Protocols in Current Issue
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0 Q&A 1369 Views Jul 5, 2023

In the environment, bacteria compete for niche occupancy and resources; they have, therefore, evolved a broad variety of antibacterial weapons to destroy competitors. Current laboratory techniques to evaluate antibacterial activity are usually labor intensive, low throughput, costly, and time consuming. Typical assays rely on the outgrowth of colonies of prey cells on selective solid media after competition. Here, we present fast, inexpensive, and complementary optimized protocols to qualitatively and quantitively measure antibacterial activity. The first method is based on the degradation of a cell-impermeable chromogenic substrate of the β-galactosidase, a cytoplasmic enzyme released during lysis of the attacked reporter strain. The second method relies on the lag time required for the attacked cells to reach a defined optical density after the competition, which is directly dependent on the initial number of surviving cells.

Key features

• First method utilizes the release of β-galactosidase as a proxy for bacterial lysis.

• Second method is based on the growth timing of surviving cells.

• Combination of two methods discriminates between cell death and lysis, cell death without lysis, or survival to quasi-lysis.

• Methods optimized to various bacterial species such as Escherichia coli, Pseudomonas aeruginosa, and Myxococcus xanthus.

Graphical overview

0 Q&A 5403 Views Jan 20, 2021

The in vivo toxicity of new metallodrugs either as Small Bioactive Molecules (SBAMs) or Conjugates of Metals with Drugs (CoMeDs) or their hydrogels such as with hydroxyethyl-methacrylate (HEMA) (pHEMA@SBAMs or pHEMA@CoMeDs) are evaluated by the brine shrimp assay. Thus individuals of Artemia salina larvae are incubated in saline solutions with SBAMs, CoMeDs, pHEMA@SBAMs or pHEMA@CoMeDs or without for 24 h. The toxicity is then determined in terms of the mortality rate of brine shrimp larvae. Brine shrimp assay is a low cost, safe, no required feeding during the assay, while it requiring only a small amount of the tested agent.

0 Q&A 4526 Views Aug 20, 2020
Fungal pathogen Candida albicans is one of the top leading causes of overall healthcare-associated bloodstream infections worldwide. Neutrophil is the major effector cell to clear C. albicans infection. Our study showed that mouse neutrophils utilize two independent mechanisms to kill C. albicans: one is CR3 downstream NADPH oxidase-dependent mechanism that kills opsonized C. albicans; the other one is dectin-2-mediated NADPH oxidase-independent neutrophil extracellular trap (NET) that kills unopsonized C. albicans. Neutrophil killing of opsonized C. albicans requires phagocytosing the organism and production of reactive oxygen species production (ROS). Most existing protocols that assay for neutrophil killing of C. albicans requires a washing step after allowing neutrophils to phagocytose the organism. By definition, NET kills organisms extracellularly. Therefore, it is important to skip the washing step and add an optimal ratio of neutrophils and C. albicans to the wells. To demonstrate the effect of NET, it is necessary to compare killing ability of neutrophils treated with micrococcal nuclease (MNase), an enzyme that digests NET, to that treated with heat-inactivated MNase. MNase is also applied to release NET-bound fungal elements for counting. This protocol can be applied to assay NET killing of other biofilm-forming organisms.
0 Q&A 7237 Views Nov 20, 2017
Infection with Trypanosoma cruzi causes Chagas disease. The methods provided here allow for the quantification of T. cruzi in the liver, heart, and blood of intraperitoneally-infected mice and analysis of the killing activity of the cells infected with T. cruzi in vitro.
0 Q&A 22231 Views Sep 20, 2014
Macrophages are key cells involved in orchestrating host defense against infections. Here, we describe the protocol for a bacterial killing assay in macrophages that can be adapted to any bacterial pathogen. Using this assay, we analyzed the survival of wild-type and mutant strains of Escherichia coli (E. coli) within RAW 264.7 cells, a widely used macrophage cell line. Bacterial mutants defective in intracellular survival within macrophages can be delineated using this assay.
0 Q&A 9481 Views Dec 20, 2013
Use of monoclonal antibodies (MAbs) is an established laboratory strategy for characterization of specific pathogens and their antigenicity. Especially, Human MAbs (HuMAbs) with neutralizing activity against specific virus could have potential therapeutic application, and provide significant information on human epitopes that could be important for developing the next generation of universal vaccines against the virus. In addition to the classical method for murine MAb preparation, several methods for the preparation of HuMAbs have been developed. Here, we describe the development of neutralizing HuMAbs against specific virus. HuMAbs are established by fusion of the peripheral blood mononuclear cells of vaccinated volunteers or patients with the fusion partner cell line, named SPYMEG. Then each of prepared HuMAbs is confirmed whether it can neutralize the specific virus by in vitro neutralization assay.
0 Q&A 9955 Views Nov 5, 2013
Stationary phase bacteria are highly tolerant to hydrogen peroxide. This protocol was developed to test the susceptibility to hydrogen peroxide killing in different Pseudomonas aeruginosa strains. This assay provides a reliable way to measure killing of stationary phase bacterial cells to hydrogen peroxide and can be adapted to test other oxidants.

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