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.

Nanoscale battery cathode materials induce DNA damage in bacteria

“…1 and S1 †). A growth-based viability (GBV) assay was performed at the time point when the bacterial population was about to enter stationary phase (8 hour for S. oneidensis and 5 hour for B. subtilis) to measure bacterial viability compared to controls, 38 as optical density does not effectively differentiate live and dead cells. Results showed that for S. oneidensis, an 1-hour short exposure slightly inhibited growth and compromised viability, while an 8 hour exposure caused signicantly decrease in both normalized optical density and viability in a dose-dependent manner, indicating that NMC toxicity is time-dependent.…”

“…At 1 and 8 hour post-exposure, a growth-based viability (GBV) test was performed with water evaporation control and randomized positions. 38 Negative control groups were used as 100% reference in building calibration curves.…”

“…ANOVA followed by Dunnett's multiple comparisons test was used on log-2 transformed viability data. 38 Comet assay. For each condition, tail lengths counted from three biological replicates of control or nano-NMC-treated bacterial suspensions were combined to generate the nal master dataset aer testing statistically indifferent among replicates.…”

Selective Modification of Streptozotocin at the C3 Position to Improve Its Bioactivity as Antibiotic and Reduce Its Cytotoxicity towards Insulin-Producing β Cells

“…In rich-medium conditions, STZ (1) showed An alternative way to asses antimicrobial activity is to identify the percentage of viable bacteria after a short treatment with the antibiotic (2 h). In this so-called growth-based viability assay, originally developed by Haynes and co-workers for the toxicity screening of nanomaterials [34], the percentage of viable cells is determined by correlating the growth (OD 600 ) of the treated samples to control samples with a known number of cells that are grown under the same conditions. The advantage of using relative viabilities is that it allows straightforward comparison of viability, even when the experiments have been performed under different growth conditions.…”

“…To better assess the impact of the STZ derivatives 2-4 on bacterial growth and viability, growth-based viability assays were performed [34]. The assay was performed in rich medium (lysogeny broth, LB) as well as in minimal medium with N-acetylglucosamine (GlcNAc) or ribose as additives to study the effect of these compounds on the uptake of the streptozotocin-analogues.…”