In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Summary

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Abstract

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

Introduction

Most bacterial cells are able to employ both planktonic (free-living) and surface-attached (sessile) modes of growth ¹. In the surface-attached mode of growth, bacterial cells secrete and encase themselves in large amounts of extracellular polymeric substances (EPS) to form biofilms. The EPS mainly consists of proteins, exopolysaccharide, extracellular DNA and is essential to biofilm formation ². It serves as a physical scaffold by which bacteria can use to differentiate spatially and protects the bacteria from harmful environmental conditions and host responses. Different components of EPS have distinct roles in biofilm formation ³ an....

Protocol

1. Biofilm Cultivation

1. Preparation of Bacterial Strains
   1. 1 day prior to biofilm cultivation, prepare planktonic bacterial cultures by inoculating 2 ml of appropriate growth medium from frozen bacterial culture. Use Luria-Broth medium (10 g L^-1 NaCl, 10 g L^-1 yeast extract, and 10 g L^-1 tryptone) for mucoid P. aeruginosa and its Δpel and Δpsl defective mutants. Incubate overnight at 37 °C and 200 rpm shaking conditions. Dilute overnight cultures to an OD₆₀₀ of 0.40 using a spectrophotometer.
   2. Assemble flow cell setup, which has been described previously ¹⁴....

Results

The local viscoelastic properties of the biofilm in different regions of the biofilm, which included the voids (medium above the biofilm), plains (undifferentiated flat layer of cells) and microcolonies (see labels in Figure 2A) were investigated. The temporal changes in viscoelastic properties of the biofilm during maturation from days 3 to 5 were also determined. The MSD of the particles in the voids was used as a control and comparable to the MSD of particles in pure medium. In contrast, particles tra.......

Discussion

Microrheology is a useful tool for local rheological measurements in heterogeneous systems, such as microbial biofilms. It is a non-destructive technique, enabling the real-time monitoring of rheological changes within the same biological sample over multiple time points. In this protocol, particle-tracking microrheology was applied to Pel and Psl exopolysaccharide mutants in order to investigate how they affect the elasticity and effective crosslinking of the biofilm matrix. Psl favors the development of elastic biofilm.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fluorspheres	Invitrogen	F-8821	1.0 μm red fluorescent (580/605) microspheres with carboxylate modification
Zeiss Axio Imager M1	Carl Zeiss		Epifluorescent Microscope
Masterflex L/S Digital Drive 07523-80	Cole-Parmer	EW-07523-80	Peristaltic pump
Flow Cell Chambers	Technical University of Denmark
Bubble Trap	Technical University of Denmark
Silicone Tubing	Dow Corning		3 mm outer diameter, 1 mm inner diameter
Clear polypropylene plastic connectors	Cole Parmer	06365-83	1/16 in. (1.588 mm)
Binder Clips			To clamp tubing
Coverslips	Thermo Scientific™ Nunc™	50 x 24 mm
Syringe 3 ml	Terumo
27  G Needle	Terumo
2  L Storage/Media Bottles	VWR®
Trolley			To hold biofilm setup