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MB Sample ID: SA151966
| Local Sample ID: | RMSCC2343 Mycelia_1 |
| Subject ID: | SU001736 |
| Subject Type: | Fungi |
| Subject Species: | Coccidioides posadasii;Coccidioides immitis |
| Taxonomy ID: | 199306;5501 |
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Collection:
| Collection ID: | CO001729 |
| Collection Summary: | All Coccidioides isolates were grown under BSL-3 containment, using conditions that induce mycelial or spherule growth. For mycelial growth, a 50 ml vented falcon tube containing 10 ml of RPMI media (filter sterilized RPMI 1640, 10% fetal bovine serum) was inoculated with a 1 cm x 1 cm 2xGYE agar plug for each strain. These plates were inoculated using 100 µl of glycerol stock, spread across the plate, and cultured for 30°C for two weeks. Control RPMI media was inoculated with a plug from sterile 2xGYE agar media. Each sample, including media control, was prepared in triplicate. Cultures were grown on a shaking incubator at 150 rpm, 30°C for 96 h. For spherule cultures, a 50 ml vented falcon tube containing 10 ml of RPMI media was inoculated to a final concentration of 1.0 x 10^5 arthroconidia/ml in 1xPBS. Arthroconidia were grown and harvested. Strains RMSCC2343 and RMSCC3505 did not produce enough conidia to achieve 1.0 x 10^5 arthroconidia/ml, and were inoculated at 7.0 x 10^4 and 4.0 x 10^4 arthroconidia/ml, respectively. Control media was inoculated with 1 ml of sterile 1xPBS. Cultures were grown on a shaking incubator at 150 rpm, at 39°C in 10% CO2 for 96 h. Mycelial and spherule cultures were spun at 12,000 x g at 4°C for 10 min to pellet the cells. The supernatant was removed and place in a Nanosep MF Centrifugal Devices with Bio-Inert® Membrane 0.2 µm spin filter and centrifuged at 3,200 x g for 4 min. The filtrate was stored at −80°C until volatile metabolomics analysis. The Coccidioides spp. culture filtrates and media blanks were allowed to thaw at 4°C overnight, and then 2 ml were transferred and sealed into sterilized 10 ml GC headspace vials with PTFE/silicone septum screw caps. All samples were stored for up to 12 d at 4°C until analyzed. |
| Sample Type: | Fungi cells |
