An Assay to Study Bile-Salt Induced Biofilm Formation through EPS Matrix Detection
Published: March 29, 2024
Abstract
Source: Nickerson, K. P., et al. Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification. J. Vis. Exp. (2018).
This video demonstrates a fluorescence-based method for quantifying bile salt-induced biofilm formation. Upon the addition of bile salts in the culture of a pathogenic enteric bacteria, biofilm production is detected using a fluorescent lectin that binds to the polysaccharides in the extracellular polymeric substance (EPS) matrix of the biofilm.
Protocol
1. Preparation of Reagents Bile salts (BS) medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile salts in 50 mL autoclaved TSB. Filter sterilize using a 0.22 µm filter. Make fresh medium weekly. Notes: The bile salts routinely used is a 1:1 mixture of sodium cholate and sodium deoxycholate isolated from ovine and bovine gallbladders. As demonstrated previously, the presence of glucose was required for bile …
Divulgazioni
The authors have nothing to disclose.
Materials
| Tryptic Soy Broth | Sigma-Aldrich | 22092-500G | |
| Concanavalin-A FITC | Sigma | C7642-10mg | |
| Glucose | Sigma | G7021-1KG | |
| Bile Salts | Sigma | B8756-100G | |
| LB Agar | Sigma | L7533-1KG | |
| 14 mL culture tubes, 17 x 100 mm, plastic, sterile | Fisher | 14-959-11B | |
| Vectashield hard-set antifade with DAPI | Vector Laboratories | H-1500 | |
| Formaldehyde | Sigma-Aldrich | F1635-500 | |
| Gluteraldehyde | Sigma-Aldrich | G6257 | |
| Flat-bottomed 96-well plates (clear) | TPP | 92696 | |
| Flat-bottomed 96-well plates (black) | Greiner Bio-One | 655076 | |
| Flat-bottomed 24-well plates (clear) | TPP | 92424 | |
| 96-well plate reader | Spectramax | ||
| Flourescent plate reader | Biotek Synergy 2 | ||
| Confocal or Fluorescent Microscope | Nikon A1 confocal microscope | ||
| 37°C Shaking Incubator | New Brunswick Scientific Excella E25 | ||
| 37°C Plate Incubator | Thermolyne Series 5000 |
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Citazione di questo articolo
An Assay to Study Bile-Salt Induced Biofilm Formation through EPS Matrix Detection. J. Vis. Exp. (Pending Publication), e22065, doi: (2024).