The overall goal of this procedure is to characterize biofilm interactions with the surrounding environment using integrated methods to do this. A double inlet microfluidic flow cell system is constructed in which glucose containing and glucose free media are pumped across a flow cell generating a glucose gradient. Bacteria are added to the system and biofilms are allowed to develop over the course of several days.
The biofilms are then imaged by 3D confocal microscopy to characterize solu transport. Within biofilms, fluorescent tracers are injected into the flow system and time-lapse imaging is performed to characterize the flow field around biofilm colonies, fluorescent microbeads are injected and time-lapse imaging is performed. Solute transport and particle tracking analysis of the resulting data demonstrate the utility of this method in assessing biofilm processes under various chemical environments within a single device and in one set of experiments.
The main advantage of this methods is that they allow for the simultaneous assessment of multiple aspects of biofilm environment interactions. The methods reported here can help answer key questions about biofilm formation in natural environments or in biofilm related infections. Working in a laminar flow hood transfer a colony of posa PAO one GFP end a colony of e coli DH five alpha from an LB plate to separate tubes containing three milliliters of lb broth.
Incubate the cultures overnight at 37 degrees Celsius while shaking at 225 RPM. This flow system is based around a double inlet microfluidic cell that facilitates observation of biofilm growth under a well-defined chemical gradient formed by mixing two solutions within the flow chamber. During flow, a smooth concentration gradient is formed in the transverse direction.
As a result of diffusion, the concentration profile is steep near the inlet and more relaxed downstream. On the day of the experiment, carefully assemble the flow system. First, clamp the tubing into the peristaltic pump, then attach one end of the tubing to a medium bottle containing 900 milliliters of FAB medium with glucose, and the other end to a bubble trap.
Repeat this process to connect a second medium bottle containing 900 milliliters of fab without glucose to a second bubble trap. Next, insert three-way valves just before the flow cell inlets and connect each of the bubble traps to the flow cell. Finally, connect the flow cell outlet to a waste bottle.
Make sure that the flow cell is placed with the cover slip side down to allow suspended cells to settle onto the cover slip. After the flow system is assembled, turn on the peristaltic pump at a flow rate of 10 milliliters per hour for each inlet to fill the system With the medium fab medium is introduced to the two inlets with a glucose provided only in one inlet. Next, working in the hood, dilute the two bacterial cultures to a ratio of one to one in one milliliter of sterile water with an equivalent OD 600 of 0.01 for each bacterium to inoculate the flow cell, use a sterile plastic syringe to inject 500 microliters of the inoculum into each of the flow cell inlets via the three-way valve.
After the injection, stop the pump and allow the bacterial cells to attach to the cover glass for one hour after the cells have attached, set the pump to 0.03 milliliters per minute for each inlet and allow the system to flow for three days at room temperature during which a biofilm develops under a gradient of glucose exposure. After three days have passed. Use a marker and a ruler to draw a grid on the cover glass side of the flow cell in preparation for imaging.
This will aid in locating the imaging regions to counter stain for viewing e coli. First, turn out the lights to darken the work area as the counter stain is light sensitive. Use a syringe to slowly inject one milliliter of diluted cell permeant red fluorescent nucleic acid stain into the three-way valve upstream of the flow chamber.
Stop the flow. Keep the flow cell stagnant in the dark for 30 minutes. After 30 minutes have passed.
Resume the flow at 0.03 milliliters per minute and wash out the unbound stain for 15 minutes. Then stop the flow and observe the biofilms using confocal microscopy with a 63 x objective. Find a field of view containing a good amount of biomass with biofilm colonies that are well separated as shown here.
Then capture 3D image stacks in the appropriate channels to map the spatial patterns of biofilm development within the flow cell image, the biofilms at three or more longitudinal or X distances along the flow cell inlet, and at three transverse or Y positions relative to the imposed nutritional gradient. To characterize solu transport patterns within the biofilm, begin with the flow cell. Following three days of biofilm development, again, working in the dark.
Stop the pump to pause the flow, open the bubble traps to release the injection pressure. Then use a syringe to inject one milliliter of diluted sci-fi tracer solution into either of the three-way valves upstream of the flow cell. After injecting the sci-fi solution, close the bubble traps.
Adjust the three-way valve and restart the pump at 0.03 milliliters per minute to deliver the sci-fi solution into the flow cell. Next, switch the confocal imaging mode to XYT with a frame rate of 0.15 hertz. A high temporal resolution is preferred for verbalizing the dye penetration, however, fast scanning decreases the image quality.
So set the scanning speed and line average to balance the time resolution of imaging and the image quality. Once the imaging parameters are set, restart the pump to resume the flow. At a rate of 0.03 milliliters per minute sci five will be delivered into the flow cell simultaneously.
Initiate time-lapse confocal imaging. Once the time-lapse imaging is complete. Performed diffusion analysis according to the instructions in the accompanying document.
To characterize the flow field around biofilms, dilute the fluorescent one micrometer microbeads in sterilized water to a final solid concentration of 0.2%and a final volume of 0.5 milliliters. Then vortex to make sure that the microbeads are well dispersed, pause the flow and then inject and deliver the diluted fluorescent microbeads into the flow cell. As before, change the flow rate to 0.01 milliliters per hour.
The low flow rate allows for accurate tracking of the particle path. Switch the confocal settings to XYT mode and instruct the software to track particle movement in one Z slice. Capture time series images as before using a frame rate of one hertz.
Repeat the particle injection procedure and image at different Z locations. After completing the imaging, follow the instructions in the accompanying document to calculate flow vectors to study bacterial interactions in a biofilm under a glucose gradient. Posa GFP and E coli were differentiated in dual species biofilms through counter staining.
With cyto 62 images were acquired in each of the nine imaging regions on the flow chamber. In the overlays shown here, posa GFP appears green or yellow, and e coli appears red. Posa dominated biofilm biomass in regions with high glucose concentrations and e coli dominated in regions with low glucose concentrations.
Thus the two species occupied distinct ecological niches. These results demonstrate the utility of the double inlet microfluidic flow cell in studying biofilm development activity and interactions in complex environments. After watching this video, you should have a good understanding of how to use a double inlet micro fluidic flow cell to grow biofilms under chemical gradients and how to obtain environmental parameters for biofilm development using the injections of fluorescent particles or chasers After the development.
These methods paved the way for researchers in the fields of environmental and clinical microbiology to explore complex interactions between microbial communities and their surrounding environment.
