Preparing biological samples for electron microscope

[Steve Drake]

Hi Guys,

Ive been doing some work with E. Coli recently and I would like to image them under a scanning electron microscope. There are lots of papers with lots of different methods to prepare them for such a feat... but has anyone here actually done it and gotten a decent image?

If so what method did you use? I have tried a few things but a lot of the methods seem too complex or require some nasty chemicals which id rather not have to order if possible.

Thanks

 

[basalt]

It depends on what kind of images you need and what type of SEM. If it's an environmental sem you may not need any major sample prep as they're designed to run with wet sample. Otherwise your cells will need dehydrating which means fixing them first or they'll shrivel up, they then need a metal shadowing to allow among other things charge dissipation.

There are plenty of stunning images of coli and other bugs out there so it clearly works but it depends on what kind of image you want. I'd turn to SEM for images of groups of cells together but if you wanted details of isolated cells showing flagelli and pilli I'd turn to TEM and do a simple negative stain which requires minimal sample prep.

 

[Steve Drake]

It depends on what kind of images you need and what type of SEM. If it's an environmental sem you may not need any major sample prep as they're designed to run with wet sample. Otherwise your cells will need dehydrating which means fixing them first or they'll shrivel up, they then need a metal shadowing to allow among other things charge dissipation.

We have all kinds of SEM (ESEM too) and TEM, right now I just want a close up of the cells, to see its shape and body. Flagella would be a plus but I don't know much about SEM or TEM prep... looking for some scientific journals to maybe copy their system but so far they are all quite complex.

There are plenty of stunning images of coli and other bugs out there so it clearly works but it depends on what kind of image you want. I'd turn to SEM for images of groups of cells together but if you wanted details of isolated cells showing flagelli and pilli I'd turn to TEM and do a simple negative stain which requires minimal sample prep.

yeah, i tried putting a liquid suspension and dry it out then image that but i couldn't see any, as you say they were probably all disintegrated... what do you steps of a negative stain comprise of?

Thanks!

 

[basalt]

A neg stain for Tem is about as simple as it gets - I spot a suspension of cells ( 10 microlitres ) onto an EM grid, leave for one minute and blot. Quickly dip into water and immediately blot ( removes much of the salt present in LB media ), then spot on the stain and blot after one minute.

For stains we have a selection - first choice is usually uranium acetate ( cheap, effective and easy to prepare ), we also use salts of phosphotungstic acid or ammonium molybdate.

Cells do come out a little shriveled as they've been dried and shoved in a vacuum but detail is preserved and they can be fixed with glutaraldehyde or treated with trehalose to improve the appearance.

Dunno if I'm allowed to pimp my own site but you'll find some images here:

http://www2.warwick.ac.uk/fac/sci/lifesci/facilities/imaging/schoolsinfo/

The animated version has a few more pictures than the web version.

cheers
Ian

 

[Ygggdrasil]

If you just want to see the shape and body, why can't you use light microscopy (dark field or DIC should give you nice images)? From what I've heard, fixation and dehydration of bacteria can be fairly difficult to do without damaging the sample (more difficult than with eukaryotic cells) .

 

[basalt]

Size basically - a stand light microscope, even a really good one can't give you much more than basic shape - we're dealing with submicron particles, you can distinguish comma shaped vibrioforms from rod shaped bacilli or spherical cocci but you can't get detail.
Our confocals can give a bit more info but to see structures such as pilli and flagella - bacterial anatomy you need something that can see structures in the tens of nanometers.

 

[Steve Drake]

Size basically - a stand light microscope, even a really good one can't give you much more than basic shape - we're dealing with submicron particles, you can distinguish comma shaped vibrioforms from rod shaped bacilli or spherical cocci but you can't get detail.
Our confocals can give a bit more info but to see structures such as pilli and flagella - bacterial anatomy you need something that can see structures in the tens of nanometers.

thanks basalt. Yes I use optical microscopy but that's just to see the bulk motions of them swimming. Even at 100x oil its hard to make out their shape over the diffraction patterns.

Basalt, you sound like you do exactly what I am after. Do you by any chance have a step by step guide as to how you prepare them? I looked around your website but could not find one. My background is in physics so when I read papers as to how people prepare them, the methods are another language to me. I need a guide written for an idiot that goes through everything in some decent detail.

Thanks!