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Chapter 2

Biological Sciences 55-237 Chapter 2: Microbiology Topic 2


Department
Biological Sciences
Course Code
BIOL 2070
Professor
tanyanoel
Chapter
2

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Microbiology Topic 2: Microbial Cells
Microscopy
As microbes are so small, there are challenges studying them
They are typically too small to see with naked eye
Want to compare them with organisms that are easier for us to relate to
Try to think small as we consider larger organisms
Even thinking about molecules and atoms that make things up in our world
Macroscopic (or larger organisms) are often measured in meters or centimeters
Microorganisms are typically measured in mm, micrometers, or nm
A micrometer is also referred to as a micron
The microscope has been an essential tool in observing microbes and it continues to
be important in clinical and research microbiology labs today
This is a basic compound light microscope
Microscopes provide magnification, resolution, and many also provide contrast in
studying specimens
Magnification by the microscope objective and ocular lenses helps make the picture
larger
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Resolution (Resolving Power)
Resolution (or resolving power) is the ability of a microscope or other type of
optical device, to separate adjacent points or objects from one another
The use of immersion oil under the 100X objective on the microscope helps us to
increase the resolution by reducing the scattering of light that normally occurs
With more light, it is easier for us to see the objects and the resolving power is
higher
Oil Immersion (100X objective)
The light microscope can be used to look at Eukaryotic microbes like fungi and
protozoa
o Usually under 10X or 40X objectives
Bacteria and Archaea, being much smaller, usually have to be viewed under the oil
immersion 100X objective
The oil immersion lens can resolve adjacent objects that are at least 0.2 micrometers
apart
Contrast
Contrast allows us to distinguish an object or structure from its surroundings
Improving contrast helps us make a better image
Most bacteria and other microbes lack pigments that provide contrast, although
there are some examples of organisms with chloroplasts or pigments
To visualize bacteria that lack these, we can stain them
Simple stains usually involve a single stain or dye
o Allows us to view basic structure and arrangement of cells
o See shape and size
o Do no usually help to differentiate cells with same shape and size
Differential staining
o More complex
o Usually involves more than one type of stain and multiple steps
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o The process can result in cells having different appearances at the end, which
allows us to differentiate between different types of microbes
o Gram stain
o Depending on the type of cell wall that is present, a bacteria may end up
purple (Gram-positive), or it may appear pink (Gram-negative)
o When identifying a bacterium, the Gram stain is usually one of the first steps
in narrowing down the type of bacterium
There are several stains that are commonly used in microbiology labs
Online tutorial shows examples
There are differences between simple and differential stains
There are certain stains that can be used to view certain structures
o Often also considered to be differential stains
Types of Light Microscopes
The simples type of light microscopy is Brightfield microscopy
o Typically used in most labs
There are some other variants
o Phase-contrast
o Darkfield
When would we use the different kinds?
o If you wanted to look at live bacteria, you would not want to stain them
o Staining usually kills cells
o Phase-contrast or Darkfield microscopy could be helpful in improving the
contrast of unstained specimens
Fluorescence Microscopy
Fluorescence microscopy is another specialized form of microscopy
Allows visualization of naturally fluorescent specimens, or, in some cases, we use
fluorescent dyes which can be quite specific to certain structures or organisms
3D Images
To see more of a 3D view under the microscope, special equipment or microscopes
are required
Differential interference contrast (DIC) microscopy or Nomarski microscopy is an
example
Eukaryotic cells shown
There are other structures that can be imaged in this way (see slides0
Confocal scanning laser microscopy (CSLM) carries out optical sectioning at various
depths in the specimen
This allows us to reconstruct 3D structures from the images
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