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MB Sample ID: SA318294
Local Sample ID: | Sample 10 |
Subject ID: | SU003046 |
Subject Type: | Cultured cells |
Subject Species: | Symbiodiniaceae |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU003046 |
Subject Type: | Cultured cells |
Subject Species: | Symbiodiniaceae |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Sample 10 | SA318294 | FL037873 | Breviolum minutum | Genotype |
Sample 10 | SA318294 | FL037873 | Untreated | Treatment |
Collection:
Collection ID: | CO003039 |
Collection Summary: | Sampled from Symbiodiniaceae cultures in F/2 media |
Sample Type: | Cultured cells |
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. |
Sample Preparation:
Sampleprep ID: | SP003052 |
Sampleprep Summary: | Seven days after the antibiotic treatment process, 3 × 1 mL aliquots were collected from each culture flask for flow cytometry cell counting of both bacteria and Symbiodiniaceae cells, bacteria community analysis via 16S rRNA amplicon sequencing analysis, and Symbiodiniaceae photophysiological measurements (methods described in more detail below). The remaining Symbiodiniaceae cells were concentrated by centrifugation at 700 × g for 10 mins at 26˚C, the media discarded, and Symbiodiniaceae pellets snap frozen in liquid nitrogen for metabolite profiling. |
Combined analysis:
Analysis ID | AN004811 |
---|---|
Analysis type | MS |
Chromatography type | GC |
Chromatography system | Shimadzu GC-2010 |
Column | Agilent DB5-MS (15m x 0.25mm, 0.25um) |
MS Type | EI |
MS instrument type | GC-TOF |
MS instrument name | Shimazdu 2030 |
Ion Mode | POSITIVE |
Units | relative abundance |
Chromatography:
Chromatography ID: | CH003636 |
Instrument Name: | Shimadzu GC-2010 |
Column Name: | Agilent DB5-MS (15m x 0.25mm, 0.25um) |
Column Temperature: | 100-320 |
Flow Gradient: | 10°C min-1 |
Flow Rate: | 1 mL/min |
Solvent A: | helium |
Solvent B: | helium |
Chromatography Type: | GC |
MS:
MS ID: | MS004557 |
Analysis ID: | AN004811 |
Instrument Name: | Shimazdu 2030 |
Instrument Type: | GC-TOF |
MS Type: | EI |
MS Comments: | Approximately 520 quantifying MRM targets were collected using Shimadzu Smart Database along with qualifier for each target which covers about 350 endogenous metabolites and multiple 13C labelled internal standards. Both chromatograms and MRMs were evaluated using the Shimadzu GCMS browser and LabSolutions Insight software. |
Ion Mode: | POSITIVE |