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Chapter 2

Biological Sciences 55-237 Chapter 2: Chapter 2

Biological Sciences
Course Code
BIOL 2070

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Chapter 2: Microbioal Cell Structure and Function
The science of microbiology has taken its greatest leaps forward as new tools are
developed and new tool are improved
Microscope is oldest and most basic tool
Many types of microscopy
2.1 Discovering Cell Structure: Light Microscopy
To see microorganisms, one needs a microscope of some sort
In general, light microscopes are used to examine cells at relatively low
magnifications, and electron microscopes are used to examine cells and cell
structures at very high magnification
All microscopes employ lenses that magnify the image
Magnification is not the limiting factor in our ability to see small objects
Resolutionthe ability to distinguish two adjacent objects as distinct and
separategoverns out ability to see the very small
Although magnification can be increased virtually without limit, resolution cannot,
because resolution is a function of the physical properties of light
For the light microscope, the limits of resolution are about 0.2 micrometers, while
that of the electron microscope is considerably greater
The Compound Light Microscope
Light microscope uses visible light to illuminate cell structures
Several types of light microscopes are used in microbiology
o Bright-field
o Phase-contrast
o Differential interference contrast
o Dark-field
o Fluorescence
With the bright-field microscope, specimens are visualized because of the slight
differences in contrast that exist between them and their surroundings, differences
that arise because cells absorb or scatter light to varying degrees
Modern compound light microscope contains two lenses, objective and ocular, that
function in combination to form the image
The light source is focused on the specimen by the condenser
Bacterial cells are typically difficult to see well with the bright-field microscope
because the cells themselves lack significant contrast with their surrounding
Cells visualized by a phase-contrast microscopy overcome these limitations
Pigmented organisms are also an exception, because the colour of the organism
itself adds contrast, which makes them easier to visualize by bright-field optics
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For cells lacking pigments, there are several ways to boost contrast
Magnification and Resolution
The total magnification of a compound light microscope is the product of the
magnification of its objective and ocular lens
Magnifications of about 2000x are the upper limit for light microscopes
o At magnifications above this, the resolution does not improve
Resolution is a function of the wavelength of light used and a characteristic of the
objective lens known as the numerical aperture, a measure of light-gathering ability
There is a correlation between the magnification of a lens and its numerical
aperture; lenses with higher magnification typically have higher numerical
The diameter of the smallest object resolvable by any lens is equal to (half the
wavelength)/(numerical aperture)
o This formula shows that resolution is highest when blue light is used to
illuminate a specimen and the objective has a very high numerical aperture
The highest resolution possible in a compound microscope is 0.2 micrometers
Two objects that are closer than 0.2 micrometers cannot be resolved as distinct and
Microscopes used in microbiology have ocular lenses that magnify 10-20x and
objective lenses that magnify 10-100x
At 1000x, objects 0.2 micrometers in diameter can just be resolved
With the 100x objective, and with certain other objectives of very high numerical
aperture, an optical grade oil is placed between the microscope slide and the
Lenses on which oil is used are called oil-immersion lenses
Immersion oil increases the light-gathering ability of a lens by allowing some of the
light rays emerging from the specimen at angles (that would otherwise be lost to the
objective lens) to be collected and viewed
2.2 Improving Contrast in Light Microscopy
In light microscopy, improving contrast improves the final image
Staining is a quick and easy way to improve contrast
Staining: Increasing Contrast for Bright-Field Microscopy
Dyes can be used to stain cells and increase their contrast so that they can be more
easily seen in the bright-field microscope
Dyes are organic compounds
Each class of dye has an affinity for specific cellular materials
Many dues used in microbiology are positively charged, and for this reason they are
called basic dyes
o Methylene blue
o Crystal violet
o Safranin
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Basic dyes bind strongly to negatively charged cell components, such as nucleic
acids and acidic polysaccharides
Because cell surfaces tend to be negatively charged, these dyes also combine with
high affinity to the surfaces of cells, and hence are very useful-general purpose
To perform a simple stain:
o Begin with dried preparations of cells
o A clean glass slide containing a dried suspension of cells is flooded for a
minute or two with a dilute solution of a basic dye, rinsed several times in
water, and blotted dry
Because their cells are so small, it is common to observe dried, stained preparations
of Bacteria or Archaea with a high-power (oil-immersion) lens
Differential Stains: The Gram Stain
Stains that render different kinds of cells different colours are called differential
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