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AFM has been widely used in the life sciences since the application of optical lever detection by Hansma and co workers in the late ‘80s so it was no surprise that when I searched the literature I found a bunch of papers describing experiments on bacteria using AFM. In case you’re interested in doing something similar here’s an annotated bibliography with most of the papers I found [pdf]. As always, the list is incomplete—especially the section on bacterial adhesion since that’s not my primary interest right now even though it forms the bulk of the literature. Never the less, you’ll get a good survey of the field if you have a look at the papers in the list and dive into the web of citations.
A universal challenge in all of these studies is bacterial immobilization. Unlike animal cells, bacteria don’t adhere readily to glass so if you want to look at them with an AFM you have to find a way to stick them. The two main reasons for this are that collecting an AFM image takes a few minutes so you don’t want things rearranging on that time scale and that the tip itself perturbs the sample although this can be reduced by using tapping or even non-contact AFM. Probably the easiest method is to dry the sample and image in air but then you drastically change the cell physiology. Another possibility is fixation with gluteraldehyde but this also kills the cells.
Here’s what I did to collect this image.
First, I freshly cleaved some mica and added a solution of poly-L-lysine with a molecular weight around 3800 that I then dried in a stream of nitrogen. Then I added ~50 ul of e. coli in Luria broth to the surface and sandwiched them with a glass cover slip and waited for a few minutes. I removed the glass and imaged the sample. The image clearly has clumps of bacteria instead of single isolated cells, but the images were stable over more than an hour. I’ll try again sometime this week to see if the adhesion is reproducible and start trying some mechanical measurements.
So far, I think it looks pretty promising.
Konichiwa from Okinawa OIST workshop recap: Fluorescence and tweezers
Biocurious is written by Andre Brown and Philip Johnson, since 2005. Content of the weblog is licensed under a Creative Commons Attribution-Share Alike 3.0 License.
Sweet – simple and effective. Doesn’t academia prefer elegance?
One could derivatize the cleaved mica with a reactive silane nitrene precursor or a layer of polymeric nitrene precursor. Add bacteria, add blue light, tack them in place.
A polymer spin coat with sulfonyl fluoride, sulfonyl chloride, isocyanate, vinyl sulfone… pendant groups would chemically grab the bugs’ membranes.