Begin with a tube containing a bacterial culture in the exponential phase, ensuring uniform and actively growing bacteria for consistent results.
Centrifuge to collect the cells; aspirate the debris-containing supernatant, and resuspend the cells.
Introduce a cell-permeable red fluorescent dye. Dye molecules penetrate the bacterial cell membrane and reach the nucleoid, binding with DNA and imparting red fluorescence to the bacteria.
Centrifuge to pellet the stained bacteria and aspirate unbound dye-containing supernatant.
Resuspend the bacteria into a growth medium to maintain cell viability.
Using a photometer, measure the absorbance of the diluted bacterial suspension at 600 nanometers.
Compare the absorbance value with a standard curve to determine bacterial concentration.
Transfer the desired volume of the stained bacterial suspension into a fresh tube containing a growth medium. This is to achieve the desired bacterial concentration for the host cell infection representing the multiplicity of infection, or MOI.
The stained bacteria with the desired MOI are ready for immunological studies.
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