Bacterial Cell Staining and Preparation for Immunological Studies

1. Bacterial growth and staining

1. Perform all bacterial work in a Biosafety Cabinet, Biosafety Level 2 laboratory.
2. Inoculate all bacterial cultures, including Pseudomonas aeruginosa (PAO1), Escherichia coli, Listeria monocytogenes,  Bacillus subtilis, etc., from the frozen glycerol stock and grow them in Tryptic Soy Broth (TSB, 3 mL) in a shaking incubator at 37 °C overnight maintained at 250 rpm.
3. The next day, use a 1:100 dilution of the overnight culture to inoculate a subculture and grow them in TSB (1 mL) for 3 h to the exponential phase, measure the optical density, OD at 600 nm (OD₆₀₀) to confirm. Ensure that the OD₆₀₀ is in the range of 0.4-0.6.
4. Prior to performing the bacteria-host adherence assay, plate serial dilutions of bacterial suspension onto TSB-agar plates, incubate them overnight at 37 °C and then establish the bacterial colony forming units, CFUs from the number of colonies. For P. aeruginosa, an OD₆₀₀ of 1.0 corresponds to 2 x 10⁸ viable bacterial cells/mL, and for L. monocytogenes, an OD₆₀₀ of 1.0 corresponds to 9 x 10⁸ viable bacterial cells/mL.
5. Harvest the bacterial cultures at exponential phase by centrifugation at 13,000 x g for 2 min at room temperature (RT), then wash them once using 1x phosphate-buffered saline (PBS 1 mL). Resuspend the bacterial pellets in 1 mL of 1x PBS and determine the concentrations by measuring OD₆₀₀ of bacterial suspensions. For example, P. aeruginosa with OD₆₀₀ of 0.5 represents a concentration of 1 x 10⁸ bacterial cells/mL.
6. Stain the bacterial suspension using either a green or a red fluorescent dye at RT for 30 min with gentle rotation in the dark. For this, add 2 μL of the 500-fold concentrated stock staining dye into 1 mL of bacterial suspension to dilute the dye 1-fold. To wash off the staining dye, centrifuge the stained bacteria at 13,000 x g for 2 min and resuspend the pellet in 1 mL of 1x PBS three times.
   NOTE: If fluorescence- (green fluorescent protein (GFP-), red fluorescent protein (RFP-), mCherry-, etc.) tagged bacteria are used in the experiment, then skip this bacterial staining step. GFP-tagged P. aeruginosa and red fluorescent dye-stained L. monocytogenes were used in this protocol.
7. Collect the stained bacterial cells or GFP-tagged bacteria by centrifugation at 13,000 x g for 2 min. Resuspend in fresh F-12K medium (1 mL) and measure the OD₆₀₀ of each culture. Subsequentially dilute the cultures to the desired concentrations based on the multiplicity of infection (MOI) and host cell concentration. The final volume used in this experiment is 500 μL.
   NOTE: For example, if the host cell concentration counted using Trypan Blue staining is 1 x 10⁵ cells/mL, the desired concentration of bacterial cells at an MOI of 100 will be 1 x 10⁷ cells/mL. Concentration of P. aeruginosa at 0.5 OD₆₀₀ is 1 x 10⁸/mL. To obtain the desired concentration, dilute the P. aeruginosa culture 10-fold, and add 50 μL of resuspended culture to 450 μL of fresh F-12K medium.