A Methods Manual for the Collection, Preparation and Analysis of ...

3. Preparation for Electron Microscopy (EM)Diatoms provide an excellent subject for electron microscope studies. Both scanning electronmicroscopy (SEM) and transmission electron microscopy (TEM) are useful but not essentialtools for diatom taxonomy. An impression of the three dimensional structure of diatoms isgained using SEM, while the higher resolving power of TEM makes it useful for examiningfine structures and small, thin-walled diatoms (e.g. Fistulifera saprophila Lange-Bertalot &Bonik). The silica frustule of diatoms is very robust and no special techniques such as thoseused for soft-bodied algae are required to prepare these cells for electron microscopy.2.2 Preparation for SEM Freshly collected diatom material can simply be air dried onto a small coverslip (Ø 12mm) or filtered through a 2.5 µm Millipore ® filter. Cleaned material can be prepared in a similar manner. When air dry the sample should be placed in a dessicator containing silica gel for 24hours to make sure that it is completely dehydrated before continuing with furtherpreparation. When dry the coverslip or filter should be mounted on an aluminium microscope stubwith carbon tape and sputter coated with gold palladium. The samples are now ready for examination. 10 – 15 kV is usually adequate voltage for examining diatoms.2.3 Preparation for TEMThe sample is suspended (or diluted) into water if possible and adsorbed onto a formvarfilm which is attached to a copper specimen grid.Once the diatom material has been adsorbed onto the film surface, the excess sample isblotted off and the grid is covered with a small drop (5 µl) of stain solution (AqueousUranyl Acetate).This is left on the grid for a few seconds and then blotted off.The sample is dried and examined in the TEM.38