Bacteria ubiquitous in complex, three-dimensional porous environments, such as biological tissues and gels and subsurface soils and sediments. Here we develop a method to 3D print dense colonies of bacteria into jammed granular hydrogel matrices to study their growth and motility in complex environments. Studies have revealed previously unknown differences in spreading characteristics of bacteria inhabiting porous environments compared to those in liquid cultures on flat surfaces.
The development of using granular hydrogel matrices comprised of jammed biocompatible hydrogel particles swollen in liquid bacterial culture as porous Petri dishes to confine cells in 3D. Previous studies were limited to small sample volumes about one ml and therefore, short experimental time skills, and were also limited in their ability to define in ocular geometries with high spatial resolution. We have established that the cell spreading characteristics of the bacteria depend on the pore size and the motility of the cell.
To begin, connect the 3D printer to a computer. Load a one milliliter disposable syringe with a needle into the 3D printed clamps. Use two M8 socket bolts to secure the clamps around the syringe.
Rotate the lead screw to manually raise the plunger, creating a two milliliter air gap in the syringe. To prepare cultures of Vibrio cholerae, inoculate the cells into three milliliters of Luria Broth with 10 sterile glass bead. Grow the cells in a shaking incubator at 37 degrees Celsius for five to six hours.
Similarly, inoculate E.coli into three milliliters of liquid Luria Broth. After overnight incubation at 37 degrees Celsius, inoculate 200 microliters of the culture in fresh Luria Broth for three hours. Transfer the culture into a 10 milliliter centrifuge tube.
Then centrifuge it for five minutes at 644 g at room temperature. Now remove the supernatant and resuspend the pellet in 10 microliters of liquid Luria Broth. Next, load an empty three milliliter plastic Luer Lock syringe into the 3D bio-printer.
Connect the syringe plunger with the lead screw. Retract the syringe manually to transfer the air gap to allow for better movement of the plunger. Attach an appropriately sized blunt needle to the syringe tip.
Manually rotate the screw to retract the syringe plunger and load the bacterial suspension into the syringe. To prepare the granular hydrogel mix, mix dry granules of cross-linked acrylic acid with 400 milliliters of 2%Lennox Luria Bertani Broth. Adjust the pH to 7.4 with 500 microliter increments of 10 molar sodium hydroxide and test the pH on a test strip.
Use a 50 milliliter sterile plastic syringe to transfer the hydrogel mixture into a 50 milliliter centrifuge tube. Centrifuge the matrix at 161 g for one minute at room temperature. Next, use a 30 milliliter syringe to transfer the required volume of the matrix to a container where printing will occur.
Then place the sample containers with the hydrogel matrix to the holders on the build platform of the printer. Launch the 3D printing software. Click on File"then press, Open File"to load a pre-programmed gcode into the software.
Enter the move length. Then move the X, Y, Z planes to center the print heads on the X/Y plane. Press on the home icon to readjust the Z axis.
Manually rotate the screw slowly to depress the syringe plunger until a small amount of the bacterial suspension is seen at the needle tip. Wipe away the excess suspension with a sterile disposable wipe. Now lower the print head at a fixed distance into the hydrogel media of any sample container.
Click on Print"to start printing. Once the printing is complete, close the sample containers, dispose off the syringe and needle appropriately, wipe down the printer with 70%ethanol. For large field of view imaging, use a camera with an attached zoom lens.
Image the cells right after printing at room temperature, then transfer the samples to an incubator at 37 degrees Celsius. Differences in the pore size of the matrix did not appear to affect non-motile cellular spreading. However, motile cells spread faster in the matrix with larger pores relative to the smaller pores.
The colony of Vibrio cholerae in hydrogel matrices with larger pores spread through with smooth diffuse plumes. The diffuse plumes were absent in non-motile cells. The colony and hydrogel matrices with smaller pores spreads only through rough fractal-like plumes for both, motile and non-motile cells.
Both cells were observed to spread at similar rates for the first 150 hours. However, over longer periods, some samples exhibited faster rates of spreading. Decreased yield stress, storage moduli and loss moduli was observed in the hydrogel after the experiment.
