A Solid Phase Adherence Assay to Assess Biofilm Formation

Abstract

Source: Nickerson, K. P. et al., Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification. J. Vis. Exp. (2018)

The video demonstrates a solid-phase adherence assay for assessing biofilm formation. In a multi-well plate, bacteria in bile salt-supplemented media adhere to solid surfaces, forming biofilms, which is confirmed by colorimetry through crystal violet staining and ethanol treatment.

Protocol

1. Preparation of Reagents

1. Bile salts 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 sterilizes 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....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tryptic Soy Broth	Sigma-Aldrich	22092-500G
Crystal Violet	Sigma	C6158-50
Bile Salts	Sigma	B8756-100G
LB Agar	Sigma	L7533-1KG
14 mL culture tubes, 17 x 100 mm, plastic, sterile	Fisher	14-959-11B
Flat-bottomed 96-well plates (clear)	TPP	92696
96-well plate reader	Spectramax
37°C Shaking Incubator	New Brunswick Scientific Excella E25
37°C Plate Incubator	Thermolyne Series 5000