The most important thing to get right is the sample preparation. The papers given above will help you. After that the imaging is simple.

It really does depend a lot on your microscope - tell us what type of EM you have.

To try and answer your questions:

acceleration voltage - EM dependent, if you have FEG-SEM you can work at 1 or 2kV (or lower) this will give you more surface sensitive information. Higher voltage will tend to penetrate further into the sample and you get less surface information and more 'transparency' effect. If you are on W gun you will need higher kV. If you can get down to 5kV, that wil be ok.

working distance - this is a trade off between depth of focus and resolution. Short WD = higher resolution/small depth of focus, Long WD = large depth of focus/poor resolution. So it depends on how topographic your sample is and at what resolution you require for the images. I generally choose the shortest WD that allows a full focus on all the specimen. This will also depend on your detector configuration as varying the WD will affect the detector efficiency.

probe current - the trade-off here is with image brightness/resolution. Large probe current = high brighteness (less image noise) / low resolution. Small probe current = high resolution / lower brightenss (more image noise). For smaller probe currents you will need to set longer scanning times to clean up the noise. So select a probe current that gives you acceptable resolution and with a level of noise that can be cleaned by slow scanning.

filament current - instrument dependent, best leave this on the default.

aperture diameter - instrument dependent, but smaller aperture will give you higher resolution and less signal (more noise). Larger aperture  = more signal (less loise) and lower resolution.

Remember it is not always about trying to get the maximum resoution all the time - You only need what is acceptable at the magnification you are using. I will often use conditions that deliberately give me poor resolution, because I don't need it at the required magnification. FEG-SEM up to x10,000 The images are then much brighter. Above about x10K I will start to introduce some resolution improvements and begin to use noise clean-up strategy. Then at x100,000 I am all out for resolution improvement at the expense of everything else.

There is nothing special about bacteria observation. All the parameters you are asking about are very microscope-dependant. Use what is better for your SEM. The only exception - accelerating voltage. Do not go too high. Usually (for most microscopes) 5 kV is a good starting point. 

I would go no more than 2kV, as low current as possible (sample damage) but to get sufficient S/N ratio, other parameters are microscope dependent.

Usually there is no need to use low current for viewing bacteria for as long as magnification does not exceed 5k-10k.  

About apertures: If you go for lower vacuum (1 Pa and more) you may need bigger aperture to get more signal.

The most important thing to get right is the sample preparation. The papers given above will help you. After that the imaging is simple.

It really does depend a lot on your microscope - tell us what type of EM you have.

To try and answer your questions:

acceleration voltage - EM dependent, if you have FEG-SEM you can work at 1 or 2kV (or lower) this will give you more surface sensitive information. Higher voltage will tend to penetrate further into the sample and you get less surface information and more 'transparency' effect. If you are on W gun you will need higher kV. If you can get down to 5kV, that wil be ok.

working distance - this is a trade off between depth of focus and resolution. Short WD = higher resolution/small depth of focus, Long WD = large depth of focus/poor resolution. So it depends on how topographic your sample is and at what resolution you require for the images. I generally choose the shortest WD that allows a full focus on all the specimen. This will also depend on your detector configuration as varying the WD will affect the detector efficiency.

probe current - the trade-off here is with image brightness/resolution. Large probe current = high brighteness (less image noise) / low resolution. Small probe current = high resolution / lower brightenss (more image noise). For smaller probe currents you will need to set longer scanning times to clean up the noise. So select a probe current that gives you acceptable resolution and with a level of noise that can be cleaned by slow scanning.

filament current - instrument dependent, best leave this on the default.

aperture diameter - instrument dependent, but smaller aperture will give you higher resolution and less signal (more noise). Larger aperture  = more signal (less loise) and lower resolution.

Remember it is not always about trying to get the maximum resoution all the time - You only need what is acceptable at the magnification you are using. I will often use conditions that deliberately give me poor resolution, because I don't need it at the required magnification. FEG-SEM up to x10,000 The images are then much brighter. Above about x10K I will start to introduce some resolution improvements and begin to use noise clean-up strategy. Then at x100,000 I am all out for resolution improvement at the expense of everything else.

Thank you for all of your response everyone!

Thank you.

I pick up the microscopic study of the effect of sucrose on bacteria cell walls of known drug control mechanism (eg penicillin, Ampiciline); fixation, dehydration standard metodս. Important factor  the vacuum evaporator  choice, coal, tungsten, gold, platinum and palladium etc.

Need to find out the SEM's resolutions.

Warm Regards,

If you have the option of using a LowTemperature Scanning Electron Microscopy (LTSEM), do it and get wonderful images of bacteria.

Please see fig 1 and 2 in the attached paper.

Regards

carmen