SEM analysis of Bacteria - (Sep/19/2012 )

Is it necessary to do fixation of bacteria which are attached to metal surface before SEM analysis? What is the purpose of fixation and what happened if do SEM analysis before fixation?

-Fiaq Khan-

Hi,

It is necessary* to fix the bacteria (or any cells for that matter) prior to viewing on TEM or SEM. As I'm not really familiar with SEM but the steps/principles running up to viewing is quite similar.

According to the protocol from the University of Oxford:

The main purpose of fixation is:
1) to cross-link cellular structures into a matrix so as to preserve the structure of the cells with the minimum of alteration from the living state (i.e. with no changes in morphology, volume, or spatial relationships, and with minimum loss of cellular constituents) and,
2) to protect and stabilize cellular structures from changes during subsequent treatments and from irradiation by the electron beam.

Fixation is the first and most important step in any EM study, since mistakes made at this point render the whole project useless.

So we see that it's got nothing to do with bacterial/cell attachment to a surface; rather, serving the purpose mentioned above. Only after fixation will the other steps (post-fixation/dehydration/further treatment/drying/coating) be done.
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* Unfixed samples are sometimes used negative staining. With unfixed specimens there is the potential problem of changes occurring due to osmotic shock (or to changes in ionic composition) since most negative stains are made up in distilled water. There are safety implications to be considered when examining unfixed bacterial or viral samples.

-Julio-Claudian-

Thanks a lot...

-Fiaq Khan-

I'm doing SEM training for microbial cells (by now I will do fungi but probably bacteria in future).

Fixation, basically, avoids structural changes of cells during dehydration and critical point drying (CPD) steps. One of the worst problems in SEM is the collapse of the cells. So, never let them dry until, CPD will do it.

Stainings like OsO4 are not routinely used anymore for SEM as they are not really necesary.

A basic protocol would be: fixation, washing fixative, dehydration in increasing concentrations of ethanol, CPD from absolute ethanol and coating/mounting.

For coating there are usually 2 alternatives depending if you want structural or compositional (EDS) information. For structure, Au or Au/Pd sputtering is the most common option. For composition, evaporative carbon coating is preferred as Au can hide some peaks with relevant information about composition, however images with C-coating are poor quality compared with Au coated samples

-El Crazy Xabi-