Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Summary

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Abstract

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Introduction

Fungal infections have become an important medical concern in recent decades, having considerably increased mainly due to a rise in the number of immunocompromised individuals such as those undergoing cancer treatment and those living with HIV/AIDS or transplanted organs^1,2. However, a very limited array of available antifungal drugs and the increasing number of reports on fungal resistance to them contribute to the major problems regarding the therapeutics of systemic mycoses³.

A potential source of new antifungal compounds are antimicrobial peptides (AMPs),....

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 cap and culture overnight at 30 &#....

Results

The MIC is defined as the lowest antimicrobial compound concentration that completely inhibits visible fungal growth at the end of the incubation period. Since the objective of this protocol is to have a fast method to screen potential antifungals, 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. However, if there is an interest in knowing whether a g.......

Discussion

Microdilution tests can analyze the potential antifungal activity of a target compound using small quantities of the compound, and at the same time test it in a range of concentrations. Accordingly, this protocol is recommended as a first step in screening for potential new antifungal compounds. The protocol presented here is based on the M27-A3 protocol, initially designed to aid in the selection of antifungal therapy in clinics, and can be adapted to a variety of new antifungal compounds. Overall, this protocol can foc.......

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) (o que a gente usa tem um sódio, completa o nome dele please)	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% for (solução de estoque? Criopreservação?)
Sterile water			Para diluição das drogas na diluição seriada
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