Chloramphenicol

This study was conducted on a KU70Δ mutant [23 (link)] derived from the wild-type strain S. apiospermum IHEM 14462, originally isolated from sputum sample from a CF patient and previously used for whole-genome sequencing [25 (link)]. Strains were maintained at 37 °C by weekly passages on potato-dextrose-agar plus chloramphenicol (PDA from Condalab, containing in g/L: dextrose, 20; infusion extract from potatoes, 4; chloramphenicol, 0.5; and bacteriological agar, 15), supplemented with 20 µg/mL phleomycin or 50 µg/mL hygromycin, for maintenance of the KU70Δ mutant or the double mutant KU70Δ/SODDΔ, respectively. All strains were stored at −80 °C as conidial suspensions in 20% glycerol. Conidia processed into any experiment were collected from 9-day-old cultures grown in PDA or yeast extract-peptone-dextrose (YPD containing in g/L: yeast extract, 10; peptone, 20; glucose, 20; and chloramphenicol, 0.5%) and resuspended in sterile water or saline before to be enumerating by hematocytometer counts.

A number of fungal isolates were obtained from air samples taken between January and June 2016 in different halls of Altamira Cave (Santillana del Mar, Cantabria, Spain). Two 500 L air samples were simultaneously taken with an air sampler collector SAS‐duo sampler (VWR) for 3 min and directly impinged onto plates with Sabouraud Dextrose Agar (Becton Dickinson) supplemented with 0.5 g/L chloramphenicol (Conda). The plates were transported in an isothermic bag to the lab followed by incubation at 26°C for 3 days.
Several fungal isolates selected for further analysis were cultivated on Potato Dextrose Broth (PDB; Conda) used for the preparation of both liquid and solid (agar) media. To obtain liquid cultures, the isolates were routinely grown in 3 ml of PDB medium supplemented with 30 μg/ml chloramphenicol (Sigma‐Aldrich) at 25°C with shaking at 90 rpm in 12 ml culture tubes. Alternatively, the fungal isolates were grown on PDB agar plates likewise supplemented with chloramphenicol (30 μg/ml). Glycerol stocks were prepared by mixing 450 μl of suspended mycelia and 450 μl of sterile 80% glycerol and were kept at −80°C in 1 ml cryovials. Further inoculations were performed by transferring 20 μl of glycerol stock to solid or liquid PDB media.

One piece of each toenail sample was cultured on Sabouraud dextrose agar plates with chloramphenicol (Condalab, Spain), a selective method for the isolation of fungi, at 30 °C during three to four weeks, or until positive for fungal growth.