The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

1. Preparation of Citrobacter rodentium Inoculum and Oral Gavage of Mice

1. Prepare and autoclave Luria Bertani broth (LB).
2. Obtain viable C. rodentium from a frozen glycerol stock and streak onto LB agar plate using a sterile inoculating loop or pipette tip. Incubate at 37 °C overnight. Inoculate 3 ml of sterile LB broth in a falcon culture tube with colonies from the LB plate using an inoculating loop or pipette tip. Preparation of the inoculum should be done using aseptic techniques.
3. Incubate the C. rodentium culture aerobically at 37 °C overnight in a benchtop incubation shaker at 200 rpm. The inoculated LB broth should appear cloudy after overnight culture.
4. Use a bulb tipped gastric gavage needle attached to a 1 ml syringe to gavage each mouse with 100 μl of the overnight C. rodentium culture. Gently scruff the mouse, by firmly grasping the loose skin over its neck and back with thumb and fingers. Pull back the animal’s head with your index finger to immobilize the head and straighten the esophagus for insertion of the gavage needle.
5. Maintain the mouse in an upright position and direct the bulb tip of the gavage needle along the side of its mouth and over its tongue. Gently pass the needle along the roof of the mouth and advance it down the esophagus. If there is any resistance felt during this procedure, remove the gavage needle and re-insert it.
6. Slowly inject 100 μl of the bacterial inoculum and gently remove the gavage needle. Monitor the breathing rate and behavior of the mouse after returning it to its cage.

Notes:

• In steps 2 and 3, also prepare a falcon culture tube using aseptic techniques with 3 ml of sterile LB broth to be cultured overnight to ensure that there is no bacterial contamination of the broth itself. The LB broth should appear clear after overnight culture.
• Gently agitate the culture within the falcon tube prior to loading syringes for gavage so that an equal dose of bacteria is delivered to each mouse, in step 4.
• Overnight culture should be discarded if it has been inoculated for over 24 hr.
• Tous C. rodentium infections and housing of infected animals should be carried out in a Biosafety Level 2 facility.

2. Measuring Colonic Epithelial Barrier Permeability in C. rodentium-infected Mice

1. Measure barrier integrity at a desired time point post-infection.
2. On the day of the assay, prepare enough 4 kDa fluorescein isothiocyanate (FITC)-dextran dissolved in phosphate buffered saline (PBS) to a concentration of 80 mg ml^-1 to gavage each mouse with 150 μl and to prepare the standard curve.
3. Scruff the mouse and gavage with 150 μl of FITC-dextran. Withdraw food from the cage at this time.
4. Anesthetize mice 4 hr after gavaging and collect as much blood as possible (~450 μl) through cardiac puncture. Add the blood to a final concentration of 3% acid-citrate dextrose in a microcentrifuge tube to deter coagulation. Euthanize the mice and collect colonic tissues in PBS for bacterial counts (steps 3.1 – 3.5) and in 10% formalin for tissue fixation and ultimately immunofluorescence staining (steps 4.1 – 4.8).
5. Spin the blood samples at 1,000 x g for 12 min in a centrifuge at 4 °C. Collect the serum and dilute to 1/10 and 1/100 in PBS. Add 100 μl of each sample at these two dilutions to a 96 well plate in replicates.
6. To prepare the standard curve, dilute the original 80 mg ml^-1 FITC-dextran used to gavage the mice in PBS to the following concentrations: 800, 400, 200, 100, 50, 25, 12.5, 6.25, and 0 μg/ml. Add 100 μl of each concentration to a 96 well plate in triplicate.
7. Quantify the fluorescence of each sample using a fluorometer at an excitation wavelength of 485 nm and 535 nm emission wavelength.
8. Analyze the raw data by plotting the standard curve. Input the raw data for each sample into the equation of the line generated by the standard curve to determine the concentration of FITC-dextran in each sample.

Notes:

• From step 4 onward, keep blood samples on ice and minimize exposure to light.
• When measuring epithelial barrier integrity a time point at, or before, 7 days post-infection is optimal, as severe tissue damage occurs after this point. Measuring barrier integrity prior to severe damage occurring allows you to determine maximal differences in permeability during C. rodentium infection between mouse strains.
• Ensure that uninfected control mice are also tested for epithelial barrier integrity.

3. Measurement of Bacterial Load in Tissues of C. rodentium-infected Mice

1. Add 1 ml sterile PBS and an autoclaved metal bead to a round bottomed 2 ml centrifuge tube using aseptic technique. Individual tissues collected from each animal will be placed in separate PBS tubes. Weigh the tubes prior to addition of the tissue.
2. Remove the cecum and colon from the mouse. Separate the cecum from the colon and push out the luminal contents using forceps. Add the contents to a PBS tube. After separating 0.5 cm sections from the distal colon and cecum for 10% formalin fixation, cut open the remaining colon and cecum longitudinally and wash with sterile PBS before placing tissues in tubes.
3. Weigh the PBS tubes with the tissue and then homogenize the samples using a bead beater for 6 min at 30 Hz.
4. Using aseptic techniques, add 180 μl sterile PBS per well to a 96 well plate. Add 20 μl of each homogenized sample to the first well in each row. Mix well and serially dilute, adding 20 μl from each previous well to obtain dilutions from 10^-1 (first well) to 10^-6. Plate 10 μl of each dilution from each sample in triplicate on a square bottom LB plate with a grid. Incubate overnight at 37 °C.
5. Count colony forming units (CFU), then average the 3 counts, and record the dilution at which each sample was counted. Multiply average CFU by 50, as 20 μl of the original 1 ml sample was used, and by the dilution factor at which the sample was counted resulting in CFU/ml. Finally, divide by the tissue weight to determine CFU/g.

4. Histological Assessment and Immunofluorescence Staining of Infected Colon Tissues

1. Collect tissues as in step 3.2. Store tissues in formalin overnight, then wash with 70% ethanol. Embed tissues in paraffin and cut 5 μm sections for staining. Stain with hematoxylin & eosin for histological assessment and proceed to the following steps for immunofluorescence staining.
2. To deparaffinize tissues prior to staining, place slides in a coplin staining jar and put in 65 °C water bath for 10 min. Next, place slides in xylene for four washes of 2 min each. Slides should then be rehydrated with two 5-minute washes in 100% ethanol, followed by one 5 min wash in each of the following: 95% ethanol, 75% ethanol and dH₂0. These washes should be done in a fume hood to avoid exposure to harmful vapors.
3. Place slides in the coplin jar with pre-warmed sodium citrate buffer and then place into a steamer for 30 min, then let sit for 30 min. This process breaks the protein cross-links formed by formalin fixation retrieving potential antigenic sites.
4. Wash with PBS azide for 5 min, three times. Dry slides and mark area around tissue with a PAP pen to create a hydrophobic barrier, keeping the staining reagents localized on the tissue.
5. Block tissue for 1 hr at room temperature with blocking buffer (e.g. α-goat serum) in an immunostaining moisture chamber.
6. Dilute primary antibody to desired concentration using antibody dilution buffer. Pour off blocking buffer and add 50-100 μl of primary antibody to tissues. Incubate at 4 °C overnight or at room temperature for 2 hr.
7. Wash with PBS azide for 5 min, three times. Add 50-100 μl of secondary antibody diluted in antibody dilution buffer to the tissues and incubate at room temperature for 1 hr in the dark. Wash with dH₂O for 5 min, three times.
8. Dehydrate slides, add DAPI Prolong Gold mounting medium and apply a coverslip. View slides under a fluorescence microscope.

Notes:

• PBS azide can be substituted with freshly prepared PBS as an alternative to avoid bacterial growth in the wash medium from step 4 onward.