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Jan 2021

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Development and Quantitation of Pseudomonas aeruginosa Biofilms after in vitro Cultivation in Flow-reactors
流动反应器体外培养铜绿假单胞菌生物膜的研制与定量    

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Abstract

Characterization of biofilm formation and metabolic activities is critical to investigating biofilm interactions with environmental factors and illustrating biofilm regulatory mechanisms. An appropriate in vitro model that mimics biofilm in vivo habitats therefore demands accurate quantitation and investigation of biofilm-associated activities. Current methodologies commonly involve static biofilm setups (such as biofilm assays in microplates, bead biofilms, or biofilms on glass-slides) and fluidic flow biofilm systems (such as drip-flow biofilm reactors, 3-channel biofilm reactors, or tubing biofilm reactors). Continuous flow systems take into consideration the contribution of hydrodynamic shear forces, nutrient supply, and physical transport of dispersed cells, which define the habitat for biofilm development in most natural and engineered systems. This protocol describes the assembly of 3 flow-system setups to cultivate Pseudomonas aeruginosa PAO1 and Shewanella oneidensis MR-1 model biofilms, including the respective quantitation and observation approaches. The standardized flow systems promise productive and reproducible biofilm experimental results, which can be further modified according to specific research projects.

Keywords: Biofilm characterization (生物膜的特性), Fluid-flow biofilm reactors (流体流动生物膜反应器), Pseudomonas aeruginosa (铜绿假单胞菌)

Background

Biofilm is the most prevalent growth mode of microbial organisms in nature, industry, and clinical habitats; it is commonly recognized as bacterial communities embedded in the self-generated matrix of extracellular polymeric substances (EPS) (Flemming et al., 2016). The spatial organization of a biofilm aggregate is highly heterogeneous and has diverse metabolic activities, rendering the biofilm robust and tolerant to numerous types of environmental stress (Van Dyck et al., 2021). Hence, biofilms are promising in industry to ferment for nutrient conversion, degrade hazardous compounds for bioremediation, and generate electricity in microbial fuel cells (Coenye and Nelis, 2010). Conversely, biofilms also raise public concerns such as biofouling and biofilm-associated infections. Biofilms are the leading cause of chronic wound infections and infections on biomedical devices, such as cardiac valves, tracheal tubes, and catheters (Del Pozo et al., 2018). An improved understanding and characterization of biofilm formation, dispersion, and activities will shed light on biofilm control strategies.


A typical biofilm life cycle usually involves 5 stages: initial reversible attachment of bacteria, stable and irreversible attachment, maturation, dispersion, and dispersed free-living bacteria (Martin et al., 2021). The biofilm communities show active social behaviors and interaction with environmental factors, which in return regulate the cellular metabolism of biofilms. Numerous methodologies and devices have been documented for the investigation of biofilm morphology and development in vitro, commonly categorized as static biofilm assays and continuous-flow biofilm systems. The static biofilm assays - such as those in microplates and on air-liquid interface coverslips or colony biofilm assays and Kadouri drip-fed biofilm assays - are prevalently applied to the screening of early events in biofilm formation (Merritt et al., 2005). The static biofilm assays are high-throughput and easily executed with common laboratory equipment; however, nutrient supply in static biofilm assays is limited with respect to developing mature biofilm communities that mimic nature.


Given the fact that biofilms are frequently observed under fluidic flow conditions in nature and engineered systems, nutrient availability, physical transport, and hydrodynamic shear forces significantly impact biofilm formation and metabolism (Mattei et al., 2018). To address the question of how biofilm responds to environmental stresses and communicating signals, it is important to create a standardized and reproducible protocol for cultivating in vitro biofilms in flow systems mimicking the in vivo fluid habitats (Cowle et al., 2020). The development of flow biofilm reactors has been advanced by emerging microfluidics manufacturers and designs, with the most common flow systems including drip-flow systems (Gonzalez et al., 2014), tubular reactors (Winn et al., 2014), planar flow-cell (Zhang et al., 2011), and 3-channel flow-cell (Sternberg and Tolker-Nielsen, 2006; Pamp et al., 2009). Further modifications, such as segmented flow cells (Karande et al., 2014), gradient-generator flow cells (Zhang et al., 2019), and flow-velocity microfluidic flow cells (Liu et al., 2019), are specifically modified and designed to meet specific research purposes.


In this protocol, we addressed three commonly applied flow-system setups for different research purposes with Pseudomonas aeruginosa PAO1 and Shewanella oneidensis MR-1 as model organisms. The tubing biofilm reactor mimics the biofilm habitats in pipelines, tracheal tubes, catheters, etc., allowing biofilm harvest with adequate biomass. The 3-channel flow-cell system is designed for the non-invasive spatiotemporal observation of biofilm morphologies with a continuous and steady nutrient supply. The recycling biofilm reactor enables the study of biofilm metabolic responses toward its bioremediation metabolites and/or environmental chemicals (Sternberg and Tolker-Nielsen, 2006; Weiss Nielsen et al., 2011; Pamp et al., 2019). Overall, the standardized flow systems promise productive and reproducible biofilm observation and quantitation, which can be further modified according to the specific research project.

Materials and Reagents

  1. Silicon tube (I.D=1 mm, O.D=3 mm, Runze, China)

  2. Silicon tube (I.D=3.2 mm, O.D=6.4 mm, Runze, China)

  3. Peristaltic pump tube (I.D=1.02 mm, O.D=2.62 mm, Pharmed BPT, Sain-Gobain)

  4. Straight connector (Runze, catalog number: DI-016)

  5. Straight connector (Runze, catalog number: DI-032)

  6. Luer connector (Runze, catalog numbers: RH-G016; RH-M016)

  7. Needle (Gauge: 26G, Hypodermic; BD, catalog number: 305111)

  8. Syringe (1 ml, BD, Luer-Lock, catalog number: 309628)

  9. Silicone glue (Advanced Silicone 2, GE sealant)

  10. Parafilm (BEMIS, catalog number: PM996)

  11. Metal foil (Maryya, , catalog number: HC081260)

  12. 10 μl Inoculation loop (Sangon Biotech, catalog number: F619312)

  13. 15 ml Centrifuge tube (Sangon Biotech, catalog number: F602888)

  14. Coverslips (Sangon Biotech, catalog number: F518117)

  15. Microplates (ThermoFisher, NuncTM, catalog number: 168055)

  16. 0.22 μm Syringe filter (Sangon Biotech, catalog number: F513163)

  17. 2 ml Microtubes (Maxyclear Snapclock, Axygen, US)

  18. LB broth (Sangon Biotech, catalog number: A507002)

  19. Calcium chloride (Sangon Biotech, catalog number: A501330)

  20. Sodium chloride (Sangon Biotech, catalog number: A100241)

  21. QIAamp DNA Micro Kit (QIAGEN, catalog number: 56304)

  22. RNeasy Mini Kit (QIAGEN, catalog number: 74104)

Equipment

  1. Peristaltic pump (200 series 16-channel pump, Watson Marlow)

  2. Microplate reader (Spark®, Tecan, Switzerland)

  3. Flowcytometer (CytoFLEX, Beckman, USA)

  4. Probe sonicator (SONICS, model: VCX750)

  5. Centrifuge (Eppendorf, model: 5418R)

  6. Biosafety Cabinet, BSC (MSC-AdvantageTM II, ThermoFisher, USA)

  7. Autoclave (Zealway, model: GR-60DA)

  8. Confocal Laser Scanning Microscope, CLSM (Zeiss, model: LSM900)

Software

  1. Imaris (Bitplane, Oxford Instrument)

  2. ImageJ (https://imagej.net/)

  3. Comstat 2 (http://www.comstat.dk/)

Procedure

  1. Tubing biofilm reactors (Figure 1, Figure S1)



    Figure 1. Illustration of the tubing biofilm reactor


    Notes:

    1. Tubing biofilm reactors contain the following parts in sequence: feeding bottle, feeding tubing (I.D=1 mm), peristaltic pump tubing (I.D=1.02 mm), syringe filter (0.2 μm), injection tubing (I.D=1 mm), biofilm tubing (I.D=3.2 mm), waste tubing (I.D=1 mm), and waste bottle.

    2. All parts of this setup, except ⑤ and ⑦, can be reused for repeated experiments. Please refer to section H for cleanup procedures.

    3. The tubing biofilm reactors do not allow the spatiotemporal imaging observation of biofilms. They particularly meet the requirement of harvesting a large amount of biomass for -omics analysis.


    1. Connect the feeding tubing to the peristaltic pump tubing with the straight connector. Make sure that the length of the feeding tubing is compatible with the height of the feeding bottle.

    2. Connect the other side of the pump tubing to the syringe filter with a female Luer fitting.

    3. Connect the other side of the filter to the injection tubing with a male Luer fitting.

    4. Connect the injection tubing to the biofilm tubing with a reducing straight connector. The length of the biofilm tubing depends on the experimental design.

    5. Connect the biofilm tubing to the waste tubing with a reducing straight connector.

    6. Wrap the assembled part (1-5) in metal foil and autoclave to sterilize.

    7. Freshly prepare one-tenth LB broth in the feeding bottle and autoclave to sterilize.

    8. Autoclave the waste bottle and pour in bleach before use.

    9. UV-sterilize pieces of parafilm (10 cm × 10 cm) and metal foil (15 cm × 15 cm) in the biosafety cabinet before use.

    10. Place the feeding tubing into the feeding bottle and make sure that the tubing reaches the bottom of the bottle. Place the waste tubing into the waste bottle and make sure that the tubing is close to the bottleneck rather than the bottom. Cover each bottle with sterilized parafilm and metal foil. This step must be carried out in the biosafety cabinet.

    11. Place the pump tubing onto the peristaltic pump and start the pump at max speed to purge the system with growth medium. Make sure that the system is free of bubbles before setting the flow rate back to normal.

    12. Shift the tubing biofilm reactor with the peristaltic pump into a temperature-controlled incubator. Make sure that the biofilm tubing is at a similar level as the bottleneck of the waste bottle to avoid any backflow.


  2. Three-Channel flow-cell biofilm reactor (Figure 2, Figure S2)



    Figure 2. Illustration of the 3-channel flow-cell biofilm reactor


    Notes:

    1. The 3-channel flow-cell system contains the following parts in sequence: feeding bottle, feeding tubing (I.D=1 mm), peristaltic pump tubing (I.D=1.02 mm), intermediate tubing (I.D=1 mm), syringe filter (0.2 μm), injection tubing (I.D=1 mm), biofilm tubing (I.D=3.2 mm), waste tubing (I.D=1 mm), and waste bottle.

    2. All parts of this setup, except ⑤, can be reused for repeat experiments. Part ⑦ can be reused by removing the coverslip and cleaning with 70% ethanol. For the reuse of other parts, refer to section H for the cleanup procedures.

    3. The 3-channel biofilm system is most commonly applied to study the biofilm formation and activities of P. aeruginosa, Staphylococcus aureus, etc. This setup is slightly modified from the traditional version; the bubble trap is replaced by a syringe filter and the air-connecting tubing in the feeding bottle is removed. According to the authors' experience, the bubble trap is the major cause of leaking, and removal of the air-connecting tubing does not impact the oxygen level in the media. This device is feasible for the non-invasive spatiotemporal observation of biofilm morphology and community compositions, although biofilm harvest is not recommended.


    1. Wash the 3-channel flow cell with ethanol and dry it in the fume hood. Make sure that the surface of the flow cell is clean and dry upon assembly.

    2. Place a thin layer of silicon glue on the walls of the 3-channel flow-cell and place a coverslip on top. Carefully press the coverslip toward the base of the flow-cell until it is fully sealed without any bubbles in the sealing area. Leave the sealed flow-cell at room temperature overnight to fully dry the glue.

    3. Connect the feeding tubing to the peristaltic pump tubing with a straight connector.

    4. Connect the other side of the pump tubing to an intermediate tubing with a straight connector. Make sure that the length of intermediate tubing allows the flow-cell unit to be moved to the stage of the confocal microscope.

    5. Connect the other side of the intermediate tubing to the syringe filter with a female Luer fitting.

    6. Connect the other side of the filter to the injection tubing with a male Luer fitting.

    7. Connect the injection tubing to one channel of the 3-channel flow-cell.

    8. Connect the waste tubing to the other side of the flow-cell channel.

    9. Assemble the tubing with the feeding bottle, waste bottle, and peristaltic pump using the same procedures as described for the tubing biofilm reactors.


  3. Recycling flow-cell system (Figure 3)



    Figure 3. Illustration of the recycling flow-cell system


    Notes:

    1. The recycling flow-cell system contains the following parts in sequence: feeding bottle, feeding tubing (I.D=1 mm), peristaltic pump tubing (I.D=1.02 mm), flow-cell reactor, waste tubing (I.D=1 mm), waste bottle with recycling tubing, and fresh medium bottle with supply tubing.

    2. All parts of this setup, except ⑤ and ⑦ can be reused for repeat experiments. Please refer to section H for cleanup procedures.

    3. The recycling flow-cell system is commonly applied to study the bioremediation of toxicants and environmental compounds with a view to mimicking the wastewater treatment plants. Part ⑦ is not limited to tubing and/or 3-channel chambers. The feeding ratio of fresh medium to waste medium should be modified by each project.


    1. Assemble the feeding parts by following the same procedures 1-5 in the section of “Assembly of the tubing reactor.”

    2. Assemble the recycling waste parts: Connect the feeding tubing to a syringe filter with a female Luer fitting; connect the pump tubing to the other side of the syringe filter with a male Luer fitting; connect the other side of the pump tubing to another feeding tubing (recycling feeding tubing) with a straight connector.

    3. Assemble the recycling fresh medium parts: Connect the feeding tubing to the pump tubing with a straight connector; connect the other side of the pump tubing to another feeding tubing (recycling feeding tubing) with a straight connector.

    4. Wrap each part individually in metal foil and autoclave to sterilize.

    5. Freshly prepare one-tenth LB broth in both the feeding and fresh-medium bottle.

    6. Autoclave the waste bottle.

    7. UV-sterilize pieces of parafilm (10 cm × 10 cm) and metal foil (15 cm × 15 cm) in the biosafety cabinet before use.

    8. Place the three feeding tubings into the feeding bottle, waste bottle, and fresh-medium bottle, respectively. Make sure that the tubing reaches the bottom of each bottle. Place the waste tubing into the waste bottle and make sure that the tubing is close to the bottleneck rather than the bottom. Place the end of the recycling feeding tubing from both the waste and fresh-medium parts into the feeding bottle. Make sure that the tubings are close to the bottleneck rather than the bottom. Cover each bottle with sterilized parafilm and metal foil. This step must be carried out in the biosafety cabinet.

    9. Place the feeding pump tubing onto the feeding peristaltic pump and place the two recycling and feeding tubings onto the recycling peristaltic pump.

    10. Adjust the rotation speed of the feeding pump to be twice that of the recycling pump.

    11. Start the feeding pump first at the highest speed to purge the system with growth medium. Make sure that the system is free of bubbles before setting the flow rate back to normal.

    12. Shift the whole system into a temperature-controlled incubator and start both pumps.


  4. Biofilm inoculation and cultivation (Figure S3)

    1. Prepare the inoculum: cultivate bacteria overnight at the appropriate temperature and dilute to the desired cell density with growth medium. Typically, 1 × 108 cells/ml for Pseudomonas aeruginosa can promise a robust biofilm.

    2. Load the bacterial inoculum into a syringe with a 26G needle. Make sure that the volume of the inoculum fills the biofilm reactor.

    3. Stop the peristaltic pump and clamp off the injection tubing before inoculation.

    4. Sterilize the insertion site on the biofilm tubing with 75% ethanol.

    5. Load the bacterial suspension into each biofilm reactor.

    6. Clamp off the effluent tubing.

    7. Remove the needle and seal the hole immediately with silicone glue.

    8. Allow attachment for 2 h without flow in the incubator. Make sure that the inner surface of the biofilm reactor faces down for initial attachment.

    9. Turn over the biofilm reactor so that the inner surface of biofilm attachment faces up.

    10. Start the flow (3-8 ml/h/channel) and remove the clamps.

    11. Cultivate the biofilm in the incubator for the desired duration before harvest.


  5. Biofilm treatment

    1. Direct perfusion

      1. Prepare the CaCl2 solution at the desired concentrations in LB broth (0 mM, 0.34 mM, and 0.68 mM) and load into a 1-ml syringe with a 26G needle.

      2. Prepare the control solution containing LB broth and the chemical vector (H2O in this case) and load into a 1-ml syringe with a 26G needle.

      3. Stop the peristaltic pump and clamp off the injection tubing before perfusion.

      4. Sterilize the insertion site on the biofilm tubing with 75% ethanol.

      5. Inject and load the chemical solution into the biofilm tubing.

      6. Remove the needle and seal the hole immediately with silicone glue.

      7. Allow static treatment for the desired duration.

      8. Start the flow (3-8 ml/h/channel) and remove the clamp, or harvest the biofilms directly.

    2. Continuous loading

      1. Prepare a new bottle of sterile medium containing CaCl2 (0.68 mM) and a bottle of sterile medium containing the vector for the drug solution (H2O).

      2. Sterilize pieces of parafilm and metal foil.

      3. Stop the peristaltic pump and clamp off the feeding tubing.

      4. Shift the feeding tubing into the new feeding bottle containing calcium chloride/vector.

      5. Cover the feeding bottle with parafilm and metal foil.

      6. Remove the clamps and start the pump; make sure that there are no bubbles inside the system.


  6. Biofilm harvest

    1. Biofilm harvest from the tubing reactor:

      1. Stop the flow and clamp off both the injection and waste tubings.

      2. Take the section including the injection tubing, biofilm tubing, and waste tubing off the system into the biosafety cabinet.

      3. Place the biofilm tubing horizontally and carefully remove the injection and waste tubings.

      4. Let the liquid contents of the biofilm tubing drip into a sterile centrifuge tube.

      5. Cut the biofilm tubing from the inlet to the outlet and scrape out the biomass using an inoculation loop. Collect all biomass into the centrifuge tube used above.

      6. Adjust the volume of each harvest sample to the same level.

      7. Water-sonicate the harvested biofilms at room temperature for 5 min to dissociate the bacterial aggregates. Repeat this step if large aggregates are present in the samples.

      8. Vortex the samples before aliquoting for different quantitation methods.

    2. Biofilm harvest from the microfluidic devices/3-channel flow-cells

      Note: Most microfluidic devices are sealed with silica glue, plasma bonding, and heat bonding, which are not easy to disassemble for biofilm harvest.

      1. Stop the flow and clamp off both the injection and waste tubings.

      2. Shift the devices carefully into the biosafety cabiniet.

      3. Replace both the injection and waste tubings with short silica tubing without losing the biofilm contents.

      4. Allow the liquid contents to drip into a sterilized centrifuge tube from one side of the device.

      5. Connect the other side of the device to a syringe containing PBS buffer.

      6. Purge the channel forward and reverse with buffer.

        1. Adjust the volume of each harvested sample to the same level.

        2. Water-sonicate the harvested biofilms at room temperature for 5 min to dissociate the bacterial aggregates. Repeat this step if large aggregates are present in the samples.

      7. Vortex the samples before aliquoting for different quantitation methods.


  7. Biofilm observation and quantitation

    1. Relative density quantitation: load 100 μl dispersed biofilm suspension into a 96-well microplate with at least 5 replicates for each sample. Read the OD600 using a microplate reader. The relative density represents the relative biomass formed in each sample.

    2. CFU counting: prepare a serial dilution (101, 102, 103, 104, 105, and 106 ×) of the dispersed biofilm suspension with LB broth. Drop 10 μl diluted suspension onto LB agar plates with at least 5 replicates for each dilution (Herigstad et al., 2001). Cultivate in a 37°C incubator for 16 h and subsequently count the colonies.

      Note: This method usually aims to selectively cultivate bacteria with specific metabolic activities in the biofilm communities.

    3. Flow cytometry analysis (Desai et al., 2019): prepare a 1,000× dilution of the dispersed biofilms in 0.9% NaCl buffer. Count and analyze the treated and control biofilms following the manufacturer’s protocol.

      Note: This method usually targets the relative quantitation of bacteria with different fluorescent tags.

    4. Omics study (Tan et al., 2014; Ding et al., 2019): Isolate and purify DNA/RNA/protein from the dispersed biofilm suspension following the manufacturer’s instructions for the isolation kit.

      1. Total genome isolation: centrifuge 1 ml dispersed biofilm suspension at 8,000 × g for 3 min and remove the supernatant. Isolate total DNA from the bacterial pellets using a DNA isolation kit (QIAamp DNA micro kit).

      2. Total RNA isolation: immediately centrifuge 1 ml dispersed biofilm suspension at 8,000 × g for 1 min; carefully wash the cell pellets twice with NaCl. Isolate the total RNA following the manufacturer’s instructions for the kit (RNeasy Mini Kit).

        Note: Snap-freeze the bacterial pellets using liquid nitrogen if the sample handling procedures exceed 10 min.

      3. Total protein isolation: aliquot 1 ml dispersed biofilm suspension in 2-ml microtubes and place in an ice-water bath. Lyse the bacteria using a probe sonicator at 250 W for 30 min with 4-s intervals per 2-s sonications. Centrifuge the total lysates at 20,000 × g for 1 h to collect the total dissolved protein in the supernatant.

    5. Biofilm observation and quantitation by confocal laser scanning microscopy (CLSM).

      Note: The biofilm reactor is only compatible with CLSM systems when its attachment surface is transparent and has a thickness less than 0.5 mm.

      1. Stop the pump and clamp off both the injection and effluent tubings of the flow-cell.

      2. Shift the whole system with the pump into the CLSM facility room using a trolly.

      3. Place the flow-cell onto the CLSM sample stage with the coverslip facing down (for reversed microscope only).

      4. Start the pump and remove the clamps.

      5. Image the 3D structures, tracing the movement of particles/bacteria, etc.

      6. Image analysis approaches:

        Software: Imaris, Comstat2, ImageJ, etc.

        Calculation: biovolume of total and each aggregate, relative fluorescence intensity of total and each aggregate, surface coverage, roughness, number of aggregates, etc.


  8. Clean-up and reuse of tubings

    1. Remove parts ⑤ and ⑦ from each system and reconnect the tubing with straight connectors.

    2. Fill the feeding bottle with 70% ethanol and flush the tubing system at 1 ml/min for 1 h.

    3. Purge the tubing system with air by placing the feeding tubing in the air with the peristaltic pump working at max speed for 10 min.

    4. Repeat steps 2-3 three times.

    5. Fill the feeding bottle with H2O and flush the tubing system at 1 ml/min for 1 h.

    6. Purge the tubing system with air by placing the feeding tubing in air with the peristaltic pump working at max speed for 10 min.

    7. Repeat steps 5-6 three times.

    8. Disconnect each part of the system, dry all parts, and wrap-up all parts in metal foil.

Data analysis

  1. Relative density quantitation

    1. Raw data (Table 1)


      Table 1. Demo results for the OD600 readings of dispersed biofilms of PAO1 wildtype and mutants

      Replicates Blank PAO1 wild-type PAO1 mutant1 PAO1 mutant2 PAO1 mutant3 PAO1 mutant4
      1 0.66 2.28 1.51 1.68 2.28 2.41
      2 0.70 2.04 1.73 1.89 1.96 2.15
      3 0.50 1.99 1.55 1.77 2.17 2.31
      4 0.34 2.01 1.90 1.80 2.21 2.41


    2. Calculate the average value and standard deviation (Table 2)


      Table 2. Demo results analysis for the OD600 readings of dispersed biofilms of PAO1 wildtype and mutants

      Blank PAO1 wild-type PAO1 mutant1 PAO1 mutant2 PAO1 mutant3 PAO1 mutant4
      Average 0.55 2.08 1.67 1.79 2.16 2.32
      Stdev 0.17 0.13 0.18 0.09 0.14 0.12


    3. Plot the results (Figure 4)



      Figure 4. Demo results showing the relative density of dispersed biofilms of PAO1 wildtype and mutants


  2. CFU counting results for the dispersed biofilms (Figure 5)



    Figure 5. Demo results for the CFU counting of dispersed biofilms


  3. Flow cytometry analysis (Figure 6)



    Figure 6. Demo illustration of the flow cytometry results for dispersed PAO1-GFP-tagged biofilms


  4. Transcriptomics analysis of the biofilms (Figure 7)



    Figure 7. Demo illustration of the transcriptomics analysis of biofilms: correlation across the significantly dysregulated genes


  5. Biofilm observation and quantitation by CLSM

    1. S. oneidensis MR1-GFP was cultivated in a 3-channel flow-cell for 7 days. Biofilm morphology was observed by CLSM on days 3, 5 and 7. Representative images are shown in Figure 8.



      Figure 8. Ortho images of S. oneidensis MR1-GFP biofilms during cultivation. The biofilm was developed from a thin layer of single microcolonies on day 3 to thick and robust flat biofilms on day 7.


    2. The S. oneidensis MR1-GFP biofilms were cultivated in microfluidic systems for three days with four different calcium concentrations. Subsequently, biofilm formation was evaluated by CLSM imaging for each concentration, followed by image analysis. As shown in Figure 9, biofilm formation is positively correlated with calcium supply, with increased biovolume and surface coverage.



      Figure 9. CLSM images and analysis of sample biofilms cultivated in microfluidic devices with different calcium supplys. A. CLSM imaging. Green: SYTO9 stain for all biomass. B. Biovolume and surface coverage were calculated from the CLSM images.


      1. Biovolume analysis of biofilms based on CLSM imaging results

        1. Open the Imaris and create a new “arena” following the manufacturer’s instructions; create respective subfolders (i.e., “Treated,” “Control”).

        2. Upload CLSM images into the subfolders. For example, load “PAO1-wild type.czi” into the subfolder “Control.”

        3. In the analysis panel, create a surface with an adjusted threshold. Make sure that the surface covers most of the fluorescence signals.

        4. Calculate the volume and surface coverage in the mounted “surface.”

        5. Export the volume information into Excel.

          Note: Each piece of volume information in the list represents the calculated biovolume of an individual microcolony. The sum of the volume list is the total volume of the image.

      2. Surface coverage and roughness analysis based on CLSM imaging results

        1. Make sure that the file format of the images is compatible with Comstat2.

        2. Start Comstat2 and add folder destinations to upload the image files.

        3. Choose the target image file and adjust to the proper threshold.

        4. Choose the target parameters and start the analysis according to the manufacturer’s instructions.

      3. Morphology observation based on CLSM imaging results

        1. Open the CLSM images in Zen.

        2. In the ORTHO panel, select the focusing point with the most representative X, Y, and Z views.

        3. Adjust the threshold of the image.

          Note: The threshold must be set the same for the same batch of experimental images to avoid imaging bias.

        4. Generate a new image from the current view.

        5. In the new image panel, add a scale bar and adjust its front and line size.

        6. In the Analyzing panel, choose Export with the target file type and location; export the Ortho image.

Acknowledgments

This work was supported by Guangdong Natural Science Foundation for Distinguished Young Scholar [2020B1515020003] and Start-up [Y01416206] Grants from the Southern University of Science and Technology (SUSTech) to LY, and the National Natural Science Foundation of China (31870060) to HL. This protocol was originally published by Sternberg and Tolker-Nielsen (2006).

Competing interests

The authors declare no competing financial interests.

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简介


[摘要]的生物膜形成和代谢活动表征是关键investigat荷兰国际集团与环境因子的生物膜相互作用和illustrat荷兰国际集团生物膜regulat ORY机制。适当的体外模型模仿小号生物膜体内栖息地有脱颖而出需求小号准确孔定量牛逼通货膨胀和生物膜的调查-相关活动。目前的方法通常涉及静态生物膜的设置(如生物膜测定小号在微孔板中,胎圈的生物膜,在玻璃载玻片生物膜,等等。)和流体流动生物膜系统(如滴流生物膜反应器,3通道生物膜反应器,管生物膜反应堆等)。连续流系统考虑流体剪切力,养分供应的贡献,并分散细胞的物理传输,它定义了最自然和工程系统生物膜发展的栖息地。该协议describ ES的组件3流动的系统设置培养铜绿假单胞菌PAO1和希瓦氏菌oneidensis中MR-1模型生物膜,与所述相应的孔定量吨通货膨胀和观察方法。标准化的流程系统保证高效和可重复的生物膜的实验结果,可以根据需要进一步修改具体的研究项目小号。

[背景]生物膜是在NATU微生物的最普遍增长方式重新,工业,和临床的栖息地,其通常认为是嵌入在胞外聚合物质(EPS)的自产生的矩阵的细菌群落(弗莱明等人。,2016 ) 。生物膜聚集体的空间组织是高度异质的,具有多种代谢活动,使生物膜坚固耐用,能够耐受多种类型的环境压力(Van Dyck等,2021 )。采取这样的进步,生物膜在有前途的产业中的微生物燃料电池,以发酵营养物转化,降解生物修复有害化合物,并产生电力(Coenye和Nelis,2010 )。相反,生物膜也引起了公众的关注,例如生物污染和生物膜相关感染。生物膜是慢性伤口感染和生物医学装置,如心脏瓣膜,气管导管感染的主要原因,和导管(波索等人。,2018 )。更好地理解和表征的生物膜形成,分散,和活动将在生物膜控制策略线索。

一个典型的生物膜生命周期通常包括 5 个阶段 -在初始翻领我BLE细菌,稳定的和不可逆转的连接,成熟,分散的附接,并分散自由生活的细菌(马丁等人。,2021 )。生物膜群落表现出积极的社会行为和与环境因素的相互作用,反过来调节生物膜的细胞代谢。许多方法和装置已被记载为investigat的离子生物膜的形态和发展体外,它们通常分类为静态生物膜测定法和连续-流生物膜系统。静态生物膜测定中,如那些在微孔板和上气-液界面盖玻片S或集落的生物膜测定和Kadouri滴加到生物膜测定法,等等,被普遍地应用于对筛选的生物膜形成的早期事件(梅里特等。 , 2005 ) 。静态生物膜测定是高通量和EAS IL与普通实验室设备执行ÿ ; ħ H但是,在静态生物膜测定营养供应被限制相对于显影成熟生物膜群落模拟性质。

鉴于生物膜经常观察到的事实下在自然界中的流体的流动条件和工程系统,营养物可用性,物理传输,和流体剪切力显著影响生物膜形成和代谢(马泰等人。,2018 )。为了解决这个问题的生物膜respon如何DS对环境压力和传送信号,这一点很重要吨ö创建一个标准化的和可再现的协议,用于培养在体外流动生物膜系统模仿体内流体的栖息地(Cowle等人。,2020 )。流生物膜反应器的发展公顷š是新兴的微流体先进小号制造- [R S和设计,以最常用的流动系统includ荷兰国际集团滴流系统小号(冈萨雷斯等人。,2014 ),管式反应器(温等人。, 2014 ),平面的流动池(张等人。,2011 ),和3通道流动-细胞(斯特恩伯格和Tolker -尼尔森,2006;玉兰等人,2009年。)。进一步的修改,如分段的流动细胞(Karande等人。,2014 ),梯度发生器流动池(张等人。,2019 ),和流速的微流体流动池(刘等人。,2019 ),具体改性和设计,以满足特定的研究目的小号。

在这个协议中,我们讨论了不同的研究目的3共同施加流动的系统设置小号与绿脓杆菌PAO1和希瓦氏菌oneidensis中MR-1作为模式生物。油管生物膜反应器模拟生物膜的栖息地在管道小号,气管导管,导管,等等,允许生物膜收获足够的生物量。3通道流动-电池系统被设计成用于所述非侵入性spati ö颞生物膜形态的与观察一个连续和稳定的营养供给。回收生物膜反应器使得能够朝向其生物膜代谢反应的研究bioremediat离子代谢物和/或环境的化学品(斯特恩伯格和Tolker -尼尔森,2006;魏斯尼尔森等人。,2011;玉兰等人,2019。 )。 总的来说,规范化,流程化系统保证高效和可重复的生物膜观察孔定量牛逼通货膨胀,可以根据需要进一步改进的具体的研究项目。

关键字:生物膜的特性, 流体流动生物膜反应器, 铜绿假单胞菌

材料和试剂
 
1.硅管(ID=1 mm,OD=3 mm,润泽,中国)      
2.硅管(ID=3.2 mm,OD=6.4 mm,润泽,中国)      
3.蠕动泵管(ID=1.02 mm,OD=2.62 mm,Pharmed BPT,Sain-Gobain)      
4.直式连接器(润泽,目录号:DI-016 )      
5.直式连接器(润泽,目录号:DI-032 )      
6.鲁尔接头(润泽,目录号小号:RH-G016 ; RH-M016 )      
7.针(规格:26G,皮下;BD,目录号:305111)      
8.注射器(1毫升,BD,Lu er-Lock,目录号:309628)      
9.硅胶胶(Advanced Silicone 2,GE密封胶)      
10.封口膜(BEMIS,目录号:PM996)   
11.金属箔(Maryya ,目录号:HC081260 )   
12. 10 μl接种环(Sangon Biotech,目录号:F619312 )   
13. 15 ml离心管(Sangon Biotech,目录号:F602888 )   
14.盖玻片小号(生工生物,目录号:F518117 )   
15.微孔板小号(热˚F isher,Nunc公司TM ,Ç atalog号:168055 )   
16. 0.22 μm注射器过滤器(Sangon Biotech ,目录号:F513163 )   
17. 2米升微型试管小号(Maxyclear Snapclock,爱思进,US)   
18. LB肉汤(Sangon Biotech ,目录号:A507002 )   
19.氯化钙(Sangon Biotech ,目录号:A501330 )   
20.氯化钠(Sangon Biotech ,目录号:A100241 )   
21. QIAamp DNA Micro Kit(QIAGEN,目录号:56304 )   
22. RNeasy Mini Kit(QIAGEN,目录号:74104 )   
 
设备
 
蠕动泵(200 系列 16 通道泵,Watson Marlow)
酶标仪(星火® ,Tecan公司,瑞士)
Flowcytomet ë R(CytoFLEX,美国Beckman公司)
探针超声波仪(SONICS,米Odel等:VCX750 )
离心机仪(Eppendorf,米Odel等:5418R )
生物安全柜,BSC(MSC-优势TM II,热电˚F isher,USA)
高压釜(Z EAL方式,米Odel等:GR-60DA )
共聚焦激光扫描中号icroscop ë ,CLSM(蔡司,米Odel等:LSM900 )
 
软件
 
Imaris (Bitplane, Oxford Instrument)
ImageJ ( https://imagej.net/ )
Comstat 2 ( http://www.comstat.dk/ )
 
程序
 
管式生物膜反应器(图 1 ,图 S1)
 
 
图1的插图中管生物膜反应器
 
注意小号:
管生物膜反应器包括以下部分中的序列:奶瓶,喂食管(ID = 1mm)的,蠕动泵管(ID = 1.02 mm)时,注射器过滤器(0.2 μ M),注射管(ID = 1 mm)时,生物膜管(ID = 3.2毫米),废液管(ID = 1mm)的,和废液瓶。
这种设置,除了各部位⑤和⑦ ,可重复使用的重复实验。有关清理程序,请参阅 H 部分。
管道生物膜反应器不允许在spati Ø生物膜的时间成像观测。他们特别满足收获的需要一个大量的生物质的-组学分析。
 
进料管连接到该蠕动泵管的直线连接器。确保送料管的长度与奶瓶的高度兼容。
使用女性 Luer 接头将泵管的另一侧连接到注射器过滤器。
所述过滤器的另一侧连接到所述与注射器阳配件注射管。
使用减少直连接器将注射管连接到生物膜管。生物膜管道的长度取决于该实验设计。
使用减少的直连接器将生物膜管连接到废物管。
包裹组装部分(1-5)在金属箔和高压釜至steriliz È 。
新鲜制备中十分之一LB肉汤的奶瓶和高压釜至steriliz È 。
高压灭菌废液瓶,并在使用前倒入漂白剂。
紫外线-在使用前对生物安全柜中的封口膜 (10 cm × 10 cm) 和金属箔 (15 cm × 15 cm) 进行消毒。
将进料管进入奶瓶,并确保该管道到达瓶底。P花边废液管到废液瓶,并确保该管道是接近的瓶颈,而不是底部。用消毒的封口膜和金属箔盖住每个瓶子。这一步必须进行的生物安全柜。
将泵管放在蠕动泵上,并以最大速度启动泵,用生长培养基清洗系统。确保该系统设置流量恢复正常之前,无气泡。
将带有蠕动泵的管状生物膜反应器转移到温控培养箱中。确保该生物膜管是在一个类似水平的瓶颈的废液瓶,以避免任何回流。
 
3通道流通池生物膜反应器(图2 ,图S2)
 
 
图2的示意图的3通道流动池生物膜反应器
 
注意小号:
Ť他3通道流动池系统包含在序列中的以下部分:奶瓶,喂食管(ID = 1mm)的,蠕动泵管(ID = 1.02 mm)时,中间管(ID = 1 mm)时,注射器过滤器( 0.2 μ M),injec灰管(ID = 1 mm)时,生物膜管(ID = 3.2毫米),废液管(ID = 1mm)的,和浪费瓶。
这种设置的所有部件,除⑤ ,可重复使用的重复实验。第⑦部分可以通过取下盖玻片并用 70% 乙醇清洗来重复使用。对于t他重用其他部分,是指H节对的清理过程。
3 通道生物膜系统最常用于研究铜绿假单胞菌、金黄色葡萄球菌等的生物膜形成和活性。此设置与传统版本略有不同;第Ë气泡捕捉器通过注射器过滤器和第取代Ë在奶瓶空气连接管被除去。根据作者的经验,气泡捕集器是泄漏的主要原因,移除空气连接管不会影响介质中的氧气含量。此装置是用于可行的非侵入性spati ö颞生物膜形态和社区的组合物,观察人虽然biof不推荐ILM收获。
 
洗3通道流动池用乙醇和干燥它的通风橱。确保该表面的流动池是干净的,在装配时干燥。
在 3 通道流通池的壁上放置一层薄薄的硅胶,并在顶部放置盖玻片。小心地将盖玻片压向流通池底部,直到它完全密封,密封区域没有任何气泡。将密封的流通池在室温下放置一夜,使胶水完全干燥。
供给管道连接的蠕动泵管具有直的连接器。
将泵管的另一侧连接到带有直连接器的中间管。确保该中间管的长度允许流动池单元被移动到的阶段的共焦显微镜。
的另一端连接所述中间管连接到所述与注射器阴配件的注射器过滤器。
所述过滤器的另一侧连接到所述与注射器阳配件注射管。
将注射管连接到 3 通道l流通池的一个通道。
将废液管连接到流通池通道的另一侧。
组装与管道的奶瓶,废液瓶,以及蠕动泵使用相同的程序作为描述用于管生物膜反应器。
 
回收流通池系统(图 3)
 
 
图3的示意图的循环流动池系统
 
注意小号:
Ť他再循环流动池系统包含在序列中的以下部分:奶瓶,喂食管(ID = 1 mm)时,蠕动泵管(ID = 1.02 mm)时,流动池反应器,废液管(ID = 1mm)的,带回收管的废液瓶和带供应管的新鲜培养基瓶。
这种设置,除了各部位⑤和⑦ ,可重复使用的重复实验。有关清理程序,请参阅 H 部分。
循环流-细胞系统通常用于研究的毒物和环境化合物的生物修复,以期以模仿王的废水处理厂。第⑦部分不限于管道和/或 3 通道室。新鲜培养基与废弃培养基的补料比例应根据每个项目进行调整。
 
Assembl Ë按照相同的程序1-5“装配的部分中的供电部的管反应器。”
Assembl Ë循环废料部分:连接进给管与一个注射器阴配件的注射器过滤器; 连接泵管到另一侧的针筒式过滤器与一个公鲁尔接头; 用直接头将泵管的另一侧连接到另一条进料管(回收进料管)。
Assembl Ë循环新鲜培养基部分:连接进给管与直连接器的泵管; 用直接头将泵管的另一侧连接到另一条进料管(回收进料管)。
单独包装每个部分中的金属箔和高压釜至steriliz È 。
在喂养瓶和新鲜培养基瓶中新鲜制备十分之一的 LB 肉汤。
对废液瓶进行高压灭菌。
紫外线-在使用前对生物安全柜中的封口膜 (10 cm × 10 cm) 和金属箔 (15 cm × 15 cm) 进行消毒。
将三个管材进入奶瓶,废液瓶,并且新鲜的介质瓶,分别。确保该管道到达每个瓶子的底部。将废液管插入废液瓶,并确保该管道是接近的瓶颈,而不是底部。将来自两个循环喂养管末端的废物和新鲜介质部件到奶瓶。确保这将油管接近的瓶颈,而不是底部。用消毒的封口膜和金属箔盖住每个瓶子。这一步必须进行的生物安全柜。
将输送泵管到喂养蠕动泵和将两个回收和进料管小号到回收蠕动泵。
调整输送泵的旋转速度成为这两次循环泵。
首先以最高速度启动进料泵,用生长培养基清洗系统。确保该系统设置流量恢复正常之前,无气泡。
将整个系统转移到温控培养箱中并启动两个泵。
 
生物膜接种培养(图S3)
制备接种物:在培养细菌过夜的适当的温度和稀到所述用生长培养基所需细胞密度。通常,铜绿假单胞菌的1 × 10 8 个细胞/ml可以保证强大的生物膜。
加载细菌接种我n要利用26G针头的注射器。确保该体积的接种体填充Š的生物膜反应器。
停止蠕动泵和接种前钳断的注射管。
用75% 乙醇对生物膜管上的插入部位进行消毒。
将细菌悬浮液加载到每个生物膜反应器中。
钳断了污水管道。
取下针头并立即用硅胶胶封住孔。
允许在培养箱中不流动的情况下连接 2 小时。确保的内表面的生物膜反应器朝下为初始附着。
改过的生物膜反应器,使生物膜附着FAC的内表面上课起来。
启动流量(3-8 毫升/小时/通道)并取下夹子。
培养在培养箱中生物膜的收获前所需的持续时间。
 
生物膜处理
直接灌注
制备吨他的CaCl 2溶液在所需浓度小号在LB肉汤(0毫米,0.34毫米,和0.68毫摩尔)和负载在以一个1 -毫升注射器用26G针。
制备含有LB肉汤和对照溶液的化学矢量(H 2 ö在这种情况下)和负载在到1 -毫升注射器用26G针。
灌注前停止蠕动泵并夹住注射管。
用75% 乙醇对生物膜管上的插入部位进行消毒。
将化学溶液注入并加载到生物膜管中。
取下针头并立即用硅胶胶封住孔。
允许静电处理的所需的持续时间。
开始流动(3-8毫升/小时/信道)并取下夹具,或收获的直接生物膜。
连续加载
制备含有氯化钙的无菌介质中的一个新的瓶2 (0.68毫摩尔)和含有该载体的瓶的无菌介质的用于药物溶液(H 2 O)。
消毒封口膜和金属箔片。
停止蠕动泵并夹住进料管。
移进给管插入包含新的奶瓶Ç alcium氯化物/载体。
覆盖奶瓶用石蜡膜和金属箔。
取下夹子并启动泵;确保有系统内部无气泡。
 
生物膜收获
从生物膜收获的管道反应器中:
下车的流体和夹具的注射挠度和废液管小号。
就拿部分包括的注油管,油管生物膜,也浪费了系统管路进关的生物安全柜。
将生物膜管水平放置并小心取出注射和废液管s 。
让液体内容小号的生物膜管滴我n要无菌离心管中。
切生物膜从管的入口到所述出口和废料Ë出使用接种环的生物质。将所有生物质收集到上面使用的离心管中。
将每个收获样品的体积调整到相同的水平。
在室温下对收获的生物膜进行水声处理 5 分钟,以分离细菌聚集体。重新泥炭此步骤,如果大AGG regates是存在于样品英寸
涡流等分对于不同的孔定量前,样品吨通货膨胀的方法。
从生物膜收获的微流体装置/ 3通道的流动细胞
注:中号OST微流体装置被密封用二氧化硅胶,等离子体接合,和热粘合,这是不容易二sassemble生物膜收获。
下车的流体和夹具的注入和废液管小号。
移位设备小心进入生物小号afety cabiniet 。
同时取代了注射和废液管小号与短石英管不失生物膜内容小号。
允许液体含量s至滴入无菌离心管从该装置的一侧上。
将设备的另一侧连接到含有 PBS 缓冲液的注射器。
使用 buffer向前和向后清除通道。
调节每次收获的体积编样品至相同的水平。
在室温下对收获的生物膜进行水声处理 5 分钟,以分离细菌聚集体。如果样品中存在大的聚集体,则重复此步骤。
涡流等分对于不同的孔定量前,样品吨通货膨胀的方法。
 
生物膜观察孔定量牛逼通货膨胀
相对密度孔定量吨通货膨胀:负载100 μ升分散生物膜悬浮液到一个96孔微板具有至少5个重复对每个样品。[R EAD的OD 600使用酶标仪。相对密度代表在每个样品中形成的相对生物量。
CFU计数:制备系列稀释液(10 1 ,10 2 ,10 3 ,10 4 ,10 5和10 6 × )分散生物膜悬浮液用LB肉汤。d ROP 10 μ升稀释的悬浮液在LB琼脂平板上具有至少5个重复的每个稀释(Herigstad等人,2001 )。培育一个37 °C培养箱孵育16小时,随后对菌落进行计数。
注:牛逼他的方法通常旨在培养选择性与生物膜社区的特定代谢活性细菌。
流式细胞术分析(Desai等人,2019 年):在 0.9% NaCl缓冲液中制备分散生物膜的 1 , 000 ×稀释液。按照制造商的协议计算和分析处理过的和控制的生物膜。
注:牛逼他的方法通常针对的相对孔定量牛逼细菌的通货膨胀不同的荧光标记小号。
组学研究(谈等人。,2014;丁等人。,2019 ):分离和纯化DNA / RNA /从以下分散的生物膜悬浮蛋白质的制造商的说明书对所述分离试剂盒。
总基因组隔离:离心机1米升以8,000分散生物膜悬浮×克为3分钟,去除上清。从使用细菌沉淀分离总DNA一个DNA分离试剂盒(QIAamp的DNA微试剂盒)。
总RNA分离:我mmediately离心1米升分散生物膜悬浮液在8000 ×克为1分钟; 用NaCl仔细清洗细胞沉淀两次。隔离总RNA中的制造商的说明小号的试剂盒(RNeasy Mini试剂盒)。
注:弹簧-冻结使用液氮如果样品处理程序超过10细菌沉淀分钟。
总蛋白质分离:等分试样1米升中分散生物膜悬浮液2 -米升在微管和地点的冰-水浴中。裂解使用该细菌一个在250瓦特探头超声波仪30分钟与4 -小号间隔小号每2 -小号超声小号。离心机吨他裂解物总小号以20,000 ×克为1个ħ以收集总dissolv编蛋白的上清液。
生物膜观察和孔定量吨通货膨胀通过共聚焦激光扫描显微镜(CLSM) 。
注:Ť他生物膜反应器仅与CLSM系统兼容当其安装面是透明的并且具有厚度小于0.5毫米。
停止泵和夹具关闭两个的注入和流出物管道小号流动池的。
移整个系统中泵入CLSM设备间使用一个小车。
放置流动池到具有CLSM样品台的盖玻片面向下(仅用于反向显微镜)。
启动泵并拆下夹子。
IMAG Ë的3D结构,追踪颗粒/细菌,运动等。
图像分析方法:
软件:Imaris、Comstat2、ImageJ等。
计算:总的biovolume和每个聚合,相对fluorescen CE总和每个骨料,表面覆盖度,粗糙度,聚集体的数目,强度等。
 
管道的清理和再利用
从每个系统上拆下s ⑤和⑦ 部件,然后用直接头重新连接管道。
填充奶瓶用70%乙醇冲洗,并在1管道系统米升/分钟为1 ħ 。
通过将进料管中吹扫与空气管道系统的与空气的蠕动泵以最大速度工作10分钟。
重复步骤2-3 3倍。
填充奶瓶与ħ 2 ö并冲洗在1管道系统米升/分钟为1 ħ 。
通过与将所述进料管中的空气吹扫与空气管道系统的蠕动泵以最大速度工作10分钟。
重复步骤5-6 3倍。
断开系统的每个部件,干燥所有部件,然后用金属箔包裹所有部件。
 
数据分析
 
相对密度孔定量牛逼通货膨胀
原始数据(表 1)
 
表1演示结果为所述OD 600读数小号PAO1野生型和突变体的分散生物膜的
 
计算的平均值和标准偏差(表2)
 
表2.演示结果分析对于在OD 600读数小号PAO1野生型和突变体的分散的生物膜的
 
3 .绘制结果(图 4)   
 
 
图4. d EMO结果示出了PAO1野生型和突变体的分散生物膜的相对密度
 
CFU计数结果为分散的生物膜(图5)
 
 
图5.演示结果为分散生物膜的CFU计数
 
流式细胞术分析(图 6)
 
 
 
图6.流动的演示插图术结果为分散PAO1- GFP -标记的生物膜
 
生物膜的转录组学分析(图 7)
 
 
图 7.生物膜转录组学分析的演示说明:显着失调基因之间的相关性
 
生物膜观察和孔定量吨通货膨胀通过CLSM
S. oneidensis中MR1- GFP进行培养在一个3通道流动-细胞为7天内进行付运。观察到生物膜的形态通过CLSM上天小号3,5和7 - [R具有代表性的图像一个在图8所示重新。
 
 
图 8. S. oneidensis MR1- GFP生物膜在培养过程中的正射图像。生物膜是由开发一个薄单一小菌落的层上一天3至厚且坚固的平的生物膜上天7。
 
在S. oneidensis中MR1- GFP生物膜我们再次在微流体系统中培养3天四种不同的钙离子浓度。随后,通过 CLSM 成像评估每个浓度的生物膜形成,然后进行图像分析。如图 9 所示,生物膜形成与钙供应呈正相关,生物量和表面覆盖率增加。
 
 
图9 。在具有不同钙供应量的微流体装置中培养的样品生物膜的 CLSM 图像和分析。A. CLSM 成像。绿色:SYTO9染色的所有生物。乙。乙iovolume和表面覆盖物Ç从CLSM图像alculated。
 
基于 CLSM 成像结果的生物膜生物体积分析
打开 Imaris 并按照制造商r的说明创建一个新的“竞技场” ;创建各自的子文件夹(即,“治疗”,“控制”) 。
将 CLSM 图像上传到子文件夹中。例如,将“PAO1-wild type.czi”加载到子文件夹“Control”中。”
在分析面板中,创建一个表面与一个调整的阈值。确保该表面覆盖小号米的fluorescen的OST CE信号。
Ç alculate在体积和表面覆盖安装“表面。”
导出卷信息到Ë Xcel公司。
注:È ACH片的列表中的音量信息表示小号单个菌落的计算biovolume。体积列表的总和就是图像的总体积。
基于 CLSM 成像结果的表面覆盖率和粗糙度分析
确保该的文件格式的图像与Comstat2兼容。
开始Comstat2并添加文件夹目的地上传的图像文件。
选择目标图像文件,并调整到了合适的阈值。
选择目标参数,并开始分析根据制造商的说明小号。
基于CLSM成像结果的形态观察
在 Zen 中打开 CLSM 图像。
在邻面板中,选择了具有聚焦点的最有代表性的X,Y ,和Z的景色。
调整图像的阈值。
注:牛逼他阈值必须设置为同一批次的实验相同的图像,以避免成像偏差。
生成一个新的形象的最新观点。
在新的图像面板中,添加一比例尺,并调整其前部和线大小。
在 Analyzing 面板中,选择 Export with the target file type and location ;导出正交图像。
 
致谢
 
这项工作是由广东省自然科学支持基金会杰出青年学者[2020B1515020003]和启动[Y01416206]拨款从南方科技大学(SUSTech)至LY的,和中国国家自然科学基金(31870060),以HL。该协议最初由Sternberg 和 Tolker-Nielsen ( 2006) 发布。
 
利益争夺
 
作者声明没有相互竞争的经济利益。
 
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