In Vitro Persister Assay: A Method to Evaluate the Effect of Osmolytes on Bacterial Persistence

On exposure to harmful environment conditions, a subset of bacteria enter a state of low metabolic activity, evading the detrimental effects. These bacteria, called persisters, can tolerate transient exposure to high concentrations of antibiotics.

To study the effect of osmolytes - small organic solutes - on bacterial persistence, first, obtain an exponential phase bacterial cell culture with pre-existing persisters below the limit of detection, suspended in media.

Next, transfer the suspension into osmolyte-containing wells of a microarray plate. Keep a set of wells without osmolyte as control. Seal the plate with a gas-permeable membrane to allow rapid gas diffusion for cell growth and incubate.

The osmolytes in the medium at appropriate concentrations induce osmotic stress, triggering some cells to reprogram their cellular metabolism from rapid to slow growth, forming persisters.

Now, transfer the osmolyte-treated and control cells to an antibiotic-containing persister assay plate. Seal the persister assay plate and incubate.

In the presence of antibiotics, normal cells undergo cell death, whereas persisters - being antibiotic-tolerant - survive. Last, collect a small fraction of the cells, serially dilute with buffer, and spot onto an antibiotic-free agar plate. Incubate the plate.

In the absence of antibiotics, the persisters resume growth and proliferate, forming colonies. Count the number of colonies on the agar plate.

A higher number of colonies than no-osmolyte control post-antibiotic treatment suggests a positive effect of osmolytes on persister formation.