Preparation of bacterial cells for SEM and TEM - (Aug/02/2012 )

Hey Guys,

Hopefully someone may be able to help me.

I am visualising my bacterial cells with a scanning electron microscope (SEM) and a transmission electron microscope (TEM). I am not carrying out the imaging myself (as my lab does not have an EM ) but I need to fix the samples prior to transport.

I will be fixing the cells in 3% gluteraldehyde buffer (equal volumes of buffer to cell suspension), however I am unsure what concentration of cells are suitable for TEM, SEM. 0.5 McFarlands, 1McFarland....4McFarlands????

If the sample is too turbid I doubt I will see individual cells....however if it's too dilute we will see no cells!

Does anybody know if there's a guideline to cell concentration prior to SEM and TEM??

Thanks in advance for your help!

Susan

Hi Susan,

I've done a fair share of fixing, cutting, and viewing for TEM so this will be based on that. The 'standard' preparation established by the varsity's EM unit requires that the amount of pellet covers the 100uL of a microcentrifuge tubes. I've worked with a pellet amount many times more but they all get fixed properly (in phosphate-buffered McDowell-Trump and then osmium tetroxide).

Given how dense the sample was during the fixing stage, I'd still see individual cells under the EM. The most important thing (for me) to keep in mind was 1) completely resuspend pellet in McDowell-Trump and 2) ensure complete resuspension in osmium tetroxide (seen as change in color dark brown to black).

Do check with the place you're sending your samples to

Thank you for your help!!