Aug 07, 2020
Screening of compounds for inhibition of Sporothix spp. growth
- 1Universidade Federal do Rio de Janeiro
- L. P. Borba-Santos
External link: https://doi.org/10.1371/journal.pone.0240658
Document Citation: luanaborba 2020. Screening of compounds for inhibition of Sporothix spp. growth. protocols.io https://dx.doi.org/10.17504/protocols.io.bje9kjh6
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 07, 2020
Last Modified: August 07, 2020
Document Integer ID: 40129
Screening of compounds for inhibition of Sporothrix spp. growth
1. Stock solutions of compounds in dimethyl sulfoxide (DMSO) at 1 mM were diluted in RPMI 1640 medium1 supplemented with 2% glucose and buffered to pH 7.2, with 0.165 M MOPS (from here on referred to as “supplemented RPMI”) to obtain concentrations of 2 µM using sterile microtubes;
2. Aliquots of 100 µl of each compound were added in four wells of a flat-bottom 96-well microplate (KASVI, Brazil);
3. 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;
4. The final concentration of compounds was 1 µM, while the final concentration of cells was 1 x 105 CFU/ml;
5. The following controls were included in the experiment: (i) antifungal control containing 1 µM itraconazole1, (ii) diluent control containing 0.1% DMSO in supplemented RPMI, (iii) negative control containing only supplemented RPMI, and (iv) positive control with Sporothrix cells growing in supplemented RPMI without compounds (all in quadruplicate);
6. Samples were incubated for 48 h, at 35 ºC, in a 5% CO2 atmosphere;
7. Fungal growth was analyzed by visual inspection in an inverted light microscope (Axiovert 100, ZEISS Company, Germany);
8. 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);
9. The absorbance value for each well was subtracted from the mean value for the negative control;
10. 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;
11. Inhibitions of more than 80% were defined as the cutoff, corresponding to clearly visible preventions of growth when samples were analyzed by visual inspection.
1Sigma Chemical Co., USA.