Universal Bac³Gel
Culturing single strains and consortia in a general-purpose 3D mucus matrix, in multi-well plates, and preparing them for downstream analysis.
Product
Reproducible and high-throughput in vitro models are essential for studying complex microbial communities. This kit is a general-purpose 3D matrix for culturing microorganisms, designed for the in vitro modelling of human mucus.
The protocol describes how to culture single strains in Universal Bac³Gel in a multi-well plate format, and how to prepare the samples for downstream analyses: cell viability, flow cytometry, metabolomics and sequencing.
Before you begin
- The protocol below is an example to guide you in developing your own. It is written for single strains, and has also been used for co-cultures and microbial consortia.
- The gel can be produced in the culture medium of your choice and contains porcine stomach type III mucin at 50 mg/mL. The composition can be tuned, and custom compositions are available.
- The cross-linking and the oxygen gradient form during production, and change as microbes grow.
- Sterilize all instruments and consumables, and work under aseptic conditions. Prepare a sterile 0.9% (w/v) NaCl solution.
Procedure
APreparing the microbial suspension~15 min
- Grow the species of interest, for example Escherichia coli, to logarithmic phase in the appropriate medium.
- Centrifuge to pellet the cells and discard the supernatant.
- Resuspend the pellet in 0.9% (w/v) NaCl or an appropriate culture medium.
- Measure the optical density at 600 nm and adjust the cell concentration as needed.
BCulturing in Universal Bac³Gel~5 min per plate
- Bring the plates to room temperature. This protocol uses a 24-well plate as the example.
- Add the microbial culture to each well at a 1:1 volume ratio with the gel: 700 µL per well in a 24-well plate. Other formats are in the table below.
- Cover the plate and incubate at 37 °C, or another appropriate temperature, aerobic or anaerobic, for up to 72 hours.
Note To test a molecule such as a biotic or an active principle, let the cells stabilize in the gel for 24 hours at 37 °C before adding it.
Tip The plates can be sealed with a breathable membrane before incubation, to prevent cross-contamination.
Critical Add the culture gently on top of the gel. Do not mix. Anaerobic conditions before culturing are not required, but they prevent the loss of obligate anaerobes.
CDissolving the gel~3 min per well
- Aspirate the supernatant gently without disturbing the gel. Wash with 0.9% (w/v) NaCl, 700 µL per well in a 24-well plate, then aspirate and discard.
- Add Bac³Gel dissolution medium (200 mM sodium citrate) at a 1:1 ratio with the gel volume, and mix until the gel has fully dissolved. Transfer to microcentrifuge tubes.
Note The wash keeps only the microbes that colonized the gel. Dissolution takes longer in larger wells and where exopolysaccharides are present.
| Plate format | Bac³Gel per well |
|---|---|
| 6-well | 3,700 µL |
| 12-well | 1,600 µL |
| 24-well | 700 µL |
| 48-well | 300 µL |
| 96-well | 100 µL |
The microbial culture and the Bac³Gel dissolution medium are each added at a 1:1 ratio with the gel, so both equal the volume shown.
Downstream analysis
Sequencing
Centrifuge at 14,000 × g for 10 minutes, then use the pellet for DNA extraction.
Metabolomics
Centrifuge at 14,000 × g for 10 minutes, then collect and lyophilize the supernatant.
Other readouts
Also suited to cell viability assessment, flow cytometry, metabolic activity and standard microbiological characterization assays.
Troubleshooting
The gel does not dissolve, or dissolves slowly
Add more 200 mM sodium citrate and mix again. Larger wells and exopolysaccharide-rich cultures take longer.
For research and development use only. Not for diagnostic or clinical use.