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MB Sample ID: SA253769
| Local Sample ID: | 2243_Ac0_3_TP4i |
| Subject ID: | SU002617 |
| Subject Type: | Bacteria |
| Subject Species: | Eggerthella lenta |
| Taxonomy ID: | 84112 |
| Genotype Strain: | DSM 2243, Valencia, AB8n2 |
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Collection:
| Collection ID: | CO002610 |
| Collection Summary: | Time course experiments were conducted in tubes in the anaerobic chamber in a 37°C incubator. For all metabolomics experiments, three independent culture replicates were included for each condition, with an equal number of uninoculated control tubes. Starter cultures and inocula were prepared as described above for growth assays. 5mLs of defined media was added to VWR glass culture tubes (53283-800) with screw caps. The PBS-washed inoculum was added to culture tubes to obtain an approximate starting OD600 of 0.001. A preliminary growth assay was conducted to define time points spanning the exponential growth phase in the tested conditions. At each time point, OD600 measurements of all inoculated tubes were first measured using a Hach DR1900 spectrophotometer, with a paired control tube to normalize for the background. 100 μL from each tube were then transferred into a 96-well microplate, which was sealed and removed from the anaerobic chamber. Plates were centrifuged at 1,928 rcf at 4°C for 8 minutes, after which supernatants were collected into fresh polypropylene tubes or plates, sealed, and flash-frozen in liquid nitrogen. Two time course experiments were carried out with stable isotope-labeled substrates. Experimental groups included conditions in which sodium acetate in the defined media was replaced with 13C2 labeled sodium acetate (Sigma-Aldrich 282014), along with a matched experimental group with the same concentration of unlabeled substrate. Intracellular extract samples were prepared with the following procedure, which was optimized for lysis of thick gram-positive cell walls: 600 μL of culture was transferred to an Eppendorf tube in anaerobic conditions and subsequently centrifuged at 10,000rcf for three minutes at 4°C, after which the supernatant was removed and the samples were immediately flash frozen to quench metabolites. 300 μL of cold methanol was then added to each pellet, followed by sonication on ice for 5 minutes and then shaking at 4°C for 4-12 hours. Samples were then centrifuged at 4°C at 15,000 rcf for 8 minutes, after which 120 μL of supernatant was transferred to fresh tubes and stored at -80°C until analysis. |
| Sample Type: | Bacterial culture supernatant |
| Collection Frequency: | at time points specified in study design table over 64 hours (full growth phase) |
| Storage Conditions: | -80℃ |
