SUMMARY

Summary

Summary

Chapter 4: Microscopy, Staining, and classification

Microscopy refers to the use of light or electrons to magnify objects.

General Principles of Microscopy

The same general principles guide both light and electron microscopy.

Magnification

Magnification is the apparent increase in size of an object and is indicated by a number followed by an “X” which is read “times.”

Resolution

Resolution (also called resolving power) is the ability to distinguish between objects that are close together.

Contrast

Contrast refers to differences in intensity between two objects, or between an object and its background.

Light Microscopy

Several classes of microscopes use various types of light to examine specimens.

Bright-Field Microscopes

The most common microscopes are bright-field microscopes, in which the background (or field) is illuminated. There are two basic types: Simple microscopes contain a single magnifying lens and are similar to a magnifying glass.

Dark-Field Microscopes

Pale objects are best observed with dark-field microscopes, which utilize a dark-field stop in the condenser that prevents light from directly entering the objective lens. Instead, light passes into the slide at an oblique angle. Only light rays scattered by the specimen enter the objective lens and are seen, so the specimen appears light against a dark background(Bauman, 2008).

Phase Microscopes

Phase microscopes use a phase plate to retard light rays passing through the specimen so that they are 1/2 wavelength out of phase with neighboring light waves, thereby producing contrast. Phase-contrast microscopes produce sharply defined images in which fine structures can be seen.

Fluorescent Microscopes

Fluorescent microscopes use an ultraviolet (UV) light source to fluoresce objects. Since UV light has a shorter wavelength than visible light, resolution is increased. Contrast is improved because fluorescing structures are visible against a black background.

Electron Microscopy

Because the shortest wavelength of visible light is about 400 nm, structures closer together than about 200 nm cannot be distinguished using light microscopy.

Staining

Both light and electron microscopy use staining—the coloring of specimens with dyes—to increase contrast.

Preparing Specimens for Staining

Preparing specimens for staining involves making a thin film of organisms—or smear—of the specimen on a slide, and then either passing the slide through a flame (heat fixation) or applying a chemical (chemical fixation) to attach the specimen firmly to the slide.

Principles of Staining

The colored portion of a dye, known as the chromophore, typically binds to chemicals via covalent, ionic, or hydrogen bonds. Anionic chromophores called acidic dyes or cationic chromophores known as basic dyes are used to stain different portions of an organism to aid viewing and identification.

Simple Stains

Simple stains are composed of a single basic dye such as crystal violet and involve no more than soaking the smear in the dye and rinsing.

Differential Stains

Differential stains use more than one dye so that different cells, chemicals, or structures can be distinguished. The Gram stain differentiates between purple-staining Gram-positive cells and pink-staining Gram-negative cells.

Special Stains

Acidic dyes are repulsed by the negative charges on the surface of cells and therefore do not stain them. Such dyes that stain the background and leave the cells colorless are called negative (or capsule) stains. Flagellar stains bind to flagella, increase their diameter, ...