Microscopy

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Microscopy
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Compound Microscope

• The Compound Microscope is the type of microscope
  we use in the lab. It uses visible light as its
  light source for illumination.
• The average compound microscope has resolving
  power of .2?m (1?m .000001m)
• The resolving power is the ability of the
  microscope to distinguish between 2 pts, or to
  show detail.
• Staining is used to make the specimen contrast
  sharply with the medium so that it is easier to
  see.

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Fluorescence Microscopy

• This particular microscope uses fluorochromes to
  attach to organisms and allow them to fluoresce.
  Fluorochromes are small molecules that only
  fluoresce once they have attached to their
  target.
• Immunofluorescence is a particular type of marker
  used in fluorescence microscopy. The
  fluorochromes are first attached to a specific
  antibody (Ab). The antibody is usually specific
  for a particular type of cell, virus, or other
  type of specimen.
• This type of microscopy is useful for detecting
  viruses and other org. within cells, tissue, or
  other clinic specimen that normally we wouldnt
  be able to see or detect otherwise.

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• For example, lets say that we suspect someone
  has Hepatitis. Hepatitis is caused by a virus.
  Viruses are so small that they cant be seen
  under a compound microscope. In addition, they
  also spend much of their replication time within
  host cells and you cant see into a cell with a
  compound microscope.
• So we take a sample of blood from the patient.
• Then we get some antibodies that are specific
  for the hepatitis virus.
• Next we put the fluorochrome on the antibody.
• .

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• Then we put the fluorochrome, antibody
  combination together on the specimen.
• If the virus is present, then the antibody will
  bind to the virus or virus infected cell. Upon
  binding, it will turn on the switch for the
  fluorochrome. The fluorochrome will now
  fluoresce indicating the specimen is positive for
  that virus.
• If the virus is not present, then the antibody
  wont be able to bind and the fluorochrome wont
  fluoresce.

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Electron Microscopes

• Electron Microscopes are used to examine objects
  smaller than .2?m. There are 2 types that are
  used.
• SEM Scanning Electron Microscope
• This type of electron microscope creates and
  image of the surface structures of the organism.
  
• It bounces a beam of electrons (e-s) off the
  organism to create the image. As the electrons
  come into contact with the specimen the e-s
  bounce off in different directions. A computer
  then records the direction it went and
  extrapolates the shape of the surface of the
  specimen by the direction that each e-s bounces
  off.

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• TEM Transmission Electron Microscopy
• The Transmission Electron Microscipe uses ultra
  thin slice of the specimen to create images of
  inside cellular structures.
• In this case the image is created by sending the
  e-s through the specimen and recording the
  trajectory of the e-s as they exit out the other
  side.
• These are both great ways for us to learn more
  about a particular organism. Unfortunately a
  live specimen can never be viewed this way
  because the preparations are toxic to the cells.

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Biological Stains Used in the Lab

• To prepare specimens to be viewed under the
  compound microscope there are two types of stains
  that are used.
• 1. Basic Stain The basic stain is composed of a
  positively () charged ion that attaches to the
  negatively () charged cell membrane.
• Crystal violet and safranin are examples of basic
  stains.
• 2. Acidic Stain An acidic stain is composed of
  a negatively (-) charged ion that is repelled by
  the negatively () charged membrane.
• Eosin and Acid fuschin are examples of acidic
  stains.

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• There are two different ways that we can use
  basic and acidic stains to better view and
  identify microbes.
• 1. Simple Stain A simple stain is when we use
  one stain, basic or acidic and color the organism
  to view under the microscope. It helps us to
  determine the shape of the organism.
• 2. Differential Stain A differential stain is
  when we combine 2 or more stains to gather more
  information.
• For example, a gram stain uses 2 stains. One
  stain indicates the organism is G and the other
  indicates the organism is G-.
• Other types of differential stains are endospore
  stains, capsule stains, acid-fast stains, and
  flagella stains.

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The Basic Gram Stain Procedure

• Lets say that you have a culture that has two
  different kinds of organisms growing in it. One
  organism is a cocci and the other is a rod. You
  want to determine what the gram reaction is for
  each.
• The first step is to heat fix the specimen. (You
  smear the specimen on the slide and wave it
  through the bunsen burner to slightly bake it
  onto the slide. You dont want it to wash off
  during the staining procedure.!)
• The second step is to apply the Primary stain.
  The primary stain is always the first stain
  applied in a differential staining procedure. In
  this case the primary stain is crystal violet.
  It is a basic stain so it attaches to all the
  cells present because they all have a (-) charged
  cell membrane.

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• The next step is to apply iodine to the specimen.
  The iodine acts as a sealant to tightly bind the
  primary stain to the G cell wall. This is
  called a mordant.
• Then the specimen is washed with alcohol. The
  alcohol dissolves lipids. Remember that the G-
  cell wall is made up of an outer membrane that
  contains lipids. The alcohol wash removes the
  cell wall from the G organism.
• Next the secondary stain is applied. In a
  differential stain, the secondary stain is the
  second type of stain used in the process. In
  this case, safranin is the secondary stain that
  is used. It is a basic stain too. It attaches
  only to the unstained cells, ie the G- organisms
  on the slide.

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• The final result is that the G cells are stained
  purple and the G- cells are stained magenta or
  pink.
• The gram stain works simply because there is a
  difference in the cell walls between G and G-
  organisms.
• See Insight 4.2 on page 98 of your textbook for
  an excellent explanation of the gram stain
  procedure.

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Endospore stain

• An endospore is a special structure made by some
  bacteria to store genetic material for a new
  cell.
• Because endospores are so resistant to many
  environmental pressures they cant be stained
  with ordinary dyes because dont penetrate the
  wall.
• The Primary stain for an endospore stain is
  malichite green. Heat is used with this stain to
  force it into endospore.
• The Secondary stain is safranin. Safranin stains
  the cell wall of the microbe giving a contrast
  between the endospore and the organism.

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Capsule Stain

• Remember that a capsule often indicates
  virulence.
• A Negative stain (India Ink) is used to show the
  outline of the capsule. It will not bind to
  capsule but it will stain around the capsule.
• The the Secondary stain, methyl-blue is applied
  to stain the organism inside the capsule.
• The clearing around the stained orgaism is the
  capsule.

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Acid-fast Stain

• Some microbes have cell walls that are
  constructed a little differently that how we have
  discussed. The organism, Mycobacterium has a
  waxy substance in its cell wall called mycholic
  acid. The mycholic acid makes it difficult to
  stain this organism using a basic dye. So a
  different type of stain is used for it.
• The acid-fast stain is used to distinguish
  mycobacterium from other types of rods.
• Carbol fuschin is the primary stain for this
  technique. Like the endospore stain, heat is
  used to force the stain on the organism.
• A Secondary stain is added after to bind to any
  organism that did not bind to the primary stain.

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Flagella Stain

• A flagella stain is a very tedious and delicate
  staining procedure.
• It uses a mordant and carbol fuschin to build up
  the diameter of the flagella until they become
  visible.
• This type of staining procedure is valuable
  because the arrangement of the flagella can be
  used as a diagnostic aid.
• No homework for this chapter.