Static Biofilm Cultures of Gram‐Positive Pathogens Grown in a Microtiter Format Used for Anti‐Biofilm Drug Discovery

Steven M. Kwasny1, Timothy J. Opperman1

1 Microbiotix, Anti‐Infectives R&D, Worcester, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13A.8
DOI:  10.1002/0471141755.ph13a08s50
Online Posting Date:  September, 2010
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An in vitro assay is presented for culturing staphylococcal biofilms and biofilms of nonmotile Gram‐positive bacteria under static conditions in microtiter assay plates, and for the quantification of biofilm growth, using a simple staining procedure that measures amounts of bacterial cells and extracellular matrix. This basic assay can be adapted readily to study several aspects of biofilm formation, for high‐throughput screening to identify small molecule inhibitors of biofilm formation or biofilm‐defective mutants, and for quantifying the anti‐biofilm activity of biofilm inhibitors. Curr. Protoc. Pharmacol. 50:13A.8.1‐13A.8.23. © 2010 by John Wiley & Sons, Inc.

Keywords: biofilm; microtiter; Gram‐positive

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Table of Contents

  • Introduction
  • Basic Protocol 1: Basic Assay for Biofilm Formation of Nonmotile Gram‐Positive Bacteria
  • Alternate Protocol 1: Biofilm Formation on Alternative Surfaces, Such as Medical‐Grade Materials
  • Basic Protocol 2: Optimizing Biofilm Formation Assay Conditions
  • Basic Protocol 3: Screening Assay for Mutants or Compounds that Inhibit Biofilm Formation
  • Basic Protocol 4: A Quantitative Assay for Anti‐Biofilm Activity: The Minimal Biofilm Inhibitory Concentration (MBIC) Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Basic Assay for Biofilm Formation of Nonmotile Gram‐Positive Bacteria

  • Gram‐positive bacterial strain(s) of interest
  • Sterile solid growth medium: tryptic soy agar (TSA; see recipe)
  • Sterile liquid growth medium: tryptic soy broth (TSB; see recipe)
  • Sterile 80% (v/v) glycerol (autoclaved; store at room temperature)
  • 40% (w/v) glucose (filter sterilize; store at room temperature)
  • Washing solution e.g., deionized H 2O, phosphate‐buffered saline (PBS; see recipe), or 0.9% saline (see recipe)
  • 95% ethanol (optional)
  • Bouin's fixative: dissolve 0.5 g picric acid in 37.5 ml deionized H 2O, then add 12.5 ml 37% formaldehyde and 2.5 ml glacial acetic acid
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.06% crystal violet (see recipe)
  • Sterile inoculating loops, toothpicks, and applicator sticks
  • 37°C bacteriological incubator containing rotary shaker or tube roller
  • Culture tubes (e.g., 16 × 150 mm)
  • 2‐ml cryogenic storage vials vial (e.g., Nalgene, cat. no. 5000‐0020)
  • 96‐well assay plates with lids (Costar 3595; flat bottomed, polystyrene, tissue culture treated, or equivalent)
  • Adhesive foil lids (Costar 6569, optional)
  • Plate washer: e.g., BioTek ELx405 or equivalent (optional)
  • Two 2‐liter beakers (optional)
  • Multichannel pipettor and pipetting reservoirs (optional)
  • Baking oven set at 60°C
  • Microtiter plate reader (Molecular Devices, optional)
  • Camera (optional)

Alternate Protocol 1: Biofilm Formation on Alternative Surfaces, Such as Medical‐Grade Materials

  • Silicone sealant (e.g., Silicone II, GE Sealants and Adhesives)
  • 30% (v/v) acetic acid
  • Nonreinforced, medical‐grade sheeting:
    • Silicone sheeting, 0.03‐in. thick (Cardiovascular Instrument Corp.;
    • Polyurethane sheeting (Pellethane 55‐D, Specialty Silicone Fabricators)
  • No. 5 (∼1‐cm‐diameter) cork borer
  • 24‐well assay plate (Costar 3526)
  • Forceps

Basic Protocol 2: Optimizing Biofilm Formation Assay Conditions

  • Library of small molecules stored in 96‐well plates (MicroSource Discovery Systems,; ChemBridge Corporation,; TimTec LLC,; ChemDiv Inc.,
  • Library of transposon‐insertion mutants stored in 96‐well plates (Tu Quoc et al., ; Boles et al., ; Xia et al., )
  • Dimethyl sulfoxide (DMSO)
  • 96‐pin replicator (Boekel Scientific,
  • Additional reagents and equipment for preparing and assaying biofilms ( protocol 1) and optimizing biofilm formation assay conditions ( protocol 3)
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Literature Cited

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