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MB Sample ID: SA121868

Local Sample ID:sample45
Subject ID:SU001507
Subject Type:Bacteria
Subject Species:Multi-species non-defined biofilm consortium

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Subject:

Subject ID:SU001507
Subject Type:Bacteria
Subject Species:Multi-species non-defined biofilm consortium

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
sample45SA121868FL014903Proline 25mMTreatment

Collection:

Collection ID:CO001502
Collection Summary:Saliva was processed and biofilms were grown using a previously published methodology with modifications (Cleaver, Moazzez and Carpenter, 2019). Saliva samples were centrifuged (5000g for 5 mins), and the supernatant was collected, pooled, boiled in sealed tubes for 20 minutes to sterilise, and left to cool to room temperature; hereafter referred to as sterile saliva. The loose pellet from the spun saliva samples was pooled and combined with enough sterile saliva to facilitate inoculation of the hydroxyapatite (HA) discs. Samples were vortexed vigorously to resuspend the pellet; this constituted the pooled saliva inoculum. The remaining sterile saliva was split into 11 aliquots. D-proline, L-arginine, and glucose were added to the aliquots to achieve concentrations of 10mM, 25mM and 50mM respectively for each (based on concentrations of proline obtained from previous publication (6)). One aliquot was also supplemented with 10mM carbon 13 labelled (C13) D-proline. Two aliquots were left unsupplemented to act as a positive and negative control. Six HA discs in a sterile microtitre plate were inoculated per treatment condition with 1 ml pooled saliva inoculum (negative control discs were inoculated with sterile saliva only to check for sterility) and incubated for 24 hours aerobically in a 40 L aerobic incubator (GenLab Ltd, UK) at 37°C. After this time the discs were washed three times with sterile phosphate buffered saline (PBS) and 1 ml of supplemented/unsupplemented saliva per condition was added to the discs. The discs were then incubated anaerobically in a 3.5 litre anaerobic jar with an Anaerogen anaerobic generator pack (Oxoid, UK) at 37°C for 72 hours. After this spent saliva from the biofilms was removed and stored at -20°C for further analysis, discs were washed three times with PBS, and refreshed with supplemented/unsupplemented saliva and further incubated anaerobically at 37°C for 72 hours. After this final incubation the spent saliva was removed and stored at -20°C for further analysis.
Sample Type:Bacterial cells

Treatment:

Treatment ID:TR001522
Treatment Summary:Sterile saliva was split into 11 aliquots. D-proline, L-arginine, and glucose were added to the aliquots to achieve concentrations of 10mM, 25mM and 50mM respectively for each (based on concentrations of proline obtained from previous publication (6)). One aliquot was also supplemented with 10mM carbon 13 labelled (C13) D-proline. Two aliquots were left unsupplemented to act as a positive and negative control.

Sample Preparation:

Sampleprep ID:SP001515
Sampleprep Summary:Samples were processed for NMR using a previously published method (6). Briefly, centrifuged spent saliva supernatant and the sterile saliva sample were each mixed with TSP buffer in a 5 mm NMR tube (Bruker, Germany). The tubes were sealed and analysed at the Biomolecular Spectroscopy Centre, King’s College London, UK on a 600 MHz spectrometer (Bruker) for 1H 1D-NMR and 1H-C13 1D- and 2D-NMR. The concentration of metabolites relative to the sterile saliva baseline were collected using Chenomix NMR Suite version 8.5 (Chenomix Ltd, Canada). The 2D C13 spectra were analysed using TopSpin version 3.6.2 (Bruker) and COLMAR (30).

Analysis:

MB Sample ID:SA121868
Analysis ID:AN002395
Analysis Type:NMR
Num Factors:11
Num Metabolites:19
Units:mM

NMR:

NMR ID:NM000166
Analysis ID:AN002395
Instrument Name:Bruker
Instrument Type:FT-NMR
NMR Experiment Type:1D-1H
Spectrometer Frequency:600Hz
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