Assessing Phage-Mediated Bacterial Growth Inhibition Using Optical Density Measurements

Begin with a multiwell plate containing a serially diluted phage suspension in nutrient-rich broth as the treatment and phage-free broth as the control.

Add E. coli to each well. Using a spectrophotometer, measure the baseline optical density (OD) to record the initial bacterial concentration.

Next, incubate the plate to allow bacterial growth and phage infection.

In the control, bacteria utilize nutrients and proliferate, increasing OD.

In the treatment, phages bind to bacterial surface receptors and inject their genomes into the cytoplasm.

Using the host cell machinery, the viral genome replicates and produces viral proteins, which assemble into new virus particles. These particles lyse the cell, releasing progeny.

Measure OD at intervals to compare bacterial growth.

Control wells show a progressive increase in OD, while phage-treated wells exhibit lower OD values, with greater reductions at higher phage concentrations.

This helps determine the phage concentration required for maximum bacterial growth inhibition.

To begin with, set up the microplate assay using filtered tips to avoid cross contamination. First, add 180 microliters of mTSB in the wells of columns one to 12 of the 96-well microplate. Then prepare serial tenfold dilutions of each phage in columns one to eight of the sterile 96-well microplates to set up the assay. Add 20 microliters of individual phages or phage cocktails to wells one to eight of the top row A of the microplate.

For phage-free and blank control, add 20 microliters of mTSB to the top well of columns nine to 12. While pipetting, mix the well contents with gentle and repeated aspiration at least five times and changing tips between dilutions. Remove 20 microliters from the last row H, then set up a reservoir for the tested bacterial strain by transferring two to three milliliters of the diluted culture into the reservoir using a one milliliter pipette.

Using a multichannel pipette, add 20 microliters of the diluted bacterial culture to each well in columns one to 10 while changing tips between each addition. Cover and incubate the microplate at 37 degrees Celsius. At two hour intervals, remove the microplate from the incubator and read the plate using a microplate reader.

Alternatively, incubate the plate in a microplate reader and read it over time.