Hi Ruby,

I would fix the cells in suspension in 3% glutaraldehyde in 0.1M phosphate buffer pH 7.2 for a minimum of 1 h. After 3 washes in buffer ( pellet cells in between washes) postfix in 1% osmium tetroxide (in H20) for 1 h and dehydrate cells in 30%, 50%, 75%, 90% and 2 x 100%, 5 min each (spin down in between). Filter cells onto a 0.2 micron black polycarbonate filter (size exclusion depending on cell size). I use a vacuum pump to suck the solution of cells in 100% ethanol through the filter. Make sure to prepare critical point drying boat filled with 100% ethanol beforehand to avoid drying out the sample. Then critical point dry pieces of the filter for a minimum of 1 hr, mount filter onto SEM stub and sputter-coat (I use 10 nm gold/palladium).

Hope that helps to give you a rough idea what should work for most bacteria. If you want to preserve the structure closer to the "native" state then I would choose cryo-SEM.

Hi Ruby,

I would fix the cells in suspension in 3% glutaraldehyde in 0.1M phosphate buffer pH 7.2 for a minimum of 1 h. After 3 washes in buffer ( pellet cells in between washes) postfix in 1% osmium tetroxide (in H20) for 1 h and dehydrate cells in 30%, 50%, 75%, 90% and 2 x 100%, 5 min each (spin down in between). Filter cells onto a 0.2 micron black polycarbonate filter (size exclusion depending on cell size). I use a vacuum pump to suck the solution of cells in 100% ethanol through the filter. Make sure to prepare critical point drying boat filled with 100% ethanol beforehand to avoid drying out the sample. Then critical point dry pieces of the filter for a minimum of 1 hr, mount filter onto SEM stub and sputter-coat (I use 10 nm gold/palladium).

Hope that helps to give you a rough idea what should work for most bacteria. If you want to preserve the structure closer to the "native" state then I would choose cryo-SEM.

Thanks a lot Christian Hacker Sir.

The idea of cryo-SEM is really good. If it is not available you can use low energy up to  3kV. It can work. The objective is not to heat up the bacteria. That will deform the bacteria. 

We tried once to see bacteria with SEM. We took a very dilute suspension of bacteria (from culture solution). Added 1 drop on a substrate, added 1 drop of very dilute glutaraldehyde, dried. Then used it in SEM, analyzed at 3kV. It worked. Long time exposure in SEM sometimes caused loss of flagella. Then we tried to analyze it fast. It worked. 

Fix with HCHO vapor in a closed container that holds Petri Dish or fresh smear on a slide in a Coplin jar.  Small amount of 4-10% HCHO (from paraformaldehyde) in the bottom via dropper of pipette.  1 hour for thin distribution of bacteria or algae.  Coating with thin coat of Au/Pd in a sputter coater works just fine.  Or one can view organisms on a cold stage in a vacuum chamber with pressure regulated with HOH vapor ("ESEM").

Taking into account the recommendations of Dr. Christian Hacker, I understand that the step of dehydration in nucleopore filter is required in order to immobilize cells for the critical point step. I would like to ask you if I could avoid this step and use air drying of bacteria in order to identify inclusion bodies.