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MB Sample ID: SA152000
| Local Sample ID: | RMSCC2010 Spherule_1 |
| Subject ID: | SU001736 |
| Subject Type: | Fungi |
| Subject Species: | Coccidioides posadasii;Coccidioides immitis |
| Taxonomy ID: | 199306;5501 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001736 |
| Subject Type: | Fungi |
| Subject Species: | Coccidioides posadasii;Coccidioides immitis |
| Taxonomy ID: | 199306;5501 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| RMSCC2010 Spherule_1 | SA152000 | FL017877 | 39°C | Culture tempurature |
| RMSCC2010 Spherule_1 | SA152000 | FL017877 | 10% CO2 | Culture conditions |
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 |
Treatment:
| Treatment ID: | TR001749 |
| Treatment Summary: | All Coccidioides isolates were grown under BSL-3 containment, using conditions that induce mycelial or spherule growth. See Collection Protocol for details |
Sample Preparation:
| Sampleprep ID: | SP001742 |
| Sampleprep Summary: | Filtrate samples were heated to 50 degrees Celsius with agitation, and volatiles were sampled for 10 minutes using solid-phase microextraction (SPME) and analyzed using comprehensive gas chromatography coupled to time-of-flight mass spectrometry. |
| Sampleprep Protocol Filename: | ehiggins_invitro_GCxGC_methods.docx |
| Extraction Method: | Solid-phase microextraction (SPME) |
Combined analysis:
| Analysis ID | AN002710 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 7890B |
| Column | Multidimensional configuration |
| MS Type | EI |
| MS instrument type | GC x GC-TOF |
| MS instrument name | Leco Pegasus 4D GCxGC TOF |
| Ion Mode | POSITIVE |
| Units | Peak areas |
Chromatography:
| Chromatography ID: | CH001999 |
| Chromatography Summary: | Samples analyzed using comprehensive two-dimensional gas chromatography (GCxGC). See attached method file. |
| Instrument Name: | Agilent 7890B |
| Column Name: | Multidimensional configuration |
| Chromatography Type: | GC |
MS:
| MS ID: | MS002507 |
| Analysis ID: | AN002710 |
| Instrument Name: | Leco Pegasus 4D GCxGC TOF |
| Instrument Type: | GC x GC-TOF |
| MS Type: | EI |
| MS Comments: | see attached MS methods |
| Ion Mode: | POSITIVE |
