Microscopy and Staining

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Microscopy and Staining
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Science relies on technology

• The progress of microbiology and many other
  disciplines relies heavily on the introduction of
  new technologies.
• Without a microscope it would be impossible to
  characterize the morphology of bacteria.

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History of the Microscope

• Development of the microscope originated in
  __________ by ________________.
• Luckily, he was a skilled observer and artist and
  produced many fine illustrations of microbes
  (_______)
• Unfortunately, he was not willing to share his
  microscopes or knowledge with other scientists
  until much later in life to help the progress of
  microbiology

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We measure specimens based on the metric system.
We will be looking at bacteria ranging from
__________.
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Wavelength and Resolution

• The shorter the wavelength, the better the
  resolution (__________________)

• Is (a) or (b) resolved?
• Which resolves images better, gamma rays or
  visible light?

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Magnification is useless without good resolution.
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Properties of Light

• Light has the ability to a_____ b_____ c_____
  d______

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• Microscopists use _________to reduce refraction
  and reflection to help increase resolving power.

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One objective lens can hold up to a dozen small
lenses inside.
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Anatomy Physiology of the microscope should be
review.

• How do you calculate total magnification?

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Types of Microscopy

• Each type of microscopy has a preferred use.
• 1. Light-field microscopy
• 2. Dark-field microscopy
• 3. Phase-contrast
• 4. Nomarski
• 5. Fluorescence
• 6. Transmission Electron
• 7. Scanning Electron
• 8. Scanning Tunneling

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What does an electron microscope use to
visualize specimens? 2. Which type of microscope
has better resolving power, bright-field or
electron?
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In lab, we use Bright-Field Light Microscopy

• We will perform the following light microscopy
  techniques
• _________
• Wet mount and hanging drop methods
• _______
• Spread, air-dry, heat fix
• _________
• Cheek cells with methylene blue (only 1 stain)
• __________
• Gram stain
• Acid-fast (time permitting)
• ___________
• Capsule stain
• Flagellar stain (prepared slides since this is a
  difficult technique)
• Endospore stain (time permitting)

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Wet mounts

• We will use the hanging drop technique to
  investigate bacterial motility
• Advantages of the hanging drop
• 1
• 2
• Disadvantages of the hanging drop
• 1
• 2
• 3

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Smears

• Performing quality smears takes practice. There
  are many steps in the preparation of a smear that
  must be performed correctly. (Think of
  Goldilocks that wants her porridge just right)
• 1. Smear bacteria onto slide from broth or agar
• ___
• 2. Air dry
• _______
• 3. Heat Fix (see next slide)

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Smears (cont.)

• 3. Heat Fix
• 1
• 2
• 3
• Heat fixation
• 1
• 2
• 3

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Simple Stains

• Before, you applied one stain (methylene blue) to
  visualize your cheek (buccal) cells.

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Differential Stains

• Allow us to differentiate between types of
  bacteria.
• 1. _________
• Gram-positive vs. Gram-negative
• See next slide
• 2. _________
• Used to identify acid-fast bacteria with a waxy,
  lipid cell wall.
• Detects Mycobacteria like M. tuberculosis and M.
  leprosae
• Most of the bacteria in our lab in Acid-Fast
  negative

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Gram Stain

• Most frequently used stain
• Dictates which type of antibiotic treatment
• Gram-Positive
• 1
• 2
• 3
• 4
• 5
• Gram-Negative
• 1
• 2
• 3
• 4

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Special Stains

• We use these special stains to identify special
  structures that some bacteria possess. We will
  learn more about these structures in Ch. 4.
• _________
• Capsule does not stain so we stain the background
• Can not heat-fix or capsule will be destroyed
• _________
• Very difficult stain, we will look at prepared
  slides
• _________
• The endospore is difficult to stain since it is
  made of a thick, hard to penetrate spore wall.
  We will boil the stain to help it penetrate the
  spore coat.

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Chapter Outline Review

• 1. Historical Microscopy
• 2. Principles of Microscopy
• 3. Light Microscopy
• 4. Electron Microscopy
• 5. Techniques of Light Microscopy