Microbiology Chapter 3Microscopy 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

• 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

• 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 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-

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