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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. Experimental Design
- Start the treatment of animals by oral gavage, after 2 days of acclimatization. Use a syringe with the oral gavage needle. Treat 16 rats with Bacillus subtilis BSB3 suspension (1 ml per rat) twice a day for two days (B. subtilis group). Treat 16 rats with phosphate-buffered saline, PBS (1 ml per rat), twice a day for two days (PBS group) (Figure 1).
- After treatment, subdivide each group (8 rats per group): Group 1-control (PBS/no stress), Group 2-B. subtilis (B. subtilis /no stress), Group 3-stress (PBS/heat stress) and Group 4- B. subtilis + stress (B. subtilis/heat stress).
- Measure each rat's rectal temperature using an electronic digital thermometer. Keep rats of groups 1 and 2 at room temperature for 25 min. Place animals of groups 3 and 4 in a climate chamber at 45 °C and 55% relative humidity for 25 min.
- Afterwards, measure the rectal temperature of each rat in all groups using an electronic digital thermometer. Place all animals at room temperature for 4 hr.
- Anesthetize rats with isoflurane (2 - 4%) in a sealable anesthesia jar and observe until the rats are deeply anesthetized (absence of response to toe pinch). Euthanize rats by rapid decapitation with a guillotine.
- Collect trunk blood from each rat (15 - 16 ml) with the apyrogenic pipettes into apyrogenic tubes to obtain serum, and immediately take samples of blood (7 µl from each rat) with a pipette for microscopic examination.
- Put the tubes with the blood in a refrigerator for at least 30 min to allow clotting. Centrifuge the tubes at 20 °C, 7,000 x g for 10 min, and quickly remove the serum with a pipette. Aliquot the serum obtained from each rat into 50 µl volumes and store at -20 °C until the assay.
2. Surgical Procedure
- Cut/trim all fur from the abdomen and thorax of the underside of the carcass with electric shears. Place the carcass in a sterile cabinet and treat the shaved surface of the carcass with 70% alcohol.
- Use a sterile scalpel to create a midline incision of the abdomen. Remove liver, mesenteric lymph nodes, spleen and small intestine from each rat using sterile tweezers.
3. Analysis of Bacterial Translocation
- Place each sample of liver, mesenteric lymph nodes, and spleen into pre-weighed sterile tubes and weigh. Calculate the weight of the sample as the difference between the weight of a tube with a sample and the weight of an empty tube.
- Add sterile PBS to the tube to obtain a 1:10 dilution (weight per volume, w/v) of the sample and homogenize each sample with a sterile glass tissue homogenizer to obtain a homogeneous suspension.
- Make serial dilutions (10-1 to 10-4) of each sample in sterile PBS. Plate 0.1 ml of all dilutions of each tissue onto the surface of MacConkey's and 5% blood agar and Brucella blood agar with hemin (0.005 g/L) and vitamin K1 (0.01 g/L) plates.
- Incubate MacConkey's and 5% blood agar plates aerobically and Brucella blood agar plates under anaerobic conditions at 37 oC.
- Count colonies of aerobic bacteria after 24 hr, and colonies of anaerobic bacteria after 48 hr of incubation using a colony counter. Express the results as the number of colony-forming units (CFU) in a gram of tissue.
