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…
Author: Michael Klaczko
He gives his blood to science.
To Pulldown or Not to Pulldown, That is the Question: Testing the Utility of a Pulldown in µSiM-CA Bacteria Assays
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…
Posted in NRG
Less is More: Lower Initial Bacteria OD Increases Transmission in uSiM-CA Bacteria Assays
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…
Posted in NRG
A Serendipitous Disaster of Interesting Proportions: High OD Staphylococcus aureus Pulldown on 0.5 µm Microporous Membranes
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…
Posted in NRG
The Big Squeeze: Unintentionally Pulling Staphylococcus aureus Through 0.5 µm Microporous Membranes
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…
Posted in NRG
Check Yourself Before You Wreck Yourself: Quality Control of 0.5 µm Microporous Membranes
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…
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Ghosts of Christmas Past: Live Imaging Bead Capture on 0.5 μm Microporous Membranes
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…
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Innocent Until Proven Guilty: Evaluating Pore Patency Across 0.5 μm Microporous Membranes for use in μSiM-CA Bacterial Assays
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,…
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Spot the Difference: Testing the Spatial Resolution of the Andor Dragonfly Confocal Microscope Using GFP Tagged Staphylococcus aureus and Nonporous Membranes
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…
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Staph Discipline: Pulling Staphylococcus aureus onto the Membrane to Encourage Timely Transmission
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…
Posted in NRG
