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

Abstract

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).

Protocol

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 ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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