Quantification of Bacterial Infection in Host Cells by the Colony-Forming Unit Assay

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparing Cells for Infection

1. Propagate mouse macrophage-like cell line RAW 264.7 in DMEM tissue culture media supplemented with 10% fetal calf serum (henceforth referred to as DMEM/FCS) using a 125 mL flask and a tissue culture incubator maintained at 37 °C/5% CO₂.
2. The day before the infection, use a cell scraper to remove cells from the expansion flask and collect in a 15 mL screw cap conical tube. Centrifuge cells at 800 x g for 10 min, decant supernatant, suspend the cell pellet in 1 mL of DMEM/FCS (without antibiotics) and determine the titer using a hemocytometer or an automated cell counter.
3. Adjust titer to 2 x 10⁶ cells/mL and add 0.10 mL (2 x 10⁵ cells) to wells of a 48-well tissue culture dish. Ensure that the cell suspension covers the entire surface of the well.
   1. Plate cells in additional wells for a later source of conditioned media. Also, place 0.10 mL of DMEM/FCS in three additional wells to serve as a 'no cell control'.
4. Incubate overnight in a tissue culture incubator at 37 °C/5% CO₂. The next day, do not remove from the incubator until the bacterium inoculum is prepared and ready to use.

2. Preparing Listeria monocytogenes (Lm) for infection

1. Inoculate 2 mL of brain-heart infusion (BHI) with a single colony from a freshly-streaked agar plate (<2 days) of Lm and incubate overnight at 37 °C with shaking at 130 rotations/min.
2. The next day, add 0.10 mL of overnight culture to 2 mL pre-warmed BHI and continue incubating at 37 °C with shaking until the optical density (OD₆₀₀) is between 0.4 and 0.6.
3. Determine approximate bacterial titer using an empirically derived conversion formula. In our lab, an exponentially growing Lm culture in BHI is approximately 1.3 x 10⁹ colony-forming units (CFU)/OD₆₀₀. Minimize the amount of time bacterial cultures are exposed to room temperature.
4. Just prior to infection adjust the titer to 5 x 10⁷ CFU/mL using pre-warmed DMEM/FCS as diluent.

3. Infection

1. Working quickly and precisely, add 20 µL (1 x 10⁶ CFU; multiplicity of infection, MOI = 5) of the freshly-prepared Lm inoculum (step 2.4 above) to wells by slightly tilting the dish and carefully placing the pipet tip in the overlying media;
   NOTE: Avoid touching the walls of the well with the pipet tip.
2. Gently pipet the contents of each well to ensure complete mixing; do not shake or significantly tilt the plate as this will spread Lm over the walls of the well. Return the cell culture dish to a 37 °C/5% CO₂ incubator.
3. Prepare a 10 µg/mL gentamicin solution using conditioned media from uninfected wells as diluent.
4. At 30 minutes post-infection (mpi), carefully add 30 µL of the gentamicin solution (final concentration 2.5 µg/mL) and gently pipet the contents of the well as before and return the cell culture dish to 37 °C/5% CO₂ incubator.
5. At 60 mpi, harvest appropriate wells for either CFU assay (60 mpi time point) or FCM analysis (as described below in sections 4 and 5, respectively).
6. For remaining wells, at various times thereafter either harvest cells as before or, for the Lm emergence assay, entirely remove media from the well and replace it with 150 µL of gentamicin-free conditioned media.

4. Sampling Processing and Analysis of Intracellular and Emergent Lm by CFU Assay

1. To harvest, slightly tilt the dish and carefully remove the entire media from the well. Add 0.50 mL of distilled sterile water (dsH₂O) to the well. After 30 s, transfer the resulting water lysate (10⁰) to a 1.5 mL microcentrifuge tube and vortex vigorously for 10 s.
2. Prepare 10^-1 and 10^-2 dilutions by adding either 4.5 µL or 50 µL of the water lysate to 450 µL of dsH₂O and briefly vortex to ensure thorough mixing.
3. Spread 50 µL of the 10⁰, 10^-1, and/or 10^-2 samples onto lysogeny broth (LB) agar plates.
4. To assay for emergent Lm (step 3.6 above), remove 10 µL of the overlaying media and divide it into two 5 µL aliquots: to one aliquot add 5 µL of DMEM/FCS and to the second aliquot add 5 µL of DMEM/FCS containing 5 µg/mL gentamicin. The latter control distinguishes between extracellular Lm (gentamicin sensitive) and intracellular Lm (gentamicin resistant) in the subsequent CFU assay.
5. After 5 min at room temperature, add 90 µL of dsH₂O, vortex vigorously for 10 s, and spread 50 µL on LB agar plates.
6. Enumerate Lm colonies on LB agar plates following 2 days of incubation at 37 °C.