Gut³Beads
Supplementing a planktonic culture with mucin beads to raise the biomass yield and viability of fastidious gut microorganisms.
Product
This kit is designed to improve the cultivation of fastidious microorganisms. Gut³Beads are an additive that raises biomass yield and microbial viability, which helps the production of probiotics and live biotherapeutics.
The protocol describes how to supplement a planktonic microbial culture with Gut³Beads, and how to prepare the samples for downstream analyses: cell viability, flow cytometry, metabolomics and sequencing.
Before you begin
- The steps below are written for single-strain planktonic cultures in laboratory-scale batch fermentation. Gut³Beads have also been used in controlled bioreactors and other dynamic culture systems.
- The beads in this kit are 4.5 to 6.0 mm across. Their composition can be tuned, and custom compositions are available.
- Sterilize all instruments and consumables, and work under aseptic conditions, ideally in a Class II biological safety cabinet. Prepare a sterile 0.9% (w/v) NaCl solution.
Procedure
APreparing the microbial suspension~15 min
- Grow the species of interest, for example Akkermansia muciniphila, to logarithmic phase in the appropriate medium.
- Centrifuge to pellet the cells and discard the supernatant.
- Resuspend the pellet in an appropriate culture medium.
- Measure the optical density at 600 nm and adjust the cell concentration as needed.
BSupplementing with Gut³Beads~5 min
- Dispense the beads from the flask with the spoon provided.
- Supplement the culture with beads at 10% of the total culture volume, measured as their hydrated volume.
- Incubate at 37 °C for up to 72 hours, aerobic or anaerobic, according to the experimental design.
Note The beads are supplied submerged in 0.9% (w/v) NaCl. Keep that solution if you need to store them.
Tip Transferring the beads into a Petri dish first makes the dose easier to control.
CDissolving the beads~3 min per sample
- Add Bac³Gel dissolution medium (200 mM sodium citrate) at a 1:1 ratio to each sample.
- Mix thoroughly until the beads have fully dissolved. The homogenized samples are ready for downstream assays.
Downstream analysis
Sequencing
Centrifuge at 14,000 × g for 5 minutes, then use the pellet for DNA extraction.
Metabolomics
Centrifuge at 14,000 × g for 10 minutes, then collect and lyophilize the supernatant.
Metabolic activity or optical density
Centrifuge at 14,000 × g for 5 minutes, discard the supernatant and resuspend in 0.9% (w/v) NaCl.
Other readouts
Also suited to cell viability assessment, flow cytometry and standard microbiological characterization assays.
Troubleshooting
For research and development use only. Not for diagnostic or clinical use.