The Study of Microbial Structure: Microscopy and Specimen Preparation Contd..

Electron Microscopy
 For centuries the light microscope has been the most important
instrument for studying microorganisms. The electron microscope
now has transformed microbiology and added immeasurably
to our knowledge. The nature of the electron microscope and
the ways in which specimens are prepared for observation are reviewed
briefly in this section.

1. The Transmission Electron Microscope
      The very best light microscope has a resolution limit of about
0.2 m. Because bacteria usually are around 1 m in diameter,
only their general shape and major morphological features are
visible in the light microscope. The detailed internal structure of
larger microorganisms also cannot be effectively studied by
light microscopy. These limitations arise from the nature of visible
light waves, not from any inadequacy of the light microscope itself.

Recall that the resolution of a light microscope increases with a
decrease in the wavelength of the light it uses for illumination. Electron
beams behave like radiation and can be focused much as light is
in a light microscope. If electrons illuminate the specimen, the microscope’s
resolution is enormously increased because the wavelength
of the radiation is around 0.005 nm, approximately 100,000
times shorter than that of visible light. The transmission electron microscope
has a practical resolution roughly 1,000 times better than the
light microscope; with many electron microscopes, points closer than
5 Å or 0.5 nm can be distinguished, and the useful magnification is
well over 100,000 . The value of the electron microscope
is evident on comparison of the photographs in figure ;
microbial morphology can now be studied in great detail.

A modern transmission electron microscope (TEM) is
complex and sophisticated , but the basic principles behind its operation can be understood readily. A heated tungsten
filament in the electron gun generates a beam of electrons that is
then focused on the specimen by the condenser . Since
electrons cannot pass through a glass lens, doughnut-shaped electromagnets
called magnetic lenses are used to focus the beam. The
column containing the lenses and specimen must be under high
vacuum to obtain a clear image because electrons are deflected by
collisions with air molecules. The specimen scatters electrons passing
through it, and the beam is focused by magnetic lenses to form
an enlarged, visible image of the specimen on a fluorescent screen.
A denser region in the specimen scatters more electrons and therefore
appears darker in the image since fewer electrons strike that
area of the screen. In contrast, electron-transparent regions are
brighter. The screen can also be moved aside and the image captured
on photographic film as a permanent record.


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