High‐Throughput Assessment of Bacterial Growth Inhibition by Optical Density Measurements

Jennifer Campbell1

1 Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100115
Online Posting Date:  October, 2010
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Abstract

The increasing incidence of antibiotic‐resistant bacterial infections both in hospitals and in the community intensifies the need for new antibacterial strategies and targets. Although high‐throughput screening against live bacteria allows rapid discovery of compounds with growth‐inhibitory activities, these efforts have failed to fill the pipeline with the anticipated antibacterial compounds because target identification is often onerous. Recently, a strategy was reported that employs a bacterial growth inhibition assay readout using optical density measurements on paired strains—both a wild‐type strain and a pathway‐null mutant—to find inhibitors of wild‐type bacterial growth that specifically target conditionally essential enzymes in the pathway of interest. Protocols are provided here for determining the robustness of an assay, screening in a high‐throughput format, and setting up dose‐response curves in paired Staphylococcus aureus strains. However, the protocols can be used to screen for growth‐inhibitory compounds in any bacterial strain of interest. Curr. Protoc. Chem. Biol. 2:195‐208 © 2010 by John Wiley & Sons, Inc.

Keywords: high‐throughput screening; Staphylococcus aureus; optical density; cell‐based assay; pathway specific screen; bacterial growth inhibition

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Z′ Determination to Assess Screening Assay Robustness
  • Basic Protocol 2: Screening for Growth‐Inhibitory Compounds
  • Basic Protocol 3: Dose‐Response Assays for Secondary Screening of Potential Bacterial Growth Inhibitors
  • Support Protocol 1: Determining Correct Bacterial Concentration for a Screening Assay
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Z′ Determination to Assess Screening Assay Robustness

  Materials
  • Sterile tryptic soy broth (TSB; 30 g/l; sterile filtered or autoclaved; BD Difco, cat. no. 211823) plus selective antibiotic, if appropriate
  • Plated bacterial strain(s) of interest (e.g., S. aureus)
  • Positive control compound that inhibits growth of bacterial strain (e.g., for S. aureus, erythromycin at 10 mg/ml in ethanol)
  • 30°C shaking incubator
  • Multichannel pipettor (volume range from 20 to 100 µl) with aerosol‐barrier pipet tips (VWR, cat. no. 53510‐106) and sterile solvent reservoirs (VWR, cat. no. 89094‐680)
  • 384‐well clear‐bottom plate (Corning, cat. no. 3702)
  • Low‐evaporation lid (Corning, cat. no. 3009)
  • Plate reader capable of measuring optical density at 600 nm (OD 600) in a 384‐well format (e.g., EnVision from PerkinElmer)

Basic Protocol 2: Screening for Growth‐Inhibitory Compounds

  Materials
  • Sterile tryptic soy broth (TSB; 30 g/l; sterile filtered or autoclaved; BD Difco, cat. no. 211823) plus selective antibiotic, if appropriate
  • Plated bacterial strain(s) of interest (e.g., S. aureus)
  • 20 ml TSB medium containing positive control compound at 2× final concentration (typically erythromycin at 20 µg/ml)
  • Compound library plates, with compounds diluted in DMSO at 5 mg/ml or 10 mM stock concentration
  • 1‐liter flasks (sterile)
  • 30°C shaking incubator
  • Multichannel pipettor (volume range from 20 to 100 µl) with aerosol‐barrier pipet tips (VWR, cat. no. 53510‐106) and sterile solvent reservoirs (VWR, cat. no. 89094‐680)
  • 384‐well clear‐bottom plates (80 per screening session; Corning, cat. no. 3702)
  • Microplate dispenser (Matrix WellMate or comparable liquid handler; see Rudnicki and Johnston, )
  • Pin transfer robot (Rudnicki and Johnston, )
  • Low‐evaporation plate lids (16–20; Corning, cat. no. 3009)
  • Plate reader capable of measuring optical density at 600 nm (OD 600) in a 384‐well format (e.g., EnVision from Perkin Elmer)

Basic Protocol 3: Dose‐Response Assays for Secondary Screening of Potential Bacterial Growth Inhibitors

  Materials
  • Sterile tryptic soy broth (TSB; 30 g/l; sterile filtered or autoclaved; BD Difco, cat. no. 211823) plus selective antibiotic, if appropriate
  • Plated bacterial strain(s) of interest (e.g., S. aureus)
  • Sterile TSB containing 2% (v/v) DMSO (plus selective antibiotic, if appropriate)
  • Sterile TSB containing positive control compound at 2× final concentration
  • 384‐well clear‐bottom plates (one per 48 compounds; Corning, cat. no. 3702)
  • Single‐channel pipettor (volume range of 0.5 to 2.0 µl)
  • Sterile solvent reservoir (VWR, cat. no. 89094‐680)
  • Multi‐channel pipettor (volume range of 15 to 30 µl); alternatively use plate filler
  • Aerosol barrier tips that fit the multichannel pipettor (VWR, cat. no. 53510‐014)
  • Low‐evaporation lids (one per plate; Corning, cat. no. 3009)
  • 30°C shaking incubator
  • Plate reader capable of measuring optical density at 600 nm (OD 600) in a 384‐well format (EnVision from PerkinElmer)

Support Protocol 1: Determining Correct Bacterial Concentration for a Screening Assay

  • TSB plates containing 1.5% (w/v) agar
  • Spreaders
  • Sterile 2‐ml tubes
  • UV‐vis spectrophotometer
  • Cuvettes
  • Graphing software
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Figures

Videos

Literature Cited

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   Weidenmaier, C., Peschel, A., Xiong, Y.Q., Kristian, S.A., Dietz, K., Yeaman, M.R., and Bayer, A.S. 2005. Lack of wall teichoic acids in Staphylococcus aureus leads to reduced interactions with endothelial cells and to attenuated virulence in a rabbit model of endocarditis. J. Infect. Dis. 191:1771‐1777.
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Internet Resources
   http://ncgc.nih.gov/pub/openhts/curvefit/
  Dose‐response curve fitting.
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