I am trying to prepare Mycobacterium tuberculosis for SEM analysis.
This is my current method, basically summarized:
Fix cells in buffer + formaldehyde and glutaraldehyde, rinse in buffer, fix in OsO4, rinse in buffer, dehydrate in EtOH series, dry using HMDS. I use HMDS since I don't have access to criticism point drying.
I have tried capturing the cells using 13 mm, 0.2 um membrane filters (Merck Millipore isopore GTTP, polycarbonate). At the end of the process, the membrane filters are brittle and break when I try to remove from the holders, furthermore, after carbon casting and viewing, there is a weird background. It almost looks like the membrane filter has dissolved.
Could I try fixing the cells to slides that have poly - l - lysine? After fixation (1 hour) on the slide, could I continue with the normal SEM prep? Will my cells adhere enough not to be dislodged during EtOH and HMDS processing?
Can someone suggest any other method?
Thanks in advance.
http://onlinelibrary.wiley.com/doi/10.1016/j.femsle.2004.09.004/pdf
Electron microscopy analysis of Mycobacterium tuberculosis
cell division
Hi there. Try only with cold glutaraldehyde (3%). and with acetone instead ETOH, starting with 25% (next 50, 70, 90 and x3-100). but only on glass not plastic or filters. wash the acetone gradients with centrifugation (only bacteria).
We don´t use fixation in OsO4. and use critical point drying ...
I think that you could try fixing the cells to slides that have poly - L - lysine and after fixation on the slides, you could continue with the normal SEM preparation. I am sure that your cells will adhere enough not to be dislodged during EtOH processing.
I am attaching a paper to the message in which I have applied that procedure for Trypanosoma. evansi
Good luck
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