For year one of the Trace grant, I will be focusing on the development of a standard operating procedure for the uSiM-CA. To begin this process, I am transferring all of my current operating procedures from the Andor dragonfly confocal scope to the new Etaluma microscope. The Etaluma scope will be automated, collecting images of…
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…
On our quest to optimize bacteria assays using µSiM-CAs, we recently discovered that higher initial bacterial ODs decrease transmission in positive controls, described here. This was something we did not initially anticipate, engendering a new hypothesis around the idea that less initial bacteria leads to more transmission across our membranes in the µSiM-CAs due to…
After titrating our pulldown volume to avoid pulling Staphylococcus aureus through our membranes, discussed here, we found that 10 µL withdrawals allow us to continue to use the pulldown modification without compromising our results by providing false positive transmissions. With newfound confidence in this pulldown modification, we took a step backwards to repeat a bacterial…
On our quest to improve the performance of bacterial assays that use the µSiM-CA by modifying our standardized protocol, our most recent modification has caused controversy. Described here, we recently decided to add a step to our protocol where we pull fluorescently tagged Staphylococcus aureus onto the membrane by withdrawing a bacteria filled solution which we load…
For the osteomyelitis project which I have been working on for quite some time now, my collaborators and I have been working solely with 0.5 µm microporous membranes. To ensure that every membrane we work with has pores of the right size and are defect free, the two following protocols were created. The first checks…
The last time I did live imaging on the confocal microscope was in December of 2019. Then I was imaging the capture of 1.24 µm diameter beads in 1 µm slit pore membranes to visualize the difference between steric and affinity based capture. This time around, all of the materials are different, but the process…
When we first began developing a standardized protocol for μSiM-CA bacterial assays (described here), bad assay performance led some to believe our issues were due to availability of pores within the membranes inside of the μSiMs. At the same time, I developed a visualization protocol to view bacterial transmission across the membrane (described here). While…
While imaging GFP tagged Staphylococcus aureus on our membranes within µSiM-CA devices, the question of whether or not we can differentiate between bacteria on the top and bottom of the membrane versus within the membrane has persisted. We know that during our experiments bacteria exist in all three of these places, therefore the question really is whether…
Since our attempt to standardize the Staphylococcus aureus µSiM-CA division assay, described here, we have moved towards investigating biological reasons as to why our assay performance is not where we want it to be. While biofilms have been suspect from the beginning, we have also thought that bacterial transmission requires two main steps: 1) Pore discovery…