The overall goal of this experiment is to evaluate the preventative effect of the B.subtilis BSB3 strain against complications after heat stress. This method can help answer key questions in the prevention of adverse effect from heat stress. The main advantage of this technique is that it can be used to evaluate different approaches to mitigate the complications of heat stress.
Demonstrating this procedure will be Ludmila Globa. And Oleg Pusovyy, research associates from our laboratory. To begin, with a single colony of Bacillus subtilis BSB3 that was grown overnight on a nutrient agar plate.
Inoculate ten milliliters of nutrient broth in a flask. Grow the culture overnight at 37 degrees Celsius. Prepare 500 milliliters of sporulation medium according to the recipe shown here.
And autoclave at 121 degrees Celsius for 20 minutes. Allow the medium to cool to 50 degrees Celsius before adding the following sterile solutions. In a sterile cabinet, cool the prepared medium at room temperature to 40 degrees Celsius for 20 minutes.
And pour 25 milliliters into each of 20 sterile petri dishes. Allow the medium to solidify overnight. Next, spread 0.5 milliliters of the overnight Bacillus subtilis BSB3 culture onto the surface of the sporulation medium plates.
Incubate the plates at 37 degrees Celsius for five days until 90%sporulation. Then use high resolution microscopy to check for phase-bright spores. To harvest the bacteria, add one milliliter of PBS to the plates, and use a sterile cell spreader to scrape the bacteria from the surface.
Store the suspension at four degrees Celsius until needed. Confirm the viability of the bacteria by plating 0.1 milliliter of the appropriate dilution of a bacterial suspension on sporulation medium plates and incubate at 37 degrees Celsius for 18 to 24 hours. Use a colony counter to count bacterial colonies and calculate the titer of bacteria in the tested suspension.
Then prepare a bacterial suspension in PBS at a final concentration of one times ten to the eighth CFUs per milliliter. 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.
Following the measurement of the rectal temperatures of each rat again, place all animals at room temperature for four hours. Then, after euthanizing the animals, collecting the trunk blood, and isolating organs according to the text protocol, place each organ sample into pre-weighed sterile tubes and weigh. 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 MacConkeys 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.
Express the results as the number of colony forming units, or CFUs, per gram of tissue. After preparing and staining sections of the small intestines according to the text protocol, use a high resolution microscope to measure the intestinal villi and total mucose wall thickness. Analyze at least four samples from each rat and make at least 20 measurements in each sample.
To carry out high resolution light microscopy of blood samples, use the vibration isolation platform as a base for the microscope system with a video camera and computer to record live images. After calibrating test images according to the text protocol, place seven microliters of freshly drawn blood from each rat on a glass slide and add a coverslip. Then photograph and record ten image frames of 72 by 53.3 micrometers squared in each sample.
Finally, using software that provides high resolution direct view optical images in real time, measure the concentration of vesicles. The mean body temperature of animals before and immediately after heat stress was 36.7 plus or minus 07 degrees Celsius and 40.3 plus or minus 0.17 degrees Celsius respectively. Exposure of rats to heat resulted in significant inhibition of villi height and total mucosal thickness.
However, treatment with the BSB3 strain before stress, protected the intestine from the harmful effect of heat. For translocation of bacteria from the gut, mesenteric lymph nodes, liver, and spleen were analyzed. As illustrated here, bacteria were isolated at a concentration of 1.7 times ten to the three plus or minus 4.6 times ten to the two CFUs per gram of tissue only in samples from mesenteric lymph nodes and liver of PBS treated rats exposed to heat.
On the other hand, all tested samples from control and heat stressed animals that received the BSB3 strain were sterile. No change in IL 1beta, IL-6, TNFalpha, or interferon gamma cytokine levels were registered in heat stressed rats. However, IL-10 and LPS levels were elevated in PBS treated animals before heat treatment.
Therefore, B.subtilis BSB3 pre treatment prevented the rise of IL-10 and LPS in serum after heat treatment. Finally, a significant increase in the concentration of free vesicles was found in the blood of rats that received PBS before heat stress, but not in the BSB3 treated animals. It's important to remember to use a pyrogenic materials for blood collections and precision to keep serum liquids at minus 20 degrees Celsius that with repeated freezing and thawing, and to follow a sterile procedure during analysis of bacterial translocation.
Following this procedure like administration of bacteria after heat stress can be performed in order to answer additional questions about complications of heat stress.
