Ex Vivo Pig Lung Model of Biofilm: A Technique to Develop Lung Model to Study Bacterial Biofilms on Pig Bronchiolar Tissue
Transcription
Place the bronchiole in the first DMEM/RPMI 1640 wash. Leave it in the wash, and harvest additional sections of bronchiole from the same lung to yield sufficient tissue sections for the planned experiment. Place any additional bronchiolar sections from the same lung into the wash, and leave them in the wash for at least two minutes.
Remove the bronchioles from the first wash and place the samples in a sterile Petri dish. Lightly, hold each bronchiole with sterile forceps, taking care not to damage the tissue. Remove as much remaining soft tissue as possible, and cut the tissue into five-millimeter wide strips using sterile dissection scissors.
Place all of the bronchiolar tissue strips into the second DMEM/RPMI 1640 wash. Leave them in the wash for at least two minutes. Then, remove the tissue strips from the second wash using sterile forceps, and place them in a clean, sterile Petri dish. Remove any remaining soft tissue attached to the bronchiole, and cut the strips into squares using sterile dissection scissors.
Add the third DMEM/RPMI 1640 wash into the Petri dish, and lightly mix the tissue pieces in the wash by swirling the dish. Pour the third wash out of the Petri dish without removing the tissue pieces.
Then, add the final SCFM wash, ensuring that all of the tissue pieces are covered. Sterilize the tissue in SCFM under UV light for five minutes. Use sterile forceps to transfer each sterilized bronchiolar tissue piece into individual wells of a 24-well plate containing SCFM agarose pads.
To infect each tissue piece with the desired bacterial strain, touch a colony grown on an agar plate with the tip of a 29-gauge needle attached to a sterile 0.5-milliliter insulin syringe. Then, touch the colony onto the tissue piece, gently pricking the surface. For the uninfected controls, gently prick the surface of the tissue piece with the tip of the needle.
Then, use a pipette to add 500 microliters of SCFM to each well. Sterilize a breathable sealing membrane for each 24-well plate under ultraviolet light for 10 minutes. Remove the lid from the 24-well plate, and replace it with the breathable membrane. Then, incubate the plates at 37 degrees Celsius without shaking.
