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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  •   FigureFigure 13.A0.1 Streaking bacteria on solid agar to obtain single, well‐isolated colonies. (A) Streaking plates. Plates can be streaked using a standard wire inoculating loop, a disposable plastic loop, a toothpick, or a long “picking stick.” The wire loop can be sterilized in a flame, but it must be cooled by touching the agar surface prior to making contact with bacteria. Single colonies can be isolated by repeated streaking as illustrated in the figure. (B) An example of the results obtained using this method. Single, well‐isolated colonies are indicated.
  •   FigureFigure 13.A0.2 Results of a biofilm assay using the strains listed in Table demonstrates the range of phenotypes of individual strains and the stimulatory effect of glucose. Biofilm cultures were grown in 0.5× TSB supplemented with 0.25% or 1% glucose (gluc) at 37°C for 18 hr. (A) Photographs of two representative rows from each tissue culture‐treated 96‐well assay that was stained with crystal violet. (B) The amount of crystal violet bound was quantified by measuring absorbance at 600 nm and plotting for each strain. Each bar represents the average of eight individual assay wells, and the error bars the standard deviation.
  •   FigureFigure 13.A0.3 Preparing assay plates for biofilm formation on alternative substrates.
  •   FigureFigure 13.A0.4 Optimization of assay conditions. (A) A biofilm growth curve of S. aureus ATCC 35556 grown in 0.5× TSB supplemented with 1% glucose. (B) Biofilms were grown in varying media conditions to identify optimal growth conditions and two methods of biofilm detection compared. Biofilms were grown at 37°C for 18 hr. Biofilms were stained and the amount of crystal violet bound to the bottom of each assay well measured directly as absorbance at 600 nm, or crystal violet was eluted from each well, diluted 1:10, and the absorbance measured at 600 nm. (C) Comparison of absorbance at 600 nm of neat versus a 1:10 dilution of eluted crystal violet demonstrates the linear range of the assay.
  •   FigureFigure 13.A0.5 An example of the screening results obtained using S. epidermidis 18972 and a mock screening plate. (A) A photograph of the crystal violet–stained mock screening plate containing nine compounds that are inhibitors of biofilm formation and two antibiotics. The remainder of the wells in columns 2 to 11 contained DMSO. Columns 1 and 12 contained the 0% INH (DMSO only) and 100% INH (100 µg chloramphenicol/ml; Cm100) controls, respectively. The wells containing anti‐biofilm and antibacterial compounds exhibited significant reductions in the intensity of crystal violet staining. (B) The OD600 values obtained for the planktonic cultures. Compounds that produced ≥40% inhibition of planktonic growth are highlighted in orange. The compound in well number C07, highlighted in blue, produced an OD600 of 2.64, which is significantly higher than those produced by the 0% INH (DMSO‐only) controls. This well contained gentian violet, an intensely colored compound that absorbs light at 600 nm. Beware of colored compounds that absorb at 600 nm, as they can mask antibacterial activity. (C) The OD600 values obtained for the crystal violet–stained biofilm cultures. Compounds that produced ≥80% inhibition of planktonic growth are highlighted in orange. (D) A table listing the compounds used in the mock screening plate, their location on the plate (row, column), and the screening call (HIT or antibacterial). Compounds that produced ≥80% biofilm inhibition and ≤40% planktonic growth inhibition were designated anti‐biofilm “HITS.” Compounds that produced ≥40% planktonic growth inhibition were designated as “Antibacterial” compounds. Seven of the anti‐biofilm compounds used in the mock screening plate were identified previously in a pilot screen using a compound library comprised of 2000 known bioactive chemicals (Spectrum Library; MicroSource Discovery Systems). MBX‐1240 and MBX‐1246 were identified in screen of compounds purchased from Chembridge Corp. (Opperman et al., ).
  •   FigureFigure 13.A0.6 An example of a Minimal Biofilm Inhibitory Concentration (MBIC) assay for eight anti‐biofilm compounds that were tested against S. epidermidis 18972. The assay plate in which biofilm cultures have been stained with crystal violet is shown in the photograph. The concentrations of each compound tested are indicated above the plate, and the compound tested in each row is indicated on the left side of the plate. The MBIC and MIC are defined as the lowest compound concentration that produces a ≥80% inhibition of biofilm growth or planktonic growth, respectively. The compounds used in this assay (Compounds 1 to 8), their chemical names and structures, and the MBIC and MIC values obtained as a result of the assay are also shown. Note that Gentian violet produced an MIC that was equal to the MBIC. Because this compound strongly absorbs light at 600 nm, the antibacterial activity of this compound became apparent only when it was diluted in the dose‐response assay. The compounds used in this assay are commercially available from MicroSource Discovery Systems. Alternatively, three of these compounds (Tomatidine, Actinonin, and Emodin) can be purchased from Sigma‐Aldrich. MBX‐1240 can be purchased from ChemBridge Corp.


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