• A simple microscope has only one lens.

Chapter 3 - Microscopy

• Multiple lenses

• Image from objective lenses is magnified again by the ocular lens.

• Total magnification =objective lenses ocular lens

• Resolution is the ability to distinguish between two points.

Compound Light Microscope

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Compound Light Microscope

• is the bending of light.

• Light may bend in air so much that it misses the small high-magnification lens.

• is used to keep light from bending.

• Dark objects are visible against a bright background.

• Light is concentrated by condenser and passes through specimen.

Brightfield Illumination

• Light objects are visible against a dark background.

• Condenser causes light to reflect off the specimen, which then enters the objective lens.

Darkfield Illumination

• Accentuates differences in the refraction of different regions of the specimen.

Phase-Contrast Microscopy

• Uses Ultra Violet light.

• Fluorescent substances absorb and emit

• Cells may be stained with fluorescent dyes (fluorochromes).

• Immunofluorescence

Fluorescence Microscopy

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http://www.microscopyu.com/articles/digitalimaging/drentdigital.html

Link to digital microscopy workstation

• Uses electrons instead of light.

• Resolution is much greater than light microscopes

• Two types: Transmission and Scanning

Electron Microscopy

• Magnifies tens of thousands of times

• Used to visualize internal structures

Transmission Electron Microscopy (TEM)

• Magnifies tens of thousands of times

• Used to scan the surface of a specimen

Scanning Electron Microscopy (SEM)

Preparation of Specimens for the Compound Light Microscope

• A thin film of a solution of microbes on a slide is a

• A smear may be to attach the microbes to the slide and to kill the microbes.

• In a basic dye, the dye is a cation, and is

• In an acidic dye, the dye is an anion, and is

• Staining the background instead of the cell is called negative staining.

Staining Smears

• Simple Staining: Use of a single basic dye to see morphology and arrangement.

• Differential Staining: Two or more dyes are used to distinguish between cell types or cell parts

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

Gram Staining

Color of Color of

Primary stain: Crystal violet Purple Purple

Mordant: Iodine Purple Purple

Decolorizing agent: Alcohol Purple Colorless

Counterstain: Safranin Purple Pink

The Gram stain classifies bacteria into gram-positive or gram-negative.

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Gram Staining

Gram Stain

• Cells that retain the primary stain in the presence of acid-alcohol are called acid-fast.

• Non–acid-fast cells lose the primary stain when rinsed with acid-alcohol, and are usually counterstained to see them.

Acid-Fast Stain

• Negative staining is useful for capsules,

• Endospore staining distinguishes endo(spores) from vegetative cells

Special Stains