The overall goal of this methodology is to show how to measure the real logical properties within the micro domains of heterogeneous flow cell and static biofilms. This method can help answer key questions on ology in biofilm field, such as how specific metrics component, different species or compound affect biofilm neurology and test its response to physical force such as shere. This is a non-destructive technique with high spatial resolution, and it can be conducted with equipment standard to biological laboratories.
After preparing overnight bacterial cultures and supplies and medium for flow cell setup, according to the text protocol, aliquot fluorescent microspheres in sterile water into micro centrifuge tubes to remove the sodium azide, spin down the tubes for five minutes under a sterile environment such as a flow hood, remove the supernat and use one milliliter of growth medium to re suspend the microspheres. Then add microspheres to the rest of the growth media for flow cell culture. Set up flow cell culture according to the text protocol for a static culture setup for biofilms grown in micro well chambers dilute overnight cultures 100 fold in micro centrifuge tubes by adding 10 microliters of overnight culture to one milliliter of growth.
Medium containing microspheres. Then use the diluted cultures to fill the microwell chambers and incubate at 37 degrees Celsius under static conditions. On days three and five, switch off flow from the peristaltic pump and bring the flow cell setup into the microscopy room.
Then clamp the tubing near the entrance and exit of the flow cell chambers to prevent drift from flow. Place the flow cell onto the stage of an upright microscope and with a 40 x oil objective and fluorescence microscopy, record the microsphere motion embedded in the biofilm at various locations such as micro colonies and flat undifferentiated layers for both temporal and spatial investigations. For events occurring within a short time scale record shorter videos with a faster frame rate for fast dynamics For longer events, capture longer videos with a slower frame rate.
For slower dynamics, save the videos in an appropriate format that can be read by Fiji image J, such as CZI or Tiff. Check the drift is not occurring. By scrolling through a finished video and confirming that the particles do not move in the same direction simultaneously, but that motion is random.
Remove the flow cell from the microscope stage and restart the peristaltic pump to continue cultivating the biofilm. To obtain particle tracking trajectories of the recorded videos, open Fiji and load a file Under the plugins menu, select tracking and trackmate. Check or adjust resolution and frame rate parameters in the initial calibration settings so that they match the video parameters To detect particles, select LOG detector and input particle diameter and threshold value.
For example, 1000. Check the preview button and scroll through the video to check that the purple circles Follow the particles throughout the video. Increase the threshold value.
If the purple circle appears in the empty space, reduce the threshold value. If not enough particles are detected, click the next button to complete the detection of particles. If the initial particle detection was satisfactory and there is no need to add or remove particles, click the next button For the next three windows, initial thresholding, select a view and select a filter without adjustment.
Next, in the option bar of the select A tracker method window, select simple LAP tracker. As particles rarely move out of focus in the biofilm and are easily tracked, use the suggested or low linking and gap closing max distance values and click.Next. Check that the particle tracking is satisfactory by scrolling through the video to see that particles follow the tracks drawn by trackmate and that there are no unwanted or missing tracks.
Various filters are provided to exclude particles based on their individual tracking results. If this is not required, go to the final window, select in action from the dropdown menu, select export tracks to XML file and click execute. Choose a folder and save the tracks in matlab.
Prepare to import the XML files of particle trajectories by selecting file set path and add the scripts folder available with the Fiji package to carry out particle trajectory analysis. Begin by downloading MSD analyzer. Extract the at msd analyzer folder and drop it into a folder that belongs to the MATLAB path.
Start MATLAB and initiate the analyzer by typing the following where micrometers and seconds are the physical space and time units in the video. Next, import the particle trajectories by typing the following where clip Z removes the Z dimension. For A 2D video and scale, T scales the units in the time dimension from frames to seconds and add the tracks to plot particle trajectories.
Input the following, compute the mean square displacement or MSDS of the particles using the commands shown here. To plot the MSDS of each particle, use figure and MA dot plot msd. Then combine particle trajectories from other videos of the same category.
By importing the trajectories using the import trajectory command, then plotting and computing msds as just demonstrated to plot the ensemble mean or the average overall curves. Type the following. Then change the Y and X axi from linear to logarithmic scale.
To remove regions of the curve with long lag times or that rise steeply from dynamic error, use the brush tool to select the end of the curve. And in the tools menu, select brushing. Remove unbred after installing easy FITT and restarting MATLAB with the easy menu in the figure window.
According to the text protocol, fit the MSD curves to the power law by selecting show fit power A times X to the N, where N is the estimated diffusive exponent alpha. Then to calculate new particle msds at other locations or time type clear to discard the current particle trajectories and msds. After calculating various MSD curves for the particles in various locations of different biofilm strains and at different time points, copy and paste curves into one graph.
For comparison, select the curve and go to tools to look at the data statistics such as the median range. The MSD of the bead is proportional to the creep compliance JFT of the material in which the bead is embedded according to the relation shown in this formula where J equals creep compliance. D equals particle diameter KB equals boatman's.
Constant uppercase T equals temperature and lowercase T equals time. In these experiments, viscoelastic properties of different biofilm regions of wild type P arosa and delta PE A mutant strains, including voids, planes, and micro colonies were investigated. The MSD of particles in the void was used as a control and was comparable to the MSD of particles in pure medium.
In contrast, particles trapped in the biofilm vibrated at fixed positions and MSD values ranged from those typical A viscoelastic materials to strongly elastic gels in biofilms formed by the wild type and delta Pele strains the MSD of particles. In the day three micro colonies was independent of time for time, legs of 0.1 seconds to 10 seconds indicating that the micro colonies were elastic. The median creep compliances calculated from the corresponding particle msds were 4.3 times 10 to the negative two pascals to the negative one and 3.6 times 10 to the negative two pascals to the negative one respectively.
By day five, the creep compliance of the micro colonies in the wild type strain increased to 2.5 times 10 to the negative one, pascals to the negative one indicating a reduction in effective cross-linking within the matrix Biofilms formed by the delta P-S-L-B-C-D mutant strain were less differentiated and delayed in development with thick planes that developed micro colonies after day three. From the MSD values, the biofilms were much less effectively cross-linked than the wild type and delta Pele strains. Sufficient stats are taken from micro domain when the analysis of more videos do not add any new information To the MSD curve.
It is important to conduct microbiology using a wide range of particles that differ in surface chemistry and sizes. Additional experiments may be added to the procedure to investigate health. Physical forces, such as sheer or chemical treatment impacts the biofilm radiology Microbiology extract structural information not easily obtained by other methods at the micrometer scale.
After watching this video, you should have a good understanding of how to track particles, analyze their msds, and obtain diffusive exponents of biofilms. Do not forget working with bacterial pathogen can be hazardous. All waste has to be autoclave and disposed properly.
