This protocol allows for evaluating the bidirectional regulating effect of probiotic supplementation on health and stable and reproducible results. An alternative DSS induced colitis model was established using pseudo germ free mice in order to avoid some laboratory limitations in using germ free mice as receptors for probiotics. To explore the beneficial effects of probiotics in this technique, it's a promising approach for developing novel treatment strategies for alleviating the symptoms of chronic inflammatory associated disorders.
To begin, prepare a cocktail of antibiotics by dissolving ampicillin, neomycin sulfate, metronidazole, and vancomycin in drinking water. After complete dissolution, store the solutions at four degrees Celsius. Put the antibiotic cocktail solutions into a water bottle.
Ensure to use brown bottles or wrap the bottles with aluminum foil to protect the antibiotic solutions from light. Place the bottle on top of the mouse cage. To make a 2.5%dextran sulfate sodium solution dissolve 2.5 grams of DSS in 100 milliliters of distilled water.
Replace the drinking water with the 2.5%DSS solution in the colitis model mice group. To assess the positive effect of B cereus on alleviating colitis, randomly assign the mice to the following three groups, the control group, the DSS induced colitis model, and probiotic B cereus supplementation for DSS induced mice. To explore the role of gut microbiota in regulating the probiotic effects of B cereus on DSS induced colitis, randomly assign the mice into the following three groups, ABX/control, ABX/DSS, and ABX/B cereus plus DSS.
Record the body weight of the mice as the baseline weight after the administration of the antibiotic cocktail. Immediately treat the male C 57 BL six mice with 2.5%DSS solution in drinking water for seven days. Prepare eight 1.5 milliliter micro centrifuge tubes filled with 900 microliters of sterile normal saline.
Pipette 100 microliters of logarithmic growth B cereus and dissolve in 900 microliters of sterile normal saline. Serially dilute B cereus using tubes. Plate 40 microliters of each dilution onto the respective LB agar plate labeled with the dilution ratio.
Repeat three times for each dilution ratio. Culture the plates in a 37 degrees Celsius constant temperature incubator for 16 hours. Select the plates with approximately 20 to 70 colonies for counting.
After the quantification of B cereus, collect one milliliter of B cereus medium containing one times 10 to the ninth CFU and centrifuge at 8, 000 G for 10 minutes. Then wash the bacterial cells two times with sterile water by centrifigation at 8, 000 G for 10 minutes and remove the supernatant. Dilute the concentrated B cereus with sterile normal saline to a final concentration of two times 10 to the eighth CFU per 200 microliters.
Pipette the dilution up and down repeatedly to acquire a dispersed B cereus suspension. Wipe the outside of the gavage needle with 70%ethanol and maintain proper dose application. To restrain the mice, pick them up by their tail and grasp the loose skin of the back firmly with the end of the tail fixed between the handler's last two fingers.
Keep the mice in a vertical position. Then insert the gavage needle carefully and gently through the mouth into the esophagus. Administer the B cereus solution, withdraw the needle gently, and return the mouse to its cage.
Measure the body weight of the mice daily and collect the feces samples immediately in a sterilized centrifuge tube. Determine the fecal occult blood by stool occult blood test paper. Collect 10 milligrams of stool sample on an applicator stick.
Put the specimen on the test card and apply a thin smear to the card. Open the back flap and apply the developer over the smear. Fix the mice supine on the operating table and open the peritoneal cavity.
Separate the entire colon surgically and measure the length with a ruler. Then gently wash the colon with a five milliliter syringe filled with pre-cooled PBS. De-wax and hydrate the colon sections.
Next, stain the colon sections with hematoxylin and eosin. Let two independent experimenters evaluate the histological scores of the sample under a light microscope, Hematoxylin and eosin staining revealed that the colon tissue of the colitis model mice presented crypt loss, goblet cell damage, inflammatory cell infiltration, and the loss of epithelium. B cereus treatment improved DSS induced histopathological damage.
The histopathological scores of colitis mice were significantly higher than in the control group. However, the histopathological scores in the B cereus group were significantly lower than in the colitis mice. Mice were randomly assigned into three groups, the body weight loss, DAI scores, and histopathology scores were higher and the colon length was shorter in the ABX/DSS group compared with mice in ABX/control group.
The body weight loss, colon length, DAI scores, and histopathology scores were not statistically different between the ABX/DSS and ABX/B cereus plus DSS groups. The present results indirectly suggest that the gut microbiota plays a key role in the positive effects of B cereus on colitis. One of the most important steps is to use Bacillus cereus by gavage method to construct the colitis modeling in mice.
The treatment of Bacillus cereus eliminated the DSS induced colitis, which led a foundation for the follow up study of probiotics for the treatment of colitis.
