An Assay to Study Bile-Salt Induced Biofilm Formation through EPS Matrix Detection

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

1. 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 salt-induced biofilm formation. TSB has added glucose relative to Luria-Bertani (LB) broth; and therefore, was sufficient to induce biofilm formation ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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