Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Summary

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

Abstract

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 µm), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Introduction

Studies aiming to understand the transport of microbes through porous media have mainly been driven by concerns of contamination¹, the transmission of disease² and bioremediation³. In this regard, bacteria have mostly been treated as particles in transport models⁴ and processes such as filtration, straining, gravitational settling or remobilization from biofilms have been identified as drivers of retention or transport of microbes⁵. However, studying the transport of bacteria through porous landscapes can also inform us on the ecological strategies u....

Protocol

1. Bacterial culture conditions

1. Working under a laminar flow hood, use 100 μL of a glycerol stock of GFP-tagged Pseudomonas putida KT2440 (1 × 10⁷ mL^-1, stored at -80 °C) to inoculate 5 mL of Luria-Bertani (LB) medium. Incubate at 30 °C while shaking at 250 rpm overnight.
2. The next day, resuspend 100 μL of the overnight culture in 5 mL LB medium and incubate under the same conditions for 5h (exponential phase). Sample a 1 mL aliquot into a 2 mL tube, allow to cool to room temperature (~15 min) and centrifuge (2300 x g for 5 min).
3. Remove the supernatant and add 1 mL motility buffer to the pellet. Vo....

Results

To illustrate the functionality of the presented workflow, we performed experiments using genetically modified Pseudomonas putida KT2440, a gram negative motile bacterium important for bioremediation and biotechnology. Genetically modified versions of this strain that express GFP production are commercially available. A non-motile strain of P. putida KT2440 which lacks the relevant structural and regulatory genes for motility is also available. Using both, motile and non.......

Discussion

Here we suggest two means to study the transport of microbes through porous systems at the single-cell and population level. While the study of transport phenomena using BTC modeling has provided valuable insights into the spread of pathogens or contaminants at the ecosystem scales, difficulties to scale from laboratory experiments to field conditions still exist. The tools described here allow researchers to experimentally resolve the spatial and temporal scales in order to better understand the ecological strategies of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EDTA	Sigma
Elastomer Sylgard 184	Dowsil	101697
Flow cytometer NovoCyte	Acea
Glucose	Sigma		https://www.makeblock.com/project/xy-plotter-robot-kit
LB broth	BD
Liquid dispenser, XY Plotter Robot Kit	makeblock
Microscope Axio Imager	Zeiss
Microscope AxioZoom v16	Zeiss
Microscope slides, 75 mm × 25 mm	Corning
Minipuls 3 peristaltic pump	Gilson
Plasma bonder Corona SB	BlackHole Lab
Potassium phosphate	Sigma
Syringe pump New Era NE 4000	New Era
Syto 13 Green Fluorescent Nucleic Acid Stain	Molecular Probes, Invitrogen
Tygon tubing	Ismatec
WF31SA universal milling machine	Mikron