An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells

Wang, Hengwei; Cheng, Hairong; Wang, Fengqing; Wei, Dongzhi; Wang, Xuedong

doi:10.1016/j.mimet.2010.06.014

Cited by 195 publications

(131 citation statements)

References 17 publications

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“…Glucose oxidation rates were corrected for OD 600 changes and averaged from the results of three independent replicates. Cellular dehydrogenase activity was estimated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (62). WT and phospholipid-altered cell suspensions were prepared by diluting (1/10 [vol/vol]) exponentially growing cultures (OD 600 of ϳ0.3) with fresh M9 minimal medium containing all the necessary supplements.…”

Section: Methodsmentioning

confidence: 99%

Impact of Membrane Phospholipid Alterations in Escherichia coli on Cellular Function and Bacterial Stress Adaptation

Rowlett¹,

et al. 2017

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Bacteria have evolved multiple strategies to sense and rapidly adapt to challenging and ever-changing environmental conditions. The ability to alter membrane lipid composition, a key component of the cellular envelope, is crucial for bacterial survival and adaptation in response to environmental stress. However, the precise roles played by membrane phospholipids in bacterial physiology and stress adaptation are not fully elucidated. The goal of this study was to define the role of membrane phospholipids in adaptation to stress and maintenance of bacterial cell fitness. By using genetically modified strains in which the membrane phospholipid composition can be systematically manipulated, we show that alterations in major Escherichia coli phospholipids transform these cells globally. We found that alterations in phospholipids impair the cellular envelope structure and function, the ability to form biofilms, and bacterial fitness and cause phospholipid-dependent susceptibility to environmental stresses. This study provides an unprecedented view of the structural, signaling, and metabolic pathways in which bacterial phospholipids participate, allowing the design of new approaches in the investigation of lipiddependent processes involved in bacterial physiology and adaptation.IMPORTANCE In order to cope with and adapt to a wide range of environmental conditions, bacteria have to sense and quickly respond to fluctuating conditions. In this study, we investigated the effects of systematic and controlled alterations in bacterial phospholipids on cell shape, physiology, and stress adaptation. We provide new evidence that alterations of specific phospholipids in Escherichia coli have detrimental effects on cellular shape, envelope integrity, and cell physiology that impair biofilm formation, cellular envelope remodeling, and adaptability to environmental stresses. These findings hold promise for future antibacterial therapies that target bacterial lipid biosynthesis.KEYWORDS membranes, metabolism, phospholipids, physiology, stress adaptation, stress response B acteria have to cope with and adapt to a wide range of environmental conditions, such as nutrient limitation or exposure to antibiotics. Therefore, the ability to sense and quickly respond to fluctuating conditions is key for bacterial survival (1-3). Bacterial stress adaptation often requires major metabolic reprogramming that involves coordinated changes in the cell transcriptome, proteome, and metabolome, along with cellular envelope remodeling (4, 5). Escherichia coli cells consist of four compartments: the cytoplasm, the inner membrane, the periplasm, and the outer membrane. The inner and outer membranes exhibit different makeups. The inner membrane is a bilayer containing ␣-helical proteins, and more than 95% of the total lipids are phospholipids; the outer membrane is an asymmetric bilayer made of both phospholipids and

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Section: Methodsmentioning

confidence: 99%

Impact of Membrane Phospholipid Alterations in Escherichia coli on Cellular Function and Bacterial Stress Adaptation

Rowlett¹,

et al. 2017

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“…The effects of GH12 at sub-MIC levels on the viability of S. mutans was assessed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) staining method as described [25,26] with some modification. Briefly, S. mutans was suspended with BHI broth containing GH12 at sub-MIC levels or sterile deionized water.…”

Section: Methodsmentioning

confidence: 99%

Antimicrobial peptide GH12 suppresses cariogenic virulence factors of Streptococcus mutans

Wang

Jiang

et al. 2018

Journal of Oral Microbiology

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Cariogenic virulence factors of Streptococcus mutans include acidogenicity, aciduricity, and extracellular polysaccharides (EPS) synthesis. The de novo designed antimicrobial peptide GH12 has shown bactericidal effects on S. mutans, but its interaction with virulence and regulatory systems of S. mutans remains to be elucidated. The objectives were to investigate the effects of GH12 on virulence factors of S. mutans, and further explore the function mechanisms at enzymatic and transcriptional levels. To avoid decrease in bacterial viability, we limited GH12 to subinhibitory levels. We evaluated effects of GH12 on acidogenicity of S. mutans by pH drop, lactic acid measurement and lactate dehydrogenase (LDH) assay, on aciduricity through survival rate at pH 5.0 and F1F0-ATPase assay, and on EPS synthesis using quantitative measurement, morphology observation, vertical distribution analyses and biomass calculation. Afterwards, we conducted quantitative real-time PCR to acquire the expression profile of related genes. GH12 at 1/2 MIC (4 mg/L) inhibited acid production, survival rate, EPS synthesis, and biofilm formation. The enzymatic activity of LDH and F1F0-ATPase was inhibited, and ldh, gtfBCD, vicR, liaR, and comDE genes were significantly downregulated. In conclusion, GH12 inhibited virulence factors of S. mutans, through reducing the activity of related enzymes, downregulating virulence genes, and inactivating specific regulatory systems.

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“…The MTT test, which determines the metabolic activities of bacteria, was done as previously described by Wang et al [25], and originally described by Mosmann [26], with slight modifications. Briefly, the bacteria were cultivated and exposed to the RMF ( f = 1-50 Hz, B = 22-34 mT) for 60 minutes.…”

Section: Methodsmentioning

confidence: 99%

The Effects of Rotating Magnetic Field on Growth Rate, Cell Metabolic Activity and Biofilm Formation by Staphylococcus Aureus and Escherichia Coli

kowski¹,

Nawrotek²,

Struk³

et al. 2013

Journal of Magnetics

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This work presents results of the study which concerns the influence of the rotating magnetic field (RMF) on the growth rate, cell metabolic activity and ability to form biofilms by E. coli and S. aureus. Liquid cultures of the bacteria were exposed to the RMF (RMF frequency f = 1-50 Hz, RMF magnetic induction B = 22-34 mT, time of exposure t = 60 min, temperature of incubation 37 °C). The present study indicate the exposition to the RMF, as compared to the unexposed controls causing an increase in the growth dynamics, cell metabolic activities and percentage of biofilm-forming bacteria, in both S. aureus and E. coli cultures. It was also found that the stimulating effects of the RMF exposition enhanced with its increasing frequencies and magnetic inductions.

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An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells

Cited by 195 publications

References 17 publications

Impact of Membrane Phospholipid Alterations in Escherichia coli on Cellular Function and Bacterial Stress Adaptation

Impact of Membrane Phospholipid Alterations in Escherichia coli on Cellular Function and Bacterial Stress Adaptation

Antimicrobial peptide GH12 suppresses cariogenic virulence factors of Streptococcus mutans

The Effects of Rotating Magnetic Field on Growth Rate, Cell Metabolic Activity and Biofilm Formation by Staphylococcus Aureus and Escherichia Coli

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