Assessing Antibiotic-Induced Filamentation in E. coli Using Plating Assay and Flow Cytometry

Abstract

Source: Cayron, J., and Lesterlin, C. Multi-scale Analysis of Bacterial Growth Under Stress Treatments. J. Vis. Exp. (2019)

This video demonstrates the procedure for assessing E. coli filamentation and DNA replication under cell division-inhibiting antibiotic treatment using plating to measure viability and flow cytometry to analyze cell size and DNA content.

Protocol

1. Plating assay

NOTE: The plating assay measures the concentration of cells able to generate a colony-forming unit (CFU) in the culture samples. This procedure reveals the rate at which one cell divides into two viable cells and allows detection of cell division arrests (e.g., an increase in the bacterial generation time of cell lysis).

1. Prepare 10-fold serial dilutions up to 10^-7 of the 200 µL of culture sample in fresh medium. Plate 100 µL of the appropriate dilution on non-selective Luria-Bertani (LB) agarose plates to obtain between 3−300 colonies after overnight incubation at 37 °C.NOTE:

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	BioRad	1613100	Certified molecular biology agarose
Attune NxT Acoustic Focusing Cytometer	ThermoFisher scientific	A24858	Cytometer
E. coli strain carrying a chromosomal insertion for a hupA-mCherry fusion			Created by P1 transduction of hupA-mCherry in E. coli MG1655
Luria-Broth agarose medium	MP Biomedicals	3002232	Growth medium for plating assay
SYTO9 Green Fluorescent Nucleic Acid Stain	ThermoFisher scientific	S34854	DNA fluorescent dye