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

JoVE Encyclopedia of Experiments

Microbiologie

0 weergaven • 3:27 min • September 26th, 2025

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Begin with an E. coli culture pre-treated with an antibiotic.

This antibiotic blocks cell division, resulting in the formation of elongated filamentous cells with increased DNA content.

Plate a portion of the culture onto a nutrient-rich agar plate and incubate.

Filamentous cells do not divide despite continued growth; only non-filamentous cells form colonies, resulting in low colony counts.

Dilute the remaining bacterial culture and add a fluorescent DNA-binding dye. Then, incubate in the dark.

The dye enters the cells and binds to DNA.

Vortex the sample and then pass it through a flow cytometer.

The amount of scattered light from each cell corresponds to its size.

Similarly, fluorescence emitted from dye-bound nucleic acids indicates the bacterial DNA content.

Antibiotic-treated cells show increased scatter and fluorescence, reflecting filamentous growth and continued DNA replication without division.

This demonstrates modifications in the bacterial physiology caused by cell division-inhibiting antibiotics.

Prepare ten-fold serial dilutions, up to 10 to the minus seventh, of the 200 microliters of culture sample in fresh medium.

Mix between each dilution by very gentle vortexing or by inverting the tube. Plate 100 microliters of the appropriate dilution on non-selected LB agarose plates and incubate overnight at 37 degrees Celsius. The next day, count the number of colonies to determine the concentration of viable cells in each culture sample.

Plot the CFU per milliliter as a function of time for the untreated and treated cell cultures. Measure the OD 600 of the culture on a spectrophotometer. Dilute the culture sample with fresh medium at four degrees Celsius to obtain a sample with OD 600 at 0.06, corresponding to approximately 15,000 cells per microliter.

For DNA staining, mix the diluted bacterial sample with a 10 microgram per milliliter solution of DNA fluorescent dye at the volume ratio of one to one and incubate in the dark for 15 minutes. Before injection of the sample, mix the sample by very gentle vortexing or by inverting the tube. Pass the sample into the flow cytometer at a flow rate of approximately 120,000 cells per minute.

Acquire forward-scattered and side-scattered light as well as DNA fluorescent dye/fluorescent signal with the appropriate settings.

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Laatst bijgewerkt: 15 augustus 2026