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

To harvest, slightly tilt the dish and carefully use a pipette to remove the entire media from the well. Add 0.5 milliliters of distilled sterile water to the well. After 30 seconds, transfer the resulting water lysate to a 1.5-milliliter microcentrifuge tube and vortex vigorously for 10 seconds.

Add 4.5 microliters and 50 microliters of the water lysate to 450 microliters of distilled sterile water to prepare the 10-fold and 100-fold dilutions. Briefly, vortex to ensure thorough mixing. Add 50 microliters of the original 10-fold diluted and 100-fold diluted samples onto lysogeny broth agar plates and spread using a plate spreader.

To assay for emergent Lm, extract 10 microliters of the overlaying media from the dish, and divide it into two 5-microliter aliquots in microcentrifuge tubes. Add 5 microliters of DMEM/FCS to one aliquot and add 5 microliters of DMEM/FCS containing 5 microliters per milliliter gentamicin to the second aliquot as control.

Wait 5 minutes at room temperature. Add 90 microliters of distilled sterile water to each aliquot and vortex the tube vigorously for 10 seconds. Then, add 50 microliters of the sample on LB agar plates, and spread using a plate spreader. Store the plates in 37-degrees-Celsius incubator for two days before enumerating Lm colonies.