title: Growth-Based Bacterial Viability Assay for Interference-Free and High-Throughput Toxicity Screening of Nanomaterials.
abstract: Current high-throughput approaches evaluating toxicity of chemical agents toward bacteria typically rely on optical assays, such as luminescence and absorbance, to probe the viability of the bacteria. However, when applied to toxicity induced by nanomaterials, scattering and absorbance from the nanomaterials act as interferences that complicate quantitative analysis. Herein, we describe a bacterial viability assay that is free of optical interference from nanomaterials and can be performed in a high-throughput format on 96-well plates. In this assay, bacteria were exposed to various materials and then diluted by a large factor into fresh growth medium. The large dilution ensured minimal optical interference from the nanomaterial when reading optical density, and the residue left from the exposure mixture after dilution was confirmed not to impact the bacterial growth profile. The fractions of viable cells after exposure were allowed to grow in fresh medium to generate measurable growth curves. Bacterial viability was then quantitatively correlated to the delay of bacterial growth compared to a reference regarded as 100% viable cells; data analysis was inspired by that in quantitative polymerase chain reactions, where the delay in the amplification curve is correlated to the starting amount of the template nucleic acid. Fast and robust data analysis was achieved by developing computer algorithms carried out using R. This method was tested on four bacterial strains, including both Gram-negative and Gram-positive bacteria, showing great potential for application to all culturable bacterial strains. With the increasing diversity of engineered nanomaterials being considered for large-scale use, this high-throughput screening method will facilitate rapid screening of nanomaterial toxicity and thus inform the risk assessment of nanoparticles in a timely fashion.
authors.affiliation: Department of Chemistry, University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Tian A
authors.identifier: 0000-0002-7254-9233
authors.initials: TA
authors.lastname: Qiu
authors.affiliation: Chemistry Department, Augsburg College , Minneapolis, Minnesota 55454, United States.
authors.forename: Thu Ha Thi
authors.initials: TH
authors.lastname: Nguyen
authors.affiliation: Department of Chemistry, University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Natalie V
authors.initials: NV
authors.lastname: Hudson-Smith
authors.affiliation: Department of Chemistry, University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Clement
authors.affiliation: Department of Chemistry, University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Dona-Carla
authors.initials: DC
authors.lastname: Forester
authors.affiliation: Chemistry Department, Augsburg College , Minneapolis, Minnesota 55454, United States.
authors.forename: Hilena
authors.initials: H
authors.lastname: Frew
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Mimi N
authors.initials: MN
authors.lastname: Hang
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.
authors.forename: Catherine J
authors.identifier: 0000-0001-7066-5575
authors.initials: CJ
authors.lastname: Murphy
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Robert J
authors.identifier: 0000-0003-3821-9625
authors.initials: RJ
authors.lastname: Hamers
authors.affiliation: Chemistry Department, Augsburg College , Minneapolis, Minnesota 55454, United States.
authors.forename: Z Vivian
authors.initials: ZV
authors.lastname: Feng
authors.affiliation: Department of Chemistry, University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Christy L
authors.identifier: 0000-0002-5420-5867
authors.initials: CL
authors.lastname: Haynes
mesh_terms: D000465:Algorithms; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001331:Automation; D001412:Bacillus subtilis / Q000187:drug effects* / Q000254:growth & development*; D057166:High-Throughput Screening Assays / Q000379:methods*; D008826:Microbial Sensitivity Tests; D049329:Nanostructures / Q000633:toxicity*; D016133:Polymerase Chain Reaction / Q000379:methods; D020592:Shewanella / Q000187:drug effects* / Q000254:growth & development*; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents