Soil Microbial Ecology - Soil Molecular Ecology Laboratory

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OBJECTIVE:
Train to use safely the basic microscopes (dissecting and compound-oil immersion) and
to observe various soil microorganisms
INTRODUCTION
Microscopes are the most important tools of any microbiology studies. They are needed
to observe structures of microorganisms that are too small to be seen by naked eye.
Magnification and resolution are the two major steps to achieve quality observation.
Most are familiar with magnification, but not all are familiar with resolution. Optic
technology in the area of electron microscope allows the achievement of millions of
times magnification of small object, but these same principles limit magnification to
about 1000 of times light microscope. Why the two systems are so different?
Resolution is the answer to this question. The eye is the ultimate receptor of any image.
Eye resolution is determined by the distance between receptor cells in the retina. Pictures
of two objects that are received on the same receptor simultaneously cannot be
distinguished from each other. However, if these two pictures are received on adjacent
receptors, they may be resolved. The optics of the eye and the spacing of receptors on
the retina make the optimum focusing distance for any eye. Regular eye can distinguish
25-cm lines as separate lines if they are 60 to 100 µm apart. Lines less 60 µm apart are
unresolved and will appear as a single solid line. Eye resolution is an individual
phenomenon and it is different from one eye to another of the same person, and from one
person to another. For practical applications, 100 µm will be considered the resolving
power of human been eye.
Microscope magnification is required to observe an object that is smaller than 100 µm or
to distinguish between two structures that are closer together than 100 µm. Microscope
is also equipped to resolve mix light rays. Achieving this mainly depends on the ability
of glass to bend light, its refractive index, and the wavelength of light used to form the
image. These factors are quantified by this equation R = 0.61λ / NA (where: R = the
resolution in µm, λ = the wavelength of light in µm, NA = the numerical aperture of the
objective lens that includes the refractive index of the lens (n) and the sine of the lens
angle (U): NA = n * sine U).
Example:
Using blue light, λ = 45 µm
The 45X lens on your microscope (color coded yellow) NA = 0.66
R = (0.61 x 0.45) / 0.66 = 0.42 µm resolution
The smallest object that can be observed in this lens is 0.42 µm. Microscope
magnification is the product of the ocular lens (10x) and the objective lens (45x) or 450