Aug 08, 2020
Version 2
Determination of minimum inhibitory concentration values (MICs) against Sporothrix brasiliensis and Sporothrix schenckii V.2
- 1Universidade Federal do Rio de Janeiro
- L. P. Borba-Santos
External link: https://doi.org/10.1371/journal.pone.0240658
Document Citation: luanaborba 2020. Determination of minimum inhibitory concentration values (MICs) against Sporothrix brasiliensis and Sporothrix schenckii. protocols.io https://dx.doi.org/10.17504/protocols.io.bjgxkjxn
Manuscript citation:
Borba-Santos LP, Vila T, Rozental S (2020) Identification of two potential inhibitors of Sporothrix brasiliensis and Sporothrix schenckii in the Pathogen Box collection. PLoS ONE 15(10): e0240658. doi: 10.1371/journal.pone.0240658
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: August 08, 2020
Last Modified: August 08, 2020
Document Integer ID: 40183
Determination of minimum inhibitory concentration values (MICs) against Sporothrix brasiliensis and Sporothrix schenckii
The minimum inhibitory concentration (MIC) of compounds was determined using the broth microdilution technique described by the CLSI1 with modifications.
1. Stock solutions of compounds in dimethyl sulfoxide (DMSO) at 1 mM were diluted in RPMI 1640 medium2 supplemented with 2% glucose3 and buffered to pH 7.2, with 0.165 M MOPS (from here on referred to as “supplemented RPMI”) to obtain solutions 4-fold concentrated using sterile microtubes;
2. Itraconazole2 (reference antifungal) was also included in the experiment;
3. Aliquots of 100 µl of compounds were added in two wells of the first column of a flat-bottom 96-well microplate (KASVI, Brazil);
4. Aliquots of 100 µl of supplemented RPMI were added in all microplate wells (including wells containing compounds) using a multichannel pipette and a serial 2-fold dilution was performed to the tenth column;
5. The eleventh column corresponded to the positive controls (containing 100 µl supplemented RPMI without compounds) and the twelfth to the negative controls (containing 200 µl supplemented RPMI);
6. A standardized suspension of Sporothrix yeasts was adjusted using a Neubauer chamber, prepared in supplemented RPMI, and 100 µl containing 2 x 105 CFU/ml was added in microplates containing compounds (except in the twelfth column);
7. Ten different concentrations of compounds were tested and their final concentration ranged from 0.002 to 1 µM, while the final concentration of yeasts were 1 x 105 CFU/ml4;
8. Samples were incubated at 35 °C for 48 h, in a 5 % CO2 atmosphere5;
9. Fungal growth was analyzed by visual inspection in an inverted light microscope (Axiovert 100, ZEISS Company, Germany);
10. After visual inspection, samples were homogenized and the optical density was quantified by spectrophotometric readings at 492 nm, in a microtiter plate reader (EMax Plus, Molecular Devices, USA);
11. The absorbance value for each well was subtracted from the mean value for the negative controls;
12. Inhibition of fungal growth (I) relative to positive controls was calculated according to the following equation: I = 100 – (A x 100/C), where A is the absorbance of treated wells, and C is the absorbance of positive controls;
13. Concentrations that inhibit 50% and 80% of fungal growth (IC50 and IC80, respectively) were estimated.
1CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 4th ed. CLSI standard M27.Wayne, PA: Clinical and Laboratory Standards Institute; 2017.
2Sigma Chemical Co., USA.
3 The RPMI medium was supplemented with 2% glucose to improve the growth of Sporothrix yeasts.
4 The inoculum of 1x105 CFU/ml was used due to the slow growth of Sporothrix yeasts.
5 Microplates were incubated in a 5% CO2 atmosphere to maintain the culture in the yeast form.