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The overall goal of the following experiment is to perform in C two measurements of the local physical properties of a bacterial biofilm at the micrometric scale. To do this, magnetic particles are introduced into a growing biofilm to serve as probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers are then used to exert a defined force on each particle.

In embedded in the biofilm film, the particle displacement induced by the magnetic pulling is monitored using video microscopy to derive the local viscoelastic parameters and provide the 3D spatial distribution of the mechanical properties of the biofilm. Results are obtained that show the mechanical heterogeneity of the e coli biofilm and gives clues as to which biofilm components support the biofilm physical properties. So the main advantage of this technique over existing methods like microscopy, cology, or whole bio vegetation, is that it reports the spatial distribution of the mechanical properties in situ without disturbing the original structure of the material.

To prepare magnetic particles for this protocol, vortex's stock vial of 2.8 micrometer particles to resuspend them. Then transfer 10 microliters to a micro centrifuge tube. Then to wash the particles, add 190 microliters of minimum medium and swirl the tube, place it on a magnetic sample rack, wait for the formation of a pellet and aspirate the supernat solution.

Repeat this wash step two more times and resuspend the beads in 190 microliters of minimum medium. Next, add 950 microliters of M 63 B one with 0.4%glucose to 50 microliters of the washed bead solution to adjust the particle concentration to approximately 5 million beads per milliliter. To prepare the channel, use a diamond glass cutter to cut two square bo silicate glass capillaries 10 centimeters long to obtain two eight centimeter long pieces.

Using the glass cutter, cut a glass slide in half. Then using a fast acting cyanoacrylate glue or super glue, attach the two capillary pieces to two glass slide pieces as shown in this diagram. Two centimeters apart with one centimeter overhang on either end autoclave the entire setup and the necessary tubing required.

For further channel connection, gather the following sterile materials under a lanar flow hood. The mounted channel tubing and connectors, two pediatric bubble filters or traps. Two clamps, 30 milliliter syringes filled with M 63 B, 1.4%glucose and the waste bottle.

Connect the two capillaries the same way. Using one of the tubes. Connect the waste bottle to one capillary end.

Then using lure lock connectors as junctions, connect the other end of the capillary to the homemade bubble trap or trap one and trap one to trap two. Then connect the 30 milliliter M 63 B one syringe to the trap two. Next to fill the setup with sterile M 63 B one with 0.4%glucose.

Install the syringe on the syringe pump and turn on the syringe pump at a rate of about five milliliters per hour higher than the experimental rates. Carefully track and eliminate all the bubbles in the circuit if needed. Disconnect and reconnect the circuit While attempting this procedure.

Be careful to remove any bubbles before and when Introducing the cell particle mixture into the tubing, it helps to place the trash above the capillaries, and this will help for the continuous growth of the biofilm. Allow the medium to flow through the system for 10 to 15 minutes. In the meantime, in a micro centrifuge tube, mix one milliliter of the bacterial suspension at optical density 0.5 with one milliliter of the washed bead solution prepared earlier.

Switch the flow off, reattach the tubing, and then close the clamps. Next, fill up a one milliliter syringe with the bacteria bead mixture and introduce it into the capillary. After the homemade bubble trap, transfer the apparatus to the microscope there.

Install the capillary on the microscope stage and place the waste container at a slightly higher level. Put the syringe pump on the countertop beside the microscope. Allow the apparatus to stand for 15 to 20 minutes to enable the bacteria to settle and attach to the surface of the capillary.

Once the bacteria have attached to the capillary, open the clamps and adjust the flow rate on the syringe pump and start the flow. Over the course of 24 to 48 hours, a biofilm will develop on the capillary surface, focus on the capillary bottom plane and start the time lapse recording of the sample images. Usually an acquisition frequency of two images per minute will adequately report the biofilm growth.

This movie shows the initial stage of the biofilm just after injection of the cell particle mixture. In the capillary acquisition, frequency is one image per second, and the movie is played at 10 frames per second. After four hours of growth, particles progressively detached from the surface to be distributed into the biofilm volume.

The blue arrows marked detachment events. Here, the acquisition frequency is two images per minute, and the movie is played at 15 frames per second. After 20 hours of growth, a dozen evenly distributed particles can be monitored in a focal plane.

This movie is taken in a middle plane, 20 micrometers over the capillary bottom. Two images were acquired per minute. The movie is played at 15 frames per second.

Once the biofilm seeded with magnetic particles is growing manually, screw the magnetic tweezers onto the microscope stage. To ensure the appropriate magnetic field gradients is generated in the observation zone, ensure that the tweezers are placed as shown in this diagram in which the measurement region shown in red is located 300 micrometers from the edges of the left right poles of the tweezers along the Y axis. The bottom of the capillary is placed 200 micrometers below the bottom of the tweezers.

To achieve this step, use the XY movement control of the microscope stage to bring the edge of the left hand magnetic pole and the left hand edge of the capillary. In the same observation field, adjust the position of the pole at 300 micrometers from the edge of the capillary using the x dial of the left hand microm manipulator of the tweezers. Do the same operation on the right side.

Then adjust the tweezers position along the Z axis with respect to the bottom of the capillary. First, align the bottom of the poles with the bottom of the capillary and shift the poles. 200 micrometers up.

Using the Z dial of the micro manipulator, set the parameters of the function generator to 24 seconds of zero signal and 16 seconds of four amps. Direct current with a trigger. Send to the bright field light shutter after 20 seconds.

For signal synchronization, connect the tweezers to the function generator via the power amplifier. Pay attention to properly connect the poles in series to acquire spatially distributed creep curves. Take a spatial origin, take the origin of the Z axis at the inside bottom of the capillary.

Make note of the microscope Z control position. Then locate the intersection of the x and Y axis, which are defined by the edge of the capillary and the edge of the pole piece respectively. This is the origin of analysis.

Make note of the coordinates. All coordinates will be taken with respect to this origin in the following. Once the biofilm has grown, using the fine focus control knob, adjust the vertical position of the capillary so that the first plane will be located four to seven micrometers above the capillary bottom.

This slice corresponds to the biofilm slice in which the origin of the spatial referential is located at the top left hand corner. Then switch on the current generator and manually trigger the image acquisition sequence. To initiate the 42nd sequence of events, the image stream acquired here corresponds to a given plane and XY field.

After the sequence has been captured, move the capillary to capture a video of the new field. Continue repeating this process to obtain all of the required videos to analyze the data. Open the images in image J or an equivalent particle tracking software for all of the stacks acquired in all the particles.

Convert the images to text files by tracking particle displacement. Next, using a data treatment software, draw the displacement curves and convert the displacement curves to compliance curves in which the total compliance of the material JFT is calculated as a function of time according to the compliance formula shown here, This formula describes the relationship between the applied stress and the material strain for a historical particle of reds, or embedded in the viscoelastic and uncompressible medium as it was previously established, and his coworkers From the adjustment of the creep compliance curve to the general burgers model for viscoelastic materials derive the viscoelastic parameters J zero J one at the zero at the one prime to provide the spatial distribution of the viscoelastic Properties of the biofilm creep curves were collected at different depths in a biofilm micro volume of approximately 200 by 200 by 40 cubic micrometers. Typical results are shown here.

The values of J zero, the elastic compliance are given as a function of the Z axis along the depth and y axis along a lateral dimension of the biofilm. Each point corresponds to a bead for which creep curve analysis has provided a J zero value. The data reveals that local compliance varies along the depth of the biofilm over almost three orders of magnitude.

Also, strong lateral heterogeneity took place at all biofilm heights. The data also provides the distribution of the compliance values, which exhibit a widespread and asymmetric shape. Comparing these results with the ones previously obtained on other biological polymers suggests that the mechanical properties of the biofilm are supported by highly cross-linked polymer gels.

In C two measurements of biofilm, local properties were also performed under different environmental conditions, such as lower flow rate as can be seen here. The compliance values of a biofilm grown at 0.1 milliliter per hour still exhibit a highly heterogeneous mechanical profile. But the clear Z depth dependence of the biofilm rigidity observed in the biofilm grown at one milliliter per hour has disappeared.

These results demonstrate the significant impact of the external conditions on the biofilm organization Watching the video. You should have a good understanding of how to map the Visco properties of the Biofilm Institute under fully controlled growth conditions.