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

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 invert