1. Microbiology Chapter 3 Microscopy and Staining

2. What’s on a Pinpoint? How many bacteria? How many are needed to start an infection? Sometimes as few as 10 bacteria are enough!

3. Historical Microscopy 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”

4. 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

5. 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

6. 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)

7. 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

8. 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

9. Total Magnification Calculations 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

10. 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”

11. 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

12. Different Types of Electron Microscopy EM uses electron beam and electro- magnets 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-

13. 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

14. 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-

15. 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

16. 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

17. 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