In Vitro Parallel-Plate Flow Model to Study Bacterial Adhesion on a Tissue Biopsy

Begin with a perforated microscope slide and place a circular tissue biopsy over the perforated area.

Next, place a rubber gasket over it.

Flip and insert this assembly into the bottom section of the metal perfusion chamber frame.

Align the top frame with the bottom and secure them.

Use a caliper to confirm the uniform chamber height.

The chamber is connected to a pump, which in turn is attached to a reservoir containing bacteria suspended in buffer.

Start the perfusion of the bacterial suspension into the chamber.

The central perforation in the slide allows the entry of the bacterial suspension into the assembly.

This enables the interaction between bacteria and the tissue surface, promoting adhesion and colonization.

This setup generates an in vitro model that mimics in vivo bacterial colonization on the tissue.

To begin the protocol, place a previously prepared tissue biopsy 10 millimeters in diameter, and the same thickness between a microscope slide and an eight millimeter circular perforation, and a rubber gasket with the inner surface facing up, to contact the bacterial suspension.

Focus on the tissue graft orientation while placed in the holder. Make sure that the inner surface of the tissue is already exposed to the bloodstream faces up to contact your experimental medium.

Next, insert your holder with the tissue into the gasket sheet that is embedded in the bottom metal frame of the chamber.

Attach the upper metal frame with the corresponding gasket sheet onto the bottom of the chamber with the previously inserted tissue holder. Then mount the entire chamber with eight screws and screw nuts. Make sure that the chamber height is always the same across grafts by using a caliper or ruler.

Next connect the flow chamber with a peristaltic pump. Then connect the 400 milliliter fluid reservoir with the tubes. Perfuse the tissues with 100 milliliter suspensions of ten to the seven fluorescently-labeled colony forming units in phosphate buffered saline with the sheer stress of three dines per square centimeter and corresponding flow rate of four milliliters per minute for one hour using the peristaltic pump and the bacterial reservoir. Recirculate continuously the 100 milliliter bacterial suspension using the same collection reservoir.