Units of Measurement

         1 m = 10–6 m = 10–3 mm

         1 nm = 10–9 m = 10–6 mm

         1000 nm = 1 m

         0.001 m = 1 nm

 

Microscopy: The Instruments

         A simple microscope has only one lens

 

Light Microscopy

         Use of any kind of microscope that uses visible light to observe specimens

         Types of light microscopy

         Compound light microscopy

         Darkfield microscopy

         Phase-contrast microscopy

         Differential interference contrast microscopy

         Fluorescence microscopy

         Confocal microscopy

 

Compound Light Microscopy

         In a compound microscope, the  image from the objective lens is magnified again by the ocular lens

         Total magnification =
objective lens ocular lens

         Resolution is the ability of the lenses to distinguish two points

         A microscope with a resolving power of 0.4 nm can distinguish between two points 0.4 nm

         Shorter wavelengths of light provide greater resolution

         The refractive index is a measure of the light-bending ability of a medium

         The light may bend in air so much that it misses the small high-magnification lens

         Immersion oil is used to keep light from bending

 

 

Brightfield Illumination

         Dark objects are visible against a bright background

         Light reflected off the specimen does not enter the objective lens

 

Darkfield Illumination

         Light objects are visible against a dark background

         Light reflected off the specimen enters the objective lens

 

Phase-Contrast Microscopy

         Accentuates diffraction of the light that passes through a specimen

 

Differential Interference Contrast Microscopy

         Accentuates diffraction of the light that passes through a specimen; uses two beams of light

 

Fluorescence Microscopy

         Uses UV light

         Fluorescent substances absorb UV light and emit visible light

         Cells may be stained with fluorescent dyes (fluorochromes)

 

Confocal Microscopy

         Cells stained with fluorochrome dyes

         Short wavelength (blue) light used to excite the dyes

         The light illuminates each plane in a specimen to produce a three-dimensional image

         Up to 100 m deep

 

Two-Photon Microscopy

         Cells stained with fluorochrome dyes

         Two photons of long- wavelength (red) light used to excite the dyes

         Used to study cells attached to a surface

         Up to 1 mm deep

 

Scanning Acoustic Microscopy (SAM)

         Measures sound waves that are reflected back from an object

         Used to study cells attached to a  surface

         Resolution 1 m

 

Electron Microscopy

         Uses electrons instead of light

         The shorter wavelength of electrons gives greater resolution

 

Transmission Electron Microscopy (TEM)

         Ultrathin sections of specimens

         Light passes through specimen, then an electromagnetic lens,
to a screen or film

         Specimens may be stained with heavy metal salts

         10,000–100,000; resolution 2.5 nm

 

Scanning Electron Microscopy (SEM)

         An electron gun produces a beam of electrons that scans the surface of a whole specimen

         Secondary electrons emitted from the specimen produce the image

         1,000–10,000; resolution 20 nm

 

Scanned-Probe Microscopy

         Scanning tunneling microscopy (STM) uses a metal probe to scan a specimen

         Resolution 1/100 of an atom

         Atomic force microscopy (AFM) uses a metal- and-diamond probe inserted into the specimen.

         Produces three-dimensional images.

 

Stains and Smears

         Staining: Coloring the microbe with a dye that emphasizes certain structures

         Smear: A thin film of a solution of microbes on a slide

         A smear is usually fixed to attach the microbes to the slide and to kill the microbes

 

Preparing Smears for Staining

         Live or unstained cells have little contrast with the surrounding medium. Researchers do make discoveries about cell behavior by observing live specimens.

         Stains consist of a positive and negative ion

         In a basic dye, the chromophore is a cation

         In an acidic dye, the chromophore is an anion

         Staining the background instead of the cell is called negative staining

 

Simple Stains

         Simple stain: Use of a single basic dye

         A mordant may be used to hold the stain or coat the specimen to enlarge it

 

Differential Stains

         Used to distinguish between bacteria

         Gram stain

         Acid-fast stain

 

Gram Stain

         Classifies bacteria into gram-positive
or gram-negative

         Gram-positive bacteria tend to be killed by penicillin and detergents

         Gram-negative bacteria are more resistant to antibiotics

 

Acid-Fast Stain

         Stained waxy cell wall is not decolorized by  acid-alcohol

         Mycobacterium

         Nocardia

 

Special Stains

         Used to distinguish parts of cells

         Capsule stain

         Endospore stain

         Flagella stain

 

Negative Staining for Capsules

         Cells stained

         Negative stain

 

Endospore Staining

         Primary stain: Malachite green, usually with heat

         Decolorize cells: Water

         Counterstain: Safranin

 

Flagella Staining

         Mordant on flagella

         Carbolfuchsin simple stain