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1. Prepare synthetic cystic fibrosis medium (SCFM2)
NOTE: Preparation of SCFM2 comprises three main stages outlined below (Figure 1).
<ol>
	<li>Sterilization of porcine mucin
	<ol>
		<li>Prepare sterile mucin at a final concentration of 5 mg/mL in SCFM2. For example, for a 5 mL volume of SCFM2, weigh 25 mg of Type II mucin in a sterile Petri dish, and place it into an ultraviolet (UV) sterilizer for 4 h, gently agitating every hour.</li>
		<...

an in vitro technique to study antimicrobial treatments on bacterial aggregates

Source: Gannon, A. D., et al. <a href="https://app.jove.com/v/62420">Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model.</a> J. Vis. Exp. (2021).
This video demonstrates an in vitro technique to study the impact of antimicrobial treatment on Pseudomonas aeruginosa (Pa) aggregates. Fluorescently-labeled Pa cells are grown as aggregates, mimicking the growth pattern during infection. Upon challenging the aggregates with a sublethal concentration of the antibiotic colistin, the real-time assessment of the bacteria-antibiotic interaction is performed via fluorescence microscopy.

<img alt="Figure 1" src="/files/ftp_upload/21646/21646_Figure1.jpg" /> 
Figure 1: Preparation and inoculation of SCFM2 medium. (A) Buffered base is prepared using salts and amino acids listed in Table 1 and Table 2. Buffered base can be stored at 4 &#176;C for up to 30 days, but must be protected from light exposure. (B) Mucin and DNA are added to an aliquot of buffered base and dissolved into solution overnight at 4 &#176;C. (C) Lipid and additional stocks are added...

An In Vitro Technique to Study Antimicrobial Treatments on Bacterial Aggregates

Source: Gannon, A. D., et al. Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model. J. Vis. Exp. (2021).
This video demonstrates ...

To study the impact of antimicrobials on bacterial aggregates, take Pseudomonas aeruginosa culture in a growth medium that promotes aggregate formation &#8212; simulating growth during infection.
The engineered bacteria express a fluorescent protein for aggregate detection under a fluorescence microscope. Image in three dimensions to compute the volume of the aggregates, or the bacterial biomass, and visualize the increase in biomass over time, indicating bacterial growth.
Add colistin &#8212; a cationic antimicrobial peptide &#8212; at a sublethal concentration that kills susceptible bacteria while selecting the antibiotic-tolerant ones.
Colistin binds to negatively-charged lipopolysaccharides, or LPS, on the bacterial outer membrane, displacing cationic bridges that stabilize the LPS monolayer &#8212; damaging membrane integrity.
Colistin enters periplasm and binds to LPS on the cell membrane, disrupting the membrane and resulting in cell lysis. As a counter mechanism, specific genetic mutations in a subset of bacteria neutralize the negative charge of LPS. This modification inhibits the electrostatic interaction of cationic colistin with less anionic LPS &#8212; preventing cell death.
Image to visualize decreased biomass with time, indicating the impact of the antimicrobial. Add propidium iodide or PI &#8212; a fluorescent dye &#8212; that enters through the damaged membrane to bind DNA, labeling only the dead cells.
Perform fluorescence-activated cell sorting, or FACS, separating PI-labeled susceptible cells and fluorescently-labeled antibiotic-tolerant cells. The cells are ready for downstream assays.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

This video demonstrates an in vitro technique to study the impact of antimicrobial treatment on Pseudomonas aeruginosa (Pa) aggregates. Fluorescently-labeled Pa cells are grown as aggregates, mimicking the growth pattern during infection. Upon challenging the aggregates with a sublethal concentration of the antibiotic colistin, the real-time assessment of the bacteria-antibiotic interaction is performed via fluorescence microscopy.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/21646/21646_Figure1.jpg" /> 
Figure 1: Preparation and inoculation of SCFM2 medium. (A) Buffered base is prepared using salts and amino acids listed in Table 1 and Table 2. Buffered base can be stored at 4 &#176;C for up to 30 days, but must be protected from light exposure. (B) Mucin and DNA are added to an aliquot of buffered base and dissolved into solution overnight at 4 &#176;C. (C) Lipid and additional stocks are added...

An In Vitro Technique to Study Antimicrobial Treatments on Bacterial Aggregates

In This Article

Overview

Protocol

Representative Results

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