The overall goal of this procedure is to establish a chronic salmonella infected mouse model. This is accomplished by first preparing a salmonella solution. The second step of the procedure is to gavage mice with streptomycin, followed by salmonella or with streptomycin only.
The third step of the procedure is to isolate salmonella from mouse fecal matter, and detect salmonella infection in mouse intestine using BL chroma plates. The final step of the procedure is to observe and record mouse body mass and to note changes in vitality following salmonella infection. Ultimately, results can be obtained that show an infection of salmonella can be established in the intestine of mice gavage with salmonella.
Hi, I'm Michelle P from the laboratory of Dr.Johnson in Department of Medicine at University of Rochester. I'm also from the Sun Lab and I'm Y from the Sun Lab. Today we will show you a procedure for establishing Alytic Lennar infected mass model.
We use this procedure in our labor to study host bacteria interaction signal transduction and the tumor genesis. So let's get started. Before experimentation, prepare enough luria, berti, or LB broth plates for each salmonella strain and to use throughout the experiment.
The plates can be refrigerated and stored for several months. Next, prepare salmonella culture. Remove a vial of stock salmonella frozen at minus 80 degrees Celsius and let it thaw on the countertop to plate salmonella flame.
An inoculating loop in a buns and burner and dip into the thawed bacterial stock. Zigzag the loop back and forth on the top third of the LB plate. Flame the loop again and rotate the plate.
Zigzag the loop to cross the prior streaks and spread bacteria over the second third of the plate. Repeat once more. Flaming the loop again.
Do not let the loop penetrate into the agar. Allow colonies to grow by storing plates in a 37 degree Celsius incubator overnight. If done correctly, the salmonella plate should yield individual colonies in at least one section.
The next day. Pick a clone from the LB plate using a sterile toothpick or bacterial loop and put into seven milliliters of LB in a 12 milliliter tube. Shake the tube at 37 degrees Celsius for about five hours.
Use 0.05 milliliters of this culture to inoculate. 50 milliliters of LB in a Falcon tube. Incubate at 37 degrees Celsius without shaking for about 18 hours.
Spin the 50 milliliter bacterial overnight culture at room temperature at 5, 000 RPM for 10 minutes and discard the supernatant. Next, add 1.5 milliliters of Hank's buffered salt solution or HBSS to the tube to resuspend the bacteria or 100 parts lb to three parts HBSS. Then bring the total volume to 15 milliliters with HBSS or a one to 10 ratio of LB to HBSS.
Each mouse requires 100 microliters of diluted salmonella culture, or between one times 10 to the six to one times 10 to the eight colony forming units before starting gavage withdrawal water and food from mice four hours prior to experimentation. Then prepare a solution of streptomycin immediately before gavage by diluting 7.5 milligrams of powdered streptomycin in 100 microliters of HBSS. For each mouse injected, make sure to prepare some extra volume to account for loss before mouse gavage.
First, prepare a feeding needle with 100 microliters of streptomycin solution. Remove the first mouse from the cage to receive gavage and hold the mouse facing towards the bench top. Grab the skin over the mouse shoulder firmly with the thumb and middle finger, stretching the head and neck with the index finger to make the esophagus straight.
Direct the ball tip of the feeding needle along the roof of the mouth and toward the right side of the back of the pharynx. Then gently pass it down into the esophagus and inject the 100 microliters of streptomycin solution. No resistance should be felt.
Repeat this process for all mice experimental and control undergoing experimentation. After about 20 hours following strp de m and gavage and four hours before salmonella gavage, withdraw water and food from mice that will receive salmonella gavage orally. Gavage each mouse in the same manner as before, using 100 microliters of salmonella solution in HBSS.
After gavage, transfer each mouse to a new cage to begin the fecal collection. Starting around eight hours after gavage, remove the fecal matter from the cage immediately after dropping. Collect 100 milligrams of mouse fecal matter as experimental sample.
Collect samples daily or weekly depending on the study length for fecal matter salmonella analysis. Transfer the fecal sample to a 1.5 milliliter of micro centrifuge tube with one milliliter of PBS and vortex vigorously centrifuge for 10 minutes at 800 RPM. Transfer the supernatant into a clean 1.5 milliliter tube centrifuge at 6, 000 RPM for five minutes.
Discard the supernatant and add 200 microliters of PBS to the pellets vortex to resuspend. Next, detect salmonella with a BB l roag plate. Use 10 to 100 microliters of fecal matter solution per plate spreading on the plate as shown in section two.
The salmonella species appear mar in color due to metabolic differences in the presence of selected chromogens. Count the colonies of salmonella on the plate to compare the level of infection among individual mice. In this experimental procedure we used specific pathogen free female C 57 black six mice that were six to seven weeks old.
Mice were infected with salmonella and streptomycin plus HBSS. Mice were controlled gavage with streptomycin only salmonella strains used include salmonella typhimurium wild type strain, A TCC 1 4 0 2 8 S FP C, non-pathogenic salmonella mutant strain, and three FPC strains from Dr.James Madera's lab carrying the virulence associated AAV R age gene in PSKW 29, low copy plasmid. Using this protocol, salmonella tyrian colonization can be detected in the mouse intestine.
Chronic salmonella infection was measured by fecal matter analysis and body weight and intestinal tissue samples were collected for immunofluorescent staining. Typically body weight loss and death occur within four weeks post-infection. The survival proportions four weeks post-infection are shown here.
Once mice survive four weeks post-infection, the mice gain body weight and incidents of death decreases in the remaining 23 weeks. Overall, over 65%of mice infected with salmonella strains, FSY fsy, A VRA null or fsy. A VRA heterozygote can survive over six months with persistent salmonella infection in the intestine.
After the acute infection, the survivors still carried salmonella, which could be detected six months post-infection using the method described in this protocol. Immunofluorescence analysis of tissue samples from mouse intestine show the invasion of salmonella in intestinal mucosa at 27 weeks post-infection. In this image, the nuclei are stained blue and the salmonella infected are shown in green.
As shown in the control group, there is no bright green staining, which indicates no salmonella colonization. Whereas in the salmonella infected mice at 27 weeks post-infection, there is bright green staining in the intestinal mucosa. This shows the persistence of salmonella infection in the streptomycin pretreated mouse.
We have just show you how to induce chronic infection of some in the mouse model. When doing this procedure, it's important to remember to prepare some culture freshly for garage, correct garage skills and detect the existence of ANA to make sure your experiment model is working. So that's it.
Thanks for watching and good luck with your experiments.
