The idea of adding a pulldown modification to our bacteria assay protocol came about after we started to believe that pore discovery was our rate limiting step of the transmission process, discussed here. Initially, it seemed to benefit our assay as it bumped our performance up significantly. Upon closer inspection we realized that we had been given some false hope as pulldowns of volumes greater than 10 μL had the tendency to physically pull bacteria through our membrane, discussed here. This left us uncertain of the utility of the pulldown modification, especially after we saw 100% transmission once we lowered our initial bacteria OD, discussed here. I set out to test this utility using two different pulldown volumes at an initial bacterial OD of 0.1, discussed below.

50 µL Pulldown at 0.1 OD

The first pulldown modification that I ever attempted used a 50 µL pulldown volume, discussed here. While it pulled bacteria through the membrane when I first used it, I was unsure if it would pull bacteria through the membrane at a lower OD, so I decided to test it out using a similar protocol as was outlined in the same post, described below.

Preparing the µSiM-CAs for Testing and Loading them with Bacteria

1. Ahead of time, prepare Tryptic Soy Broth (TSB) by adding 30 g of BBL Trypticase Soy Broth to 1 L of purified water
2. Mix thoroughly and warm gently until powder is completely dissolved
3. Autoclave at 121°C for 15 minutes
4. Add 250 µl of USA300 glycerol stock into 50 ml of TSB (in a 500 ml flask)
5. Add 50 uL Chloramphenicol (CLM) antibiotic to the same flask
6. Put the culture in a shaker and let grow at 37°C while shaking until it measures an OD of ~0.1
7. While the culture is growing, thoroughly examine each µSiM-CA device you will use for any defects such as improper sealing between components or broken membranes
8. Prepare petri dishes to hold µSiM-CA devices by placing 2 Kim Wipes moistened with sterile dH₂O along the perimeter of each petri dish
9. Separate and label µSiM-CA devices into two groups: the first used for withdrawing the bacteria onto and potentially through the membrane and the second to display any bacteria that is pulled through the first group of devices
10. Pair the devices so one device from each group corresponds to another in the second group
11. Pipette ~15 µL of TSB media by inserting the pipette tip into one of the two open ports of the µSiM-CA and depressing the plunger of the pipette; the media should flow from this port through the bottom channel and out the opposite, open port. Remove the pipette tip before releasing the plunger of the pipette to avoid sucking injected fluid back out of the bottom channel of the device
12. With the bottom channel wet, block one of the open ports using a 3M double-sided tape sticker
13. Fill the well of the µSiM-CA by pipetting 100 µL of fresh TSB into it; care is taken to not create air bubbles and/or remove them by by withdrawing injected media and injecting it again until no air bubbles are visible
14. In the same manner as above, pipette ~40 µL of TSB media into the open port and look to see the well fill with little resistance. If resistance is appreciable, discard the device and prepare a new one
15. Remove all liquid from the well (~140 µL), then add 15 µL of TSB into the open port
16. Add 35 µL of TSB media to the wells of all of the devices in group 2 so that their total well volume is 50 µL
17. Next, add 100 µL of the desired OD subculture to the well of each device in group 1 (the devices used for withdrawal), withdrawing and injecting the subculture again if air bubbles are present until they are gone
18. Withdraw 50 µL of fluid from the open port of the devices in group 1, sucking fluid from the well through the membrane, into the bottom channel and out the port
19. Immediately take the withdrawn media from the group 1 device and inject this into the well of the corresponding device in group 2
20. Replenish the well volume of group 1 devices to 100 µL by pipetting 35 µL of TSB directly into the well
21. Allow the all devices to incubate at 37°C for 6 hours

After loading, I imaged the group 1 devices at different time points up to 6 hours using confocal imaging and the method described below. Using the same method below, I also imaged the group 2 devices after 6 hours of incubating in order to allow any bacteria that may have been pulled through the membrane the chance to divide substantially, increasing our odds of seeing them even if only a handful of bacteria is pulled through.

Confocal Imaging µSIM-CA Devices Containing GFP Tagged Staphylococcus aureus 

1. PDMS blocks (~5 mm thick) were laid on top of loaded µSiM-CA devices in order to allow them to be imaged in the microscopy core while maintaining gas permeance
2. Any liquid pushed out to the sides of the device is wiped away with a Kim Wipe
3. To create an adequate seal, the interface between the PDMS bottom and the acrylic top of the device was sealed with nail polish
4. Upon sealing, devices were stored in petri dishes filled with wet Kim Wipes for transport over to river campus
5. Once at the microscopy core, images were taken with the 488 nm laser was used with the 525 nm filter
6. While imaging, devices were stored in an incubator stage attached the confocal microscope which is held at 37°C
7. In between imaging sessions, devices were stored in an oven held at 37°C containing two 1 L beakers of DI water

Results

By the end of our assay, bacteria was in the bottom channel of all ten devices that we used in this experiment. Transmission over time can be seen in Figure 1 below, reminiscent of similar plots in our previous experiments.

While the transmission was great, we also saw that we pulled bacteria through two of the membranes again. This can be seen in the last column of Figure 2 below along with all of the other images I took of each device at our three time points.

Seeing that we pulled bacteria through our membrane using a 50 µL pulldown once again, we realized that our pull through issue was in fact due to the volume that we were using and not the initial concentration of bacteria. To correct this, we tried the same experiment but this time using a 10 µL pulldown which we had already verified did not pull bacteria through the membrane, discussed here.

10 uL Pulldown at 0.1 OD

This experiment followed essentially the same protocol as above, with a few changes highlighted in red below.

Preparing the µSiM-CAs for Testing and Loading them with Bacteria

*Protocol changes shown in red

1. Ahead of time, prepare Tryptic Soy Broth (TSB) by adding 30 g of BBL Trypticase Soy Broth to 1 L of purified water
2. Mix thoroughly and warm gently until powder is completely dissolved
3. Autoclave at 121°C for 15 minutes
4. Add 250 µl of USA300 glycerol stock into 50 ml of TSB (in a 500 ml flask)
5. Add 50 uL Chloramphenicol (CLM) antibiotic to the same flask
6. Put the culture in a shaker and let grow at 37°C while shaking until it measures an OD of ~0.1
7. While the culture is growing, thoroughly examine each µSiM-CA device you will use for any defects such as improper sealing between components or broken membranes
8. Prepare petri dishes to hold µSiM-CA devices by placing 2 Kim Wipes moistened with sterile dH₂O along the perimeter of each petri dish
9. Separate and label µSiM-CA devices into two groups: the first used for withdrawing the bacteria onto and potentially through the membrane and the second to display any bacteria that is pulled through the first group of devices
10. Pair the devices so one device from each group corresponds to another in the second group
11. Pipette ~15 µL of TSB media by inserting the pipette tip into one of the two open ports of the µSiM-CA and depressing the plunger of the pipette; the media should flow from this port through the bottom channel and out the opposite, open port. Remove the pipette tip before releasing the plunger of the pipette to avoid sucking injected fluid back out of the bottom channel of the device
12. With the bottom channel wet, block one of the open ports using a 3M double-sided tape sticker
13. Fill the well of the µSiM-CA by pipetting 100 µL of fresh TSB into it; care is taken to not create air bubbles and/or remove them by by withdrawing injected media and injecting it again until no air bubbles are visible
14. In the same manner as above, pipette ~40 µL of TSB media into the open port and look to see the well fill with little resistance. If resistance is appreciable, discard the device and prepare a new one
15. Remove all liquid from the well (~140 µL), then add 15 µL of TSB into the open port
16. Add 75 µL of TSB media to the wells of all of the devices in group 2 so that their total well volume is 90 µL
17. Next, add 100 µL of the desired OD subculture to the well of each device in group 1 (the devices used for withdrawal), withdrawing and injecting the subculture again if air bubbles are present until they are gone
18. Withdraw 10 µL of fluid from the open port of the devices in group 1, sucking fluid from the well through the membrane, into the bottom channel and out the port
19. Immediately take the withdrawn media from the group 1 device and inject this into the well of the corresponding device in group 2
20. Replenish the well volume of group 1 devices to 100 µL by pipetting 35 µL of TSB directly into the well
21. Allow the all devices to incubate at 37°C for 6 hours

After loading, I imaged the group 1 devices at different time points up to 6 hours using confocal imaging and the method described above. Using the same method above, I also imaged the group 2 devices after 6 hours of incubating in order to allow any bacteria that may have been pulled through the membrane the chance to divide substantially, increasing our odds of seeing them even if only a handful of bacteria is pulled through.

Results

We again saw great transmission in these devices. After 6 hours all nine devices had bacteria in their bottom channels. This, along with the number of devices that had bacteria in their bottom channels after 3 hours is shown in Figure 3 below.

Fortunately this time we did not pull any bacteria through the membrane. Not only did this mean we could continue to use the pulldown modification if it was found to be useful, but it also strengthened our belief that a pulldown using 10 µL or less volume did not pull bacteria through the membrane. The images within each device at both time points are shown in Figure 4 below along with the images of the pull though membranes.

At the 6 hour time point, I imaged each device using a 20X objective thinking the lower magnification would give me a better representation of bacterial transmission across a larger area of the bottom channel in one image. While this was the case, it also made finding bacteria very challenging, especially when there were only a handful of bacteria in the bottom channel of some of the devices. As a result, I will avoid using the 20X objective in the future for this purpose. This being said, it did give me fantastic images of biofilms which had formed on the top of the membranes in two of the devices, shown in Figure 5 below.

Conclusions and Future Directions

Overall, we did get fantastic performance out of the pulldown modification and found that the 10 µL pulldown is usable going forward. Our only issue is we had the same amount of success at the same OD (0.1) without using the pulldown modification, albeit not as quickly (shown here). Therefore, we were still left wondering if the pulldown modification actually gave us an advantage in our assays. Since there was seemingly no use for the pulldown modification at lower ODs, we wondered if it could still be useful at higher ODs where biofilm formation happens more rapidly. This is what we set out to test next.