Download Microscopy Chapter 1

Chapter
Microscopy
1
Microscopy
Light Microscopes – uses light passed through a specimen
Types include:
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Brightfield
Darkfield
Phase-Contrast
Differential Interference Contrast
Flourescence (UV Light)
Confocal (Laser light)
Electron Microscopes – use a beam of electrons which are either
passed through or bounced off of a specimen
Types include:
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Transmission (TEM)
Scanning (SEM)
Scanning Tunneling (STEM)
Atomic Force (SEM also)
Light Microscopy
see images on pg 64-65
Bright-field: dark image against a brighter background (what we use in
lab)
Dark-field: only refracted light enters objective, making specimen
bright, background dark (useful for observing living cells, particularly
spirochetes)
Phase-contrast: converts slight changes in refraction into easily
detected variations of light intensity (useful in observing living cells)
Fluorescence: specimen exposed to ultraviolet light, compounds in the
sample fluoresce, giving off visible light
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Microscope
Compound Lens Microscope : a microscope with a series of lenses
Total magnification: calculated by multiplying the objective lens
magnification by the ocular lens magnification
Resolution: ability to distinguish between two points a specified
distance apart. The smallest distance that can be distinguished equals
the resolving power of the scope.
About 0.2 microns is the limit of white light resolution
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Visible
to eye
Light
microscope
Electron
microscope
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Ocular lens
Prism
bends light
look in here!
Second magnification
Objective Lens
First magnification
Stage
Move specimen
Focus knob
Specimen
Condenser
Focuses light
Illuminator
‘the light’
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Oil Immersion Lenses for
High Magnification
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BUT, if our specimen
is too small for light
Must use electron microscope
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TEM
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SEM
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Table 3.2 (7 of 7)
Staining
Fixation: process by which internal and external structures of cells are
preserved
Heat-fixation - fix an air-dried thin film (smear) by passing through flame
Dyes: have chromophore groups (give color) and bind to cells by ionic,
covalent, or hydrophobic bonding
Hydrophobic – e.g. Sudan black, stains lipids
Ionic stains can be either: (remember ions?)
Basic (+ charged dye attracted to – charged bacteria) OR
Acidic (- charged dye attracted to + background)
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Staining continued…
Simple Stain: uses one staining agent
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Methylene blue
Crystal violet
Safranin
Carbolfuchsin
Mordant – chemical added to make the dye bind more tightly
(increased affinity for specimen)
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Differential Stains
divide bacteria into separate groups based on different staining
properties – different groups stain differently
Gram stain - developed by Dr. Christian Gram, 1884
Our most important/useful staining procedure:
Crystal Violet - 1 minute (primary stain)
Gram's Iodine - 1 minute (this is a mordant!)
95% ethanol - 5-10 sec (Rinse to decolorize)
Safranin - 1 minute (secondary stain)
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Gram Staining
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Interpreting Gram Stains
Purple cells = Gram Positive (G+)
Gram positive bacteria - trap crystal violet-iodine complex, due to
thick layer of peptidoglycan in cell wall
Red cells = Gram Negative (G-)
Gram negative bacteria - large amount of lipids in cell wall dissolved
by the 95% ethanol, allowing crystal violet-iodine complex to escape
leaving cells colorless. The safranin counterstain turns these cells red
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Acid Fast Stain
Acid-fast stain: Carbolfuchsin
used to stain Mycobacterium, which have high content of mycolic acid
(waxy substance)
Must use steam heat to force carbolfuchsin stain into cells
Acid-alcohol decolorizer removes stain from non-acid fast cells
counterstain with methylene blue
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Negative/Capsular Stain:
reveals capsule layer around cells.
Capsules often indicate a bacterium’s virulence, or ability to cause
disease!
Dye = Nigrosin or India Ink
mix bacteria with the dye & spread on clean slide, dry
Bacteria are then often stained with a simple stain
The capsule shows as a clear halo around the bacterial cells
useful to observe Klebsiella pneumonia & other capsule producers
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Spore Stain
used to stain endospores of Clostridium and Bacillus
Endospores do not readily take up dye, must be ‘forced’ but once it
penetrates the stain is not easily decolorized
heat smear over steam with Malachite green stain
Then rinse with water & counterstain with safranin (red stain)
RESULT = Endospores - Green; Vegetative cells - Red
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Flagellar Stain
Some bacteria have flagella, whip-like structures with which they
move. Some stains specifically stain these structures
Flagella of bacteria are coated with tannic acid or potassium alum and
stained with basic fuchsin (Gray Method)
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