In studies of the pathogenesis of ventilator-associated pneumonia, a close correlation has been found between bacterial biofilms of tracheal tubes and ventilator-associated pneumonia. Bacterial biofilm is a growth pattern formed by bacteria adhering to the surface of active or affective materials as opposed to planktonic cells, and consists of bacteria and their own secreted extracellular matrix, leading to difficulty in clearing infections in vivo. Bacterial colonization of tracheal ducts and formation of biofilms leads to increased bacterial drug resistance and difficulty in clearing the infection, thus becoming a persistent source of infection and an important cause of chronic infection.
Appliance
It is used to study the in vivo pathogenic mechanism of biofilm and to develop and study drugs against tracheal duct-associated bacterial biocoating and screening of their drugs.
Operation method
Establishment of an animal model of tracheal tube-associated bacterial biofilm infection
Materials and Instruments
Equipment: Move The modeling method for establishing an animal model of intravenous catheterization infection is divided into two categories with and without catheters, and the specific steps are as follows: A. Pick a single colony of Pseudomonas aeruginosa clinical isolate and inoculate it into L-Broth culture medium, incubate it at 37 ℃ and 200 r/min overnight. B. Determine the OD value of the bacterial liquid by spectrophotometer and adjust the OD value to 0.5. C. Anesthetize rats with 10% chloral hydrate 0.3 mL/100 g. Fix the limbs and head of the rats, cut off the fur from the lower jaw to the two front legs, disinfect the skin with iodine from the middle to the periphery, and then deiodinate with ethanol. D, Lift the skin with forceps and cut a small incision with small scissors, and then cut a small incision about 1.5 cm long from submandibular to chest right in the middle of the sterilized area. The superficial fascia is cut with scissors until the muscle is exposed, and the muscle is bluntly separated from the center with curved forceps until the trachea is exposed. E. Separate the tissues around the trachea, put the small tube pad under the trachea, cut a small opening of about 0.5 cm long vertically with small scissors, put the sterilized catheter into the trachea, and then use a small suture needle to pass through the nearest intercondylar muscle at the distal end of the tracheal incision and through the catheter, and then pass through the most distal intercondylar muscle at the proximal section of the incision. Tighten the suture first, observe the rat's breathing, and ligate if smooth. F, The bacterial fluid was slowly injected into the lungs through the catheter, and then the subcutaneous tissue was straightened and covered, and the skin was sutured and the wound was sterilized with ethanol. G, Execute the rats at different time points to remove the catheter to study the changes in the lungs. A. Pick a single colony of Pseudomonas aeruginosa clinical isolate and inoculate it into L-Broth culture medium, incubate it at 37 ℃, 200 r/min vibration overnight.
One set of routine surgical instruments, catheters, disposable infusion sets.
Reagents:
① strain; ② culture medium; ③ anesthetic (chloral hydrate); ④ sterile saline.
B. Determine the OD value of the bacterial solution by spectrophotometer and adjust the OD value to 0.5.
C. Inoculate the bacterial solution into a 24-well cell culture plate with a sterile catheter (about l cm, as a carrier for BF growth), 1 mL per well, and change the solution every other day to obtain mature BF after 7 days.
D. Anesthetize rats with 10% chloral hydrate 03 mL/100 g. Fix the limbs and head of rats. Fix the limbs and head of the rat, cut off the fur from the lower jaw to the two front legs, disinfect with iodine from the middle to the periphery, and then deiodize with ethanol.
E, Lift the skin with forceps and cut a small incision with small scissors, then cut a small incision about 1.5 cm long from the submaxilla to the chest right in the middle of the sterilized area. Cut the superficial fascia with scissors until the muscle is exposed, and bluntly separate the muscle from the center with curved forceps until the trachea is exposed.
F. Separate the tissues around the trachea, put the small tube pad under the trachea, cut a small opening of about 0.5 cm long vertically with small scissors, put the catheter with BF length into the trachea, and then use a small suture needle to pass through the nearest intercartilage muscle at the distal end of the tracheal incision and through the catheter, and then pass through the farthest intercartilage muscle at the proximal section of the incision. Tighten the suture first and observe the rat's respiration. If smooth, it can be ligated.
G. The subcutaneous tissue was straightened and covered, the skin was sutured and the wound was sterilized with ethanol. The rats were executed at different time points to remove the catheter and study the lung changes.For more product details, please visit Aladdin Scientific website.