Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA318293

Local Sample ID:	Sample 24
Subject ID:	SU003046
Subject Type:	Cultured cells
Subject Species:	Symbiodiniaceae

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Treatment:

Treatment ID:	TR003055
Treatment Summary:	Symbiodiniaceae cultures Two Symbiodiniaceae cultures were targeted from existing stocks at the University of Technology Sydney, Symbiodinium microadriaticum (ITS2: A1, culture ID: RT61), and Breviolum minutum (ITS2: B1, culture ID: RT2, CCMP2463) as preliminary trials allowed these cultures to be maintained for extended periods in an extracellular bacteria-free state. Each Symbiodiniaceae species was sub-cultured (n = 10 per Symbiodiniaceae species) by adding 10 mL of original cultures in 90 mL of autoclaved and filter sterilised (0.22 µm) artificial seawater (ASW) and F/2 media. Cultures were grown for one month (to achieve a minimum cell density of 106 cells/mL) at 26˚C with an irradiance of 85 ± 15 µmol photons m-2 s-1 (Philips TLD 18W/54 fluorescent tubes, 10 000 K on a 12h:12h light:dark cycle). Before use, cells were centrifuged at 700 × g for 10 mins at 26˚C and rinsed twice with ASW to remove residual media solution. Cells were resuspended in 100 mL ASW + F/2 media in sterile culture flasks. Untreated cultures Untreated Symbiodiniaceae cultures (n = 5) were maintained as above alongside the Ab+Tx treatments (below). Antibiotic treatment (AbTx) Each antibiotic treatment subculture (n = 5 per Symbiodiniaceae species) was provided with TritonX-100 detergent added to a final concentration of 20 µg/mL and placed on a shaker at mid speed for 30 s. All cultures (Ab+Tx treatment and untreated) were immediately centrifuged at 700 × g for 10 mins at 26˚C, and the supernatant discarded. Cells were rinsed in 20 mL ASW and centrifuged at 700 x g for 10 mins at 26˚C. Cells were transferred to sterile culture flasks and resuspended in 9 ml ASW+F/2. An aliquot of 1 mL custom antibiotic mix (Penicillin at 31.25 µg mL-1, Streptomycin and Kanamycin at 50 µg mL-1, and Neomycin, Ciprofloxacin and Ampicillin all at 100 µg mL-1; Ab+Tx) was added to each antibiotic treatment flask (and 1 mL ASW added to each untreated flask), and flasks were replaced in the incubator. After 48 hours, 90 mL ASW + F/2 was added to all cultures. Cultures were allowed to recover for 5 days prior to metabolism quenching.

Treatment ID:

TR003055

Treatment Summary:

Symbiodiniaceae cultures Two Symbiodiniaceae cultures were targeted from existing stocks at the University of Technology Sydney, Symbiodinium microadriaticum (ITS2: A1, culture ID: RT61), and Breviolum minutum (ITS2: B1, culture ID: RT2, CCMP2463) as preliminary trials allowed these cultures to be maintained for extended periods in an extracellular bacteria-free state. Each Symbiodiniaceae species was sub-cultured (n = 10 per Symbiodiniaceae species) by adding 10 mL of original cultures in 90 mL of autoclaved and filter sterilised (0.22 µm) artificial seawater (ASW) and F/2 media. Cultures were grown for one month (to achieve a minimum cell density of 106 cells/mL) at 26˚C with an irradiance of 85 ± 15 µmol photons m-2 s-1 (Philips TLD 18W/54 fluorescent tubes, 10 000 K on a 12h:12h light:dark cycle). Before use, cells were centrifuged at 700 × g for 10 mins at 26˚C and rinsed twice with ASW to remove residual media solution. Cells were resuspended in 100 mL ASW + F/2 media in sterile culture flasks. Untreated cultures Untreated Symbiodiniaceae cultures (n = 5) were maintained as above alongside the Ab+Tx treatments (below). Antibiotic treatment (AbTx) Each antibiotic treatment subculture (n = 5 per Symbiodiniaceae species) was provided with TritonX-100 detergent added to a final concentration of 20 µg/mL and placed on a shaker at mid speed for 30 s. All cultures (Ab+Tx treatment and untreated) were immediately centrifuged at 700 × g for 10 mins at 26˚C, and the supernatant discarded. Cells were rinsed in 20 mL ASW and centrifuged at 700 x g for 10 mins at 26˚C. Cells were transferred to sterile culture flasks and resuspended in 9 ml ASW+F/2. An aliquot of 1 mL custom antibiotic mix (Penicillin at 31.25 µg mL-1, Streptomycin and Kanamycin at 50 µg mL-1, and Neomycin, Ciprofloxacin and Ampicillin all at 100 µg mL-1; Ab+Tx) was added to each antibiotic treatment flask (and 1 mL ASW added to each untreated flask), and flasks were replaced in the incubator. After 48 hours, 90 mL ASW + F/2 was added to all cultures. Cultures were allowed to recover for 5 days prior to metabolism quenching.