Material and Equipment:
1. Ethanol
2. foil
3. E.Coli culture
4. Gold Sputter
5. Scanning Electron microscope
6. Dessicator

Step 1 Sample preparation: The cells or non-biological material for SEM analysis needs to place on a small piece of allumium foil.

Step 2 Fixation: Biological samples are fragile and fixation of biological sample is required for two purpose. (1) Stopping biological actrivity and (2) it stops the relative movement of cellular components and intracellular macromolecules. Sample is fixed with 2-5% glutaraldehyde and 1% osmium tetraoxide in 50mM sodium cacodylate pH 7.4 for overnight at 40C.

Step 3 Dehydrartion: Biological samples are fragile and contains large amount of water. Water present in the biological sample diffract electron rays and may increase the background signal. Following osmium fixation, water is chemically extracted from the specimen using a graded series of ethanol.

It is performed in the following steps:

• Sample is incubated with 50% ethanol for 30mins.

• Sample is incubated with 70% ethanol for 30mins.

• Sample is incubated with 80% ethanol for 30mins.  

• Sample is incubated with 90% ethanol for 30mins.  

• Sample is incubated with 95% ethanol for 30mins.  

• Sample is incubated with 100% ethanol for 30mins. 

• Sample is incubated with anhydrous ethanol for 30mins.  

Step 4 Drying: During dehydration, sample needs to prevent air drying. Once the dried material is removed, it needs to be stored in a desiccated environment until viewing.