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: Serial dilution in fresh medium must be performed rapidly to limit bacterial divisions. Alternatively, researchers might consider using a saline solution without a carbon source to prevent cell divisions during the dilution process.
2. The next day, count the number of colonies to determine the concentration of viable cells (CFU/mL) in each culture sample. Plot the CFU/mL as a function of time for untreated and treated cell cultures.

2. Flow cytometry

NOTE: The following section describes the preparation of cell samples for flow cytometry analysis. This analysis technique reveals the distribution of cell size and DNA content for a large number of cells. When possible, it is recommended to process the flow cytometry samples immediately. Alternatively, samples can be kept on ice (for up to 6 h) and analyzed simultaneously at the end of the day, once plating and microscopy imaging have been performed.

1. Dilute the 250 µL of culture sample to obtain 250 µL at a concentration of ~15,000 cells/µL (corresponding to an OD_600nm ~0.06) in fresh medium at 4 °C.
   NOTE: Incubation on ice will limit the growth and morphological modification of the cells. Alternatively, the user might consider performing fixation of the cells in 75% ethanol, as is usually recommended for flow cytometry.
2. For DNA staining, mix the bacterial sample with a 10 µg/mL solution of DNA fluorescent dye (ratio 1:1) and incubate in the dark for 15 min before analyzing the sample.
3. Pass the sample into the flow cytometer with a ~120,000 cells/min flow rate. Acquire forward-scattered (FSC) and side-scattered (SSC) light as well as DNA fluorescent dye fluorescence signal (FL-1) with the appropriate settings.
4. Plot the FSC and FL-1 cell density histograms to represent the distribution of cell size and DNA content in the cell population.

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