This article discusses the development of a bacterial biofilm model using pig bronchiolar tissue to study chronic lung infections. The methodology includes tissue preparation, bacterial inoculation, and biofilm formation under controlled conditions.
Bronchioles are the tubular airways of the lungs. During chronic infection, the bronchioles get clogged due to the accumulation of mucus. The nutrient-rich mucus provides a suitable environment for opportunistic bacteria to grow. These bacteria get embedded in a self-produced extracellular polymeric matrix and proliferate to form a biofilm.
To mimic bacterial biofilm development on the lungs, take a pig bronchiolar tissue specimen in a Petri dish. Dissect the tissue into pieces of the desired shape. Add a biofilm culture media to the Petri dish. Sterilize the dish under UV light to remove any surface contaminants.
Transfer the sterilized tissue pieces into a multiwell plate containing biofilm culture media and agarose pads, which serve as an optimum substrate for bacterial adhesion. Next, prick a needle tip containing a colony of the desired bacteria onto each tissue piece to introduce planktonic bacteria into the tissue. Seal the plate with a UV-sterilized, porous membrane that eases uniform oxygen exchange throughout the plate surface. Incubate the sealed plate.
During incubation, bacteria adhere irreversibly to the epithelial surface through pili and begin to secrete the extracellular matrix. Within the matrix, bacteria utilize the energy sources, mucin, and free DNA from the culture media to form biofilms. The bacterial biofilm model is ready for further testing.
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.
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