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The overall goal of this procedure is to establish persistent long-term chronic pseudomonas aerogen airway infection in mice. This is accomplished by first culturing the selected pseudomonas aerogen bacterial strain. The second step is to embed bacteria into small agar beads by mixing bacteria with agar and mineral oil at 50 degrees Celsius, and then cooling down the mixture at four degrees Celsius under slow, continuous stirring.

Next, the mineral oil is removed by several washes with PBS and an aliquot of the agar beads is homogenate and plated on agar plates To determine the bacterial content, the final step is to inject a few microliters of the agar bead suspension into the trachea of mice to establish a chronic pseudomonas aerogen lung infection. Ultimately, bronchoalveolar lavage fluid and lungs can be recovered at different time points from challenge and plated on agar plates to detect the percentage of chronically infected mice and the bacterial load in the airwaves of survived mice. The main advantage of these aga bits methods over the existing methods is that it allows a stable long term chronic infection in a high percentage of mice leading to persistence of a stable bacterial load up to one month.

The AGA bit methods provide in the mouse lung the micro aerobic condition that allows bacteria to grow in the form of micro colonies. Similarly to the growth in the mucus of cystic fibrosis patients, these methods can help researchers in addressing key question on the pathogenesis of cystic fibrosis and other respiratory diseases and testing novel therapies against pseudomona sgin chronic lung infection. The methods can also be used to achieve chronic infection with other pathogens, including bur XO Apache or Staphylococcus Iris co-infection with different pathogens or competition Experiments are also possible demonstrating the procedure will be either the pheno, a postdoc, and Camil Riva, A PhD student in my laboratory Three days prior to the mouse challenge, inoculate a loop full of pseudomonas air gen from the minus 80 degrees Celsius stock culture to the try toay soy agar plate and incubated at 37 degrees Celsius overnight.

The day after. Pick a single colony inoculated in five milliliters of try toay soy broth and incubated at 37 degrees Celsius overnight in a shaking incubator at 200 RPM the next morning dilute a small aliquot of bacterial overnight culture in phosphate buffered saline at one to 50. Then measure the optical density at 600 nanometers.

Next, dilute the overnight bacterial culture by adding two OD of the bacterial suspension in a new tube containing 20 milliliters of fresh TSB incubated at 37 degrees Celsius for approximately three to four hours to log phase in a shaking incubator at 200 RPM until a total of 10 to 50 OD is reached. Once pseudomonas aerosa reaches the log phase, collect the bacterial cells by centrifugation at 2, 700 times, gravity for 15 minutes at four degrees Celsius and discard the supernatant. After that.

Resus suspend the bacterial pellet in one milliliter of sterile PBS and vortex thoroughly to resuspend it completely. Then mix one milliliter of the bacterial suspension with nine milliliters of liquid TSA pre equilibrated to 50 degrees Celsius. Add this mixture to the 50 degrees Celsius prewarm heavy mineral oil.

Immediately stir for six minutes at room temperature. The agitation must produce a visible vortex in the oil. Subsequently cool the mixture to four degrees Celsius, stirring at the minimum speed for 35 minutes.

Then rest in ice for an additional 20 minutes. Afterward, transfer the agar beads into 50 milliliter falcon tubes and centrifuge at 2, 700 times. Gravity for 15 minutes at four degrees Celsius.

Accurately remove the mineral oil and wash the agar beads pellet with sterile PBS. Repeat the procedure six times after three washes. The beads can be pelleted by gravity instead of using the centrifuge after the last wash.

Resuspend the agar beads in 20 to 30 milliliters of PBS aseptically homogenized 0.5 milliliters of the beads. Next, dilute 100 microliters of the homogenized beads in 900 microliters of sterile PBS and serially. Dilute the beads six times down to 10 to the minus sixth at a ratio of one to 10.

Then plate serial dilution on the TSA plates in including undiluted. Sample down to 10 to the minus sixth and incubate the plates at 37 degrees Celsius. Measure bead diameter using an inverted light microscope in several fields.

Bead diameter must be between 100 and 200 microns before mice challenge. Count the number of colony forming units on the TSA plates in order to determine the number of CFU per milliliter in the agar bead suspension. Then adjust the volume of the agar beads to two to four times 10 to the seventh CFU per milliliter to reach an optimal inoculum of one to two times 10 to the sixth in 50 microliters.

Place a six to eight week old male mouse previously anesthetized with ketamine and xylazine in a supine position on a heating pad, disinfect its coat with 70%ethanol. Expose the trachea by a vertical cut of the skin. Next, use tweezers to separate the muscles that are covering the trachea.

Then take up to 50 microliters of agar bead suspension by a one milliliter syringe. After that, intubate the trachea with a catheter and attach the one milliliter syringe with agar bead suspension to the catheter. Gently push the plunger of the syringe to allow the beads to be implanted into the lung.

Then close the incision using suture clips. In this step. Place the euthanized mouse in the supine position and disinfect its coat with 70%ethanol.

Next, expose the trachea and the thoracic cage by a vertical cut of the skin. After that, expose the lungs by cutting the diaphragm. Insert a suture thread under the trachea using tweezers and intubate the trachea with a catheter.

Then pull the two ends of the suture thread to bind the catheter to the trachea and not the thread around the trachea. Now take one milliliter of the cell culture medium using a one milliliter syringe and attach it to the catheter. Push the plunger of the syringe to wash the lungs immediately recover the liquid and store it in a 15 milliliter tube.

Repeat this step three times with a total of three milliliters of medium, and then store the bronchoalveolar lavage fluid on ice for the collection and analysis of the lungs. Excise the lungs from the mouse. Rinse them in sterile PBS.

Then separate the lobes. Put them in a round bottom tube with two milliliters of sterile PBS and store on ice. Aseptically homogenize the lungs and serially.

Dilute the homogenate in PBS at a ratio of one to 10. Then plate serial dilutions, including the undiluted sample on the TSA plates and incubate at 37 degrees Celsius overnight. Take the BAL fluid from the ice and dute it with Turk solution at a ratio of one to two under an inverted light optical microscope.

Count the total number of cells in a burker cell count chamber for differential cell count centrifuge the BAL fluid at 330 times gravity for eight minutes at four degrees Celsius. Next, discard the supernatant and resuspend the pellet at a concentration of 1 million cells per milliliter in cell culture medium containing 10%fetal bovine serum place. The microscope slides and filters in the appropriate slots in the cyto spin with the cardboard filters facing the center of it.

Pipette 150 microliters of each sample into the appropriate wells of the cytosine and centrifusion is cyto centrifuge at 300 times gravity for five minutes. Then a spot of cells should be observed after staining of the spot differential count can be performed at the microscope. This figure shows the time course of pseudomonas aerogen chronic infection with PA oh one reference strain and RP 73.

Clinical strain for each time point. Histograms represent the percentage mortality induced by bacteremia in red and survival in gray, or the percentage of animals that cleared the infection in white, and those able to establish a chronic infection in green. The pseudomonas aerogen RP 73 clinical strain is more efficient in establishing a chronic infection compared to the PA oh one laboratory strain.

Surviving mice were euthanized at the indicated time points and the lungs were harvested, homogenate and cultured on TSA plates to determine the bacterial load. The growth curves of P oh one and RP 73 strains in the murine lungs show that the amount of bacteria at three days post infection is higher for P oh one compared to RP 73. Then from seven days onwards when chronic infection is established, the bacterial load stabilizes at between 10 to the fourth and 10 to the fifth CFU per lung for both strains after seven days of chronic infection with RP 73 clinical strain, the inflammatory cell recruitment in BAL fluid was evaluated.

The number of total leukocytes is considerably higher in RP 73 infected mice compared to uninfected mice. The most represented cells are neutrophils indicating a considerable response by the innate immune system to chronic infection. In the box below, arrows indicate neutrophils and a macrophage as seen in a spot on a slide after cyto spin.

Shown here are hematin and eosin staining on histological sections of mouse lungs after seven days of chronic infection with rp. 73 clinical strain beads indicated by arrows can be observed in the bronchial lumen and micro colonies of bacterial cells. In the infected areas, the bronchia and the pulmonary parenchyma are characterized by massive inflammatory cell recruitment.

While the airways of an uninfected mouse were clear from inflammatory cells, The diameter and the final concentration of the BES are very important details. To achieve a good result, it's important to remember that be size is affected by the steering rate during the cooling phase, and that the starting amount of bacteria can affect the final dilution of the beads. Moreover, the selection of an appropriate pseudomona strain, which causes low mortality of mice and high percentage of chronic infection, will minimize the number of mice used.

After the infection of mice, bacterial count is performed. In addition, inflammatory response in terms of total and differential cell count In the bronchial VIR level, fluid cytokine analysis, melo peroxidase assay and histological analysis can be performed in order to study the ulcer response to bacterial infection. Don't forget that working with opportunistic pathogens like pseudomonas can beard and applications must be always taken, such as wearing gloves, handling bacteria under a biosafety cabinet, and using sterile techniques.

The development of appropriate animal models allowed the preclinical testing of novel antibacterial and anti-inflammatory molecules. This is an important step for future therapeutic interventions of lung infection in patients affected by cystic fibrosis or other respiratory diseases.