Super-resolution Imaging of Proteus mirabilis Biofilm by Expansion Microscopy

Summary

This article presents a comprehensive protocol for PmbExM, an Expansion Microscopy technique designed specifically for Proteus mirabilis biofilms. PmbExM utilizes a stepwise enzymatic treatment of biofilm samples to achieve an isotropic, 4.3-fold expansion, enabling super-resolution analysis of the spatial organization of cellular and subcellular structures within these sessile microbial communities.

Abstract

Accessing detailed visual information and quantitative data from microbiological samples using conventional optical microscopy is limited by the diffraction barrier. One solution to enhance resolution is Expansion Microscopy (ExM), an innovative and cost-effective super-resolution technique that physically enlarges samples by approximately four times their original size. For successful expansion, it is essential to homogenize the mechanical properties of the biological material. Biofilms are bacterial communities, adhering to a surface and embedded in an extracellular matrix they produce; they require ExM protocols to be adapted to accommodate their unique structural components. This article presents Proteus mirabilis biofilm ExM (PmbExM), a specialized variant of ExM that enables super-resolution visualization of P. mirabilis biofilms grown for 48 h. The protocol focuses on the targeted degradation of key structural components of samples through serial enzymatic digestions, optimized near their theoretical conditions. PmbExM utilizes a combination of enzymes, including α-amylase, cellulase, and lyticase glycoside-hydrolases for polysaccharide hydrolysis; mutanolysin for peptidoglycan hydrolysis; and proteinase K for protein hydrolysis. These digestion procedures are independent of the gelation process, allowing modifications to meet specific homogenization requirements in different biofilm models. This adaptability offers great potential for application across various bacterial species and growth conditions. ExM has been applied to different biofilm species with overall suboptimal expansion factors. In contrast, PmbExM achieves the theoretical maximum expansion factor of the standard acrylamide-acrylate ExM hydrogel, without significant distortion of morphology or topology. The aim of this work is to provide an accessible super-resolution protocol for visualizing the architecture, assembly, and cellular and intracellular features of P. mirabilis biofilms.

Introduction

Fluorescence microscopy is a widely used tool for studying biofilm architecture, composition, and function¹. However, optical microscopy's ability to resolve the structural, cellular, and subcellular features of biofilms is limited by the diffraction barrier. Expansion Microscopy (ExM) is an innovative, accessible, and easy-to-use super-resolution technique that has great potential for revealing biofilm structure beyond the diffraction limit^2,3. ExM enhances resolution by isotropically expanding biological specimens approximately four times their original size. The method involves the in-situ polymerization of a swellable ionic hydrogel throughout the sample and preserving the relative spatial arrangement of molecular targets when applied with care^4,5.

For ExM to work properly, homogenizing the mechanical properties of the biological material is a crucial step⁶. Standard ExM variants were developed upon mammal cell cultures and tissues, where they efficiently achieved homogenization only through proteinase K proteolytic digestion^4,5 or soft protein denaturation with detergents and autoclaving⁷, yielding isotropic 4-fold expansion. However, in the case of bacteria and bacterial biofilms, the peptidoglycan cell wall and EPS matrix elements, such as structural polysaccharides, are obstacles against expansion^2,3,8,9.

This article describes the procedures of Proteus mirabilis biofilm Expansion Microscopy (PmbExM), a tailored ExM variant designed to expand biofilms of P. mirabilis, a clinically relevant gram-negative bacillus associated with high antibiotic resistance and catheter-associated urinary tract infections¹⁰. PmbExM uses a combination of mutanolysin, α-amylase, cellulase, lyticase, and proteinase K enzymatic treatments to hydrolyze the peptidoglycan cell wall, polysaccharides, and proteins, respectively. The results demonstrate that this method achieves a 4.3-fold isotropic expansion of 48-h-old P. mirabilis biofilms. The technique enables super-resolution visualization of P. mirabilis biofilm architecture, assembly, and intracellular features through double staining³. Furthermore, PmbExM can be adapted to other biofilm models by adjusting the enzyme treatments to target species-specific biofilm components.

The objective of this article is to provide a detailed description of the procedures involved in gelation, digestion, expansion, and mounting of biofilm samples for PmbExM. Additionally, this work outlines bacterial growth conditions, sample formats, and fluorescent staining techniques used by the authors, along with a data processing and analysis routine that can be easily applied by researchers with limited experience in image processing. For a more comprehensive and in-depth guide on image processing and analysis, please refer to Castagnini, D. et al.³.

Protocol

The overall process of PmbExM is summarized in Figure 1. Please refer to Supplementary File 1 to find the composition of all named solutions (i.e., monomer solution, proteinase K digestion solution, etc.) used in this work. Likewise, refer to Supplementary File 2, Supplementary File 3, and Supplementary File 4 for a simplified data processing and analysis routine that can be used by researchers without vast experience in image processing. The protocol can be comfortably performed over a period of 2.5-3 weeks: the first week dedicated to coverslip cleaning and sterilization, biofilm growth, and sample fixation; the second week to sample staining and the PmbExM protocol itself, and the third for sample mounting and image acquisition. Additionally, consider 1 week of prior preparations for the required materials and solutions. Details about the reagents, materials, equipment, and software employed in this study are listed in the Table of Materials.

Figure 1: Anchoring, polymerization, digestion, and dialysis of the PmbExM protocol for successful expansion, observation, and analysis of P. mirabilis biofilm. The left column identifies six states of the sample at selected steps during the expansion protocol. It starts from the fixed, permeabilized, and stained biofilm sample, following four intermediate states until the final gelified and expanded state, ready for microscopy and image analysis on super-resolution. The EPS matrix of the biofilm is shown in green, the bacterial cell wall is represented by purple lines, the general protein cell structure is shown in gray, and the acrylamide-acrylate ExM polymer is displayed as undulated blue lines. All enzymes involved in the process are represented by Pac-Man symbols: 3 glycoside-hydrolases (green) for exopolysaccharide hydrolysis: α-amylase, cellulase, and lyticase; mutanolysin (purple) for degradation of the peptidoglycan cell-wall; and proteinase K (gray) for general protein digestion. The colors of the Pac-Man symbols match the respective target structures. All objects shown in low opacity or dashed borders represent a state of enzymatic degradation. This figure is modified from Castagnini, D. et al.³. Please click here to view a larger version of this figure.

1. Cleaning and sterilization of glass coverslips for biofilm formation

1. Ultrasonicate 12 mm glass coverslips in 1 M sodium hydroxide for 20 min at room temperature and then rinse with abundant deionized water.
2. Ultrasonicate the coverslips in 70% (v/v) ethanol for 15 min at room temperature and then rinse with 95% (v/v) ethanol.
3. Allow the coverslips to air-dry at room temperature.
4. Sterilize by autoclaving.

2. Proteus mirabilis biofilm growth

1. Grow P. mirabilis in 10 mL of Luria Bertani (LB) broth for 24 h at 37 °C without shaking.
   NOTE: A commercially available P. mirabilis type strain (see Table of Materials) was acquired and employed in this study, but the use of any P. mirabilis strain that is readily available is encouraged.
2. Take 1 mL from the culture and add it to 9 mL of fresh and sterile LB broth to generate a bacterial suspension of 1:10 dilution.
3. Check the Optical Density (OD) at 600 nm of the 1:10 bacterial suspension to be 0.1. Adjust if needed by adding small volumes of fresh LB or P. mirabilis culture until it reaches OD₆₀₀ = 0.1.
4. Introduce 12 mm glass coverslips (previously cleaned and sterilized) in a sterile polystyrene 24 well-plate (one coverslip per well).
   NOTE: Ensure that the entire coverslip surface touches the bottom of the well. The biofilm will form on the side of the coverslip, which is facing upwards.
5. Seed the wells of the plate with 350 µL of the OD₆₀₀ = 0.1 bacterial suspension.
   NOTE: It is recommended to seed only the inner wells of the plate, while filling the outer wells (i.e. wells in rows A and D and in columns 1 and 6) with sterile water or LB broth. This is because the outer wells are prone to higher liquid evaporation rates than the inner wells, which affects biofilm growth.
6. Set the seeded plate inside an incubator and position it with 45 degrees of inclination.
   NOTE: Use common laboratory material such as 50 mL centrifuge tubes or micropipette tip boxes. The tilted position will allow the formation of an air-liquid interface on the surface of the coverslips at which the P. mirabilis biofilm will establish its thickest region.
7. Allow biofilms to form for 48 h at 37 °C, without shaking.
   NOTE: This biofilm growth setup is not suitable for incubation times longer than 48 h due to biofilm desiccation because of evaporation of the medium.

3. Biofilm sample fixation and storage

1. Carefully remove the culture medium from the wells and gently wash the biofilm-bearing coverslips twice with 300 µL sterile Phosphate Buffer Saline (PBS) for 5 min at room temperature with very mild agitation.
   NOTE: Avoid destruction of the biofilms with repetitive fluid exchange by using small volumes for incubations or washing. 300-400 µL of liquid is enough to submerge the biofilms in a 24-well plate. Slow and methodical pipetting is strongly suggested, as well as avoiding hitting the biofilms directly with the exchanged solutions.
2. Carefully remove the PBS and replace it with 400 µL of 4% (w/v) paraformaldehyde (PFA) in PBS for 20 min at room temperature for biofilm fixation.
3. Remove the PFA and replace it with 300 µL of 1% PBST (1% v/v Triton X-100 in PBS) for cell permeabilization. Incubate for 20 min at room temperature.
4. Replace the 1% PBST solution with 300 µL of 0.3% PBST (3% v/v Triton X-100 in PBS). Incubate for 30 min at room temperature.
5. Remove the 0.3% PBST and replace it with 300 µL of a 50:50 mixture of methanol and 0.3% PBST for sample dehydration. Incubate for 5 min at room temperature and then replace the mixture with 1 mL of absolute methanol.
   NOTE: Add 1 mL of methanol slowly, directing it against the walls of the well. This higher volume of methanol is used to prevent its complete evaporation during sample storage.
6. Store the samples at -20 °C for at least 1 day and up to a month.
   NOTE: Sealing the plates using a flexible wax film is recommended to delay methanol evaporation and desiccation of the samples.
7. Upon usage, sequentially rehydrate the samples by replacing the methanol with 300 µL of a 50:50 mixture of methanol and 0.3% PBST, and then with 300 µL 0.3% PBST, at room temperature.

4. Biofilm fluorescent staining

1. Incubate the rehydrated biofilm samples with 300 µL of 50 µg/mL Wheat-germ agglutinin fluorescent lectin conjugate (hereafter just referred to as WGA conjugate; see Table of Materials) in PBS for 20 min at room temperature for cell-wall/capsule/exopolysaccharides staining.
   NOTE: Please see relevant topics regarding fluorescent staining in the Discussion section. From now on, protect the samples from light.
2. Remove the WGA conjugate solution and gently wash twice with 300 µL of PBS for 10 min at room temperature.

5. PmbExM steps

1. MA-NHS anchoring and acrylamide-acrylate polymerization
   1. Incubate the stained biofilm samples with 400 µL of 1 mM methacrylic acid N-hydroxysuccinimidyl ester (MA-NHS) in PBS for 1 h at room temperature with mild agitation.
      NOTE: MA-NHS is an activated ester that is prone to hydrolysis in an aqueous medium, so treat the samples immediately after preparing the 1 mM in PBS solution.
   2. Perform three gentle washes of 10 min with 300 µL of PBS at room temperature.
   3. Remove the PBS and replace it with 300 µL of monomer solution. Incubate overnight (O.N.) at 4 °C.
   4. Construct the gelation pre-chambers. Use a glass slide as a base and place on it two pieces of folded-over double-sided tape to act as 400 µm spacers. Arrange the spacers 8-10 mm from each other.
      NOTE: See Figure 2A for further clarity on how to construct the gelation pre-chamber. The double-sided tape used here was approximately 100 µm thick, so it was trimmed and folded on itself to create 3 mm x 5 mm x 0.4 mm spacers. Measure the thickness of available double-sided tape and arrange it accordingly. Absorption of the gelling solution by the tape spacers is negligible. As an alternative to double-sided tape, 12 mm round glass coverslips can be adhered together with drops of water and piled up until the required height.
   5. Arrange wet chambers to protect samples during polymerization from desiccation.
      NOTE: A common box of micropipette tips can be easily converted into a wet chamber by removing the plastic scaffold of the tips and replacing it with wet paper towels.
   6. Prepare a fresh stock volume of gelling solution by thoroughly mixing volumes of monomer solution, 10 % (w/v) N, N, N', N'-tetramethylethylenediamine (TEMED), 0.5 % (w/v) 4-hydroxy-TEMPO (4-HT) and 10 % (w/v) ammonium persulfate (APS) in a 47:1:1:1 proportion, respectively. Mix the APS with the rest of the gelling solution reagents only when steps up to 5.1.5 are done.
      NOTE: The total volume of stock gelling solution to prepare will depend on the amount of biofilm samples that are being processed. For example, to process three biofilm samples, prepare 900 µL of gelling solution by mixing 846 µL of monomer solution with 18 µL of 10 % (w/w) TEMED, 18 µL of 0.5 % (w/w) 4-HT and 18 µL of 10 % (w/w) APS. To prevent premature polymerization, APS must be added last to the mix and only when samples, gelation pre-chambers, and wet chambers are ready to be used. Maintain all the reagents mentioned and the recently prepared gelling solution cooled during this step.
   7. Immediately after preparing the gelling solution, gently remove the monomer solution and replace it with 300 µL of the former. Incubate at 4 °C for 5 min.
   8. While step 5.1.7 is ongoing, remove the remaining protective cover of the double-sided tape stripes arranged on the glass slides, and in the space between them, place 40 µL of gelling solution.
   9. After step 5.1.7 has finished, remove each biofilm-bearing coverslip from its well and place it on top of the 40 µL drop of gelling solution previously laid on a glass slide. Orient the coverslip so that the biofilm on its surface contacts the gelling solution.
   10. Finish the construction of the gelation chamber by gently pressing on the biofilm-bearing coverslip using tweezers to secure its adherence to the double-sided tape spacers.
       NOTE: This sandwich-like setup consists of what is called the gelation chamber (Figure 2B).
   11. Let the samples polymerize inside a wet chamber and incubate for 2 h at 37 °C without agitation.
   12. Visualize the samples under fluorescence microscopy (see step 6.2.1) without disassembling the gelation chambers for pre-expansion image capture.
       NOTE: Do not leave the gelled samples out of the wet chamber for more than an hour, as pre-expanded gels are prone to desiccation, which renders the sample unusable. Please see step SF2.3 (Supplementary File 2) for relevant pre-expansion acquisition procedures.
   13. Disassemble the gelation chambers and trim the excess gel around the region of interest in the biofilm sample using a surgical blade.
       NOTE: Trimming the gels is highly recommended to facilitate the handling and mounting of expanded samples. Additionally, to keep track of the spatial orientation of the region of interest within samples, trim the gels into an asymmetric shape or leave a characteristic cut-out in the gel for easy recognition of correct orientation.
   14. Place the coverslips carrying the trimmed gels inside a new 24-well plate, with the gel facing upwards.
       NOTE: At this stage, the gelled samples should remain adhered to their original glass coverslip.
2. Enzymatic digestion I-III and DNA staining
   NOTE: Homogenization of the mechanical properties of P. mirabilis biofilm samples follows a series of enzymatic digestions that the authors have categorized in 3 steps, based on the biological structure being targeted for degradation: polysaccharide hydrolysis (Digestion I), peptidoglycan hydrolysis (Digestion II) and protein hydrolysis (Digestion III).
   1. Digestion I
      1. Immerse the samples in 1 mL of α-amylase digestion solution. Incubate O.N. at 50 °C.
         NOTE: Large volumes (i.e., 0.5-1 mL) of enzymatic solution are used to prevent sample desiccation due to the high incubation temperature.
      2. Remove the α-amylase solution and wash the gels for 5 min at room temperature with 300 µL of cellulase digestion buffer.
      3. Remove the cellulase digestion buffer and replace it with 500 µL of cellulase digestion solution. Incubate for 2 h at 45 °C.
      4. Remove the cellulase digestion solution and wash the gels for 5 min at room temperature with 300 µL of lyticase digestion buffer.
      5. Remove the lyticase digestion buffer and replace it with 500 µL of lyticase digestion solution. Incubate for 2 h at 30 °C.
   2. Digestion II
      1. Remove the lyticase digestion solution and wash the gels for 5 min at room temperature with 300 µL of mutanolysin digestion buffer.
      2. Remove the mutanolysin digestion buffer and replace it with 1 mL of mutanolysin digestion solution. Incubate O.N. at 55 °C.
   3. Digestion III
      1. Remove the mutanolysin digestion solution and wash the gels for 5 min at room temperature with 300 µL of proteinase K digestion buffer. Remove the proteinase K digestion buffer and replace it with 500 µL of proteinase K digestion solution. Incubate for 2 h at 37 °C.
   4. (OPTIONAL) DNA staining
      1. Remove the proteinase K solution and wash the gels with 300 µL of 10x PBS for 5 min at room temperature.
      2. Remove the 10x PBS and replace it with 300 µL of 5 µM red fluorescent nucleic acid stain in 10x PBS for 30 min at room temperature.
         NOTE: See the Discussion section for comments about DNA staining for ExM procedures.
3. Expansion
   1. Remove the proteinase K solution (or the DNA staining solution if the optional step 5.2.4 was performed) and transfer the gels to a 60 mm Petri dish. Place one gel per Petri dish.
      NOTE: Use a small flat paintbrush to push the gel onto its glass coverslip. With laboratory tweezers, grab the glass coverslip and use it as a platform for the gel. Prevent the gel from falling off the coverslip with the help of the brush when lifting it from the well of the plate. Avoid the gels from folding onto themselves.
   2. Fill the Petri dish with excess deionized water, fully submerge the gel, and incubate under gentle agitation at room temperature for 20 min. Exchange the water 4-5 times.
      ​NOTE: Note that the expanding gel becomes virtually invisible when submerged in water. Position the Petri dish against the light to facilitate locating the gel to avoid damaging it with the suction from the Pasteur pipette or micropipette during water exchanges.

Figure 2: Gelation chamber and sample mounting. (A) Pre-gelation chamber. On a slide, position 2 double-sided tape spacers (yellow). A Glass coverslip ( = 12 mm) sits atop the as a distance reference. (B) Gelation chamber. The bacterial biofilm (green) is formed on the lower coverslip surface. 400 µm-thick spacers (orange) are made from 100 µm-thick double-sided tape cut-outs. Spacers allow the gel to embed the entire sample and avoid overflow of gelling solution from the edges of the chamber, preventing the sample from being squashed between the slide and the coverslip. (C) 3D-Printed imaging chambers (visit the following link to access the .stl files https://github.com/scianlab/ExM). An expanded gel sample (left) and an expanded gel sample immersed in deionized water (right). Expanded gels become virtually invisible when immersed. Please click here to view a larger version of this figure.

6. Observation and analysis

1. Mounting of expanded samples
   1. Prepare the imaging chambers with poly-lysine coating for sample immobilization during imaging¹¹: Soak the glass surface of the imaging chamber (See Table of Materials) with 0.1% (w/v) poly-L-lysine for 20 min at room temperature.
   2. Remove the poly-L-lysine solution and perform two washes with excess deionized water. Allow to air dry for 1 h or place inside an incubator at 37 °C to accelerate the drying process.
   3. After the fifth dialysis cycle from step 5.3.2, remove the water covering the gels and use a small flat brush to push the sample onto a 24 mm x 50 mm glass coverslip. Use the rectangular coverslip as a platform for the gel and lift it from the Petri dish.
   4. Remove the excess water from the gel using tissue paper to prevent water from blocking direct contact between the gel's surface and the poly-lysine coating on the glass surface of the imaging chamber.
   5. Carefully place the gel onto the glass surface of the imaging chamber, avoiding the retention of air bubbles between the gel and glass.
      NOTE: The glass surface of the imaging chamber should be completely dry. If pre-expansion images were taken, consider a correct orientation of the expanded gel to facilitate finding the previously captured fields of view in the pre-expanded sample.
   6. Add deionized water until the gel is completely submerged (see Figure 2C).
      NOTE: The expanded gels must remain hydrated during the entirety of the imaging process to avoid shrinkage during capture.
2. Image acquisition
   1. Visualize and capture images of the samples on an available diffraction-limited fluorescent microscope.
      NOTE: The authors used a spinning disk confocal microscope, with a 63x water immersion objective of 1.2 numerical aperture (NA). For fluorophore excitation, a solid-state laser line at 488 and 647 nm was used. Captures were made with a 16-bit digital CMOS camera controlled by proprietary software (see Table of Materials). Voxel size was set to 64.4 mm x 64.4 mm x 200 nm (XYZ axes).
3. Image processing
   1. Please refer to Supplementary File 2 to find a digital image processing routine for the segmentation of bacteria (section SF2.1), cell width measurement (section SF2.2), EF determination (section SF2.3), and image matching for pre-expanded & expanded conditions (section SF2.4).

Results

PmbExM expanded the P. mirabilis biofilm 4.3 fold and preserved morphology and topology
Figure 3A-D evidences the physical magnification of P. mirabilis biofilm cells and the increased resolution brought up by the technique. Moreover, this was not only observed in thin biofilm regions where cells organize in a monolayer-like layout, but also in thicker regions of densely populated arran...

Discussion

Critical steps of PmbExM
As with any ExM variant, one of the most critical steps of the protocol is the homogenization of the sample's mechanical properties. In the case of PmbExM, this is achieved through a series of enzymatic treatments. Insufficient digestion can result in low EFs and potential damage to biofilm morphology and/or topology, leading to suboptimal resolution and unreliable results. Conversely, overly aggressive digestions, particularly proteolytic treatments, can cause signific...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
α-Amylase from Bacillus sp.	Sigma-Aldrich	10070	Polysaccharide digestion
0.1% (w/v) poly-L-lysine water solution	Sigma-Aldrich	P8920	Sample immobilization for imaging
12 mm glass coverslips	Chemglass Life Sciences	CLS-1760	Gelation chamber component
16-bit C13440-20CU camera	Hamamatsu Photonics	Not available	CMOS digital camera for image acquisition
24 mm x 50 mm glass coverslips	Chemglass Life Sciences	CLS-1764	Sample manipulation/handling
3D-printed imaging chamber	In-house design	-	Imaging chamber option
4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-hydroxy-TEMPO)	Sigma-Aldrich	176141	Gelling solution component
6-Well glass-bottom plate with black walls	CelVis	P06-1.5H-N	Imaging chamber option
Acetic acid	Sigma-Aldrich	818755	Enzyme buffer component
Acrylamide	Sigma-Aldrich	A4058	Monomer solution component
Ammonium persulfate	Sigma-Aldrich	A3678	Gelling solution component
Axiovert 200 microscope	Zeiss	Not available	Confocal microscope
Calcium chloride	Sigma-Aldrich	C8106	Enzyme buffer component
C-Apochromat 63x 1.2 NA water immersion objective	Zeiss	Not available	Microscope objective
Cellulase from Aspergillus niger	Sigma-Aldrich	22178	Polysaccharide digestion
Chamlide CMB 35 mm dish magnetic imaging chamber	Live Cell Instrument	Not available	Imaging chamber option
Dimethyl sulfoxide	Sigma-Aldrich	472301	MA-NHS anchoring reagent stock solution
Disodium phosphate	Sigma-Aldrich	S3264	Enzyme buffer component
Ethanol	Sigma-Aldrich	E7023	Cleaning and sterilization
Ethylenediaminetetraacetic acid	Merck	324503	Enzyme buffer component
Falcon polystyrene 24 well-plates	Corning	351147	General procedures platform
FIJI Software	National Institutes of Health	-	Image processing software
Glycerol	Sigma-Aldrich	G7893	Enzyme buffer component
Guanidine hydrochloride	Thermo Scientific	24115	Enzyme buffer component
Huygens Software	Scientific Volume Imaging	-	Deconvolution software for image restoration
Hydrochloric acid	Supelco	1.00317	Buffer preparation
Kimwipes tissue paper	Fisher Scientiffic	06-666	Excess water removal
Laboratory tweezers	Electron Microscopy Sciences	M5E	General sample manipulation/handling
Luria Bertani broth	Invitrogen	12795-027	Biofilm growth medium
Lyticase from Arthrobacter luteus	Sigma-Aldrich	L4025	Polysaccharide digestion
Magnesium chloride	Sigma-Aldrich	D5652	Enzyme buffer component
Methacrylic acid N-hydroxysuccinimidyl ester	Sigma-Aldrich	730300	Anchoring reagent
Methanol	Sigma-Aldrich	32213	Sample dehydration & storage
MicrobeJ	-	-	Image processing software plugin
Microscopy glass slides	Chemglass Life Sciences	CG-8221	Gelation chamber component
Monosodium phosphate	Sigma-Aldrich	S9638	Enzyme buffer component
Mutanolysin from Streptomyces globisporus	Sigma-Aldrich	M990	Peptidoglycan digestion
N, N, N', N'-tetramethylethylenediamine	Sigma-Aldrich	T7024	Gelling solution component
N,N′-methylenebisacrylamide	Sigma-Aldrich	M1533	Monomer solution component
Nunc 40.4 mm glass-bottom Petri dishes	Thermo Fisher	12-567-400	Imaging chamber option
Paraformaldehyde	Sigma-Aldrich	158127	Sample fixation
Phosphate buffer saline	Sigma-Aldrich	79383	Monomer solution component, Washing, Enzyme buffer
Proteinase K from Engyodontium album	Invitrogen	25530-031	Protein digestion
Proteus mirabilis ATCC 12453	Microbiologics	0440P	Study subject
Small flat paint brush	Local hardware store	-	Sample manipulation/handling
Sodium acetate	Sigma-Aldrich	241245	Enzyme buffer component
Sodium acrylate	Sigma-Aldrich	408220	Monomer solution component
Sodium bicarbonate	Sigma-Aldrich	S5761	Enzyme buffer component
Sodium chloride	Sigma-Aldrich	S9888	Monomer solution component, Enzyme buffer component
Sodium hydroxide	Sigma-Aldrich	221465	Glass slides cleaning
Solid-state laser line	Omicron	Not available	488, 568 and 647 nm Excitation laser line
SYTO 61 nucleic acid staining	Invitrogen	S11343	Fluorescent dye
Tris(hydroxymethyl)aminomethane hydrochloride	SAFC	108219	Enzyme buffer component
Triton X-100	Sigma-Aldrich	X100	Sample permeabilization, Enzyme buffer component
Volocity 7.0.0 capture software	Quorum Technologies	-	Microscoy acquisition controller software
Volocity ViewVox spinning disk system	Perkin-Elmer	Not available	Spinning disk system for confocal microscope
Wheat-germ agglutinin – Alexa Fluor 488 fluorescent lectin conjugate	Invitrogen	W11261	Fluorescent dye