Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

This method can help answer key questions, about biofilm development, such as how to identify key environmental conditions, that affect biofilm growth and behavioral characteristics during development. The main advantage of this technique, is that the multi-channeled, micro-fluidic plates, allow for the quick acquisition of statistically significant results. Though this method can provide insight into biofilm structure, it can also be applied to the study of antibiotic treatments, and bioremediation.

Generally, individuals new to this method will struggle, because careful attention to the details of the protocol and meticulous skills, are needed. To avoid an error of the connection of the instrument to the software, turn on the PC work station first, followed by the fluorescence module. Make sure the fluorescence shutter is on, and turn on the hardware controllers.

Next, turn on the imaging system controller and the CCD camera. Then turn on the imaging station. When all of the equipment is ready, start the control application, and enter the plate number, located on the label on the side of the plate.

For priming, first remove the 48 well micro-fluidic plate from the packaging, without touching the glass surface at the bottom of the plate, and clean the glass slide at the plate bottom, with a lens tissue. To prime the micro-fluidic channels, pipette 200 microliters of 37 degrees celsius minimal medium into the output well, taking care to avoid bubbles. Then place the plate onto the plate stage.

Wipe the interface with ethanol, sealing it onto the plate stage once the ethanol has dried. In manual mode on the control module, set the fluid as Luria-Bertani broth at 37 degrees celsius, and max sheer as five dym per square centimeter. Click the output wells to activate the flow from the output to the input well, to prime the channels.

After five minutes, pause the flow to prepare for seeding, and carefully remove the plate from the stage. Then, aspirate any residual medium from the output well without removing any medium from the inner circle that leads to the micro-fluidic channels. To seed the experimental channels, add 300 microliters of minimal medium into the input well, followed by the addition of 300 microliters of bacterial culture, into the output well.

Return the plate to the imaging stage making sure to wipe the interface with ethanol before placing it onto the plate, and use the live camera feed to focus on a single channel. While visually monitoring the live feed, resume the flow at 1 to 2 dym per square centimeter for approximately 2 to 4 seconds, To allow the cells to enter the experimental channel but not the serpentine channels, and leave the plate on the temperature controlled stage for one hour, to allow the cells to attach. At the end of the attachment period, carefully remove the plate from the stage and aspirate the bacteria from the output well without disturbing the channel.

Then, use a new pipette tip to remove the medium from the input wells. In the software, open multiple dimensional acquisition to control the microscope image acquisition, and select time lapse, multiple stage positions, and multiple wavelengths. In the saving tab, create a simple base name, making sure that incroments base name if file exists, is checked.

Include any essential details of the experiment in the description. Click select directory, to select the folder in which all of the files will be saved Under the time lapse tab, adjust the duration of the experimental time, to 24 hours, and set the time interval to acquire images every five minutes throughout the experiment. Use the live camera feed and the 10x objective to set the stage positions, focusing on the center of the channel, located above or below the channel numbers engraved on the plate.

Switch to the 20x objective, and locate the optimal viewing area and focal plane within the channel. Then, add the stage position to the list with the new settings. Under the wavelengths menu, set the number of wavelengths to 3, and set the first wavelength to FITC 100%camera, with a ten millisecond exposure time, the second wavelength to brightfield 50%camera 50%visible, with a minimal exposure time of 3 milliseconds and the third wavelength to all closed, so no light remains on the last channel between the acquisition times.

In the edit AutoRun menu, open the protocol setup tab and set a new protocol with a 24 hour duration with the flow set in the forward direction at the appropriate experimental shear rate. Click add and save as to save the protocol and open the sequence setup tab. To set up a new sequence, select Luria-Bertani broth at 37 degrees, as the default fluid for all of the channels.

Under step iteration 1 for channels 1 through 12, select the protocol with the first experimental shear rate and enable all of the channels. For channels 13 through 24, select the protocol with the second experimental shear rate and enable all of the channels. Then select Apply and Save As, to save the sequence and open the AutoRun menu to select the saved sequence to be used for the AutoRun.

To set up the timed growth biofilm experiment, add up to 1300 microliters of sterile minimal medium into the input well of the micro-fluidic plate, and return the plate to the imaging stage. Then, wipe the interface with ethanol, and seal the plate. Confirm that the protocols and sequences are set up correctly.

Select start, to start the AutoRun, and immediately click acquire to start the microscope image collection. At the end of the first image acquisition, click pause and select the live image mode in the brightfield wavelength. Select go to to view each stage position, and select set to current, to set the new settings.

Then, click resume, before beginning the next scheduled acquisition. In this representative timed biofilm growth experiment, the biofilm coverage, or percent threshold surface area, was different for all three shear settings, indicating that the shear had a direct influence on the biofilm surface coverage. The total relative measurement of the biofilm accumulation, increased as a function of time, with the growth rate declining from the highest shear stress to the lowest shear stress.

Under each condition, there was also a clear period of exponential growth from which a quantitative growth rate could be calculated. Overall, the roughness coefficient decreased over time under all shear conditions, indicating that all of the biofilm surfaces became smoother. However, compared to the lowest shear, the higher shear settings resulted in a smoother surface over time, indicating that a faster shear flow contributes to a smoother and more even surface.

Further, the textural entropy, or the randomness in the morphology, increased over time for all of the shear conditions. While performing this procedure it's important to remember to follow the specified sequence and to take the time to perform each step precisely. The needed skills can take a few runs to master.

Following this procedure, other methods such as HBLC or GCMS, can be performed to answer additional questions about the concentrations of substraights, such as glucose, being consumed. After it's development, this technique paved the way for researchers in the field of biofilms to explore general flow environments in real time with controlled experimental conditions and hydrotherapy sampling. Don't forget, that working with BSL2 bacterial strains can be extremely hazardous, and that precautions such as wearing proper protective equipment and using appropriate waste protocols should always be taken while performing this procedure.