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Microfluidic-based Growth and Imaging of Bacterial Biofilms

JB James M Brown
BB Birte Blunk
Paul Williams Paul Williams
Kim R Hardie Kim R Hardie

Published: Dec 20, 2019 DOI: 10.21769/BioProtoc.3460 Views: 3920

Edited by: Juan Facundo Rodriguez Ayala Reviewed by: Vinai Chittezham Thomas

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Abstract

Biofilms are a ubiquitous form of growth for bacteria on surfaces in most environments, natural or manmade. Here we present a protocol using the Bioflux microfluidic system to investigate the organized structure and development of these multicellular communities. Microfluidic systems present an opportunity to grow biofilms in a stable, physiologically maintained environment that is readily observable via time-lapse microscopy.

Keywords: Biofilms search Microfluidics search Bioflux search Microscopy search Pseudomonas aeruginosa search Staphylococcus aureus search eDNA search EPS search Matrix search Expolysaccharides search Extracellular DNA search

Background

The Bioflux microfluidic system is a commercially available system that consists of a series of input and output wells linked by microfluidic channels set in a standard microplate format. Growth medium is flowed through the channel by application of controllable pneumatic pressure. The system allows for high throughput, reproducible growth of biofilms whilst avoiding the lengthy preparation required for ‘home constructed’ microfluidic set-ups.

The complex and heterogenous biofilms formed by bacteria have long proved challenging to cultivate and visualize. Traditional methods of biofilm growth, such as the use of abiotic surfaces placed in a bacterial growth medium suffer from myriad artifacts such as nutrient depletion and disturbance of the biofilm. Home-made flow systems alleviate many of these issues but remain labor intensive and unwieldy. The nature of the traditional flow systems renders them difficult to use in combination with time lapse microscopy, particularly as the large channel sizes means the continuous use of stains is costly. As a result, the structure and architecture of bacterial biofilms has so far been probed using ‘snapshots’ at arbitrary time points.

Here, we describe a simple protocol for the effective use of the Bioflux microfluidic systems, with a focus on the use of fluorescent probes to examine changes in the structure and architecture of the model biofilm-forming bacteria, Pseudomonas aeruginosa and Staphylococcus aureus.

This protocol can be easily adapted to different fluorescent stains, bacterial species, including complex multispecies biofilms such as oral biofilms. Data obtained are reproducible and biofilms are in line with the architectural features and dimensions reported in the literature.

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

Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > Microbial biofilm > Biofilm culture

Microbiology > Antimicrobial assay > Antibacterial assay

Microbiology > Microbe-host interactions > Ex vivo model

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