R.K.B. acknowledges support from the Presidential Postdoctoral Research Fellows Program. This material is also based upon work supported by NSF Graduate Research Fellowship Program Grant DGE-2039656 (to A.M.H.). A.S.D.-M. and H.N.L. acknowledge support from the Lidow Independent Work/Senior Thesis Fund at Princeton University. We also thank the laboratory of Bonnie Bassler for providing strains of V. cholerae. S.S.D. acknowledges support from NSF Grants CBET-1941716, DMR-2011750, and EF-2124863, as well as the Eric and Wendy Schmidt Transformative Technology Fund, the New Jersey Health Foundation, the Pew Biomedical Scholars Program, and the Camille Dreyfus Teacher-Scholar Program.

This approach is to convert a commercial 3D fused deposition modeling printer into a 3D bioprinter using a previously established protocol by Tashman et al.34. In brief, Tashman et al. replaced a commercial extruder head with a custom-made syringe pump extruder. This extruder enables the printing of highly concentrated liquid suspensions of bacterial cells in 3D, with its extruded volume and 3D position controlled by the G-code programming language. The extruded volume is specified in the software by the extruder step (E-step) and is additionally calibrated as described further below. These bacterial suspensions are thereby printed directly into a granular hydrogel matrix, which acts as a 3D support for the cells. Below, the protocol also describes how to prepare matrices with different polymer concentrations, characterize the resulting changes in pore size and rheological properties, and characterize subsequent bacterial motility and growth using direct imaging.
1. Conversion of a commercial 3D printer into a 3D bioprinter
<ol>
	<li>Remove the extruder and the heater from a commercial 3D printer (see Table of Materials).</li>
	<li>Follow previous protocols to fabricate the syringe pump extruder34, with an additional modification to accommodate a disposable Luer lock syringe. Mount the syringe pump extruder onto the printer. 
	NOTE: The CAD files required for modifying the syringe pump extruder for plastic syringes are provided in&#160;Supplementary Files 1-3.</li>
	<li>Install and open the 3D printer software (see Table of Materials) on a computer. Connect the 3D printer to the computer.</li>
	<li>Load a 1 mL disposable syringe with an appropriately sized needle into the 3D-printed clamps by aligning the top and bottom halves of the mechanism (Figure 1). Secure the clamps around the syringe using three M8 socket bolts and thin steel hex nuts (see Table of Materials). The syringe plunger connects with the lead screw in the syringe pump extruder. Manually raise the plunger by rotating the lead screw to create an 0.5 mL of air gap in the syringe.</li>
	<li>If contamination is a concern for the experiment, transport the syringe-clamp complex to a biohood and sterilize with 70% ethanol spray upon entry before completing the following steps.</li>
</ol>
2. Preparation of the bacterial suspension
<ol>
	<li>For V. cholerae and E. coli, grow&#160;overnight on a 2% Lennox LB (Luria Broth, see Table of Materials) agar plate at 37 &#176;C.
	<ol>
		<li>For V. cholerae, inoculate the cells into 3 mL of liquid LB with ten sterile glass beads. Grow the cells in a shaking incubator at 37 &#176;C for 5-6 h until the mid-exponential phase to an optical density (OD) 600 of ~0.9.</li>
		<li>For E. coli, inoculate the cells into 3 mL of liquid LB. Grow the cells in a shaking incubator at 37 &#176;C overnight. Innoculate 200 &#181;L of the overnight culture in fresh LB for 3 h until the OD reaches 0.6.</li>
	</ol>
	</li>
	<li>Transfer the culture into a 10 mL centrifuge tube. Centrifuge the culture for 5 min at 5,000 x g at room temperature to form a pellet. Remove the supernatant. Resuspend with ~10 &#181;L of liquid LB to achieve a cell density of ~9 x 1010 cells per mL.</li>
</ol>
3. Loading the bacterial suspension into the syringe
NOTE: Two methods are provided for loading the bacteria into the syringe (step 3.1 and step 3.2). Step 3.1 works for loading small volumes of bacterial suspensions, &#60;200 &#181;L, and step 3.2 works for loading larger volumes of bacterial suspensions, &#62;200 &#181;L. Step 3.1 was utilized for the representative results shown here.
<ol>
	<li>Load an empty 1 mL plastic Luer lock syringe into the 3D bioprinter. Connect the syringe plunger with the lead screw. Manually retract the syringe to add 0.2 mL of the air gap to provide space for the plunger to move in the syringe as a small volume of cells ~20-50 &#181;L are used for each batch of experiments.
	<ol>
		<li>Attach a blunt needle to the syringe tip with the needle size necessary for the size of print features required. Here, a 2-inch 20 G needle is used.</li>
		<li>Load the bacterial suspension into the syringe by placing a 10 mL centrifuge tube with the bacterial inoculum below the needle. Manually rotate the screw to retract the syringe plunger and load the cells into the syringe. The bacterial cell volumes are so small that often, the cells are only loaded into the needle.</li>
	</ol>
	</li>
	<li>Remove the plunger from the syringe-clamp complex and use another syringe and needle to carefully load the syringe-clamp complex with the desired bacterial suspensions, being careful to avoid trapping air bubbles. The syringe-clamp complex should be filled slightly over the brim with the desired bacterial suspension and then transferred to the bioprinter.
	<ol>
		<li>Carefully insert the syringe-clamp complex without the plunger into the corresponding socket on the main core of the bio-printer extruder.</li>
		<li>Ensure that the printer carriage is approximately halfway up the lead screw, and a collection dish is present under the loaded syringe. Then, carefully insert the plunger through both the carriage and the syringe-clamp complex until it catches on the carriage. Depress the plunger slowly into the bacterial suspension to avoid trapping air bubbles in the syringe.</li>
		<li>Slide the adaptor clamp onto the carriage over the back of the plunger to secure it in place for both extrusion and retraction maneuvers.</li>
	</ol>
	</li>
</ol>
4. Calibration of the extruder step to deposited volume
<ol>
	<li>To calibrate the extruder step (E-step) to the deposited volume, first set up the bio-printer with the exact syringe, syringe needle, and depositing bacterial suspension that will be used in the experiment. Here, a 1 mL Luer lock syringe is used.</li>
	<li>Determine an estimated E-step range to calibrate over by extruding an arbitrary E-step number (~200) and note the plunger volume change with syringe volume markers.</li>
	<li>Use this coarse E-step to volume ratio to determine the E-step settings to perform the calibration sweep over. For example, if an E-step of 200 extrudes approximately 20 &#181;L by visual inspection and one wants to deposit 10-200 &#181;L, test E-steps between 100-2000.</li>
	<li>To perform the linear calibration sweep, first label and measure the dry mass of twenty 1.5 mL sampling tubes on an analytical balance with 0.1 mg sensitivity.</li>
	<li>Extrude bacterial suspension into the pre-measured 1.5 mL tubes. For each E-step, perform at least 2 replicates. Repeat for all E-steps over the linear range, replacing bacterial suspension as necessary. If contamination is a concern for the experiment, wipe the exterior of the syringe needle with a lint-free wipe saturated with 70% ethanol after each sample.</li>
	<li>Measure the mass of all 1.5 mL tubes with the same analytical balance. Subtract the first mass value from the second to obtain a net mass of bacterial suspension deposited.</li>
	<li>Convert the mass of bacterial suspension into a volume with the material density. For many bacterial suspensions composed primarily of water, 1 g/mL is an appropriate density approximation.</li>
	<li>Perform a linear fit between the E-step and the extruded volume to finish the calibration process.</li>
</ol>
5. Preparation of the granular hydrogel matrix
<ol>
	<li>In a biosafety cabinet, add dry granules of cross-linked acrylic acid/alkyl acrylate copolymers (see Table of Materials) to 400 mL of 2% Lennox Luria-Bertani (LB) to keep the matrix sterile; however, other liquid cell culture media can also be used to swell the hydrogel matrix. 
	NOTE: The weight percent of granules added to the LB depends on the pore size that one is aiming for. In the present study, for a 0.9% granular hydrogel matrix, 3.6 g of dry granules are added to the LB and for a 1.2% granular hydrogel matrix, 4.8 g of dry granules are added to the LB. The hydrogel granules are homogenously dispersed by mixing them in a stand mixer for 2 min.</li>
	<li>Once mixed, adjust the pH to 7.4 by adding 20 to 500 &#181;L increments of 10 M sodium hydroxide (NaOH) to ensure cell viability. After each addition of NaOH, measure the pH by dipping a pipette tip into the mixture and then wiping the hydrogel matrix onto a pH test paper. 
	NOTE: As the NaOH is added, the viscosity of the mixture will increase as the hydrogel granules start to swell. The swollen hydrogel granules are ~5 &#181;m to 10 &#181;m in diameter and jammed together in a hydrogel matrix. The internal mesh size of the granules is ~40 nm to 100 nm, as previously established32. The mesh size is large enough for small molecules (e.g., oxygen and nutrients) to freely diffuse but small enough for bacteria to be confined between the interstitial pores.</li>
	<li>Next, transfer the granular hydrogel matrix to a 50 mL centrifuge tube using a 50 mL sterile plastic syringe. Centrifuge the hydrogel matrix at 161 x g for 1 min at room temperature to remove the bubbles formed during the mixing process.</li>
	<li>Allow the hydrogel matrix to sit for at least 2 days at room temperature to ensure that no contamination has occurred. The contamination appears as microcolonies suspended in the hydrogel matrix. After two days, centrifuge the hydrogel matrix at 161 x g for 1 min to remove any additional bubbles that formed. 
	NOTE: The protocol can be paused here by storing the hydrogel matrix at room temperature for up to a week.</li>
	<li>In the biosafety cabinet, using a 30 mL sterile plastic syringe, transfer the desired amount of hydrogel matrix to the container where printing will occur (here ~20 mL for a 20 mL tissue culture flask or 1 mL for 1 mL plastic micro cuvettes were used).</li>
</ol>
6. Characterization of the granular hydrogel matrix rheological properties
<ol>
	<li>Load ~3 mL of the hydrogel matrix into a shear rheometer (see Table of Materials) with a 1 mm gap between roughened 50 mm diameter parallel plates to measure the rheological properties.</li>
	<li>Quantify the yield behavior using unidirectional shear measurements on the shear rheometer by measuring the shear stress as a function of a logarithmic sweep of shear rate from 10-4 s-1 to 102 s-1 (e.g., Figure 2A). 
	NOTE: At low shear rates, the hydrogel matrix will have a constant shear stress (the yield stress) that is independent of the shear rate. At high shear rates, the shear stress will increase with a power-law dependence on the shear rate, indicating the fluidizing of the hydrogel matrix. This yield-stress behavior allows bacteria to be 3D printed within the granular hydrogel matrix29.</li>
	<li>Measure the storage and loss moduli, G&#39; and G&#39;&#39; respectively, as a function of frequency using small amplitude oscillatory rheology with a strain amplitude of 1% and frequencies between 0.1 to 1 Hz (e.g., Figure 2B). 
	NOTE: The ideal granular hydrogel matrix for 3D printing should have a storage modulus larger than the loss modulus, which indicates the medium acts as a jammed elastic solid29.</li>
</ol>
7. Characterization of the granular hydrogel matrix interstitial pore size
<ol>
	<li>Sonicate 100 nm carboxylated fluorescent polystyrene nanoparticles (~3.6 x 1013 particles/mL, see Table of Materials) in their packaging for 15 min to resuspend to break up any aggregations/clusters of particles. Transfer 50 &#181;L of nanoparticles to a 1.5 mL microcentrifuge tube.
	<ol>
		<li>Centrifuge for 10 min at 9,500 x g at room temperature until the pellet forms and the supernatant is clear. Remove the supernatant and resuspend the pellet in 1 mL of the liquid growth media (here LB) used to prepare the granular hydrogel matrix. 
		NOTE: The protocol can be paused here by storing the resuspended nanoparticles at 4 &#176;C for up to 3 months.</li>
	</ol>
	</li>
	<li>Sonicate the resuspended nanoparticles for 30 min. Transfer 1 mL of granular hydrogel matrix to a 1.5 mL microcentrifuge tube. Add 1 &#181;L of the resuspended nanoparticles to the granular hydrogel matrix and mix them with a pipette tip. After mixing, centrifuge for 30 s at 161 x g at room temperature.</li>
	<li>Transfer the hydrogel matrix and the nanoparticle mixture to a 35 mm diameter Petri dish with a 0.1 mm thick glass bottom well. The well is 20 mm in diameter and 1 mm in depth. Place a glass coverslip on top and press down to disable flow and evaporation during imaging. An alternative to the glass coverslip is to add 1 mL of paraffin oil on the top.</li>
	<li>Image the nanoparticles using a confocal microscope with a 40x oil objective with 8x additional zoom in the imaging software (see Table of Materials). 
	NOTE: A higher magnification objective could be used instead of using additional digital zoom in the software.
	<ol>
		<li>Image a time loop with no delay (ideally ~19 frames/s) in a single z-plane for 2 min with at least four nanoparticles within the field of view. Repeat 15 to 20 times in different locations to collect enough data for statistics (100 to 200 nanoparticles).</li>
	</ol>
	</li>
	<li>Use a particle tracking software to analyze the displacement of the particles. Here, a custom-written script is used based on the classic Crocker-Grier algorithm to track the center of mass of the nanoparticle35 (see Supplementary File 7).
	<ol>
		<li>From the particle tracking, calculate the mean square displacement (MSD). The MSD will exhibit free diffusion in the pore space at short lengths and time scales and transition to sub-diffusive scaling at large lengths and time scales due to confinement35.</li>
	</ol>
	</li>
	<li>Identify the length scale where the transition to sub-diffusive scaling occurs to estimate the local pore size. Calculate pore size by adding this length scale to the nanoparticle diameter. Repeat the pore size analysis for every nanoparticle measured. This will yield a pore size distribution from which a mean pore size can be calculated (e.g., Figure 3).</li>
</ol>
8. 3D printing process
<ol>
	<li>3D-print custom-made holders for the sample containers (see Supplementary Files 4-6 for the CAD files). Here, holders for the tissue culture flasks and microcuvettes are used. The holders allow for programming the printer to print multiple samples in a single print session. Place the sample containers with hydrogel media in the holders on the build platform.</li>
	<li>Open the 3D printing software. Load a pre-programmed g-code into the software. For the representative results, step 3.1 is used to load the bacterial suspension into the 3D printer. 
	NOTE: An example of a g-code for printing linear vertical geometries is given in Table 1.</li>
	<li>Through the 3D printing software, move the x-y-z planes to center the print head on the x-y plane to be the first container and then home the z-axis. The homing z-axis will raise the print head. Manually rotate the screw slowly to depress the syringe plunger until a small amount of the bacterial suspension can be seen at the tip of the needle.
	<ol>
		<li>Lightly wipe the excess bacterial suspension off with a sterile disposable wipe. Based on the height of the sample holder, needle, and syringe, using the 3D printing software, lower the print head a fixed distance into the hydrogel media in the sample container of choice. Start the printing process by clicking on Print.</li>
	</ol>
	</li>
	<li>Once the printing is complete, close the sample containers. Wipe down the printer with 70% ethanol. Properly dispose of the syringe and needle.</li>
</ol>
9. Growing and imaging the V. cholerae
<ol>
	<li>For large field-of-view imaging, use a camera with a zoom lens attachment to image cell growth with a lightbox. Image the samples right after printing at room temperature and then transfer them to an incubator. Maintain samples at 37 &#176;C in a stationary incubator between imaging sessions during the experiment.</li>
	<li>Capture images over a desired amount of time to observe the growth behavior at long periods in the granular hydrogel matrix.</li>
</ol>

Bioprinting Bacteria in Hydrogel Matrices for the Study of Motility and Growth

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+semi-solid+agar+for+the+detection+of+bacterial+motility.">The use of semi-solid agar for the detection of bacterial motility.</a> Journal of Bacteriology. 31 (6), 575-580 (1936).</li><li>Bhattacharjee, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyelectrolyte+scaling+laws+for+microgel+yielding+near+jamming.">Polyelectrolyte scaling laws for microgel yielding near jamming.</a> Soft Matter. 14 (9), 1559-1570 (2018).</li><li>Bhattacharjee, T., Datta, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confinement+and+activity+regulate+bacterial+motion+in+porous+media.">Confinement and activity regulate bacterial motion in porous media.</a> Soft Matter. 15 (48), 9920-9930 (2019).</li><li>Bhattacharjee, T., Datta, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+hopping+and+trapping+in+porous+media.">Bacterial hopping and trapping in porous media.</a> Nature Communications. 10 (1), 2075 (2019).</li><li>Bhattacharjee, T., Amchin, D. B., Ott, J. A., Kratz, F., Datta, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+migration+of+bacteria+in+porous+media.">Chemotactic migration of bacteria in porous media.</a> Biophysical Journal. 120 (16), 3483-3497 (2021).</li><li>Bhattacharjee, T., Amchin, D. B., Alert, R., Ott, J. A., Datta, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+smoothing+of+collective+migration.">Chemotactic smoothing of collective migration.</a> eLife. 11, e71226 (2022).</li><li>Tashman, J. W., Shiwarski, D. J., Feinberg, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+performance+open-source+syringe+extruder+optimized+for+extrusion+and+retraction+during+FRESH+3D+bioprinting.">A high performance open-source syringe extruder optimized for extrusion and retraction during FRESH 3D bioprinting.</a> HardwareX. 9, e00170 (2021).</li><li>Crocker, J. C., Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+digital+video+microscopy+for+colloidal+studies.">Methods of digital video microscopy for colloidal studies.</a> Journal of Colloid and Interface Science. 179 (1), 298-310 (1996).</li><li>Chatterjee, T., Chatterjee, B. K., Chakrabarti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+of+growth+kinetics+of+Vibrio+cholerae+in+presence+of+gold+nanoparticles:+Effect+of+size+and+morphology.">Modelling of growth kinetics of Vibrio cholerae in presence of gold nanoparticles: Effect of size and morphology.</a> Scientific Reports. 7 (1), 9671 (2017).</li><li>Mart&#237;nez-Calvo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+instability+and+roughening+of+growing+3D+bacterial+colonies.">Morphological instability and roughening of growing 3D bacterial colonies.</a> Proceedings of the National Academy of Sciences. 119 (43), e2208019119 (2022).</li><li>Lehner, B. A. E., Schmieden, D. T., Meyer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Straightforward+approach+for+3D+bacterial+printing.">A Straightforward approach for 3D bacterial printing.</a> ACS Synthetic Biology. 6 (7), 1124-1130 (2017).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+cell+ordering+in+confined+bacterial+biofilms.">Morphogenesis and cell ordering in confined bacterial biofilms.</a> Proceedings of the National Academy of Sciences. 118 (31), e2107107118 (2021).</li></ol>

The experimental platform used to 3D print and image bacterial communities in this publication is the subject of a patent application filed by Princeton University on behalf of Tapomoy Bhattacharjee and S.S.D. (PCT Application number PCT/US/2020/030213).

Critical steps in the protocol 
It is important to ensure that when preparing each hydrogel matrix, the matrix is made in a sterile environment. If not, contamination can occur, which manifests as, e.g., microcolonies (small spheroids) in the matrix after several days. During the mixing process, it is important that all the dry granular hydrogel particles are dissolved. Additionally, when adjusting the pH of each hydrogel matrix with the NaOH, the granules will start to swell, which increases the viscosity of the hydrogel matrix, leading to mixing being more difficult. Using the stand mixer will help ensure that the NaOH is well mixed into the hydrogel matrix. During the loading of each bacterial suspension, air pockets can form in the needle. To avoid this issue, ensure that the needle tip is always sitting in the bacterial suspension in the centrifuge tube and not at the bottom of the tube or near the top surface. Another way to overcome this issue is to grow large volumes of cells and thus have larger volumes of the bacterial suspension for printing.
Limitations 
Currently, during printing, the low viscosity of the bacterial suspension limits the geometries that can be printed and often leads to a biofilm-forming and growing on the top of the hydrogel matrix surface due to trace cells. There are a few potential methods for overcoming this limitation, including increasing the viscosity of the bacterial suspension or further optimizing the 3D printer settings. To increase the viscosity of the bacterial suspension, one could mix the bacterial suspension with another polymer - for example, alginate, which has been used prior for the 3D printing of bacteria onto flat surfaces38. The printer settings can be further optimized to enable retraction of the syringe plunger during the withdrawal of the needle from the granular hydrogel matrix, which would have the potential to stop cells from being deposited during the removal of the needle from the hydrogel matrix.
The significance of the method with respect to existing/alternative methods 
The method described here allows for the printing of bacterial colonies into granular hydrogel matrices. The granular hydrogel matrices allow the study of the impact of external environmental factors (e.g., pore size, matrix deformability) on the motility and growth of bacteria. Additionally, while in this work, LB is used as the liquid growth medium to swell the hydrogel matrix, the hydrogel matrix can be swollen with other liquid growth media, including media with antibiotics. Previous methods for studying bacteria in confined environments were limited by the length of experimental time, the polymer mesh size, and surrounding hydrogel matrix stiffness37,38. Protocols already exist for making granular hydrogel matrices out of different polymers, so the potential for studying the impacts of different environmental conditions on the motility and growth of bacteria is vast. This method allows for the study of bacteria in control environments that more readily recapitulate the environments that bacteria inhabit in the real world, such as host mucus or soil. Another limitation of many other methods is the opacity of the surrounding matrix; however, this approach using optically transparent materials provides the ability to explore, e.g., optogenetic control and patterning of bacteria in 3D.
Beyond studying motility and growth, the 3D printing method described here overcomes the limitation of many other bioprinting methods that require the deposition of a bioink on a substrate and are, therefore, limited in the height of the engineered living material they can produce. In the future, this bioprinting protocol can be further expanded to fabricate biohybrid materials by mixing polymers with biofilm-forming cells. The granular hydrogel matrices provide support for 3D printing thicker, larger-scale engineered living materials and more complex geometries than many other current bacteria bioprinting methods. While this work only used V. cholerae and E. coli, other species, such as Pseudomonas aeruginosa, have also successfully been 3D printed37. Beyond printing, the printer can be adapted to do a controlled sampling of bacteria after growth to see if there have been any genetic changes, for example.

Bacteria often inhabit diverse, complex 3D porous environments ranging from mucosal gels in the gut and lungs to soil in the ground1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21, 
22,23,24,25. In these settings, bacterial movement through motility or growth can be impeded by surrounding obstacles, such as polymer networks or packings of solid mineral grains-influencing the ability of the cells to spread through their environments26, access nutrient sources, colonize new terrain, and form protective biofilm communities27. However, traditional lab studies typically employ highly simplified geometries, focusing on cells in liquid cultures or on flat surfaces. While these approaches yield key insights into microbiology, they do not fully recapitulate the complexity of natural habitats, leading to dramatic differences in growth rates and motility behavior compared to measurements performed in real-world settings. Therefore, a method to define bacterial colonies and study their motility and growth in 3D porous environments more akin to many of their natural habitats is critically needed.
Inoculating cells into an agar gel and then visualizing their macroscopic spreading by eye or using a camera provides one straightforward way to accomplish this, as first proposed by Tittsler and Sandholzer in 193628. However, this approach suffers from a number of key technical challenges: (1) While the pore sizes can, in principle, be varied by varying the agarose concentration, the pore structure of such gels is poorly defined; (2) Light scattering causes these gels to be turbid, making it difficult to visualize cells at the individual scale with high resolution and fidelity, particularly in large samples; (3) When the agar concentration is too large, cell migration is restricted to the top flat surface of the gel; (4) The complex rheology of such gels makes it challenging to introduce inocula with well-defined geometries.
To address these limitations, in previous work, Datta&#39;s lab developed an alternate approach using granular hydrogel matrices - comprised of jammed, biocompatible hydrogel particles swollen in liquid bacterial culture - as &#34;porous Petri dishes&#34; to confine cells in 3D. These matrices are soft, self-healing, yield-stress solids; thus, unlike with cross-linked gels used in other bioprinting processes, an injection micronozzle can move freely inside the matrix along any prescribed 3D path by locally rearranging the hydrogel particles29. These particles then re-densify rapidly and self-heal around injected bacteria, supporting the cells in place without any additional harmful processing. This process is, therefore, a form of 3D printing that enables bacterial cells to be arranged - in a desired 3D structure, with a defined community composition - within a porous matrix having tunable physicochemical properties. Moreover, the hydrogel matrices are completely transparent, enabling the cells to be directly visualized using imaging.
The utility of this approach has been demonstrated previously in two ways. In one set of studies, dilute cells were dispersed throughout the hydrogel matrix, which enabled studies of the motility of individual bacteria30,31. In another set of studies, multicellular communities were 3D-printed in centimeter-scale gels using an injection nozzle mounted on a programmable microscope stage, which enabled studies of the spreading of bacterial collectives through their surroundings32,33. In both cases, these studies revealed previously unknown differences in the spreading characteristics of bacteria inhabiting porous environments compared to those in liquid culture/on flat surfaces. However, given that they were mounted on a microscope stage, these previous studies were limited to small sample volumes (~1 mL) and, therefore, short experimental time scales. They were also limited in their ability to define inocula geometries with high spatial resolution.
Here, the next generation of this experimental platform that addresses both limitations is described. Specifically, protocols are provided by which one can use a modified 3D printer with an attached syringe extruder to 3D print and image bacterial colonies at large scales. Moreover, representative data indicates how this approach can be useful for studying the motility and growth of bacteria, using the biofilm-former Vibrio cholerae and planktonic Escherichia coli as examples. This approach enables bacterial colonies to be sustained over long times and visualized using various imaging techniques. Hence, the ability of this approach to study bacterial communities in 3D porous habitats has tremendous research and applied potential, impacting the treatment and study of microbes in the gut, the skin, the lung, and the soil. Moreover, this approach could be used in the future for 3D printing bacteria-based engineered living materials into more complex freestanding shapes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL cuvettes</td>
			<td>VWR</td>
			<td>97000-586</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Luer lock syringe</td>
			<td>BH Supplies</td>
			<td>BH1LL</td>
			<td></td>
		</tr>
		<tr>
			<td>10 M NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>72068</td>
			<td></td>
		</tr>
		<tr>
			<td>100 nm carboxylated fluorescent polystyrene nanoparticles (FluoSpheres)&#160;</td>
			<td>&#160;Invitrogen, (ThermoFischer Scientific)</td>
			<td> 
			F8803</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>ThermoFischer Scientific</td>
			<td>14-955-237</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G blunt needle</td>
			<td>McMaster Carr</td>
			<td>75165A252</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL tissue culture flasks</td>
			<td>VWR</td>
			<td>10861-566</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>Lulzbot</td>
			<td>LulzBot Mini 2</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printing software</td>
			<td>Cura</td>
			<td>Cura-Lulzbot</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>ThermoFischer Scientific</td>
			<td>14-955-239</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbomer Granular Hydrogel Particles</td>
			<td>Lubrizol&#160;</td>
			<td>Carbopol 980NF</td>
			<td>dry granules of crosslinked acrylic acid/alkyl acrylate copolymers</td>
		</tr>
		<tr>
			<td>Centrifuge (2 mL tube capacity)</td>
			<td>VWR</td>
			<td>2405-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (50 mL tube capacity)</td>
			<td>ThermoFischer Scientific</td>
			<td>75007200</td>
			<td>Sorvall (brand) ST 8 (model)</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>A1R+ inverted laserscanning 
			confocal microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom petri dish</td>
			<td>Cellvis</td>
			<td>D35-10-1-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Lennox LB (Lubria Broth)</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>M8 &#215; 1.25 mm, 150 mm long, Fully Threaded Socket Cap</td>
			<td>McMaster Carr</td>
			<td>91290A478</td>
			<td></td>
		</tr>
		<tr>
			<td>M8 &#215; 1.25 mm, Brass Thin Hex Nut</td>
			<td>McMaster Carr</td>
			<td>93187A300</td>
			<td></td>
		</tr>
		<tr>
			<td>Open-source syringe pump</td>
			<td>Custom-made</td>
			<td>Replistruder 4</td>
			<td>https://www.sciencedirect.com/science/article/pii/S2468067220300791</td>
		</tr>
		<tr>
			<td>Petri dish (60 mm round)</td>
			<td>ThermoFischer Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Shear Rheometer</td>
			<td>Anton Paar</td>
			<td>MCR 501</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>VWR</td>
			<td>97043-992</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d printing bacteria to study motility and growth in complex 3d porous media

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. However, the majority of previous work has focused on studies of cells in bulk liquids or at flat surfaces, which do not fully recapitulate the complexity of many natural bacterial habitats. Here, this gap in knowledge is addressed by describing the development of a method to 3D-print dense colonies of bacteria into jammed granular hydrogel matrices. These matrices have tunable pore sizes and mechanical properties; they physically confine the cells, thus supporting them in 3D. They are optically transparent, allowing for direct visualization of bacterial spreading through their surroundings using imaging. As a proof of this principle, here, the capability of this protocol is demonstrated by 3D printing and imaging non-motile and motile Vibro cholerae, as well as non-motile Escherichia coli, in jammed granular hydrogel matrices with varying interstitial pore sizes.

Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices

3D Printing Bacteria to Study Motility and Growth in Complex 3D Porous Media

Bacteria are 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 scales, and were also limited in their ability to define inocula 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.

Watch this Scientific Journal Video about Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices at JoVE.com

This protocol describes a procedure for three-dimensional (3D) printing of bacterial colonies to study their motility and growth in complex 3D porous hydrogel matrices that are more akin to their natural habitats than conventional liquid cultures or Petri dishes.

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. ...