Microfluidic-based Growth and Imaging of Bacterial Biofilms   

Abstract

Materials and Reagents

1. 48 or 24 well Bioflux plate (Fluxion Biosciences, catalog numbers: 910-004 and 9210-009)
2. Fluorescent dyes and stains
   To stain the biofilms several different fluorescent markers can be employed. Possible options include:
   1. Fluorescent proteins. This protocol was developed using the red and green fluorescent proteins mCherry and eGFP (Zacharias et al., 2002; Shaner et al., 2004)
   2. Live/dead BacLight dyes (Syto9 and Propidium iodide) (Invitrogen, catalog number: L7007)
   3. Concanavalin A (Thermo Fisher, catalog number: C860), used at a concentration of 50 μg/ml. Stock solutions prepared to 1 mg/ml in PBS
   4. Wheat germ agglutinin (Thermo Fisher, catalog number: W849), used at a concentration of 10 μg/ml. Stock solutions prepared to 1 mg/ml in PBS
   5. YOYO-1 (Thermo Fisher, catalog number: Y3601), used at a concentration of 0.1 μM. Stock solution prepared to 10 μM in PBS
   6. HHA and WFL (EY labs, R-8008-1 and F-3101-2), used at a concentration of 50 μg/ml. Stock solutions prepared to 1 mg/ml in PBS at room temperature with gentle agitation
3. Glycerol stock of Pseudomonas aeruginosa (P. aeruginosa). The protocol was developed using the strain PAO1-N
4. Glycerol stock of Staphylococcus aureus (S. aureus). The protocol was developed using the strains SH1000 and USA300 JE
5. Na₂HPO₄·7H₂O (Acros Organics, catalog number: 424380010)
6. KH₂PO₄ (Sigma-Aldrich, catalog number: P0662)
7. NaCl (Sigma-Aldrich, catalog number: S7653)
8. NH₄Cl (Sigma-Aldrich, catalog number: 11209)
9. MgSO₄ (Sigma-Aldrich, catalog number: 20894)
10. Glucose (Sigma-Aldrich, catalog number: G5400)
11. CaCl₂ (VWR, catalog number: IB110)
12. Tryptic soy broth (TSB) (Oxoid, catalog number: CM0129)
13. 70% v/v ethanol (Fisher Scientific, catalog number: 10517694)
14. M9 salts (see Recipes)
15. M9 minimal medium (see Recipes)
    

Equipment

1. Bioflux 200 system (Fluxion Biosciences, see https://bioflux.fluxionbio.com/systems)
   Note: This consists of an interface, moisture trap, tubing and pump.
2. Bunsen burner (Fisher Scientific, catalog number: 12842154) or flow hood (Fischer Scientific, catalog number: 10795194)
   Note: All manipulations of the Bioflux plate with the interface detached should be performed under aseptic conditions (i.e., manipulations should be performed inside a flow hood or adjacent to a lit Bunsen burner).
3. Heating stage (included with Bioflux 200 kit or bespoke temperature hood (see the webpage for guidance, https://www.microscopyu.com/references/live-cell-imaging-and-perfusion-chambers)
4. Appropriate personal protective equipment (PPE)
   Note: PPE should be worn throughout and consist of a lab coat (Fisher Scientific, catalog number: 12841388), safety goggles (Fisher Scientific, catalog number: 11952815), and nitrile gloves (Starlab microflex supreno su-int-I).
5. Inverted fluorescent microscope (we use a Nikon Ti Eclipse, https://www.microscope.healthcare.nikon.com/en_EU/products/inverted-microscopes/eclipse-ti2-series)
   Note: An inverted fluorescent microscope with the appropriate filter sets, a 20x objective and a motorized stage.
6. Computer (Hewlett Packard model z440)
   Note: A computer connected to the microscope system and running the listed software for control, acquisition and image analysis.
7. Incubator (Panasonic, model MIR-154_PE)
   Note: For the bacteria used in this protocol, temperature of 37 °C was used, and both a shaking and a static incubator would be required for overnight cultures and plates respectively.
8. Autoclave (Prestige, model: Medical ED2A Classic, https://www.prestigemedical.co.uk/products/classic-standard-121)
   

Software

1. ImageJ (https://imagej.nih.gov/ij/) or its packaged version FIJI (https://fiji.sc/)
2. NIS elements (Nikon)
3. Bioflux control software (Fluxion Biosciences)
   

Procedure

This protocol is for the growth of either P. aeruginosa or S. aureus in the Bioflux system. Where differences in the protocols are used, those for S. aureus are listed in square brackets.

1. Prepare all solutions, growth medium and reagents at least a day in advance of the experiment.
2. Place Bioflux plate and growth medium into a 37 °C incubator overnight to degas. Degassing is important to prevent formation of air bubbles in the microfluidic channels during the experiment.
3. Prepare cultures from a single colony on an agar plate to grow for 16 h at 37 °C with 250 rpm shaking.
4. The next day, pipette 100 μl M9 minimal medium [TSB] into the center of the outlet well (Figure 1) of a Bioflux plate in sterile conditions. Care should be taken to avoid introduction of air bubbles into the system when pipetting.
   
   
   Figure 1. Layout and securing of a Bioflux plate. A. Growth medium should always be pipetted directly into the inner section of the outlet and inlet wells as indicated by the black arrow. This prevents entry of air bubbles into the system as the medium will completely cover the channel entrance. B. The positions to attach Blu tack to secure the Bioflux plate to the heated stage are indicated by the red arrows.
   
   
5. Sterilize the interface by wiping with 70% v/v ethanol and attach to the Bioflux plate using the screws.
6. Attach the Bioflux unit tubing to the appropriate port on the interface gasket.
7. Start the Bioflux control software (Figure 2), and flow from the outlet well at a shear rate of 2 dyn/cm² for 4 min. A droplet of media should form in the inlet wells. 
8. Dilute the overnight culture to OD₆₀₀ = 0.05 [0.1] in 0.5 ml growth medium. Pipette 50 μl of the inoculum into the outlet well. Add 200 μl M9 minimal medium [TSB] to the inlet well(s).
9. Re-attach the interface to the plate and then to the tubing.
10. Seed the channel by flowing from the outlet well at a rate of 2 dyn/cm² for 4 s. Care should be taken to ensure the bacteria being seeded does not contaminate the inlet well. Incubate the plate at 37 °C for a minimum of 45 min.
    
    
    Figure 2. The Bioflux control software interface. Screenshot of the Bioflux software for the 48-well plate (Panel A) and also the 24-well plate (Panel B).
    
    
11. Add the appropriate concentrations of fluorescent stains to the growth medium as indicated in the Materials and Reagents. The volume of media required per well varies by the experimental design and can be calculated by multiplying the shear rate by the number of hours the experiment will be run. For example, a typical experiment with P. aeruginosa run at 0.25 dyn/cm² for 16 h would require a minimum of 400 μl medium. However, to ensure the well does not run dry it is recommended to supplement the calculated volume with 100-200 μl extra medium. Place the prepared medium at 37 °C.
12. After 45 min, remove the interface from the Bioflux plate and pipette the prepared growth medium into the inlet wells being used. Sterilize the interface with 70% ethanol prior to attachment.
13. Securely fasten the Bioflux plate to the heated stage (or to the stage if using a temperature hood), ensuring the Bioflux plate sits flat on the stage.
14. Ensure the tubing connecting the Bioflux to the pump is free of obstruction and unlikely to get snagged during the experiment.
15. For a simple growth experiment using the 48-well plates, use the Bioflux control software to set a flow rate of 0.25 dyn/cm² [0.6 dyn/cm²]. Run for 16 h, growth of microcolonies should be obvious. The run length can be altered depending on how rapidly a strain grows as a biofilm.
    Note: More complex experiments such as antibiotic-killing of the biofilm can be performed using the Bioflux 24 well plates:
    
    1. Prepare growth medium containing antimicrobial agent of interest or other supplements as well as the required dyes as Step 11.
    2. Pipette this medium into inlet well 2 at Step 12.
    3. The Bioflux control software can be used to set up an automated program of flow. Flow from inlet well 1 at 0.25 dyn/cm² for the first 12 h, then set to run from inlet well 2 at 0.25 dyn/cm² for the remainder of the experiment.
16. Set the microscope to acquire images every 20 min for 16 h with the appropriate filter sets. Figures 3 and 4 illustrate the structures and staining that can be obtained using these techniques.
17. Return periodically to ensure plane of focus of the microscope is maintained. This is important if only a heated stage is being used due to temperature fluctuations over time.
18. At the conclusion of the experiment, stop the Bioflux flow. Image acquisition automatically ends if the program is set up to end. Detach the interface and sterilize with 70% v/v ethanol.
19. Analysis of the data can be performed in ImageJ. For example, if doing live/dead analysis, the images obtained can be converted to an 8-bit binary image. Thresholds for images can set to remove the background fluorescence. The ratio of live to dead cells can also be presented for each time point.
    
    
    Figure 3. Pseudomonas aeruginosa PAO1 biofilm grown in M9 minimal media with the Bioflux for 16.5 h and visualized using Concanavalin A-TRITC and YOYO-1
    
    
    Figure 4. Staphylococcus aureus SH1000 biofilm grown in TSB with the Bioflux for 16 h and visualized using the Live/Dead stains Syto9 and propidium iodide

Notes

1. -80 °C freezer stocks of bacterial strains were prepared in 25% v/v glycerol.
2. Autoclaving of materials and reagents was at 121 °C for 15 min.
3. Constitutively expressed fluorescent proteins provide the easiest means to track the cells within a biofilm. Newer generation fluorescent proteins are recommended (Barbier and Damron, 2016).
4. The system can be used to explore gene expression in a biofilm through the construction of promoter fusions to a fluorescent protein such as GFP.
5. The stains Syto 9 and propidium iodide enables staining of dead and live cells. An alternative is to pair propidium iodide with a strain constitutively expressing GFP.
6. The lectin Concanavalin A is specific for α-D-mannose and α-D-glucose residues. Binds to the P. aeruginosa exopolysaccharide, Psl (Polysacharide synthesis locus) can be conjugated to a range of different fluorophores.
7. The lectin Wheat Germ Agglutinin is specific for sialic acid and N-acetylglucosamine residues found in the Gram-positive cell envelope and in the S. aureus exopolysaccharide, poly-N-acetylglucosamine.
8. YOYO-1 is a cell impermeant double stranded DNA dye that can be used to stain extracellular DNA with minimal staining of dead cells.
9. The lectins HHA and WFL are specific to the P. aeruginosa exopolysaccharides Psl and Pel respectively. The lectins that can be conjugated to different fluorophores, but commercially available conjugated to TRITC and FITC.
10. A small amount of medium is added to the inlet wells at Step 8 to counter the effect of gravity on the flow of the bacteria from the outlet well during seeding.
11. Blu tack (Bostik) can be used to ensure the Bioflux plate fits securely on the microscope stage. Apply small amounts of the blu tack to the sides of the plate and not the base to ensure the plate is level.
12. A higher shear rate of 0.6 dyn/cm² is used for S. aureus experiments to prevent blockage of the microfluidic channel.
    

Recipes

1. M9 salts
   64 g Na₂HPO₄·7H₂O
   15 g KH₂PO₄
   2.5 g NaCl
   5.0 g NH₄Cl
   Adjust to 1,000 ml with distilled H₂O
   Sterilize by autoclaving
2. M9 minimal medium
   Measure ~700 ml of dH₂O (sterilized by autoclaving)
   Add 200 ml of M9 salts
   Add 2 ml of 1 M MgSO₄ (sterilized by autoclaving)
   Add 20 ml of 20% w/v glucose (or other carbon sources: sterilized by autoclaving)
   Add 100 μl of 1 M CaCl₂ (sterilized by autoclaving)
   Adjust to 1,000 ml with dH₂O (sterilized by autoclaving)
   

Acknowledgments

This work has been funded by the EMPIR program co-financed by the Participating States and from the European Union’s Horizon 2020 research and innovation program (grant reference 15HLT01 MetVBadBugs). This protocol was adapted from the manufacturer’s instructions (Fluxion Bioscience).

Competing interests

A small number of Bioflux plates were provided by Fluxion Bioscience.

References

1. Barbier, M. and Damron, F. H. (2016). Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling. PLoS One 11(3): e0146827.
2. Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E. and Tsien, R. Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12): 1567-1572.
3. Zacharias, D. A., Violin, J. D., Newton, A. C. and Tsien, R. Y. (2002). Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296(5569): 913-916.