Real-Time Visualization of Nutrient Media Impact on Pseudomonas aeruginosa Biofilm Development Using a Microfluidic System

Summary

This protocol evaluates the influence of rich (TSB) and minimal (FAB) nutrient media compositions on Pseudomonas aeruginosa PAO1 and PA14 biofilm development in a steady state flow environment in a microchannel.

Abstract

The pathogenic bacterium Pseudomonas aeruginosa is a major cause of numerous nosocomial infections, and its growing antimicrobial resistance has led to it posing a significant public health threat. This article presents a comprehensive protocol detailing the use of a microfluidic system for the real-time visualization and quantification of biofilm development in two key P. aeruginosa strains, PAO1 and PA14. The method employs optically transparent, multichannel microchannel plates to subject bacterial cultures to a continuous, steady flow of media, including tryptic soy broth (TSB) or modified minimal fastidious anaerobic broth (FAB) with varying carbon source concentrations, thereby mimicking conditions found in the clinical environments. Over a 24-h period, automated real-time imaging captures the growth and maturation of biofilms in the form of biofilm surface area coverage, thickness, and surface roughness in a highly reproducible manner. The experimental objective is to use the results to demonstrate that biofilm formation for both strains is significantly impacted by changes in nutrient media composition. The goal of this visualized protocol is to provide a method for researchers to study biofilm dynamics under steady laminar flow conditions, and the insights gained could be leveraged to develop alternative, non-antimicrobial strategies for eradicating early-stage P. aeruginosa biofilms in nosocomial settings.

Introduction

The goal of this method is to observe the impact of two different culture media, i.e., tryptic soy broth (TSB) and modified minimal fastidious anaerobic broth (FAB), on the biofilm growth of two Pseudomonas aeruginosa strains, namely PAO1 and PA14. Though P. aeruginosa PAO1 and PA14 are both common laboratory reference strains, the PA14 strain infects a greater proportion of hosts from plants to invertebrates¹. The TSB nutrient broth is a general-purpose culture medium with 2.5 g/L of glucose as a carbon source and is able to cultivate a wide range of bacteria due to its composition of casein and soy peptones for organic nitro....

Protocol

1. Glassware cleaning

1. Submerge two 200 mL glass bottles to be used for media and agar, and three 1 L glass bottles in a 10% hydrochloric acid (HCl) bath for approximately 5 min at room temperature.
   CAUTION: Hydrochloric acid is corrosive. Perform all acid handling in a chemical fume hood while wearing appropriate personal protective equipment (lab coat, gloves, and eye protection).
2. Subsequently, remove glassware from the HCl bath and rinse twice with tap water and once with distilled water (dH₂O).

2. Tryptic soy broth media preparation

1. Prepare the TS....

Results

This section outlines the representative outcomes when applying the above protocol procedure. All experiments were performed with at least three independent biological replicates, each consisting of six parallel channels, and data are presented as mean ± standard deviation. Error bars in all graphs represent standard deviation, as defined in the figure legends.

The growth of the PAO1 and PA14 strains at 37 °C, in TSB media, and under 0.01 Pa hydrodynamic wall shear stress over a 24 h .......

Discussion

The several critical steps that collectively form this protocol enable a more comprehensive and accurate analysis of biofilm formation than alternative, more traditional methods. A key component is the use of a 48-well microchannel plate, with microscale channels of dimensions 350 µm × 70 µm × 4 mm (W × H × L). These dimensions specifically replicate the flow conditions and microenvironments found in hospital settings, such as urinary catheters or the lung bronchioles of cystic fibrosis pati.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(NH4)2SO4	Acros Organics	7783-20-2	For modified FAB medium
48-well 0-2 Pa microchannel plate - glass coverslip	Cell Microsystems	910-0047-5Pack	Microchannels for media flow and biofilm growth with real-time imaging
-80 °C Freezer	Thermo Fisher Scientific	1.16057E+15	To store glycerol stocks until further use
Agar, Bacteriological Grade	Acros Organics	443570010	For Agar plates - culturing bacteria
Analytical Balance	VWR	VWRI611-2297	To measure components of media used
Autoclave	Priorclave	4304	To sterilize glassware
BioFlux 1000HT	I&L Biosystems	https://il-biosystems.com/cell- microsystems/device/bioflux-1000ht- shear-flow-system/	For Microchannel flow and biofilm growth experiments
BioFlux Control Module	I&L Biosystems	https://il-biosystems.com/cell- microsystems/device/bioflux-1000ht- shear-flow-system/	To set up and monitor media flow and biofilm growth within the microchannels
BioFlux Montage Software	I&L Biosystems	https://il- biosystems.com/app/uploads/2025/01/Bi oFlux_Overview_.pdf	To set up and monitor media flow and biofilm growth within the microchannels
CaCl2	Fisher Scientific	10043-52-4	For modified FAB medium
CaSO4.2H2O	ChemCruz	A1719	For 1 L minimal metal solution
Clear Polystyrene 96-well Plate	Corning, Costar	CLS7007	For viable cell count
CoSO4.7H2O	Sigma-Aldrich	10026-24-1	For 1 L minimal metal solution
CuSO4.5H2O	Honeywell	209198-100G	For 1 L minimal metal solution
Eppendorf Tubes 3810X	Eppendorf	30125150	For P. aeruginosa glycerol stocks
FeSO4.7H2O	VWR International Ltd.	284005E	For 1 L minimal metal solution
Fiji (ImageJ)	National Institutes of Health	https://imagej.net/software/fiji/downloa ds	Z-stack image processing
Glucose	Sigma-Aldrich	50-99-7	For glucose modified FAB Medium
Glycerol	Sigma-Aldrich	56-81-5	For P. aeruginosa glycerol stocks
H3BO3	Fisher Scientific	10043-35-3	For 1 L minimal metal solution
Hamamatsu Orca-Flash 4.0 Camera Model C11440- 42U	Hamamatsu	C11440-42U	For Microchannel flow and biofilm growth experiment real-time imaging
KH2PO4	Fisher BioReagents	7778-77-04	For modified FAB medium
Methylated-Spirit	Sigma-Aldrich	2857	To clean microbiology cabinet before and after use
MgCl2	Sigma-Aldrich	M8266-100G	For modified FAB medium
Microbiology Cabinet Class II	Thermo Fisher Scientific	42111226	To carry-out protocol steps without contaminating bacteria, broth or agar
Mini Incubator	Labnet International	sn03171014	To thaw PAO1 and PA14 after -80 °C freezer and for initial 24 h bacterial agar plate growth
MnSO4.H2O	Sigma-Aldrich	10034-96-5	For 1 L minimal metal solution
Na2HPO4.2H2O	Sigma-Aldrich	10102-40-6	For modified FAB medium
NaCl	Sigma-Aldrich	S5886-500G	For modified FAB medium
NaMoO4.H2O	Sigma-Aldrich	10102-40-6	For 1 L minimal metal solution
Precision Balance	VWR	VWRI611-2299	To measure components of media used
Refrigerated Centrifuge	Heraeus	40289841	Separating P. aeruginosa culture from supernatant for glycerol stocks
Semi-Micro Cuvettes	Alpha Laboratories	X72053	For optical density measurements
Shaking Incubator	INFORS HT	https://infors- ht.com/en/products/incubator- shakers/multitron-standard	For overnight cultures
Sodium citrate	Sigma-Aldrich	71498-250G	For Sodium citrate modified FAB medium
Sterile Plastic Inoculation Loops	Microspec	15782105	For P. aeruginosa PAO1 and PA14 innoculation into agar plates and falcon tubes
Tryptic Soy Agar, vegitone	Millipore	14432-500G-F	For Agar plates - culturing bacteria
Tryptic Soy Broth	Sigma-Aldrich	T8907	Dehydrated culture media - 6 g/200 mL
Type 1 Water Dispenser	Thermo Fisher Scientific	sn42103311	For distiled water
ZnSO4.7H2O	Fisher Scientific	7446-20-0	For 1 L minimal metal solution