Assessing Bacteria-Host Cell Interactions Using Biomimetic Beads

Samenvatting

Source: Stones, D. H., et al. Biomimetic Materials to Characterize Bacteria-host Interactions. J. Vis. Exp. (2015)

This video demonstrates the use of bacteriomimetic beads to quantify bacterial adhesion to host epithelial cells under competitive conditions. It outlines the steps involved in treating the cells, lysing them, and plating serial dilutions to measure colony-forming units.

Protocol

1. Measurement of bacterial adhesion
   1. At indicated time points (e.g., 1 hr post-infection), remove media from the cell layer.
   2. Thoroughly wash the cell layer with sterile, pre-warmed phosphate-buffered saline (PBS) (at least 3-4 washes of 1 ml PBS each) to remove any unattached cells.
   3. Lyse host cells by adding 1 ml of a sterile 1 % v/v Triton X-100 solution in PBS per well. Incubate the plate at 37 °C for 5 min.
   4. Pipette each sample up and down several times before transferring the contents of each well to separate 1.5 ml tubes. Prepare 10-fold serial dilutions of samples into sterile PBS (e.g., use 100 ml of sample and 900 ml of PBS).
   5. Plate 100 μl of each sample on marine Luria-Bertani (MLB) agar and spread using a cell spreader. Optimize which dilutions to plate depending on the bacterial strains and time point.
      Note: For the described experimental setup, 10⁵ or 10⁶ fold dilutions give a suitable number of colony-forming units (CFUs).
   6. Incubate plates at 37 °C O/N and enumerate bacteria by colony counting.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Reagents
Carboxylate-modified polystyrene beads	Sigma	CLB9	harmful; a range of alternative particle sizes and colors is available
Dulbecco’s Modified Eagle’s Medium - high glucose - With 4500 mg/L glucose and sodium bicarbonate, without L-glutamine, sodium pyruvate, and phenol red, liquid, sterile-filtered, suitable for cell culture	Sigma	D1145	for infection experiments
DMEM	Sigma	D6429	for maintenance of cultured host cells
Fetal Bovine Serum, heat inactivated	Sigma	F9665	supplement for tissue culture medium
Penicillin-Streptomycin -Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, sterile-filtered, BioReagent, suitable for cell culture	Sigma	F9665	supplement for tissue culture medium
PBS	Sigma	D8537
Plasticware
24-well plates	Greiner	662160
Equipment
Tissue culture facilities
Multichannel pipette