CHAPTER 3



CHAPTER 3

MICROSCOPY AND STAINING

( Chapter Overview

Microbiology as a science could not begin until the development of a microscope powerful enough to observe microorganisms. Furthermore, it required an inquisitive mind to observe these "animalcules" and record their appearance and behavior. Once they were discovered, it still required many years for the connection between microbes and disease to be made. The beginning of the process was the development of the microscope.

The ability to magnify and visualize any object is based on many fundamental principles of light. These principles are often overlooked even though an understanding of these principles is essential to an understanding of some of the more complex microscopic techniques, such as phase-contrast microscopy, dark-field microscopy, and the electron microscope.

Following the presentation of the principles of microscopy, a survey of the most commonly used light microscopes in microbiology is provided. This survey is followed by a thorough review of electron microscopes, whose advent helped open and/or improve such fields as virology, immunology, histology, and cell biology.

The last section is devoted to a presentation of the principles of some of the most important stains used in microbiology, including the Gram, acid-fast, and endospore stains. Much of this last material can and should be reemphasized and demonstrated in the laboratory.

( Chapter Objectives

• Describe briefly the evolution of microscopic instruments and their relationship to the progress made in microbiology.

• List the appropriate metric units that are used to measure bacteria, viruses, fungi, protozoa, and helminths.

• Contrast the differences and relationships among the following: wavelength, resolution, and numerical aperture.

• Discuss the relationship of microbiology to the following properties of light: transmission, absorption, fluorescence, luminescence, phosphorescence, reflection, refraction, and diffraction.

• Locate and describe the function of each major part of a compound light microscope.

• List and describe the special adaptations of bright-field, dark-field, phase-contrast, differential interference contrast, and fluorescence (UV) microscopy.

• Briefly explain the principles of transmission, scanning tunneling, and scanning electron microscopy, noting especially how each differs from light microscopy.

• Compare the advantages and limitations of electron microscopy with those of light microscopy.

• List and describe the techniques used to prepare and heighten contrast in specimens to be viewed with a light microscope.

• Define the term stain; list and describe the features and purpose of at least four common types of stains used in microbiology.

• List the steps in the Gram stain and explain each of their functions and results.

( Web Destinations

•

Microbiology staining techniques

•

Nikon microscopy web site

•

Microscopy tutorial

•

On-line microbiology textbook

•

The Microscope Mania website is directed towards younger learners but still has good information about microscopes and how they can be used.

•

Secret Worlds: The Universe Within is an interesting website that starts out very large (the universe) and goes very small (atoms) by powers of 10.

•

Olympic digital microscope information, tutorials, and image gallery.

( Discussion Topics

• Some bacteria found deep in the ocean are phosphorescent. What value does there seem to be for these microbes, since there is no light down that deep?

• Why are heavy metals such as gold used in staining specimens for electron microscope work?

( Track It Down

• What wavelengths of visible light are used in microscopy? How are those wavelengths selected?

• Can viruses be identified with fluorescent dyes? Which viruses are identified and how do the dyes work?

( Chapter Outline

I. Historical Microscopy

A. Early history

B. Anton van Leeuwenhoek

II. Principles of Microscopy

A. Metric units

1. Micrometer

2. Nanometer

3. Angstrom

B. Properties of light: wavelength and resolution

1. Wavelength

2. Electromagnetic spectrum

3. Resolution

a. Factors affecting resolution

b. Resolving power

c. Numerical aperture

C. Properties of light: light and objects

1. Reflection

2. Transmission

3. Absorption

a. Luminescence

b. Fluorescence

c. Phosphorescence

5. Refraction

a. Index of refraction

b. Effect of immersion oil

6. Diffraction

III. Light Microscopy

A. Basic features

B. The compound light microscope

1. Basic parts

a. Eyepieces (ocular lens)

b. Base

c. Condenser

d. Iris diaphragm

e. Objective lens

f. Body tube

g. Mechanical stage

h. Adjustment knobs

2. Magnification

a. Calculation

b. Parfocal

3. Microscopic measurement

C. Dark-field microscopy

D. Phase-contrast microscopy

E. Nomarski (differential interference contrast) microscopy

E. Fluorescence microscopy

1. Fluorescent dyes

2. Fluorescent antibody staining

F. Confocal microscopy

G. Digital microscopy

IV. Electron Microscopy

A. Basic features

B. Transmission electron microscope (TEM)

1. General features

2. Shadow casting

a. Freeze-fracturing

b. Freeze-etching

3. Electron micrographs

C. Scanning electron microscope

1. General features

2. Scanning electron micrographs

D. Scanning Tunneling Electron Microscope

1. General features

2. Atomic force microscope

V. Techniques of Light Microscopy

A. Preparation of specimens for the light microscope

1. Wet mounts

2. Hanging drop

3. Smears

4. Heat fixation

B. Principles of staining

1. Stains

a. Cationic (basic) dyes

b. Anionic (acidic) dyes

2. Types of stains

a. Simple stains

b. Differential stains

3. The Gram stain

a. Basic steps

b. Gram-variable reactions

4. The Ziehl-Neelsen Acid-Fast Stain

5. Special staining procedures

a. Negative staining

b. Flagellar staining

c. Endospore staining

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download