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).
Protocol
1. Solutions and Media 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 Store all fungal strains as frozen stocks in 35% glycerol at -80 °C, until needed. Perform the following steps before each experiment. For Candida albicans</em…
Divulgazioni
The authors have nothing to disclose.
Materials
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