Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Summary

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Abstract

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

Introduction

Microbial communities are shaped by both deterministic factors, such as the structure of the environment, and stochastic processes, which are associated with cell death, division, protein concentration, number of organelles, and mutation¹. Within the natural environment, it can be nearly impossible to parse the individual impact of these influences on community composition and activity. Obscured by natural structures and buried within a chemical and biological milieu, identifying community members and further resolving their spatiotemporal distribution within the natural environment is extremely challenging. Nonetheless, recent efforts have und....

Protocol

1. Silicon Microwell-array Fabrication

1. Parylene coating
   1. Deposit between 1-1.5 µm of parylene N on silicon wafers using a commercially available parylene coating system according to the manufacturer's specifications and instructions (settings: vaporizer set point = 160 °C; furnace set point = 650 °C).
      NOTE: Approximately 6 g of parylene N loaded into a chamber yields coatings 1-1.5 µm thick.
2. Photolithography
   1. Spin-coat the parylene N-coated wafers with adhesion promoter, 20% hexamethyldisilazane (HMDS), and 80%propylene glycol monomethyl ether acetate (PGMEA) (see the

Results

The experimental platform presented here is designed for high-throughput and high-content studies of bacterial communities. The design enables thousands of communities, growing in wells of various sizes, to be analyzed simultaneously. With this microwell array design, the dependence of the final community composition on initial seeding densities, well size, and chemical environment can be determined. This work demonstrates the growth of a two-member community in the microwell array and pu.......

Discussion

This article presented a microwell array device and experimental protocols designed to enable high-throughput and high-content live-cell imaging-based analysis of bacterial community development. While the focus of the demonstration here was to study the effects of contact-mediated Type VI secretion on community development, the arrays were designed to be flexible and accommodate the study of a broad range of microbial communities and microbe-microbe interactions. The work here focuses solely on the use of bacteria that .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Parylene N	Specialty Coating Systems	CAS NO.:1633-22-3
Parylene coater	Specialty Coating Systems	Labcoter 2 Parylene Deposition Unit PDS2010
Silicon Wafer	WRS Materials	100mm diameter, 500-550μm thickness, Prime, 10-20 resistivity, N/Phos<100>,
adhesion promoter	Shin-Etsu Microsci	MicroPrime P20 adhesion promoter
postive tone photoresist	Rohm and Haas Electronics Materials LLC (Owned by Dow)	Microposit S1818 Positive Photoresist (code 10018357)
Quintel Contact Aligner	Neutronix Quintel Corp	NXQ 7500 Mask Aligner
Reactive Ion Etching Tool	Oxford Instruments	Plasmalab System 100 Reactive Ion Etcher
R2A Broth	TEKnova	R0005
Bovine Serum Albumin	Sigma	A9647
Multimode Plate Reader	Perkin Elmer	Enspire, 2300-0000
Fluorescent Microscope	Nikon	Eclipse Ti-U
Automated Stage	Prior	ProScan III
CCD camera	Nikon	DS-QiMc
Stage-top environmental control chamber	In Vivo Scientific	STEV ECU-HOC
Phosphate Buffered Saline	ThermoFisher Scientific	14190144
UltraPure Agarose	ThermoFisher Scientific	16500500
25 x 75 mm No. 1.5 coverslip	Nexterion	High performance #1.5H coverslips
Fluorescence Reference Slides	Ted Pella	2273
Physical Stylus Profilometer	KLA Tencor	P-6
lab wipes	Kimberly Clark	Kimipe KIMTECH SCIENCE Brand, 34155
commercial software	Nikon	NIS Elements
Zeiss 710 Confocal Microscope	Zeiss
filter cubes	Nikon	Nikon FITC (96311), Nikon Texas Red(96313)