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

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings 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

Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings 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 Primary stain: Crystal violetPurple Mordant: IodinePurple Decolorizing agent: AlcoholPurpleColorless Counterstain: SafraninPurplePink The Gram stain classifies bacteria into gram-positive or gram-negative.

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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