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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Salmonella Typhimurium ΔAroA cultures for oral gavage of mice
- From a frozen glycerol stock of S. Typhimurium ΔAroA, prepare a streak plate using Luria-Bertani agar (LB agar) containing 100 μg/mL streptomycin with a sterile inoculating loop. Incubate overnight at 37 °C. Streak plates can be stored up to one week at 4 °C.
- One day prior to infection, prepare the antibiotic by dissolving 0.5 g of streptomycin in 2.5 mL of water. After filter sterilizing the streptomycin solution, orally gavage the mice using a bulb-tipped 22G gavage needle and 1 mL syringe with 100 μL of streptomycin solution (20 mg streptomycin/dose). Using an inoculating loop, inoculate 3 mL of LB broth (50 μg/mL streptomycin) in a culture tube with a single colony. Incubate Salmonella culture aerobically at 37 °C overnight with shaking at 200 Revolutions Per Minute (RPM).
- On the day of infection, prepare the final infection dose by performing 2 consecutive 1/10 dilutions of overnight Salmonella cultures in sterile Phosphate Buffered Saline (PBS). This would result in a 100 μL inoculum containing approximately 3 x 106 Colony Forming Units (CFU).
- Using a bulb-tipped 22G gavage needle and a 1 mL syringe, gavage each mouse with 100 μL of the prepared Salmonella.
NOTE: Prepare Salmonella cultures using aseptic techniques. Final inoculation concentration of Salmonella may be verified by plating serial dilutions on LB agar with streptomycin.
2. Assessment of Salmonella burdens in tissues
- Prepare 2 mL safe-lock, round-bottom microtubes with 1 mL of sterile PBS and an autoclaved stainless steel bead. Pre-weigh the tubes prior to tissue collection.
- Resect the cecal and splenic tissues from mice euthanized by carbon dioxide exposure. Collect tissue from individual animals in separate tubes. Weigh the tubes to determine tissue weights.
- Homogenize using a mixer mill apparatus for 15 min at 30 Hz. Transfer 900 μL of PBS per well in a 96-well 2-mL mega block. Pipette 100 μL of tissue homogenate into the first well, mix well, and perform serial dilutions by adding 100 μL to subsequent wells until a 10⁻⁶ dilution is obtained. Plate 10 μL of each dilution in triplicate onto LB agar containing 100 μg/mL streptomycin.
- Count and multiply average CFU by a factor of 100 since 10 μL of the 1000 μL sample was plated, and the appropriate dilution factor. Divide the tissue weight total CFU by the tissue weights to determine CFU per gram of tissue.
NOTE: Keep all samples on ice or at 4 °C during tissue processing. Use wide-orifice pipette tips when performing serial dilutions with tissue homogenates. Prepare the 96-well megablock containing PBS in advance.