Protocols in Current Issue
    Development and Quantitation of Pseudomonas aeruginosa Biofilms after in vitro Cultivation in Flow-reactors
    Authors:  Yingdan Zhang, Jingru Zhao, Hang Cheng, Jing Wang, Liang Yang and Haihua Liang, date: 08/20/2021, view: 1104, Q&A: 0

    Characterization of biofilm formation and metabolic activities is critical to investigating biofilm interactions with environmental factors and illustrating biofilm regulatory mechanisms. An appropriate in vitro model that mimics biofilm in vivo habitats therefore demands accurate quantitation and investigation of biofilm-associated activities.

    Analysis of Pseudomonas aeruginosa c-di-GMP High and Low Subpopulations Using Flow-assisted Cell Sorting (FACS) and Quantitative Reverse Transcriptase PCR (qRT-PCR)
    Authors:  Catherine R. Armbruster and Matthew R. Parsek, date: 01/20/2021, view: 2081, Q&A: 0

    Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger signaling molecule that drives the transition from planktonic to the biofilm mode of growth in many bacterial species. Pseudomonas aeruginosa has at least two surface sensing systems that produce c-di-GMP in response to surface attachment, the Wsp and Pil-Chp systems. We recently

    Murine Acute Pneumonia Model of Pseudomonas aeruginosa Lung Infection
    Authors:  Xiaolei Pan and Weihui Wu, date: 11/05/2020, view: 1932, Q&A: 0
    [Abstract] Animal infection models play significant roles in studying bacterial pathogenic mechanisms, host pathogen interaction as well as evaluating drug and vaccine efficacies. We have been utilizing an acute pneumonia model to study bacterial colonization in lungs and assess virulence to the host by determination of bacterial loads and survival assays, ...
    Evaluation of Viable Cells in Pseudomonas aeruginosa Biofilms by Colony Count and Live/Dead Staining
    Authors:  Magdalena Pezzoni, Ramón Augusto Pizarro and Cristina Susana Costa, date: 09/20/2020, view: 3022, Q&A: 0
    [Abstract] Pseudomonas aeruginosa is a human pathogen capable to form robust biofilms. P. aeruginosa biofilms represent a serious problem because of the adverse effects on human health and industry, from sanitary and economic points of view. Typical strategies to break down biofilms have been long used, such as the use of disinfectants or ...
    Cryo-transmission Electron Microscopy of Outer-inner Membrane Vesicles Naturally Secreted by Gram-negative Pathogenic Bacteria
    [Abstract] A protocol was developed to visualize and analyze the structure of membrane vesicles (MVs) from Gram-negative bacteria. It is now accepted that these micrometric spherical vesicles are commonly produced by cells from all three domains of life, so the protocol could be useful in the study of vesicles produced by eukaryotes and archaea as well as ...
    Extraction and Quantification of Polyphosphate (polyP) from Gram-negative Bacteria
    Authors:  Jan-Ulrik Dahl, Lihan Xie and Ursula Jakob, date: 09/20/2018, view: 5023, Q&A: 0
    [Abstract] Polyphosphate (polyP), a universally conserved biomolecule, is composed of up to 1,000 phosphate monomers linked via phosphoanhydride bonds. Reaching levels in bacteria that are in the high nmoles per mg protein range, polyP plays important roles in biofilm formation and colonization, general stress protection and virulence. Various protocols for ...
    Detection of Catalase Activity by Polyacrylamide Gel Electrophoresis (PAGE) in Cell Extracts from Pseudomonas aeruginosa
    Authors:  Magdalena Pezzoni, Ramón A. Pizarro and Cristina S. Costa, date: 06/05/2018, view: 7243, Q&A: 0
    [Abstract] Bacteria in nature and as pathogens commonly face oxidative stress which causes damage to proteins, lipids and DNA. This damage is produced by the action of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), singlet oxygen, superoxide anion and hydroxyl radical. ROS are generated by antimicrobials, environmental ...
    Quantification of Bacterial Twitching Motility in Dense Colonies Using Transmitted Light Microscopy and Computational Image Analysis
    Authors:  Benjamin Smith, Jianfang Li, Matteo Metruccio, Stephanie Wan, David Evans and Suzanne Fleiszig, date: 04/20/2018, view: 5841, Q&A: 0
    [Abstract] A method was developed to allow the quantification and mapping of relative bacterial twitching motility in dense samples, where tracking of individual bacteria was not feasible. In this approach, movies of bacterial films were acquired using differential interference contrast microscopy (DIC), and bacterial motility was then indirectly quantified ...
    Measurement of Intracellular Calcium Concentration in Pseudomonas aeruginosa
    Authors:  Manita Guragain, Anthony K. Campbell and Marianna A. Patrauchan, date: 12/05/2016, view: 7898, Q&A: 0
    [Abstract] Characterization of the molecular mechanisms of calcium (Ca2+) regulation of bacterial physiology and virulence requires tools enabling measuring and monitoring the intracellular levels of free calcium (Ca2+in). Here, we describe a protocol optimized to use a recombinantly expressed Ca2+-binding protein, ...

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