All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Bacterial Inoculum
- Streak a tryptic soy agar (TSA) plate with an inoculating loop for isolation of a single colony from a freezer stock of Escherichia coli O1:K1:H7-lux that stably expresses luciferase and carries kanamycin resistance. Incubate overnight at 37 °C.
- The following day, allow Luria broth (LB) to come to room temperature (25 °C) in a biosafety cabinet.
- Under a biosafety cabinet hood, identify a single colony from the streaked plate and inoculate it in 3 mL of LB supplemented with kanamycin (30 μg/mL). Incubate overnight at 37 °C with shaking at 220 revolutions per minute (rpm). This is the starter culture.
- Dilute the starter culture 1:100 into a fresh 3 mL of LB under a biosafety cabinet hood and return it to the incubator for 2-3 h at 37 °C with shaking (220 rpm). This is the stock culture.
- Read the optical density (OD) of both the blank and stock culture at 600 nm using a spectrophotometer. Add 100 µL of LB (containing no bacteria) into one well of a 96-well flat-bottom assay plate; this is the blank. Then add 100 µL from the stock culture to a separate well. Repeat for two additional replicates. The absorbance is read using a plate reader.
- Subtract the blank absorbance from the stock culture absorbance value (the OD value) and compare to a previously generated and validated growth curve to determine an approximation of the bacterial density in the stock culture for the preparation of the infectious dose.
- Generate target inocula depending on the research question. Target inoculum of 2 x 10⁶ (low) and 7 x 10⁶ (high) colony-forming units (CFUs) per mouse (/mouse) were used for this study.
- Divide the target dose per mouse (DoseT) by the estimated concentration of bacteria in the stock culture (Stock) to get the volume of bacteria needed from the stock tube (VS).
- Multiply VS by the number of mice (NM) that need to be infected, along with enough for 5-10 extras, for the total amount of bacteria required for the infection, plus 5-10 additional doses. Remove this volume from the stock tube and add it to a new centrifuge tube.
- Use the equation below:DoseT/Stock = VS x NM = total volume (VT) of bacteria to be removed from the stock tube.
- Centrifuge the bacteria at 2,000 x g for 5 min at 4 °C and resuspend the bacterial pellet in 50 µL of phosphate-buffered saline (PBS) (pH 7.2-7.6) per mouse to be infected (e.g., for 10 doses of 2 x 10⁶ bacteria each dose, the pellet of 2 x 10⁷ bacteria would be resuspended in 500 μL PBS). Again, it is recommended to prepare more inoculum than is needed. Prepare an equal volume of PBS only for control inoculations. Maintain the infectious inoculum and PBS control on ice until infection.
- Perform seven ten-fold serial dilutions into PBS in a 96-well plastic-bottom dilution plate, and plate 25 μL of the dilutions in duplicate onto quadrant TSA plates supplemented with kanamycin (30 µg/mL) to enumerate the actual amount of bacteria administered. Incubate at 37 °C overnight for colony formation prior to enumeration.
2. Animal identification
- Arrange a sufficient number of breeding pairs such that litters may be synchronized for age-matched pups. Age variability of ± 1 day is acceptable.
- Identify a pregnant C57BL/6 female mouse and monitor for the birth of the litter in advance of the planned experiment to accurately determine age.
- To distinguish between control and infected 3- or 4-day-old pups, use a pair of small, fine-tipped, iris scissors to snip the ends of the tails of the control pups only. The infected pups do not receive tail snips. Before cutting the tail, disinfect the skin with a cotton ball doused in 70% ethanol. Apply pressure to the end of the tail with a cotton ball or gauze as needed.
NOTE: This procedure is performed under a biosafety cabinet hood. A tail snip of approximately 1/8 of an inch is sufficient. - To identify pups within the control and infected groups, use a 1 mL insulin syringe with a 28 G x ½’’ permanent needle to tattoo the tails of the pups. Before tattooing, disinfect the skin with a cotton ball doused in 70% ethanol. This procedure is performed under a biosafety cabinet hood.
- To tattoo the tail, apply animal tattoo ink to the tip of the needle. Next, carefully restrain the pup with one hand, with its tail fully exposed. Gently insert the needle under the skin, while maintaining a superficial level of depth, and move the needle parallel with the skin a few millimeters until a small marking, or dot, has been created. Wait a few seconds before slowly removing the needle from under the skin, to avoid excess ink being released from under the skin.
- Apply pressure to the wound with a cotton ball or gauze as needed. Remove excess tattoo ink on the surface of the skin with 70% ethanol.
- Repeat this process with subsequent mice in the infected and control groups, while adding an additional dot with each successive pup tattooed (e.g., pup 1 will have 1 dot on their tail, pup 2 will have two dots on their tail, etc.).
NOTE: For an additional layer of identification, it is recommended to use separate colors of animal tattoo ink for the control and infected groups.
3. Subscapular inoculation
NOTE: For this study, 2 experiments were performed with a low-dose and high-dose group designated for each experiment. In the first experiment, 7 pups were given the low-dose inoculum (4 pups were used as controls), and 5 pups from a separate litter were given the high dose (3 pups were used as controls). The pups from experiment 1 provided data for only the 24 h time point. In the second experiment, 8 pups were given the low-dose inoculum (2 pups were used as controls), and 6 pups were given the high-dose inoculum (2 pups were used as controls). Pups from experiment 2 provided data for the 0, 10, and 24 h timepoints.
- Age match pups ≤ 1 day. Assign each litter as either a low-dose or a high-dose litter. Within a litter, randomly assign pups as a control or an infected pup.
- On day 3 or 4 postnatal, record the weights of all pups prior to inoculation with E. coli-lux or the PBS control. Separate the dam from the pups during this time to ensure they are not moved during the infection.
- Within a biosafety cabinet using an insulin needle, aspirate either PBS or the E. coli-lux inoculum. For this work, inocula of 2 x 10⁶ and 7 x 10⁶ CFUs per mouse were used. Keep both infectious inoculum and PBS on ice until administration via subscapular injection.
- Place the neonate on a clean surface in the biosafety cabinet hood and raise the skin at the nape of the neck as if to scruff the pup.
- In the space now created between the skin and the muscle of the animal, insert the needle, bevel up, just beneath the skin, and inject 50 µL of PBS or E. coli-lux. Simultaneously release the pinched portion of skin to prevent injection backflow.
- Remove the needle slowly and with care. Place pups back with dams after injections are finished.NOTE: Due to their anatomical stage in development, it is technically challenging to administer a tail vein or intraperitoneal injection to neonatal pups at day 3-4. Thus, the subscapular infection route was chosen for this study due to the ease of execution.
