A 3D-Printed Silicone-Coated Device to Model Catheter-Associated Urinary Tract Infection

Take a multi-well plate containing sterile human urine.

Inoculate the test wells with bacteria. The control wells contain urine alone.

Take a 3D-printed, silicone-coated device mimicking a catheter’s inner surface, where biofilms develop during catheter-associated urinary infections.

Place it into the wells, seal the plate, and incubate it.

Bacteria attach to the device surface, utilize nutrients from the urine, and form biofilms.

Rinse the device with a buffer to remove loosely bound bacteria.

Place the device into a multi-well plate containing crystal violet, which interacts with the biofilm to stain it.

Rinse with water and dry it.

Immerse the device in a multi-well plate containing an acid solution to disrupt dye-biofilm interactions and release the dye into the solution.

Measure the absorbance of the solutions in each well using a spectrophotometer.

Higher absorbance in the test wells compared to the control wells confirms biofilm formation on the device, indicating its suitability to model catheter-associated urinary tract infection.

To begin, obtain pooled human urine sample. Aliquot 135 microliters of pooled human urine into each on-test well of a 96-well plate following the designated layout.

Inoculate each on-test well with 15 microliters of normalized overnight bacterial culture to achieve a final volume of 150 microliters per well. Reserve at least 100 microliters of this culture for subsequent serial dilution validation steps.

Prepare negative control wells by adding 150 microliters of sterile pooled human urine into designated wells.

Now, insert the pegs into their corresponding wells. Press down the peg lid until it sits flush on the plate and secure it with the microplate lid, taping it down if necessary.

Then place the peg models in a sealed plastic tub or bag containing a damp blue roll to prevent evaporation. Incubate statically for 24 hours at 37 degrees Celsius.

For inoculum validation, aliquot 180 microliters of PBS into rows 2 through 6 of the first column of a 96-well plate before adding the reserved culture.

Now, transfer 20 microliters from the first well into the second well, mixing gently by pipetting up and down. Continue serial dilutions until the initial inoculum has been diluted to the final well.

Then, plate 10 microliter spots from each dilution onto appropriate solid media in triplicate. After overnight incubation at 37 degrees Celsius, count the colonies from each spot.

For biofilm quantification, remove the peg model from the microplate and invert it under a flame or inside a microbiological safety cabinet, with pegs facing upwards. Discard the used microplates.

Transfer the peg lid to a fresh microplate containing 180 microliter aliquots of sterile PBS to rinse off unadhered cells.

Next, working aseptically, place a sterile glass test tube rack onto the bench and attach the fitted rack top that holds the peg lid.

With a micropipette, aliquot 600 microliters of sterile PBS into each test tube. Now, insert the peg lid into the rack, ensuring the pegs are aligned with the test tubes. Remove any autoclave tape from the lid.

Use sterile forceps to push each peg through the lid into the test tubes directly below. Then replace the test tube caps and vortex each tube for 60 seconds to dislodge biofilm-associated cells.

Remove 100 microliters from each vortexed tube and perform serial dilutions in sterile PBS as done previously.

To stain the cells with crystal violet, pipette 180 microliters of 0.1 percent weight per volume crystal violet in sterile distilled water into the 24 on-test wells of a 96-well plate. Insert the rinsed peg model into the corresponding wells and incubate at room temperature for 20 minutes.

After staining, remove the peg model from the plate and discard the used plate. In a clean tub, rinse the entire peg model in running tap water with gentle agitation.

After 2 more washes, invert the peg model and let it dry completely at room temperature. Then add 180 microliters of 30% acetic acid to a new 96-well plate. Insert the dried peg model into the corresponding wells.

When incubation is complete, remove the peg model from the microplate. Measure the optical density of the stained wells at 540 nanometers using a microplate reader or spectrophotometer.