Microscopy

Copyright © 2011 Pearson Education Inc.
Units of Measurement
Table 4.1

History of the Microscope
•1590 –first compound microscope

• 1655 – Robert Hooke
used a compound
microscope to observe
pores in cork
– He called them
“cells”

• Antonie van
Leeuwenhoek
–1st
to see
single-celled
organisms in
pond water

Microscopy
• General Principles of Microscopy
– Wavelength of radiation
– Magnification
– Resolution
– Contrast

The electromagnetic spectrum
Figure 4.1

What is magnification?
Magnification is defined by the
magnification by the objective
x
the magnification by eyepiece
BUT maximum magnification does not mean maximum resolution!

wavelength of visible light – 400 nm
detail smaller than 200nm (approximately half the wavelength of light)
cannot be resolved by the light microscope
400 nm
200 nm
25 nm
Rule: limit of resolution is half the wavelength
800nm
220
nm

Light refraction and image magnification
Figure 4.2

The limits of resolution
Figure 4.3

Microscope Resolution
• ability of a lens to separate or distinguish small objects that are close
together
• wavelength of light used is major factor in resolution
shorter wavelength ⇒ γρεατερ ρεσολυτιον

•working distance
— distance between the front surface of lens and
surface of cover glass or specimen

Microscopy
• General Principles of Microscopy
– Contrast
– Differences in intensity between two objects, or between an
object and background
– Important in determining resolution
– Staining increases contrast
– Use of light that is in phase increases contrast

Contrasting techniques
Brightfield
Darkfield
Phase contrast
DIC
Taken from: http://fig.cox.miami.edu/~cmallery/150/Fallsyll.htm

Microscopy
• Light Microscopy
– Bright-field microscopes
– Simple
– Contain a single magnifying lens
– Similar to magnifying glass
– Leeuwenhoek used simple microscope to observe
microorganisms

Microscopy
– Bright-field microscopes
– Compound
– Series of lenses for magnification
– Light passes through specimen into objective lens
– Oil immersion lens increases resolution
– Have one or two ocular lenses
– Total magnification = magnification of objective lens X
magnification of ocular lens
– Most have condenser lens (direct light through specimen)

A bright-field, compound light microscope
Figure 4.4

The effects of immersion oil on resolution
Figure 4.5

Microscopy
– Dark-field microscopes
– Best for observing pale objects
– Only light rays scattered by specimen enter objective lens
– Specimen appears light against dark background
– Increases contrast and enables observation of more details

The light path in a dark-field microscope
Figure 4.6

Four kinds of light microscopy
Figure 4.8

Microscopy
• Electron Microscopy
– Light microscopes cannot resolve structures closer than 200 nm
– Electron microscopes have greater resolving power and
magnification
– Magnifies objects 10,000X to 100,000X
– Detailed views of bacteria, viruses, internal cellular structures,
molecules, and large atoms
– Two types
– Transmission electron microscopes
– Scanning electron microscopes

A transmission electron microscope (TEM)
Figure 4.11

Scanning electron microscope (SEM)
Figure 4.12

SEM images
Figure 4.13

Microscopy
• Probe Microscopy
– Magnifies more than 100,000,000 times
– Two types
– Scanning tunneling microscopes
– Atomic force microscopes

Probe microscopy
Figure 4.14

Staining
• Increases contrast and resolution by coloring specimens with stains/dyes
• Smear of microorganisms (thin film) made prior to staining
• Microbiological stains contain chromophore
• Acidic dyes stain alkaline structures; more commonly, basic dyes stain
acidic structures

Preparing a specimen for staining
Figure 4.15

Staining
• Simple stains
• Differential stains
– Gram stain
– Acid-fast stain
– Endospore stain
• Special stains
– Negative (capsule) stain
– Flagellar stain

Simple stains
Figure 4.16

The Gram staining procedure
Figure 4.17

Ziehl-Neelsen acid-fast stain
Figure 4.18

Schaeffer-Fulton endospore stain
Figure 4.19

Negative (capsule) stain
Figure 4.20

Flagellar stain
Figure 4.21

Staining
• Staining for Electron Microscopy
– Chemicals containing heavy metals used for transmission
electron microscopy
– Stains may bind molecules in specimens or the background

Staining
Animation: Staining

Classification and Identification of Microorganisms
• Taxonomy consists of classification, nomenclature, and identification
• Organize large amounts of information about organisms
• Make predictions based on knowledge of similar organisms

• Linnaeus, Whittaker, and Taxonomic Categories
– Linnaeus
– System classified organisms based on characteristics in
common
– Grouped organisms that can successfully interbreed into
categories called species
– Used binomial nomenclature in his system

Levels in Linnaean taxonomic scheme
Figure 4.22

– Whittaker
– Linnaeus proposed only two kingdoms
– Whittaker proposed taxonomic approach based on five
kingdoms
– Animalia, Plantae, Fungi, Protista, and Prokaryotae

Whittaker’s five-kingdom taxonomic scheme
Figure 4.23

– Linnaeus’s goal was classifying organisms to catalogue them
– Modern goal is understanding relationships among groups of
organisms
– Goal of modern taxonomy is to reflect phylogenetic hierarchy
– Greater emphasis on comparisons of organisms’ genetic
material led to proposal to add domain

• Domains
– Carl Woese compared nucleotide sequences of rRNA subunits
– Proposal of three domains as determined by ribosomal
nucleotide sequences
– Eukarya, Bacteria, and Archaea
– Cells in the three domains also differ with respect to many other
characteristics

• Taxonomic and Identifying Characteristics
– Physical characteristics
– Biochemical tests
– Serological tests
– Phage typing
– Analysis of nucleic acids

Two biochemical tests for identifying bacteria
Figure 4.24

One tool for the rapid identification of bacteria
Figure 4.25

An agglutination test, one type of serological test
Figure 4.26

Phage typing
Figure 4.27

• Taxonomic Keys
– Dichotomous keys
– Series of paired statements where only one of two “either/or”
choices applies to any particular organism
– Key directs user to another pair of statements, or provides name
of organism

Use of dichotomous taxonomic key
Figure 4.28

Animation: Dichotomous Keys: Overview

Microscopy

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