Spring 11:Chemotaxis Assay

Introduction

For my project I designed and developed an automated microfluidic system using Seymour et al’s microinjector microfluidic device to provide a high through-put method of acquiring bacterial chemotaxis parameters. The microinjector device was chosen for its simple design, diverse application to both fluid-flow and flow-free approaches, and for its ability to measure individual cell kinetics. The development of a faster and systematic device is especially useful in the research of marine microbes’ response to settling dissolved organic matter.

Objective

Further develop Stocker’s microfluidic system that will allow researchers to run chemotaxis assays more efficiently for high though-put assays. The main improvement to Stocker's system will be the use of pneumatic pressures instead of syringe fluid pressures to control fluid flow to set up the chemoatractant and microorganism bands. The pneumatic pressure will be computer controlled to to allow precise and reproducible experiments as well as increased experimental throughput.

Spring 20.345 Project Assignments

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Readings and Resources

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Microinjector Microfluidic Device

Flow-free, or ‘stopped-flow’, chemotaxis assays allow for the analysis of individual bacterial movement in the absence of shear forces and relies on natural diffusion to generate a gradient. In this approach, fluid flow is only used to set up an initial gradient, but is stopped, allowing the gradient to evolve by diffusion alone. These devices produce unsteady gradients that are advantageous in representing environmental conditions of nutrients especially in marine bacteria and in characterizing bacterial responses to wide range of concentration and gradients within a single experiement. The bacterial distribution is measured by recording the kinetic paths of individual bacteria using videomicroscopy. An example of this approach is Seymour et al’s microinjector microfluidic device

Figure :Microinjector microfluidic device (Seymour 2009). The device has two inlets; one for a chemoeffector (inlet B) and one for a bacterial or microorganism suspension (inlet A). Bacteria and chemoeffector band created by microinjector are flowed to setup initial gradient. (B) The band width of the chemoeffector band can be adjusted by changing the flow rates into inlets A & B.

Microscope

The imaging microscope will be a brightfield and fluorescent microscope as shown in Fig 3. A CCD camera will acquire images at a given rate and the kinetic paths will be tracked using particle tracking algorithms.

Figure 3: Bright field and Fluorescent Microscope Block Diagram

• Camera (Allied Manta G032B): 7.4um sqr pixels, 656 x 492 pixels
• Field of View: 484um x 366um (10x objective w/200mm lens => 10x magnification)

Pneumatic System Design

The pneumatic system is composed of manual pressure regulators

Basic Design

The fundamental principle behind this system is that changes in pressures causes fluid flow and when the pressure equalize to the same 'ground' pressure, fluid flow should stop. By this approach

Figure 4: Pressure states of fluid flow

Experiment 1:

weekly reports

- By Emmanuel Quiroz