation.
Medium | Preparation |
2X Roswell Park Memorial Institute (RPMI) 1640 medium – 500 mL | 10.4 g RPMI-1640 (supplemented with L-glutamine and phenol red; without bicarbonate) |
330 mM 3-(N-morpholino) propane sulfonic acid (MOPS) | |
Adjust to pH 7.0 with NaOH | |
Sterilize by filtration (0.22 µM filter) | |
Phosphate buffered saline (PBS) | 137 mM NaCl |
2.7 mM KCl | |
10 mM Na2HPO4 | |
2 mM KH2PO4 | |
Autoclave at 121°C for 15 minutes. | |
Sabouraud dextrose broth | 15 g of the powder in 500 mL of distilled water. |
Adjust to pH 7.0 with NaOH | |
Autoclave at 121°C for 15 minutes. | |
Sabouraud Dextrose Agar | 32.5 g of the powder in 500 mL of distilled water. |
Adjust to pH 7.0 with NaOH | |
Autoclave at 121°C for 15 minutes. |
The authors have nothing to disclose.
Media and Reagents | |||
RPMI 1640 medium with l-glutamine, without sodium bicarbonate | Thermo Fisher | 31800-022 | |
3-(N-morpholino) propane sulfonic acid (MOPS) | Sigma-Aldrich | Use to buffer 2X RPMI medium | |
Sodium chloride (NaCl) | Dinâmica | 1528-1 | 137 mM for Phosphate buffered saline (PBS) |
Potassium chloride (KCl) | J.T.Baker | 3040-01 | 2.7 mM for Phosphate buffered saline (PBS) |
Sodium phosphate dibasic (Na2HPO4) | Sigma-Aldrich | V000129 | 10 mM for Phosphate buffered saline (PBS) |
Potassium dihydrogen phosphate (KH2PO4) | Sigma-Aldrich | 60230 | 2 mM for Phosphate buffered saline (PBS) |
BD Difco Sabouraud dextrose broth | BD | 238230 | |
BD Difco Sabouraud Dextrose Agar | BD | 210950 | |
Glycerol | Sigma-Aldrich | V000123 | 35% |
Sterile water | |||
Antifungal drugs | |||
Amphotericin B | Sigma-Aldrich | A2942 | |
Fluconazole | Sigma-Aldrich | F8929 | |
Caspofungin | Sigma-Aldrich | PHR1160 | |
Plastics | |||
50 mL conical tube | Sarstedt | 62.547.254 | |
Dish petri | J.Prolab | 0304-5 | |
96 well plate | Corning | 3595 | |
Sterile Solution Reservoir | KASVI | K30-208 | Use to pippet the solutions using the multichannel pippet |
Equipment and other materials | |||
Optical microscope | Nikon | E200MV | |
Centrifugue | Thermo Fisher | MegaFuge 16R | |
Incubator | Ethik Technology | 403-3D | Set to 37° C |
Shaker | New Brunswick Scientific | Excella E25 | Set to 37° C, 200 RPM |
Cell counting chamber, Neubauer | BOECO Germany | BOE 13 | |
Multichannel pipette | HTL | 5123 |