In Vivo Assay to Correlate Bacterial Bioluminescence with Cell Density

Take bacterial colonies transformed with a plasmid containing an inducible gene.

Pick a single colony to inoculate fresh liquid media.

Incubate with shaking for oxygenation and homogeneous bacterial growth.

Use a diluted culture sample to measure absorbance and estimate cell density.

Take a black-walled, glass-bottom well plate to prevent light cross-talk between samples, ensuring accurate luminescence quantification.

Add the bacterial culture with the desired cell concentration into the wells.

Add an antibiotic to maintain plasmid retention.

Add an inducer to initiate gene expression.

The gene encodes an enzyme that reacts with a specific bacterial protein to produce light, giving the bacteria bioluminescent properties.

Place a lid on the plate to prevent evaporation before placing it in a temperature-controlled plate reader with constant shaking mode.

At regular intervals, record absorbance to measure cell number and light intensity to monitor bioluminescence. Correlate cell density with bioluminescence.

Inoculate 3 milliliters of medium with a single colony from the overnight plate, and incubate the cells at 28 degrees Celsius, and 180 RPM for approximately one to two hours.

Measure the cel