This study investigates the effects of beneficial bacteria on gut barrier integrity during physiological stress in rats. The research highlights how these bacteria can reduce bacterial translocation into internal organs.
Begin with an experimental rat pre-treated with beneficial bacteria and a control rat administered phosphate-buffered saline or PBS.
Expose both rats to high temperatures to induce physiological stress.
In the control rat, heat stress disrupts the gut epithelial barrier, allowing gut-derived bacteria to translocate and spread into internal organs.
However, in experimental rats, beneficial bacteria release signaling molecules that preserve epithelial junctions and reduce bacterial translocation.
Take tubes containing the harvested liver samples from each rat.
Add PBS and homogenize the samples to release bacteria into the buffer.
Spread the diluted homogenate onto nutrient-rich agar plates.
Incubate the plates to allow bacterial colonies to form. Then, count the colonies.
Higher colony counts in the control sample indicate gut-barrier disruption and bacterial translocation.
In contrast, fewer colonies in the experimental sample indicate the effect of beneficial bacteria in limiting bacterial translocation into the gut.
After treating rats by oral gavage with B.subtilis BSB3 or PBS for two days, then subdividing the animals and taking their temperature, keep rats from groups one and two at room temperature for 25 minutes. Place animals of groups three and four in a climate chamber at 45 degrees Celsius and 55% relative humidity for 25 minutes.
Then, after euthanizing the animals, collecting the trunk blood, and isolating organs according to the text protocol add sterile PBS to each tube to obtain a one to ten weight per volume dilution of the sample, and use a sterile glass tissue homogenizer to generate homogeneous tissue suspensions.
Next, use sterile PBS to make serial dilutions of each sample. Then, plate 0.1 milliliters of all dilutions onto the surface of MacConkey's and 5% blood agar, and Brucella blood agar with hemin and vitamin K1. After incubating the plates, use a colony counter to count the colonies on each group of plates.
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