flow cytometry
MethodsDevelopment of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag ProteinsUp to this point, assays and methods involving fluorescent reporter proteins were unavailable or limited in Clostridium organisms and other strict anaerobes. Green fluorescent protein (GFP), mCherry, and flavin-binding proteins (and their derivatives) have been used only in a few clostridia with limited success and yielded low fluorescence compared to aerobic microbial systems. Recently, we reported a new strong fluorescent...
MethodsProtein A-Mediated Binding of Staphylococcus spp. to Antibodies in Flow Cytometric Assays and Reduction of This Binding by Using Fc Receptor Blocking ReagentThis study characterizes a widespread but little-studied problem associated with the antibody-based detection of microbes—the Staphylococcus protein A (SpA)-mediated binding of IgG antibodies—and offers a solution: the use of commercial FcR blocking reagent. A common source of false-positive signals in the detection of microbes in clinical, food, or environmental samples can be eliminated by applying this study’s findings....
BiotechnologyMultifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics SimulationMultidrug-resistant bacteria are a major threat to global health, and the Pseudomonas bacterium with the ability to form biofilms is considered one of the main causative agents of nosocomial infections. Traditional antibiotics have failed because of increased resistance. Thus, finding new biocompatible antibacterial drugs is essential. Antimicrobial peptides are produced by various organisms as a natural defense mechanism...
Food Microbiology | SpotlightSuboptimal Bacillus licheniformis and Bacillus weihenstephanensis Spore Incubation Conditions Increase Heterogeneity of Spore Outgrowth TimeSporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of Bacillus spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that...
Food MicrobiologyHeat and Chemical Treatments Affect the Viability, Morphology, and Physiology of Staphylococcus aureus and Its Subsequent Antibody Labeling for Flow Cytometric AnalysisFCM data indicated that cells conventionally considered to be dead and which would not give rise to CFU in a plate count assay, e.g., cells heated to 80°C, were labeled by antibody staining. This finding suggests that without the inclusion of a live/dead discriminating dye, these cells would be erroneously detected as viable within an FCM assay. Reductions in antibody staining due to physicochemical treatment were strain related,...
BiotechnologyA Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)FAST in association with the fluorogenic ligand HMBR is characterized as a successful, highly fluorescent reporter system in C. acetobutylicum. FAST can be used to distinguish between promoters in live cells using flow cytometry or a fluorescence microplate reader and can be used to tag and examine protein localization in live, anaerobically grown cells. Given that...
Environmental Microbiology | SpotlightCoculturing Bacteria Leads to Reduced Phenotypic HeterogeneitiesLaboratory studies have shown the impact of phenotypic heterogeneity on the survival and functionality of isogenic populations. Because phenotypic heterogeneity plays an important role in pathogenicity and virulence, antibiotic resistance, biotechnological applications, and ecosystem properties, it is crucial to understand its influencing factors. An unanswered question is whether bacteria in mixed communities influence the phenotypic...
Environmental MicrobiologyUse of a Fluorescent Analog of Glucose (2-NBDG) To Identify Uncultured Rumen Bacteria That Take Up GlucoseWe introduce a method for identifying what substrates are consumed by bacteria in natural communities. Our method offers significant improvement over existing methods for studying this characteristic. Our method uses a fluorescently labeled substrate which clearly labels target bacteria (glucose consumers in our case). Previous methods use isotope-labeled substrates, which are notorious for off-target labeling (due to cross-feeding of...