Méthodes de caractérisation de la Co-développement de biofilms et Habitat Hétérogénéité
Summary
Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.
Abstract
Les biofilms sont des communautés microbiennes attachée surface qui ont des structures complexes et produisent hétérogénéités spatiales importantes. le développement du biofilm est fortement réglementé par le flux environnante et l'environnement nutritionnel. la croissance du biofilm augmente également l'hétérogénéité du microenvironnement local en générant des champs d'écoulement complexes et des structures de transport de solutés. Pour étudier le développement de l'hétérogénéité dans les biofilms et les interactions entre les biofilms et leur micro-habitat local, nous avons grandi biofilms mono-espèces de Pseudomonas aeruginosa et les biofilms double-espèces de P. aeruginosa et Escherichia coli sous gradients nutritionnels dans une cellule d'écoulement microfluidique. Nous fournissons des protocoles détaillés pour la création de gradients de nutriments dans la cellule d'écoulement et pour la culture et la visualisation de développement du biofilm dans ces conditions. Nous avons également des protocoles actuels pour une série de méthodes optiques de quantifier la répartition spatiale de la structure du biofilm, distri circulerbutions sur les biofilms, et le transport de masse autour et à l'intérieur des colonies de biofilm. Ces méthodes prennent en charge des enquêtes approfondies de la co-développement du biofilm et l'habitat hétérogénéité.
Introduction
Microorganisms attach to surfaces and form biofilms — cell aggregates enclosed in an extracellular-polymer matrix1. Biofilms behave very differently from individual microbial cells, because biofilms have dramatic spatial heterogeneity resulting from a combination of internal solute transport limitations and spatial variations in cellular metabolism2,3. Oxygen and nutrient concentrations drastically decrease at the interface between biofilm and surrounding fluid and get further depleted within in the biofilm2. Spatial variations in biofilm respiration and protein synthesis can also occur as a response to localized oxygen and nutrient availability2.
In aquatic and soil environments, most bacteria dwell in biofilms. Natural biofilms carry out important biogeochemical processes including cycling carbon and nitrogen and reducing metals4,5. Clinically, biofilm formation is responsible for prolonged pulmonary and urinary infections6. Biofilm-associated infections are highly problematic because cells in biofilms have extremely high resistance to antimicrobials compared to their planktonic counterparts6. Because biofilms are important in diverse settings, a substantial amount of research has been focused on understanding the environmental factors that control biofilm activities and the spatial heterogeneity in biofilms and the surrounding microenvironment.
Previous studies have found that biofilm development is strongly regulated by a number of environmental factors: biofilms develop different morphologies under various flow conditions; oxygen and nutrient availability influence biofilm morphology; and hydrodynamic shear stress affects the attachment of planktonic cells to surfaces and the detachment of cells from biofilms7-9. Furthermore, external flow condition influences the delivery of substrates into and within biofilms10. The growth of biofilms also alters surrounding physical and chemical conditions. For example, biofilm growth leads to local depletion of oxygen and nutrients2; biofilms accumulate inorganic and organic compounds from the surrounding environment11; and biofilm clusters divert flow and increase surface friction12,13. Because biofilms interact with their surrounding environment in very complex ways, it is critical to simultaneously obtain information on biofilm properties and environmental conditions, and multi-disciplinary approaches need to be used to comprehensively characterize biofilm-environment interactions.
Here we present a series of integrated methods to characterize spatial patterns in microbial growth within mono-species and dual-species biofilms under an imposed nutritional gradient, and to observe the resulting modification of the local chemical and fluid microenvironment. We first describe the use of a recently developed double-inlet microfluidic flow cell to observe biofilm growth under well-defined chemical gradients. We then demonstrate the use of this microfluidic flow cell to observe the growth of two species of bacteria, Pseudomonas aeruginosa and Escherichia coli, in biofilms under a range of nutritional conditions. We show how in situ visualization of fluorescent tracer propagation into biofilm colonies can be used to quantitatively assess patterns of solute transport in biofilms. Finally, we show how microscale particle tracking velocimetry, performed under confocal microscopy, can be used to obtain local flow field around the growing biofilms.
Protocol
Representative Results
Discussion
We demonstrated a suite of methods to characterize three important biofilm-environment interactions: biofilm response to chemical gradients, effects of biofilm growth on the surrounding flow microenvironment, and biofilm heterogeneity resulting from internal transport limitations.
We first showed the use of a novel microfluidic flow cell to impose a well-defined chemical gradient for biofilm development. To generate a well-defined chemical gradient within the flow cell, it is important to main…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Matt Parsek at the University of Washington (Seattle, WA) for providing P. aeruginosa and E. coli strains and Roger Nokes at the University of Canterbury (New Zealand) for providing access to Streams software. This work was supported by grant R01AI081983 from the National Institutes of Health, National Institute of Allergy and Infectious Diseases. Confocal imaging was performed at the Northwestern Biological Imaging Facility (BIF).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Peristaltic Pump | Gilson | Miniplus 3 | Flow cell setup and inoculation |
| PUMP TUBING 0.50MM OVC, Orange/Yellow | Gilson | F117934 | Flow cell setup and inoculation |
| Three-way Stopcock w/ Swivel male Luer lock | Smiths Medical | MX9311L | Flow cell setup and inoculation |
| Sylgard 184 Solar Cell Encapsulation for Making Solar Panels | ML Solar LLC | Flow cell setup and inoculation | |
| Pyrex Medium Bottle, 1L, GL45 | VWR | 16157-191 | Flow cell setup and inoculation |
| C-FLEX Tubing | Cole-Parmer | 06422-02 | Flow cell setup and inoculation |
| 1 mL TB Syringe | BD | 309659 | Flow cell setup and inoculation |
| Polymer Tubing | IDEX | 1520G | Flow cell setup and inoculation |
| Sterile Intramedic Luer Stub Adapter | Clay Adams | 427564 | Flow cell setup and inoculation |
| PrecisionGlide Needle | BD | 305195 | Flow cell setup and inoculation |
| Spectrophotometer | HACH | Flow cell setup and inoculation | |
| Syringe filters- sterile (0.2 μm) | Fisherbrand | 09-719A | Flow cell setup and inoculation |
| MAXQ Shaker | Thermo Scientific | Flow cell setup and inoculation | |
| Ammonium sulfate | Sigma Aldrich | A4418 | Growth media |
| Sodium phosphate dibasic anhydrous | Sigma Aldrich | RES20908-A7 | Growth media |
| Monobasic potassium phosphate | Sigma Aldrich | P5655 | Growth media |
| Sodium chloride | Sigma Aldrich | S7653 | Growth media |
| Magnisium chloride | Sigma Aldrich | M8266 | Growth media |
| Calcium chloride | Sigma Aldrich | C5670 | Growth media |
| Calcium sulfate dihydrate | Sigma Aldrich | C3771 | Growth media |
| Iron(II) sulfate heptahydrate | Sigma Aldrich | 215422 | Growth media |
| Manganese(II) sulfate monohydrate | Sigma Aldrich | M7634 | Growth media |
| Copper(II) sulfate | Sigma Aldrich | 451657 | Growth media |
| Zinc sulfate heptahydrate | Sigma Aldrich | Z0251 | Growth media |
| Cobalt(II) sulfate heptahydrate | Sigma Aldrich | C6768 | Growth media |
| Sodium molybdate | Sigma Aldrich | 243655 | Growth media |
| Boric acid | Sigma Aldrich | B6768 | Growth media |
| Dextrose | Sigma Aldrich | D9434 | Growth media |
| Luria Bertani Broth | Sigma Aldrich | L3022 | Growth media |
| TCS SP2 Confocal Microscopy | Leica | Fluorescent imaging | |
| SYTO 62 | Life Technology | S11344 | Fluorescent imaging |
| Cy5 | GE Healthcare Life Sciences | PA15100 | Fluorescent imaging |
| Red Fluorescent (580/605) FluoSphere | Life Technology | F-8801 | Fluorescent imaging |
| BioSPA | Packman Lab | Image Processing | |
| ImageJ | NIH | Image Processing | |
| Volocity | PerkinElmer | Image Processing | |
| Streams 2.02 | University of Cantebury | Image Processing |
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