Transcript Microbiology Chapter 3 Microscopy and Staining

Microbiology Chapter 3
Microscopy and Staining
What’s on a Pinpoint?
• How many bacteria?
• How many are needed to start an
infection?
• Sometimes as few as 10 bacteria are
enough!
Historical Microscopy
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Anton van Leeuwenhoek-1670’s
1st to see micro-organisms
lens maker, simple scopes 100x to 300x
Single lens, like a magnifying glass
Studied “animalcules”
Principles of Microscopy
• Metric units- powers of 10
• Microscopy- technology of making very
small things visible to naked eye
• Measurements in:
- micrometers (microns) um 0.000001m=
10-6 m
- nanometers nm= 10-9 m
- angstroms- (A) 10-10 m
Properties of Light:
Wavelength and Resolution
• Wavelength- length of a light ray
• Resolution- ability to see 2 objects as
separate & discrete units (not fuzzy)
• Visible light = 550nm (NG)
• UV light= 100-400 nm better for resolution
• Electron microscopy- .005 nm high reso
• Resolving power of lens- numerical
measure of lens, smaller distance from
lens to slide =greater resolving power
Properties of Light:
Light and Objects
• Reflection-light strikes an object &
bounces
back
• Transmission- light passes through object
• Absorption- light rays taken up by object
• Luminescence-absorbed UV rays are changed
to longer wave & reemitted
• Fluoresce- luminescence only occurring during
irradiation
• Phosphorescent- object emits light when light
rays no longer strike it (some bacteria)
Properties of Light:
Light and Objects
• Refraction- bending of light as it passes from
one medium to another
• Index of refraction- measure of the speed at
which light penetrates
• Immersion oil- used for better resolution
because oil as the same index of refraction as
glass.
• Diffraction- light waves bend around an opening
and could cause blurry slides
• Iimit = oil immersion with 10 x eyepiece=1000X
Light Microscopy and Types of
Microscopes
• Microscope that uses visible light to
observe specimen
• Hooke’s compound microscope had more
than 1 lens
• The Compound Light Microscope:
- monocular- 1 eyepiece, binocular-2
• Survey of microscope parts and their
functions – pg 58
Total Magnification Calculations
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Scanning power -4x X 10x (ocular)= 40x
Low power 10x X 10x(ocular) = 100 x
High dry power 40x X 10 x = 400 X
Oil immersion 100x X 10 x = 1000x
Parfocal- in focus on one power, simple
rotate nosepiece and its should focus on
next power
• Ocular micrometer- measure size of
sample
Light Microscopy and Types of
Microscopes
• Dark-Field Microscopy- condenser causes
light to reflect off specimen at an angle
and increases the contrast
• Phase-Contrast Microscopy-to observe
live and unstained specimens by
increasing refractive index and shows
different degrees of brightness
• Nomarski Microscopy- differential
interference contrast and looks “3D”
Light Microscopy and Types of
Microscopes
• Flourescence- UV light is used to excite
molecules, longer wavelengths= bright
• Confocal Microscopy- usesbeams of UV
lases light and computer reconstructs
images, up to 40% better. Can study
microbes alive or not.
• Digital Microscopy-have built in digital
camera and can be viewed on screen
Different Types of Electron
Microscopy
• EM uses electron beam and electromagnets not lenses- high resolution
• Photos taken – Electron micrographs
• Transmission Electron Microscopy- (TEM)
better view of internal structures up to
500,000x magnification
- shadow casting- freeze fracturing- freeze etching-
Different Types of Electron
Microscopy
• Scanning Election Microscopy (SEM)- Image of the surface “3D” 50,000x mag
• Scanning Tunneling Microscopy (STM’s)- 1980 can be used with liver specimens
and under water
• Atomic force microscope-(AFM)advanced 3d from atomic size to 1 micron
- used to study DNA, proteins
Techniques of Light Microscopy
• Preparation of Specimens for the Light
Microscope:
• 1) Wet Mounts- drop of medium with
microbes is spread on a slide
• 2) Smears- microbes from a loopful of
medium are spread on a slide, then
heat fixed to kill microbes
- heat fixation-
Principles of Staining
• Stain- dye that binds to a cellular structure
and gives it color
• + charge-basic= methylene blue, crystal
violet, safranin and malachite green
• - charge-acidic= eosin and picric acid
• Simple stain- single dye and reveals basic
cell shapes and structures
• Differential stain- 2 or more dyes: Gram
stain, Ziehl-Neelsen acid fast and spore
Gram Stain
• Gram Stain- 1884 crystal violet (+) and
iodine and ethanol decolorizer, and
counterstained with safranin (-)
• Gram +=purple
• Gram - = red
• Gram non reactive= no stain
• Gram Variable= stain unevenly
Special Staining Procedures
• Ziehl-Neelsen Acid-Fast Stain
- 1882 modification of Ehrlich staining
method
- Acid fast retain red color in cell walls
• Negative staining-capsule is present and
won’t take up stain
• Flagellar staining- coats flagella so they
can be seen
• Endospore staining- Schaeffer-Fulton stain