Preparation of a Microfluidic Plate with Bacterial Cells

Abstract

Source: Sutlief, A. L., et al. Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System. J. Vis. Exp. (2019).

This video demonstrates the preparation of a microfluidic plate for shear stress studies in bacterial cultures. After priming the channels with media, bacterial cells are introduced into the experimental channel and incubated for attachment. Residual media and culture are removed to prepare the system for subsequent shear stress experiments.

Protocol

1. Media Preparation

1. Prepare minimal media (MM) with 0.25% glucose. To make 1 L of MM with 0.25% glucose, add 200 mL of sterile M9 salt solution, 2 mL of sterile 1 M magnesium sulfate (MgSO₄), 100 µL of sterile 1 M calcium chloride (CaCl₂), and 12.5 mL of sterile 20% (w/v) glucose to water (dH₂O) at a final volume of 1 L.
2. Transfer required media to a sterile bottle using sterile techniques. Prepare the amount required depending on the number of channels being used. Typically, each channel requires 200 µL for priming, 300 µL for seeding and 1300 µL for the 24 h experiment.
3. Place the bottle in a....

Results

Figure 1: Individual components of the Microfluidic System. The individual components are listed from left to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ammonium Chloride, ACS	VWR	BDH9208-500G	Part of the minimal media composition
BioFlux 1000 48 Well Low Shear Plate	Fluxion Biosciences	910-0047
BioFlux 1000Z Microfluidic Imaging System	Fluxion Biosciences	BF 1000Z
Calcium Chloride Dihydride, ACD Grade	VWR	97061-904	Part of the minimal media composition
Dextrose, Anhydrous, ACS	VWR	BDH9230-500G	Part of the minimal media composition
Magnesium Sulfate ACS Grade	VWR	EM-MX0070-1	Part of the minimal media composition
Potassium Phosphate Monobasic, ACS Grade	VWR	BDH9268-500G	Part of the minimal media composition
Pseudomonas Aeurginosa GFP	ATCC	15692GFP	Pseudomonas aeurinosa bacterial strain PA01 with GFP modification used for this study.
Sodium Chloride, ACS	VWR	BDH9286-500G	Part of the minimal media composition
Sodium Phosphate, Monobasic, Anhydrous, Reagent Grade	VWR	97061-942	Part of the minimal media composition