Method Article

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

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DOI:

10.3791/69997

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May 15th, 2026

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1School of Mechanical, Aerospace and Civil Engineering, University of Sheffield, 2School of Chemical, Materials and Biological Engineering, University of Sheffield

Corresponding Authors: Niamh Corkery-Hayward <ncorkery-hayward1@sheffield.ac.uk>

In This Article

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 nitrogen and a natural sugar supply, as well as sodium chloride to maintain osmotic equilibrium2. The minimal FAB medium, in contrast, is formulated with minimal concentrations (50-fold reduction) of a single carbon source, at levels of 0.05 g/L to explore growth requirements of anaerobic bacteria. Both the PAO1 and PA14 strains were exposed to unidirectional, laminar flow, resulting in a low wall shear stress of 0.01 Pa. This shear stress value is often present in P. aeruginosa-colonized locations, including the bronchi of cystic fibrosis patients and the gastrointestinal tract. The biofilm growth was measured over a 24-h period by assessing real-time biofilm percentage surface area coverage, thickness, and arithmetic mean of the surface roughness.

P. aeruginosa biofilms are responsible for a significant percentage of nosocomial infections and are a leading cause of death for people with cystic fibrosis3,4,5. Previous investigations into the impact of carbon source levels on bacterial biofilm development have neglected the critical role of hydrodynamic flow during biofilm development, used shear stress values that are not representative of P. aeruginosa infection sites, or have undertaken only biofilm kinetic and morphological analysis post-experiment6,7. These limitations are addressed in this method by utilizing real-time imaging of biofilm development, under steady state flow conditions within a microfluidic channel that closely replicates physiological environments found in cystic fibrosis patients.

This method offers significant advantages over alternative techniques that are used to study biofilm development. The application of real-time imaging during the 24-h biofilm growth period ensures that the developing biofilm is undisturbed, as no drying or chemical alterations are required, avoiding changes to its composition and yielding more representative results than methods that require sample manipulation8. Additionally, by applying steady hydrodynamic flow conditions and varying the carbon source concentrations over a large magnitude, this procedure more accurately replicates the physiological environments found at sites of P. aeruginosa nosocomial infections. This makes it a more representative method compared to those utilizing static assays, such as 96-well microtiter plates or coupons, which fail to capture the critical impact of fluid flow on biofilm development9,10,11.

The microfluidic device employed was specifically chosen as its dimensions are similar to parts of the lung, such as bronchioles and bronchi12. Furthermore, P. aeruginosa infections are a major concern for patients with severe burn wounds, where glucose levels can fluctuate significantly from the patient's normal range13,14.

While this protocol specifically uses two strains of P. aeruginosa, the method is also applicable to investigations into the early-stage development of other bacteria that cause infections or colonize areas under similar conditions. Additionally, other nutrient media compositions as well as flow conditions (e.g., pulsatile and oscillating flows, or higher wall shear values) could also be investigated using the same methodology.

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 TSB solution, weigh 6 g/200 mL of TSB powder using a clean weighing boat, and a precision balance.
    NOTE: The composition of the TSB powder used is as follows: Dextrose (2.5 g∙L-1), Dipotassium phosphate (2.5 g∙L-1), Pancreatic digest of casein (17 g∙L-1), Papaic digest of soy meal (3.0 g∙L-1), and Sodium chloride (5.0 g∙L-1).
  2. Measure 200 mL of dH2O using a measuring cylinder and pour it into one of the previously cleaned glass bottles.
  3. Add the 6 g of TSB into the clean glass bottle containing the 200 mL of dH2O, seal the bottle tightly and mix for approximately 2 min to dissolve the powder, creating the TSB solution media.
  4. Autoclave TSB media solution for 15–20 min at a temperature of 121 °C and pressure of 1.5 atm, to sterilize the solution.
    NOTE: (PAUSE POINT) TSB solution can be stored at room temperature (approximately 20 °C) between uses and used for future experiments.

3. Fastidious anaerobic broth media preparation

  1. Prepare FAB media trace metal solution.
    1. Use separate, clean weighing boats and a spatula, and a precision balance when weighing masses between 0.5–210 g, and an analytical balance when weighing masses between 0.001–0.5 g. Measure out the following components and their quantities: CaSO4.2H2O (200 mg∙L-1), FeSO4.7H2O (200 mg∙L-1), MnSO4.H2O (20 mg∙L-1), CuSO4.5H2O (20 mg∙L-1), ZnSO4.7H2O (20 mg∙L-1), CoSO4.7H2O (10 mg∙L-1), NaMoO4.H2O (10 mg∙L-1), and H3BO3 (5 mg∙L-1)7,9.
    2. Add each of the components into a 1 L cleaned glass bottle containing 1 L of dH2O and mix thoroughly with the dH2O.
    3. Add 1 mL of the mixture to each of the two remaining 1 L sterilized glass bottle with 1 L of dH2O and mix thoroughly.
  2. Prepare 20 mM sodium citrate-supplemented FAB for overnight cultures
    1. Measure out the following components and their quantities using separate, clean weighing boats and a spatula, a precision balance when weighing out masses between 0.5-200 g and an analytical balance when weighing out masses between 0.001-0.5 g,: Sodium citrate (20 mM), (NH4)2SO4 (2 g∙L-1), Na2HPO4.2H2O (6 g∙L-1), KH2PO4 (3 g∙L-1), NaCl (3 g∙L-1), MgCl2 (93 mg∙L-1) and CaCl2 (11 mg∙L-1)7,9.
    2. Add all components into one of the remaining 1 L sterilized glass bottles containing 1 L of dH2O with 1 mL of trace metal solution.
    3. Mix thoroughly.
  3. Prepare 0.3 mM Glucose-supplemented FAB for biofilm growth.
    1. Replace the 20 mM of sodium citrate with 0.3 mM of glucose. Use the same components and masses listed in step 3.2.1.
    2. In the remaining cleaned 1 L glass bottle, add the 0.3 mM glucose FAB components to 1 mL of dH2O with 1 mL of trace metal solution and mix well.
  4. Autoclave for 15-20 min at a steam temperature of 121 °C and pressure of 1.5 atm, to sterilize the media.
    NOTE: (PAUSE POINT) FAB medium solution can be stored at room temperature (approximately 20 °C) between uses and used for future experiments.

4. TSB agar preparation

  1. Measure out 8 g/200 mL of TS agar using the precision balance and washed weighing boats and spatula.
  2. Measure out 1 g/200 mL of across bacteriological grade agar, on a separate weighing boat, using the same precision balance.
  3. Using a measuring cylinder, measure 200 mL of dH2O and pour it into the final, previously cleaned glass bottle.
  4. Pour both agar components into the 200 mL of dH2O and mix well.
  5. Autoclave for 15–20 min at a temperature of 121 °C and pressure of 1.5 atm, to sterilize the medium.
    NOTE: (PAUSE POINT) TSB agar can be stored at room temperature (approximately 20 °C) between uses and requires re-heating before use in future experiments.

5. Petri dish Pseudomonas Aeruginosa culturing

  1. Melt agar in the microwave for approximately 5 min, or until the solution is liquid.
  2. Open and disinfect a class II safety cabinet surface with 70% ethanol solution, ready for use. Undertake all the steps below in this section within this class II safety cabinet.
  3. Measure 25 mL of agar solution into a 50 mL vial and pour it into a 90 mm Petri dish, and wait for the agar to set.
  4. Retrieve P. aeruginosa PAO1 and PA14 frozen stocks from -80 °C freezer. Place stocks in the incubator at 37 °C for approximately 5 min, or until contents are completely thawed.
  5. Dip the inoculation loop inside either of the thawed P. aeruginosa stocks and spread the sample across the agar.
  6. Repeat step 5.5 for the other P. aeruginosa strain, creating two separate P. aeruginosa Petri dish cultures, one with the PAO1 strain and the other with the PA14 strain.
  7. Label each Petri dish with the strain of P. aeruginosa within it, date of revival, and initials, then place in incubator for 16–18 h at 37 °C.
    NOTE: (PAUSE POINT) P. aeruginosa PAO1 and PA14 Petri dishes can be stored at 4 °C for a storage period of up to 4 weeks and used for future experiments.

6. Pseudomonas aeruginosa PAO1 and PA14 glycerol stocks

  1. Open and disinfect a class II safety cabinet for use, select eight 50 mL vials to contain the overnight cultures.
  2. Using the previously fabricated TSB and sodium citrate FAB media (section 3), pour 5 mL of TSB medium into four of the eight vials and the sodium citrate minimal FAB medium into the remaining four.
  3. Using the previously incubated Petri dishes and an inoculation loop, touch five separate colonies in the P. aeruginosa PAO1 Petri dish and transfer to one of the TSB vials. Repeat this for the sodium citrate FAB medium and for the P. aeruginosa PA14 strain Petri dish.
  4. Submerge a clean inoculation loop directly into the remaining four TSB and sodium citrate FAB media vials, forming controls.
  5. Incubate all eight vials in a shaking incubator for 16–18 h at 200 RPM and 37 °C and clearly label.
  6. Retrieve the culture vials, ensure that those inoculated with P. aeruginosa are turbid and those used as controls remain clear.
  7. Centrifuge the four P. aeruginosa-inoculated vials at 4000 × g for 5 min at 20 °C to separate the P. aeruginosa from the culture media. Ensure that the centrifuge is balanced.
  8. Disregard the supernatant and resuspend the P. aeruginosa pellet after centrifugation with 1 mL of solution containing 15% glycerol, with 150 µL glycerol and 850 µL fresh TSB or sodium citrate FAB media.
  9. Transfer resuspended cultures to 1 mL vials and place in the -80 °C freezer.
    NOTE: (PAUSE POINT) Glycerol stocks can be stored at -80 °C for long-term storage and used for future experiments.

7. Measuring optical density of overnight cultures

  1. Repeat steps 6.1–6.5, for two TSB medium vials only, with one as a control and the other with the PAO1 P. aeruginosa strain.
  2. Pipette 1 mL of TSB medium into a 1 mL spectrophotometer vial, within a class II safety cabinet, and transfer 0.9 mL of TSB medium into the second vial, supplementing with 0.1 mL of the overnight culture.
  3. Set the spectrophotometer to a light wavelength of 600 nm and, using the 1 mL TSB spectrophotometer vial to calibrate the machine, measure the optical density of the 0.1 mL suspension vial15.
    1. If the absorbance measured does not read 0.1, recalculate the suspension volume needed.
      NOTE: The volume of suspension needed to achieve an absorbance reading of 0.1 (V1) can be calculated using the initial 0.1 mL suspension vial absorbance reading, C1, the desired absorbance that the suspension is being adjusted to, C2, and the volume of concentrated overnight suspension wanted, V2:
      C1V1 = C2V2
      In the example above, if C1 = 3.59, C2 = 0.1 and V2 = 10 mL, the volume of overnight suspension wanted would be 0.279 mL in a 10 mL solution.
  4. Measure 10 mL of TSB medium into a 50 mL vial and remove the same volume of TSB medium as the volume of suspension needed to create a 10 mL 0.1 OD 600 nm suspension. Using the overnight culture, add the suspension volume calculated and vortex for approximately 5 s to mix.
  5. Transfer 1 mL of the 10 mL adjusted suspension into a spectrophotometer vial and re-measure the absorbance to ensure it is 0.1.
  6. Repeat the above 7.1–7.5 for PA14 and TSB medium and for both P. aeruginosa strains and the sodium citrate FAB medium.

8. Viable cell count

  1. Transfer dH2O into a square Petri dish within a class II safety cabinet and use a multi-channel pipette to transfer 180 µL into columns 2–9 of a clear 96-well polystyrene plate16.
  2. Transfer the 10 mL 0.1 OD 600 suspension into a separate square Petri dish and, using the multichannel pipette, transfer 200 µL of the suspension into column 1 of the 96-well, polystyrene plate.
  3. Transfer over 20 µL of suspension using the multichannel pipette from column 1 of the 96-well, polystyrene plate into channel 2, followed by transferring 20 µL of diluted suspension in channel 2 to channel 3, repeating to channel nine, creating suspension dilutions from 10-1 to 10-8.
  4. Transfer 50 mL of TS agar (section 4 and step 5.1), in a class II safety cabinet using a vial, into a square Petri dish and leave until agar becomes turbid.
  5. Transfer 10 µL from each of the wells along columns 2–9 onto the TS agar Petri dish in a column formation, using a multichannel pipette, ensuring the columns do not contact each other.
  6. Incubate TS agar for 24 h at 37 °C, after waiting until the transferred dilutions have settled.
  7. Calculate colony-forming units (CFU) per milliliter (mL) by determining if the Petri dish is countable (between 30 and 300 colonies).
  8. Determine the dilution factor of the 10 mL suspension used; in this case, the suspension was diluted to 1/10 (0.1 OD 600).
  9. Calculate the individual dilution factors of each plated column. This is the amount of diluted suspension added to each column divided by the total volume of the column after the suspension is added. In the case of column 2 of the 96-well plate (column 1 of the Petri dish), 20 µL/200 µL = 1/10.
  10. Calculate the total series dilution factor by multiplying each of the columns' individual dilution factors together.
    NOTE: In this case, since 20 µL was transferred from each column to the next, making the total volume of each column well 200 µL, each individual series dilution was 1/10. Making the total series dilution 1 × 10-8.
  11. Determine the plating dilution factor as the dilution is transferred from the 96-well plate to the Petri dish. To determine CFU/mL, divide the volume of each column plated (10 µL) by 1 mL. In this case: Decimal multiplication equation: 0.01/1 × 10/10 = 1/100..
  12. Determine the final dilution factor by multiplying the original suspension dilution factor (0.1) by the total series dilution factor (1 × 10-8) and the plating dilution factor (Fraction 1/100, mathematical concept, ratio representation, elementary arithmetic education.).
  13. Calculate the CFU∙mL-1 in the original sample, multiply the CFU on the plate by the inverse of the final dilution factor.
    NOTE: In this case, if 200 CFU were counted, this would mean carrying out 200 × 1/1 × 10-8 resulting in the CFU/mL being 2 × 1010 CFU∙mL-1 17.

9. Microfluidic system setup and experimental run

  1. Inoculate 5 mL broth of culture medium with each strain of P. aeruginosa to form an overnight culture and incubate for 16–18 h at 37 °C.
  2. Set up the microfluidic system and associated software.
    1. Activate the microfluidic system components in the following order: Power supply, microfluidic system controller, CMOS camera, enclosure heaters, microscope, computer, and associated monitors.
    2. Activate the microfluidic software and initiate the accompanying control module interface by inputting the 48-well 0–2 Pa multichannel plate-associated barcode when prompted, found on the side of the multichannel plate.
  3. Set P. aeruginosa multichannel plate well inoculation procedure.
    1. Adjust the optical density of the overnight culture to 0.1 OD600.
    2. Transfer 50 µL of 0.1 OD 600 suspension into the outlet well of each of the six channels in parallel inside a class II safety cabinet.
      ​NOTE: Six channels were run in parallel at any one time to maximize the likelihood of anomalous result identification.
    3. Replace the 48-well multichannel plate cover and transfer it to the movable stage situated inside the system incubator and secure it.
  4. Set P. Aeruginosa multichannel plate channel inoculation procedure.
    1. Seed the microfluidic channels using the microfluidic system control module by selecting: Manual > Columns 1–4 > Fluid: TSB at 37 °C > Max Shear (Pa): 0.1 (1 dyne∙cm-2 = 0.1 Pa) and select an outlet well of the 48-well 0–2 Pa multichannel plate diagram.
    2. Select the abort button to stop the flow after 2–4 s and leave the plate for 30 min, to ensure the cells attach to the channel substrate before starting the continuous flow.
      NOTE: A 30 min duration allows gravitational force to help adhere the planktonic P. aeruginosa bacteria to the bottom channel wall. This time will vary depending on the bacteria and the type of nutrient media.
    3. Remove the multichannel plate from the movable incubator stage and transfer 1 mL of fresh TSB into the inlet well of each of the six channels in a class II safety cabinet.
    4. Transfer the multichannel plate to the movable stage inside the incubator and secure it in place.
  5. Set up AutoRun.
    1. Ensure the flow used throughout the 24 h experiments is relevant to where P. aeruginosa is found in nature, by navigating to manual > shear stress: 0.01 Pa, and record the volume flow rate (µL⋅h-1) displayed. Ensure this value, for the 48-well microchannel plate is 8.2 µL⋅h-1.
    2. Convert the volume flow rate to m3∙s-1, calculate the channel cross-sectional area (m2), and determine the flow velocity when at a hydrodynamic shear stress of 0.01 Pa.
      NOTE: By using and rearranging the fluid volume flow rate equation, the fluid velocity through the channel can be calculated:
      Heat transfer equation, \( \dot{Q} = UA \), formula for thermal energy exchange calculation.
      Where U is the fluid velocity (m∙s-1) and A is the microchannel cross-sectional area (m2) of the channels in the multichannel plate.
    3. Calculate the flow Reynolds number using the density, ρ, and dynamic viscosity, µ, of the TSB medium, and ensure it is within a range of fluid flow that P. aeruginosa biofilms are found. For a hydrodynamic shear stress of 0.01 Pa, ensure the Reynold's number is 0.0117 and 0.0165 for TSB and FAB media, respectively. If outside of this range, check that the fluid conditions are set to TSB.
      Reynolds number equation, Re=ρUDh/μ, fluid dynamics formula, educational diagram.
      Where Dh is the hydraulic diameter of the microchannel used, obtained using:
      Darcy-Weisbach equation, hydraulic diameter formula, \(D_h = \frac{2 \times w \times h}{w + h}\) diagram.
      Where, w, and h, represent the width (m) and height (m) of the microchannel used.
    4. Calculate the fluid development length to ensure the fluid flow within the image acquisition region is fully developed.
      NOTE: The development length for laminar fluid through a channel can be calculated using the Reynolds number, Re, and the channels' hydraulic diameter, Dh:
      Ldev = 0.06ReDh
    5. Set up the autorun: Edit AutoRun > Protocol Setup > time duration: 24 h, with a repeat of 1 and a shear stress of 0.01 Pa. Save the Protocol.
    6. Create a new sequence in the Sequence Setup tab by applying the newly created protocol with one repetition, to channels 1–6 and select fluid: TSB at 37 °C. Save this sequence.
  6. Set up image acquisition using the Multi Dimensional Acquisition window.
    1. Select Multi Dimensional Acquisition using the system software, then the live button (bottom left of Multi Dimensional Acquisition window). Navigate the microscope stage to view channels 1–6 using the live view button and camera magnifications 5× to 40× on the microscope touchscreen, showing one channel in the camera field of view at a time.
    2. Prepare the camera for image acquisition: Multi Dimensional Acquisition > Saving: select a directory for the images to be saved to > Timelapse: Duration: 24 h, the same duration as the hydrodynamic flow > Time Interval: Time between each acquired image, set to 10 min.
    3. Input the camera settings by selecting Wavelength > Illumination: brightfield 50% camera 50%: black and white > auto expose: every acquisition > acquire: every time point > auto focus: never.
    4. Determine the camera positions to capture the biofilm images by setting the magnification to 20×, producing a field of view with dimensions 665.6 µm × 665.6 µm and navigate to the middle of the channel viewing region, where the channel number will be engraved at the center.
    5. Add the stage position so it is captured by the camera, navigate to Stage label the position, and select the addition button to add it to the directory.
      1. Repeat this for upstream points 0.9984 mm and downstream points 3.0016 mm from the inlet of the channel viewing region, where the position represents the middle point of the camera field of view.
        NOTE: These imaging positions were selected to provide representations of biofilm growth along the viewing region, whilst ensuring the flow within is fully developed. This results in three images acquired for each microchannel, in this case, resulting in 18 images taken every 10 min.
  7. Perform experimental run
    1. In the system control module window select AutoRun > Run Setup > Sequence: select the 0.01 Pa sequence created, then select start.
    2. Immediately after, navigate to the Multi Dimensional Acquisition window > Acquire. This begins the acquisition of the 18 images every 10 min of the 24 h experimental procedure, producing 2610 images when the experiment terminates.
      ​NOTE: The above section was reproduced over a further six microchannels approximately 48-h following the initial experiment with identical conditions.

10. Z-stack accumulation

  1. At the end of each experimental run, select three channels and obtain z-stacks of three sections along them at 40× magnification, by opening the Multi Dimensional Acquisition > Main: untick all options except 'Z Series'.
  2. Navigate to Z-series after a section along a channel has been selected within Multi Dimensional Acquisition and alter > Interactive settings: Increment: 0.5 and untick both tick boxes.
  3. Use the microscope focusing knob and rotate it just until the biofilm becomes out of focus, and input the z-distance by selecting Set Top To Current. Repeat this procedure, but by rotating the focusing knob in the opposite direction and selecting Set Bottom To Current. This gives the microscope the range to capture the z-stack.
  4. Select a folder within the Saving window to save the z-stack and select Acquire to the bottom right of the window to start acquisition.

11. Post experiment image and data analysis

  1. Obtain biofilm percentage surface area coverage data.
    1. Load the acquired images into the system software by choosing: Analysis tools > Review Multi Dimensional Data > Select Base File > Select Directory: navigate to and select the folder with the saved images > View > Wavelength 1, and in the central square section horizontally displaying the number of images acquired, right-click the small, gray box in the top, left-hand corner.
    2. Load the images of each channel section, one by one, choose Stage position: select the position to view > Load Image(s). Save the image series as a TIFF file.
    3. Threshold the image by selecting Threshold Image > Auto Threshold for Dark Objects highlighting the biofilm, then border the image using the Rectangular Region tool.
      NOTE: The slider on the left-hand side of the thresholded image can be moved up and down to alter threshold intensity to better differentiate between biofilm and background. If the image has shadows, border the section without, as the shadows may have been highlighted by default, altering measurement values.
    4. Obtain data on the percentage surface area coverage of the biofilm in the image over the 24 h experiment by selecting MM Standard > Measure > Region Measurements in the system software window to generate a table of data on the highlighted biofilm within the rectangular border, including Threshold Area %. Select Open Log to open and save a log file and F9: Log Data to input the data into the log file.
    5. Open a spreadsheet and, using the 24 h Biofilm data for each channel section, create a graph showing the biofilm percentage surface area coverage over 24 h.
  2. Obtain biofilm thickness and arithmetic mean surface roughness.
    1. Saved the z-stack to Fiji (ImageJ) by dragging the saved file into the Fiji control bar and convert to and 8-bit image: Image > Type > 8-bit.
    2. Increase the contrast between the biofilm and the channel surface to eliminate any unwanted noise: Image > Adjust > Brightness/Contrast.
    3. Threshold the image, setting the biofilm pixel value 255 (white) and the background pixel value 0 (black): Image > Adjust > Threshold.
    4. Set the scale of the image, Analyze > Set Scale > Distance in Pixels: 2048 > Known distance: 332.8 > Pixel aspect ratio: 1.0 > Unit of length: µm.
      NOTE: The known distance is 332.8 µm, as the Hamamatsu camera used has a 2048 × 2048 pixel field of view, with each pixel having a size of 6.5 µm and images were taken at 40 × magnification.
    5. Analyze the Z-stack using a custom macro18 (Supplementary File 1) which calculates and sums the surface area of biofilm on each z-stack slice and uses the input scale values to calculate the series' thickness, enabling the volume to be found. Alternatively, find the biofilm thickness by dividing the calculated volume by the surface area.
    6. Obtain the arithmetic mean surface roughness values of the biofilm from each z-series by downloading ImageJ (not Fiji) plugin SurfCharJ and save it in the plugins folder.
    7. Upload the z-stack to ImageJ by dragging the saved stack to the control bar and converting it to a 32-bit image: Image > Type: 32-bit.
    8. Create a surface plot, showing the variation in gray values across the surface of the stack: Analyze > Plot Profile > More: High-Resolution Plot. Convert the high-resolution plot to 32-bit.
    9. Obtain the arithmetic mean surface roughness value by initiating the SurfCharJ plugin: Plugins > SurfCharJ > Roughness calculation: Mean sampling length (): 10.
      NOTE: The SurfCharJ plugin uses the various gray values along the surface of the z-stack to calculate the arithmetic mean surface roughness. A table containing surface roughness parameters will be produced, the only parameter of interest is the arithmetic means surface roughness, Ra. The table values will need to be converted to micrometer from pixels.

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 period is presented in Figure 1. The figure shows that the PAO1 strain colonizes a larger proportion of the substrate surface in the initial stage of the experiment than PA14. The larger initial attachment rate found for the PAO1 strain is in agreement with current literature, suggesting that this strain is of a phenotype that forms surface attachments relatively quickly19. However, the surface area colonized by PA14 surpasses that of the PAO1 strain approximately 11-h into the experiment, while the area of substrate colonized by the PAO1 strain started to decline. The reduction in substrate colonization by PAO1 could be due to cell detachment and/or cell death. The large proportion of surface colonized by PA14 is characteristic of the strains' generational attachment process, explaining the lower biofilm formation in the initial stages of the experiment and increased levels as the experiment continues1. Following this crossover, the PA14 strain substrate surface area colonization reaches a plateau and does not decrease much afterwards. As a result of this behavior, the substrate surface area colonized by the PAO1 and PA14 biofilms after 24 h is 66% ± 25% and 83% ± 5.4%, respectively.

Statistical comparisons between groups were performed using a t-test, with significance defined as p < 0.05.

Figure 2 displays the growth of the PAO1 and PA14 P. aeruginosa strains in the modified FAB medium under the same conditions as the TSB medium experiments. The initial rate of substrate surface area colonization by the PA14 strain is significantly lower than that of the PAO1 strain. It is also seen that the initial growing rate is significantly higher for PAO1 and it is significantly lower for PA14 in FAB medium compared to TSB. This increase in growth for PAO1 as a response to FAB media has also been reported for wild-type PAO1 when studied in microtiter plates7. The percentage of the substrate surface occupied by PAO1 biofilms proceeds to decline significantly as a result of bacterial dispersal.

The impact of the TSB and modified FAB media on the growth of P. aeruginosa PAO1 strain under 0.01 Pa of hydrodynamic wall shear stress can be more easily seen in Figure 3. The initial rate of biofilm substrate surface attachment in the modified FAB medium is higher than that in the TSB medium. However, at approximately 7 h into the experiment, the surface area colonized by PAO1 in TSB medium surpasses that of PAO1 in modified FAB medium. After this crossover point, the surface area coverage in the TSB medium peaks at around 90% ± 11%, while the peak coverage in the modified FAB medium occurs earlier and remains lower, around 80% ± 11%. Following these peaks, the colonized substrate surface area for PAO1 begins to decline in both media. However, the coverage in the TSB medium remains substantially greater than in the modified FAB medium, measuring 66% ± 11% and 31% ± 12% at the 24-h mark, respectively.

Figure 4 provides a parallel comparison, illustrating the growth of the P. aeruginosa PA14 strain in both TSB and FAB media over 24 h under the identical flow conditions. As can be seen in this figure, the substrate surface area colonized by PA14 in TSB medium surpasses that in modified FAB medium approximately 3 h into the experimental procedure and proceeds to colonize a larger substrate surface area as the experiment progresses. After 24 h, the percentage of substrate surface area colonized by PA14 biofilm in TSB medium is 83% ± 5.3% as opposed to 45% ± 0.43% in the modified FAB medium.

Figure 5 and Figure 6 compare the thickness and surface roughness, respectively, of the PAO1 and PA14 biofilms after the 24-h experimental period in both the TSB and FAB media. The surface roughness of the biofilm was measured, as it has been linked to enhanced adhesion of other bacterial species, such as L. pneumophila, by creating larger low stress zones20. As shown in Figure 2, the thickness of the two P. aeruginosa biofilms formed over 24 h is very similar in TSB medium, at values of 13 µm ± 1.6 µm and 14 µm ± 3.8 µm for PAO1 and PA14, respectively. However, in modified FAB medium, the PA14 strain has formed a significantly thicker biofilm than the PAO1 strain at 34 µm ± 11.2 µm. Looking at biofilm thickness for the same bacterial strain across the two growth media, Figure 5 clearly indicates that thicker biofilms are consistently formed in the modified FAB medium compared to the TSB medium. Figure 3 then shows that both strains also exhibit greater biofilm surface roughness in the modified FAB medium compared to the TSB medium, at values of 39 µm ± 0.58 µm and 39 µm ± 0.86 µm compared to 31 µm ± 0.66 µm and 35 µm ± 4.09 µm for PAO1 and PA14 strains, respectively. However, when PAO1 and PA14 are compared within the same medium, their surface roughness values are similar.

Furthermore, Figure 7 reveals that both the PAO1 and PA14 P. aeruginosa strains form biofilms composed of more isolated microcolonies when grown in modified FAB medium. Figure 7B also shows interconnected, lattice-like structures formed by PA14 in TSB medium, and Figure 7A shows PAO1 forms biofilms evenly across the substrate surface in TSB medium also. The difference in microcolony formation can also be seen in Supplementary File 2. The cross-sections for each biofilm birds-eye view image graphically present the associated biofilm thickness.

The results of our study confirm that the protocol is a highly reproducible and effective method for the high-throughput analysis of bacterial biofilms under dynamic conditions. The success of the method is demonstrated by the continuous biofilm growth under steady shear conditions, generating reproducible kinetic curves across replicates from consistent and interpretable data, capturing a range of outcomes from robust growth to suboptimal development between media conditions.

The protocol's reproducibility is evidenced by the tight clustering of data from multiple technical replicates. For the P. aeruginosa PAO1 strain, performing at least three technical replicates in TSB medium yielded a maximum mean surface area coverage of approximately 93% ± 4.3%. Similarly, under the nutrient-limited conditions of the modified FAB medium, the protocol produced highly consistent growth curves, reaching a maximum mean coverage of 79% ± 2.1%. The minimal variability between these technical replicates suggests that the method can reliably produce repeatable results. Furthermore, its successful application to another strain, P. aeruginosa PA14, confirms that the techniques are broadly applicable to different strains or bacterial species, highlighting their versatility and high-throughput potential.

The protocol's true value lies in its capacity to generate comprehensive kinetic data that reveals a biofilm's full lifecycle, not just an endpoint measurement. By comparing the results from the nutrient-rich TSB medium (a successful experiment) with those from the nutrient-limited modified FAB medium (a suboptimal experiment), the protocol's power is clear. The data from TSB, for example, illustrates a classic, vigorous biofilm formation, characterized by rapid growth followed by a stable, mature phase. Conversely, the data from the modified FAB medium shows a slower, more limited accumulation of biomass. These distinct outcomes demonstrate that the protocol is sensitive enough to differentiate the impact of environmental factors, such as nutrient availability, on biofilm development.

Beyond kinetic curves, the method's success is further confirmed by its ability to concurrently measure key morphological characteristics, such as biofilm thickness and arithmetic mean surface roughness. This dual capability, combined with the protocol's unique feature of measuring biofilm kinetics under a constant hydrodynamic shear stress, provides a more complete and representative picture of biofilm development than traditional endpoint assays.

Biofilm growth graph, PAO1 vs. PA14 strains, time-dependent analysis, mean coverage, hours 0-24.
Figure 1: P. aeruginosa PAO1 and PA14 mean biofilm percentage surface area coverage (%) over a 24 h period in tryptic soy broth (TSB). Blue line: PAO1. Red line: PA14. Please click here to view a larger version of this figure.

Biofilm growth dynamics; line graph; PA14 + FAB vs PAO1 + FAB; time vs surface area coverage.
Figure 2: P. aeruginosa PAO1 and PA14 mean biofilm percentage surface area coverage (%) over a 24 h period in modified fastidious anaerobic broth (FAB) medium. Blue line: PAO1. Red line: PA14. Please click here to view a larger version of this figure.

Biofilm growth analysis chart; PAO1+TSB (red), PAO1+FAB (blue) over 24 hours.
Figure 3: P. aeruginosa PAO1 mean biofilm percentage surface area coverage (%) over a 24 h period in tryptic soy broth (TSB) and modified fastidious anaerobic broth (FAB) media. Blue line: FAB medium. Red line: TSB medium. Please click here to view a larger version of this figure.

Biofilm growth over 24 hours in PA14+FAB and PA14+TSB; line graph with error bars for analysis.
Figure 4: P. aeruginosa PA14 mean biofilm percentage surface area coverage (%) over a 24 h period in tryptic soy broth (TSB) and modified fastidious anaerobic broth (FAB) media. Blue line: FAB medium. Red line: TSB medium. Please click here to view a larger version of this figure.

Biofilm thickness comparison; bar chart shows P. aeruginosa PAO1/PA14 in TSB and Modified FAB Media.
Figure 5: P. aeruginosa PAO1 and PA14 biofilm thickness (µm), measured at the end of the 24 h growth period using z-stacks of 0.5 µm thickness and ImageJ software. Please click here to view a larger version of this figure.

Surface roughness experiment; bar graph; TSB vs. Modified FAB medium; P. aeruginosa; p<0.01 significance.
Figure 6: P. aeruginosa PAO1 and PA14 average surface roughness deviation from the mean value (µm), measured at the end of the 24 h growth period using z-stacks and ImageJ software. Please click here to view a larger version of this figure.

Microscopy results showing surface morphology at 10 μm scale; microstructure analysis diagram.
Figure 7: Birds-eye view and cross-sectional view of representative biofilms for PAO1 and PA14 in tryptic soy broth (TSB) and fastidious anaerobic broth (FAB) media. Obtained by processing z-stacks obtained at end of 24 h experimental procedure and processed using Fiji (ImageJ) software. (A) P. aeruginosa PAO1 in TSB medium. (B) P. aeruginosa PA14 in TSB medium. (C) P. aeruginosa PAO1 in modified FAB medium. (D) P. aeruginosa in modified FAB medium. Please click here to view a larger version of this figure.

Supplementary File 1: Macro code used to calculate volume and thickness of P. aeruginosa PAO1 and PA14 biofilms using captured z-stacks. The stacks were binarized using Fiji (which is just ImageJ), giving the biofilm a value of 255 and the background a value of zero. The macro code was applied, finding the surface area of each slice, adding them together, and multiplying them by the thickness from the initial to final slice, calculated using scale inputs. Adapted from VISIKOL18.Please click here to download this file.

Supplementary File 2: Timelapse videos showing P. aeruginosa PAO1 and PA14 strains colonizing the microchannel over a 24 h period in tryptic soy broth (TSB) or modified fastidious anaerobic broth (FAB) media steady flow. The three videos represent upstream, middle, and downstream locations of the channel observed. Please click here to download this file.

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 patients12,21,22,23. Six parallel channels per experimental run were also used, with each run repeated at least three times. This repetition is crucial to ensure data integrity due to the inherent stochastic nature of bacteria24,25. By precisely replicating these microscale conditions and running multiple replicates, the findings of this study are significantly more relevant and applicable to in vivo environments than those obtained using a few replicates and macroscale channels. Despite this, the 48-well microchannel plate is a consumable, with each plate having a limited capacity for a maximum of 24 replicates before a new one is required, as they cannot be autoclaved and reused26.

Another essential step is the combination of the optically transparent microchannel and the microfluid system's coupled scientific complementary metal-oxide semiconductor camera (sCMOS). This combination allows real-time, in situ observation and analysis of biofilm growth kinetics. This approach eliminates the need to remove the biofilms for imaging, a technique used in previous studies, which risks disturbing or altering the biofilm's composition through drying, or the application of stains and buffers6,11,27,28,29,30. With the ability to continuously monitor development over time without disruption, a more accurate and dynamic picture is provided, as biofilms contain a large proportion of water31 and the drying process required from imaging alters their composition and disrupts the development of the remaining biofilm.

A final critical characteristic of this protocol is the ability of the microfluidic system to investigate biofilm growth under a constant hydrodynamic wall shear stress of 0.01 Pa. This value was intentionally selected as it is representative of values in the bronchioles of cystic fibrosis patients and lumens of indwelling urinary catheters32,33. This holds major advantages compared to more traditional methods, such as those using 96-well plates29,34, as these often do not account for fluid flow. As fluid flow is a common characteristic in environments where bacterial infections frequently occur, such as lungs or urinary and blood catheters33,35, applying a constant shear stress makes our findings substantially more applicable. The continuous shear stress has also been found to influence bacterial adhesion and biofilm maturation, providing a more realistic and clinically relevant understanding of the growth and development process.

The protocol is adaptable, with some steps requiring modification depending on the bacterial strain or nutrient media used. For instance, in a similar set of experiments with a genetically modified, fluorescent P. aeruginosa strain PAO135, the channel seeding process was extended from 30 min to 1 h. This adjustment was necessary because the different nutrient media had varying components and quantities compared to those used in the original protocol, which altered the time required for successful cell adherence.

A limitation of this method is the relatively narrow hydrodynamic wall shear stress range (0−20 Pa) of the microfluidic system and the specific microchannel dimensions. These factors make the protocol unsuitable for replicating turbulent or transitional flow environments found in some natural or engineering settings, such as rivers or pipelines36,37, due to the system's low Reynolds number range. The liberty was also taken to equate the bronchioles of the lung, consisting of flowing mucus and air, to the hydrodynamic conditions used based on their respective flow rates. The thresholding procedure further presents a potential limitation. Random areas of shadow in a captured image, unrelated to the biofilm or fluidic channels, can be mistakenly identified as sections of biofilm by the software. This can result in biofilm percentage surface area coverage data that is artificially inflated. This relatively common imaging issue38, was mitigated by excluding any regions containing shadows from the analysis. Images where a shadow spanned the entirety of the viewing area were discarded. A more reliable solution to this issue would be to use fluorescent stains or genetically modified fluorescent bacteria, which would provide a clearer signal for the thresholding software. Additionally, during the experiment, a pocket of air within the flowing media could enter the microchannel and disrupt the continuous flow over the biofilm, making the findings unusable. The likelihood of this occurring was reduced by storing the medium at room temperature (approximately 20 °C), thereby ensuring that the difference between storage temperature and experiment temperature conditions was reduced, preventing the formation of air pockets.

While this procedure used specific nutrient media with the modified FAB and TSB media, which contain widely differing concentrations of glucose and P. aeruginosa strains, the versatility of the method extends to a range of research applications. The protocol can be easily adapted to study other bacterial genotypes, alternative hydrodynamic shear stress values, and different hydrodynamic conditions and nutrient media. This adaptability is critical for accurately replicating the unique flow environments found in specific clinical sites. This flexibility allows researchers to tailor the experimental setup to a wide variety of physiological niches, making the method a useful tool for developing targeted interventions and treatments for biofilm-related infections in diverse clinical contexts.

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors would like to thank the School of Mechanical, Aerospace and Civil Engineering (MAC) at the University of Sheffield for funding.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
(NH4)2SO4Acros Organics7783-20-2For modified FAB medium
48-well 0-2 Pa microchannel plate - glass coverslipCell Microsystems910-0047-5PackMicrochannels for media flow and biofilm
growth with real-time imaging
-80 °C FreezerThermo Fisher Scientific1.16057E+15To store glycerol stocks until further use
Agar, Bacteriological GradeAcros Organics443570010For Agar plates - culturing bacteria
Analytical BalanceVWRVWRI611-2297To measure components of media used
AutoclavePriorclave4304To sterilize glassware
BioFlux 1000HTI&L Biosystemshttps://il-biosystems.com/cell- microsystems/device/bioflux-1000ht-
shear-flow-system/ 		For Microchannel flow and biofilm growth experiments
BioFlux Control ModuleI&L Biosystemshttps://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 SoftwareI&L Biosystemshttps://il-
biosystems.com/app/uploads/2025/01/Bi oFlux_Overview_.pdf		To set up and monitor media flow and biofilm growth within the microchannels
CaCl2Fisher Scientific10043-52-4For modified FAB medium
CaSO4.2H2OChemCruzA1719For 1 L minimal metal solution
Clear Polystyrene 96-well PlateCorning, CostarCLS7007For viable cell count
CoSO4.7H2OSigma-Aldrich10026-24-1For 1 L minimal metal solution
CuSO4.5H2OHoneywell209198-100GFor 1 L minimal metal solution
Eppendorf Tubes 3810XEppendorf30125150For P. aeruginosa glycerol stocks
FeSO4.7H2OVWR International Ltd.284005EFor 1 L minimal metal solution
Fiji (ImageJ)National Institutes of Healthhttps://imagej.net/software/fiji/downloa
ds		Z-stack image processing
GlucoseSigma-Aldrich50-99-7For glucose modified FAB Medium
GlycerolSigma-Aldrich56-81-5For P. aeruginosa glycerol stocks
H3BO3Fisher Scientific10043-35-3For 1 L minimal metal solution
Hamamatsu Orca-Flash 4.0 Camera Model C11440-
42U		HamamatsuC11440-42UFor Microchannel flow and biofilm growth
experiment real-time imaging
KH2PO4Fisher BioReagents7778-77-04For modified FAB medium
Methylated-SpiritSigma-Aldrich2857To clean microbiology cabinet before and after
use
MgCl2Sigma-AldrichM8266-100GFor modified FAB medium
Microbiology Cabinet Class IIThermo Fisher Scientific42111226To carry-out protocol steps without
contaminating bacteria, broth or agar
Mini IncubatorLabnet Internationalsn03171014To thaw PAO1 and PA14 after -80 °C freezer and
for initial 24 h bacterial agar plate growth
MnSO4.H2OSigma-Aldrich10034-96-5For 1 L minimal metal solution
Na2HPO4.2H2OSigma-Aldrich10102-40-6For modified FAB medium
NaClSigma-AldrichS5886-500GFor modified FAB medium
NaMoO4.H2OSigma-Aldrich10102-40-6For 1 L minimal metal solution
Precision BalanceVWRVWRI611-2299To measure components of media used
Refrigerated CentrifugeHeraeus40289841Separating P. aeruginosa culture from
supernatant for glycerol stocks
Semi-Micro CuvettesAlpha LaboratoriesX72053For optical density measurements
Shaking IncubatorINFORS HThttps://infors- ht.com/en/products/incubator- shakers/multitron-standardFor overnight cultures
Sodium citrateSigma-Aldrich71498-250GFor Sodium citrate modified FAB medium
Sterile Plastic Inoculation LoopsMicrospec15782105For P. aeruginosa PAO1 and PA14 innoculation into agar plates and falcon tubes
Tryptic Soy Agar, vegitoneMillipore14432-500G-FFor Agar plates - culturing bacteria
Tryptic Soy BrothSigma-AldrichT8907Dehydrated culture media - 6 g/200 mL
Type 1 Water DispenserThermo Fisher Scientificsn42103311For distiled water
ZnSO4.7H2OFisher Scientific7446-20-0For 1 L minimal metal solution

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