• How many bacteria?
• How many are needed to start an infection?
• Sometimes as few as 10 bacteria are enough!

• Anton van Leeuwenhoek-1670’s
• 1 st to see micro-organisms
• lens maker, simple scopes 100x to 300x
• Single lens, like a magnifying glass
• Studied “animalcules”

• 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

• 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

• 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-

• 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- 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

• 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