In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

1. Preparation of Materials

1. Preparation of Medium Tubing
   1. Serum Bottles (one needed per biofilm culture and three needed per growth curve)
      Note: As mentioned in the introduction, any growth medium suitable to provide the nutrients needed for the strain of bacteria of interest can be utilized for this procedure; in this case, "nanowires" media and TSB without dextrose medium was used for the growth of S. oneidensis MR-1 GFP.¹³
      1. Deposit 20 mL of growth medium into one 70 mL serum bottle, cap the stopper and crimp the bottle. Cover the top with a piece of clean sterile aluminum foil.
      2. Autoclave the bottle with a liquid cycle for 30 min at 121 °C. Afterward, store the sterilized serum bottle at room temperature.
   2. Anaerobic Culture Tubes
      1. Deposit 5 mL of growth medium into an anaerobic culture tube with air headspace, put on the stopper and crimp the bottle. Cover the top with sterile foil.
2. Preparing Bubble Trap Tubing
   1. Using a 22 gauge needle, carefully punch a hole through a serum bottle rubber stopper.
   2. Cut 20 in of 1/32" polytetrafluoroethylene (PTFE) tubing with a razor such that one end of the segment of tubing is pointed.
   3. Using the pointed end, thread the PTFE tubing through the hole in the top portion of the rubber stopper. Push the tube through until roughly 2 cm is through the stopper and cut the pointed end of the PTFE tube with a razor to have a flat end.
   4. Remove the plunger of a 5 mL syringe and fit the rubber stopper from step 1.2.3 into the open end of the syringe. The 2 cm end of PTFE tubing is within the barrel of the syringe. This is the bubble trap.
   5. Wrap the bubble trap (the PTFE tubing, rubber stopper, and syringe) made in step 1.2.4 with aluminum foil and secure with autoclave tape. Autoclave the foil package to sterilize the tubing with a gravity cycle at 121 °C for 30 min. Store the sealed foil package at room temperature until ready for use in step 2.4.
3. Bacterial Growth Curve
   NOTE: This protocol uses S. oneidensis MR-1 GFP as a model organism for growing the biofilm in SALVI. However, this procedure can be adapted to accommodate other bacteria growing aerobically or anaerobically. Depending on which strain is used, growth time and environment may need to be adjusted accordingly.
   NOTE: -80 °C bacterial freezer stocks consisted of a 1:1 mixture of TSB without dextrose and glycerol. The growth curve example shown in Figure 1B was prepared using a starter culture of TSB with no dextrose, transferred in respective 0.2 mL quantities three times to 70 mL serum bottles with 20 mL "nanowires" medium to remove traces of TSB. However, this protocol assumes that only one growth medium will be used, and that subsequent transfers will not be conducted, as this was done with these particular medium solutions for S. oneidensis. Although not outlined in this protocol, extra transfers like this are recommended as initially depositing the bacteria to the rich TSB helps the frozen cells recover; and three subsequent transfers to "nanowires" ensures that all TSB is gone and that typical growth dynamics are occurring.
   1. Inoculating the Starter Culture
      1. Remove the bacterial glycerol stock from the -80 °C freezer and warm the stock with a gloved hand to thaw as quickly as possible. Move this freezer stock to the biological safety cabinet (BSC).
      2. Within the BSC, use a 1 mL syringe with a 22G needle attached to transfer 0.1 mL of freezer stock to a capped and crimped anaerobic culture tube containing 5 mL of growth medium with no dextrose, prepared in step 1.1.2. Dispose of the remaining freezer stock in the appropriate biological waste container, as freezing again could shock the cells.
      3. Incubate the starter culture in a shaker/incubator at 30 °C at 150 rotations per minute (rpm) and be sure to take an optical density at 600 nm (OD₆₀₀) reading when ready for use. Record the time of growth and OD₆₀₀ such that it can be done at that same time for every subsequent use, such that results are reproducible. As an example, this starter culture was allowed to grow for 15 h and used at an OD₆₀₀ of approximately 1.0.
   2. Inoculating Growth Medium
      1. Take the three serum bottles prepared in step 1.1.1.2, and the starter culture prepared in 1.3.1.3, and bring both to the BSC.
      2. Within the BSC, using a sterile 1 mL syringe with a 22G needle attached, transfer 0.1 mL of solution from the starter culture to each of the serum bottles, respectively.
      3. Sterilize the top of the serum bottles with 70% ethanol and cover with sterilized aluminum foil, label appropriately, and transfer to an orbital shaker within an incubator. Set the orbital shaker to 150 rpm and set the incubator to 30 °C. Incubate until the first OD₆₀₀ data point is taken, within step 1.3.3.
   3. Obtaining OD ₆₀₀ Data Points
      1. Prepare a blank by depositing 100 µL of filter-sterilized distilled deionized (DI) water into a sterile cuvette. Wrap plastic paraffin film around the top of the cuvette so that the water will not be contaminated. Store at room temperature. Use this blank with the UV/Vis Spectrophotometer before taking any data points for the growth curve by inserting the cuvette and pressing "blank".
         NOTE: Every 48 h a new blank should be prepared to avoid incorrect reading, as regularly replacing a blank is good practice.
      2. For each OD₆₀₀ data point, remove the serum bottles prepared in step 1.3.2.3 from the incubator and transfer to a sterilized BSC. Take 0.1 mL of the inoculated medium from each serum bottle and deposit into three separately labeled sterile cuvettes.
      3. After blanking, insert cuvette containing the culture into the ultraviolet visible (UV/Vis) Spectrophotometer and read the OD₆₀₀ one at a time, and record all three readings for that time point.
         NOTE: For S. oneidensis MR-1, data points were taken at 1, 14, 28, 32, 41, 57, 81, and 105 h after inoculation. The growth is complete when the bacteria has completed the death phase. These points should be adjusted accordingly if there is lack of knowledge on the particular growth time and tendency of bacteria used in this protocol. If there is uncertainty about the growth tendency, data points should be collected more frequently, for example, every three h for the first 12 h, every six h for subsequent 36 h, at 12 h intervals for the following 100 h, and at 24 h intervals for the final 124 h.
      4. Using the OD₆₀₀ data points as the y-axis and the time as the x-axis, graph a curve of the average of the three points taken with standard deviation error bars for each time point using a graphing software. The S. oneidensis MR-1 growth curve is displayed in Figure 1B in the representative results section.
      5. Using the graph prepared in step 1.3.3.4, identify the timeframe of the log-phase section of the growth curve. For Shewanella, this is between 12 and 33 h of growth; therefore it can be inferred that at 24 h of growth, Shewanella will be within its log-phase of growth, assuming that the bacteria will be cultured using the same media and treatment before use that was used for the growth curve.

2. Culturing the Bacteria

1. Day One: Inoculating the Agar Plate
   NOTE: This section of the procedure was used with an agarose plate to take one colony-forming unit (CFU) at log-phase, instead of the liquid starter culture used for the growth curve. This could be assumed to reproduce the same growth conditions, due to the fact that TSA without dextrose was used and subsequently transferred to "nanowires" medium in the growth curve procedure, and TSA has the same ingredients that comprise TSB.
   1. Remove S. oneidensis MR-1 GFPbacteria stock from -80 °C freezer and place into an ice bucket, place this bucket inside of sterilized BSC.
   2. Within the BSC, use a sterile 1 µL inoculation loop to scrape the surface of the frozen bacteria stock and use the loop to T-streak the surface of an agarose plate.
   3. After sealing the sides of the plate with plastic paraffin film, invert the plate and store in a 30 °C incubator for 24 h, until individual colonies appear.
2. Day Two: Inoculating the Serum Bottle
   1. Remove the plate from the incubator and open within the BSC.
   2. Within the BSC, clean the surface of the rubber stopper on a prepared serum bottle from step 1.1.1 with 70% ethanol in DI water.
   3. Select an individual CFU from the agar plate and, using a sterile syringe with an attached 22 gauge needle, deposit enough growth media to dislodge the colony from the plate without touching any neighboring colonies and mix the colony with the medium with the tip of the needle.
      NOTE: A singly colony should be selected that is far enough away from other bacteria on the plate, such that medium can be deposited onto it without touching any other colonies.
   4. Using the same syringe, extract the liquid from the surface of the plate.
   5. After tapping out bubbles within the syringe, inject the liquid into the serum bottle, and place onto an orbital shaker within 30 °C set at 150 rpm for 24 h.
      NOTE: Tapping the bubbles out of the syringe is important in order to avoid introducing more oxygen to the serum bottle, as introducing more oxygen to the bottle can change experimental conditions and reduce consistency between growth times for bacteria.
3. Day Two: Sterilization of the SALVI Microchannel
   NOTE: To promote sterility of the tubing system, steps that require detaching the syringe from the tubing and replacement with a new syringe should be done within the BSC. To do this, simply detach the syringe from the syringe pump by unscrewing the metal syringe holder, and bring the syringe with tubing attached to a sterile BSC. When tubing system is mentioned in the procedures, this refers to the syringe containing the liquid reservoir, attached with a polyetheretherketon (PEEK) injector to the PTFE tubing, attached to the drip chamber, which has a PEEK injector fitting attached to the SALVI inlet tubing, as well as the outlet container that the SALVI outlet tubing is sealed to.
   1. Use a new SALVI device and attach a PEEK fitting and injector to one end of a PTFE tubing.
      NOTE: SALVI devices are prepared fresh for each experiment following the device fabrication detailed in previous research and patents.^5,6,8,15
   2. Take 2 mL of 70% ethanol into a syringe and connect to the PEEK fitting on the SALVI. After connecting the syringe to a syringe pump and attaching the outlet of the SALVI to a waste bottle, allow the ethanol DI water solution to run through the SALVI at 20 µL/min for 1.5 h.
   3. Take 4 mL of sterilized DI water into a syringe and connect to the inlet of the same SALVI. After connecting the syringe to a syringe pump, allow the water to run through the SALVI at 20 μL/min for at least 3 h.
   4. Within a BSC, open the foil packet prepared in step 1.2.5 and connect a sterile PEEK injection fitting to the end of the 5 mL syringe and sterile PEEK fittings to the end of the TFE tubing. Take ~ 3 mL of sterile medium into a syringe and attach to the TFE tube. Invert the 5 mL drip chamber and, using a sterile syringe, inject the growth medium into the drip chamber until it reaches a total volume of 1 mL.
   5. Connect the end of the 5 mL syringe drip chamber to the inlet of the SALVI.
   6. Take 10 mL of growth medium into a sterile syringe and connect to the inlet of the drip tubing. After connecting the syringe to a syringe pump, allow the medium to run through the SALVI at 20 µL/min for 12 h (or overnight). Use adhesive tape to secure these parts. Cover the outlet bottle with foil or plastic paraffin film to minimize the probability of dust particles and organisms contained therein to contaminate the medium. A detailed depiction of how this setup should look can be found in Figure 1A.
      1. Allow the drip tubing to be vertical, meaning that the syringe pump should be placed on an elevated surface with drip tubing secured with tape, and with the SALVI secured on a flat surface below.
      2. Run medium through the SALVI for 12 h to ensure that all traces of ethanol have been removed from the microchannel and tubing of the SALVI before inoculating with bacteria.
      3. For added protection, keep the SALVI microchannel chamber within a sterilized Petri dish, with the sides cut to fit the inlet and outlet tubing. Additionally, keep tape always on the window to protect the SiN membrane and channel prior to imaging analysis.
4. Day Three: Inoculation of the SALVI Microchannel
   1. Remove the whole tubing system (syringe connected to drip chamber connected to SALVI connected to the waste bottle) from the syringe pump and bring it to a sterilized BSC. Additionally, remove the serum vial from step 2.2.5 from the incubator and bring it to the BSC.
   2. Clean the surface of the serum bottle stopper with 70% ethanol to prevent any contamination, and then use a sterile syringe with an attached sterile 22G needle to extract 4 mL of bacteria from the bottle.
   3. Detach the SALVI from the 5 mL chamber of the drip tubing, and connect the syringe with inoculated medium directly to the inlet of the SALVI. Leave the drip tubing within a sterile aluminum foil packet or within the BSC.
   4. Attach the syringe with SALVI and outlet bottle to the syringe pump to run at 20 µL/min for 3 h to inoculate the channel of the SALVI, in order to allow for multiple volume changes of the liquid contained within SALVI.
   5. After inoculation, disconnect the syringe with SALVI from the syringe pump and bring to the BSC. After attaching the inlet of the SALVI back to the 5 mL drip chamber from step 2.4.3, take 20 mL of growth medium into a sterile syringe and attach to the inlet of the drip tubing.
   6. Bring the tubing attached to the SALVI from the BSC to the syringe pump and allow the medium to pass through the tubing at a rate of 2 µL/min for six to ten days, or until fluorescence imaging (discussed in step 3) shows favorable visible biofilm growth for ToF-SIMS analysis. Refill fresh medium as it runs out within the BSC by filling a new sterile syringe with 10mL of growth medium and attaching to the SALVI after the previous growth medium has run out.
      NOTE: After beginning inoculation at 2 µL/min, the flow rate should never be increased or decreased, as changing the flow rate would create shear-stress within the channel and detach the biofilm. It is critical that this flow rate should never be changed; to prepare for this, ~24 h before SIMS, observe the tubing closely to make sure there are no bubbles so that there is no need to push more medium at a faster rate throughout the tubing.
      1. Avoid bubbles in the microchannel, as they can push the biofilm out of the channel. It is important to be cautious of bubbles within the bottom of the 5 mL drip tubing where it connects to the SALVI tubing. Fresh medium can be injected into the end of the PEEK injector to ensure that no air will be forced into the SALVI.

3. Optical Imaging of the Biofilm within the SALVI Microchannel

1. Fluorescence Microscopy Imaging
   NOTE: The Shewanella used as a model organism within this protocol is mutated to express GFP; as such, it does not require staining. If the bacteria require staining, this should always be done at the same flowrate (e.g., 2 µL/min) to avoid biofilm detachment. Additionally, when cells go without nutrient availability, they will detach. Therefore, if any additional staining is required, the stain should be injected to the growth medium rather than water before supplying to stain the cells.
   1. Detach the SALVI from the drip tubing inside the BSC and close the SALVI by screwing the PEEK fittings to finger-tighten the PEEK union.
   2. Tape the tubing of the SALVI microchannel chamber securely onto a glass slide, such that the window is completely flat and facing upward. Remove the protective tape from the window. Clamp the slide to the stage of the microscope by placing the glass slide between the stage clamps on the platform.
   3. Lower the stage of the microscope such that the top of the SALVI is positioned close enough to the end of the 10X objective, where adjustment of the focus will not cause the lens to touch the window.
   4. Switch on the backlight and find the channel using the 10x microscope objective by adjusting the focus. At this time, make sure the presence of bacterial attachment to the SiN window of the SALVI is apparent in comparison to the window of a control channel with no inoculated bacteria. For closer imaging of cells, switch to the 20x objective. When compared to an empty SALVI channel, the presence of bacteria attached to the window should have a strong green fluorescence.
   5. Turn off the backlight and turn on the microscope mercury source. Wait 2-3 min and switch to the FITC filter set on the microscope to view and capture images of the biofilm attached to the window. As an example of what the matured biofilm will look like when ready, refer to Figure 1C.
   6. Turn off the mercury source and detach the SALVI from the slide. If needed, attach to 2 µL/min medium flow once again to allow for more growth before imaging again, or transfer the SALVI to secondary containment use for ToF-SIMS analysis. This is needed if biofilm thickness is concluded to not be thick enough, and more time for growth is required before ToF-SIMS analysis. To attach to medium flow once again, refer to step 2.3.6.
      NOTE: If put back under medium flow, fluorescence imaging should be done again before ToF-SIMS analysis.
      1. Give the biofilm growth medium to feed it until ToF-SIMS analysis can be conducted. While not recommended, if needed, the closed SALVI can be stored within 4 °C overnight, but should be allowed to warm to room temperature before inserting to the vacuum chamber of the ToF-SIMS. However, analyze the biofilm immediately after detached from medium flow to reduce biofilm detachment.

4. ToF-SIMS Data Acquisition

NOTE: This section serves as an overview, and is discussed in greater detail within our earlier protocol describing adsorbed protein molecule liquid SIMS analysis.¹²

1. Install SALVI into the ToF-SIMS Loadlock Chamber
   NOTE: Gloves should be worn at all times when handling the SALVI device and installing it onto the ToF-SIMS stage to avoid potential contaminations during surface analysis.
   1. Mount SALVI onto the stage and fix it with several screws. Make sure that the SiN window is flat by adjusting screw tightness and inserting silicon wafer pieces at the bottom of the PDMS microchannel chamber. Remove the protective tape from the window, then load the stage into the ToF-SIMS loadlock chamber.
   2. Open the ToF-SIMS loadlock, hold and set the stage horizontally onto the loading platform, and close the loadlock door. Initiate the vacuum pump and wait to ensure that suitable vacuum is reached.
   3. Move the SALVI into the main chamber once the vacuum is stabilized to 1×10^-7 mbar.
2. Depth Profiling and Collecting Data Points
   1. Find the microfluidic channel using the optical microscope equipped in ToF-SIMS.
   2. Select positive or negative mode before data acquisition. Use the 25 keV Bi₃⁺ beam as the primary ion beam in all measurements, and use the electron flood gun to neutralize the surface charging during all measurements.
   3. Scan the Bi₃⁺ beam with 150 ns pulse width on a round area with a diameter of ~2 µm with 64 pixels by 64 pixels resolution. After punching through the 100 nm SiN window, continue to scan for another 150 s to collect high intensity data for imaging. After punch-through, the counts will increase significantly within the depth profiling region. After stabilizing, this can be referred to as the high-intensity region.
   4. Reduce the pulse width to 50 ns for data collection to acquire spectra with better mass resolution. Continue this acquisition for about another 200 s.
   5. Repeat these steps for collecting at least three positive and three negative data points.
      NOTE: Be sure to space the punch-through areas such that the SiN window will not break and leak into the chamber of the vacuum from compromised window integrity.

5. ToF-SIMS Data Analysis

1. Mass Calibration using IonToF Software
   1. Open the analysis software of ToF-SIMS (Measurement Explorer) and then click the "profiles", "spectra" and "image" buttons, respectively, to process depth profile, m/z spectrum, and image data.
   2. Open the data files obtained throughout ToF-SIMS data acquisition.
   3. Select the depth profiling data of interest and reconstruct the spectra according to the depth profile temporal series.
   4. Press "F3" to open the "mass calibration" window. Choose peaks for calibration based on chemical compounds which are expected to exist in the specific sample.
2. Peaks of Interest Selection
   1. As peak selection is necessary for sample analysis, determine characteristic peaks of the sample according to literature or other previous findings, if any. Compare the intensity of peaks of interest in the m/z spectra. If necessary, add new peaks or delete interference peaks in the peak list.
   2. Click on a peak, find the red lines at the left bottom window. Move the red lines to enclose the whole peak.
   3. Select a peak matching the chemical formula in the main spectrum window, and then to click the "add peak" button above the window.
3. Export Mass Calibrated Data
   1. To export a peak list, in the "Peak List" menu, select "Save…" the peak list in the "itmil" format.
   2. To export a depth profile, in the "Profile" window, click on the "File" menu, then select "Export", and then select "Save as" a ".txt" file.
   3. To export an m/z spectrum file, in the "Spectra" window, click on the "File" menu, then select "Export", and then select "Save as" a ".txt" file.
   4. To export an image file, in the "Image" window, print screen and save as the image file.

6. ToF-SIMS Data Plotting and Presentation

1. Use a graphic tool to import the data.
2. Make a plot using the m/z as the x-axis and peak intensity as the y-axis to show the reconstructed spectrum. An example is given in Figure 2A.
3. Combine reconstructed two-dimensional (2D) images of different m/z and form a matrix to show either positive or negative ion mapping. An example is given in Figure 2B.