This article details a protocol for staining bacterial cultures to prepare them for immunological studies. The method involves using a cell-permeable fluorescent dye to visualize bacterial DNA and determining bacterial concentration for host cell infection.
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.
First, harvest the bacterial cultures at an exponential phase by centrifugation at 13,000 times g for two minutes at room temperature. After washing the cell pellet with one milliliter PBS, resuspend the bacterial pellet in one milliliter PBS.
Determine the concentrations of bacterial suspension by measuring optical density at 600 nanometers. Then, for staining the bacterial suspension, add two microliters of the 500-fold concentrated stock green or red staining dye into one milliliter of bacterial suspension to dilute the dye one-fold. Incubate the cells at room temperature for 30 minutes with gentle rotation in the dark.
After 30 minutes, centrifuge the stained bacteria at 13,000 times g for two minutes, and resuspend the pellet in one milliliter of PBS. Collect the stained bacterial cells by centrifugation at 13,000 times g for two minutes. Resuspend the pellet in one milliliter of fresh F-12K medium, and measure the optical density of each culture at 600 nanometers. Subsequentially dilute the cultures to the desired concentrations, based on the multiplicity of infection and host cell concentration.
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