Chapter 4

Microscopy,
Staining, and
Classification

Lecture prepared by Mindy Miller-Kittrell

North Carolina State University
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Microscopy and Staining

ANIMATION Microscopy and Staining: Overview

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Table 4.1 Metric Units of Length
Microscopy

• General Principles of Microscopy

– Wavelength of radiation
– Magnification
– Resolution
– Contrast

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Figure 4.1 The electromagnetic spectrum

400 nm 700 nm

Visible light

X UV Infra- Micro- Radio waves and

Gamma rays
rays light red wave Television
Increasing wavelength
10–12m 10–8m 10–4m 100m 103m

Crest One wavelength

Trough
Increasing resolving power
Figure 4.2 Light refraction and image magnification by a convex glass lens-overview

Light

Air Glass

Focal point

Specimen Convex Inverted,

lens reversed, and
enlarged
image
Figure 4.3 The limits of resolution of the human eye and of various types of microscopes
Diameter Typical bacteria
of DNA Ribosomes and archaea Flea

Large
protozoan
Atoms Proteins Viruses Chloroplasts (Euglena) Chicken
egg

Amino Human red

acids Mitochondrion blood cell

Scanning tunneling microscope

(STM) 0.01 nm–10 nm

Transmission electron microscope (TEM) Unaided human eye

0.078 nm–100 µm 200 µm–

Scanning electron microscope (SEM)

0.4 nm–1 mm

Atomic force Compound light microscope (LM)

microscope (AFM) 200 nm–10 mm
1 nm–10 nm
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

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Microscopy

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

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Microscopy

• Light 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 (objective lens X ocular
lens)
– Most have condenser lens (direct light
through specimen)
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Figure 4.4 A bright-field, compound light microscope-overview

Ocular lens Line of vision

Remagnifies the image formed by
the objective lens
Body Ocular lens
Transmits the image from the
objective lens to the ocular lens
Path of light
using prisms
Arm Prism
Objective lenses Body
Primary lenses that
magnify the specimen Objective
Stage lenses
Holds the microscope
Specimen
slide in position
Condenser
Condenser
Focuses light
lenses
through specimen
Diaphragm Illuminator
Controls the amount of
light entering the condenser
Illuminator
Light source
Coarse focusing knob
Moves the stage up and
down to focus the image
Fine focusing knob
Base
Figure 4.5 The effect of immersion oil on resolution-overview

Microscope Microscope
objective Lenses objective

Refracted light More light

rays lost to lens enters lens
Immersion oil
Glass cover slip Glass cover slip

Slide Slide

Specimen Light source Light source

Without immersion oil With immersion oil
Microscopy

• Light 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

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Figure 4.6 The light path in a dark-field microscope
Objective

Light refracted
by specimen

Light unrefracted
by specimen

Specimen

Condenser

Dark-field stop Dark-field stop

Microscopy

• Light Microscopy
– Phase microscopes
– Examine living organisms or specimens that
would be damaged/altered by attaching them to
slides or staining
– Contrast is created because light waves are out of
phase
– Two types
– Phase-contrast microscope
– Differential interference contrast microscope

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Figure 4.7 Principles of phase microscopy-overview

Rays in phase Rays out of phase

Phase plate

Bacterium
Ray deviated by Deviated ray
specimen is 1/4 is now 1/2
wavelength out wavelength
of phase. out of phase.
Figure 4.8 Four kinds of light microscopy-overview
Nucleus

Bacterium

Bright field Dark field

Phase contrast Nomarski

Microscopy

• Light Microscopy
– Fluorescent microscopes
– Direct UV light source at specimen
– Specimen radiates energy back as a visible
wavelength
– UV light increases resolution and contrast
– Some cells are naturally fluorescent; others must
be stained
– Used in immunofluorescence to identify
pathogens and to make visible a variety of
proteins

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Figure 4.9 Fluorescent microscopy-overview
Figure 4.10 Immunofluorescence-overview

Antibodies Fluorescent dye

Antibodies
carrying dye
Bacterium
Cell-surface
antigens

Bacterial cell with

bound antibodies
carrying dye
Microscopy

• Light Microscopy
– Confocal microscopes
– Use fluorescent dyes
– Use UV lasers to illuminate fluorescent chemicals
in a single plane
– Resolution increased because light passes
through pinhole aperture
– Computer constructs 3-D image from digitized
images

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Microscopy

ANIMATION Light Microscopy

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Microscopy

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

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Figure 4.11 A transmission electron microscope (TEM) -overview

Light microscope Column of transmission

(upside down) electron microscope

Lamp Electron gun

Condenser Condenser lens

lens (magnet)
Specimen Specimen

Objective lens Objective lens

(magnet)

Eyepiece Projector lens

(magnet)

Final image Final image on

seen by eye fluorescent screen
Figure 4.12 Scanning electron microscope (SEM)

Electron gun

Magnetic Beam
lenses deflector coil

Scanning
Primary
circuit
electrons

Secondary
electrons
Photo-
Specimen multiplier Monitor
Specimen Detector
holder

Vacuum
system
Figure 4.13 SEM images-overview
Microscopy

ANIMATION Electron Microscopy

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Microscopy

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

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Figure 4.14 Probe microscopy-overview

DNA Enzyme
Staining

• Principles of Staining
– 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
– Basic dyes stain acidic structures

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Figure 4.15 Preparing a specimen for staining

Spread culture in
thin film over slide

Air dry

Pass slide through

flame to fix it
Staining

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

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Figure 4.16 Simple stains-overview
Figure 4.17 The Gram staining procedure-overview

Slide is flooded with crystal Slide is flooded with iodine

violet for 1 min, then rinsed for 1 min, then rinsed with
with water. water.

Result: All cells are stained Result: Iodine acts as a

purple. mordant; all cells remain
purple.

Slide is flooded with solution Slide is flooded with safranin

of ethanol and acetone for for 1 min, then rinsed with
10–30 sec, then rinsed with water and blotted dry.
water.
Result: Gram-positive cells
Result: Smear is decolorized; remain purple, Gram-negative
Gram-positive cells remain cells are pink.
purple, but Gram-negative
cells are now colorless.
Figure 4.18 The Ziehl-Neelsen acid-fast stain
Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis
Staining

• Differential Stains
– Histological stain
– Two popular stains for histological specimens
– Gomori methenamine silver (GMS)
– Hematoxylin and eosin (HE)

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Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae

Bacterium

Capsule

Background
stain
Figure 4.21 Flagellar stain of Proteus vulgaris

Flagella
Staining

• Staining for Electron Microscopy

– Transmission electron microscopy uses
chemicals containing heavy metals
– Absorb electrons
– Stains may bind molecules in specimens or the
background

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Staining

ANIMATION Staining

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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

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Classification and Identification of Microorganisms

• Linnaeus and Taxonomic Categories

– Linnaeus
– Classified organisms based on characteristics
in common
– Organisms that can successfully interbreed
called species
– Used binomial nomenclature in his system
– Linnaeus proposed only two kingdoms

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Figure 4.22 Levels in a Linnaean taxonomic scheme-overview
Domain

Bacteria Archaea Eukarya

Kingdom

Animalia Plantae Fungi

Phylum

Chordata Arthropoda Platyhelminthes Nematoda

(vertebrates) (joint-legged animals) (tapeworm) (unsegmented roundworms)

Class

Insecta Crustacea Arachnida

Order

Scorpionida Acariformes Parasitiformes Araneida

(mites) (mites and ticks)

Family

Ixodidae Argasidae
(hard ticks) (soft ticks)

Genus

Dermacentor Ixodes Rhipicephalus

Species

l. scapularis l. pacificus l. ricinus

(deer tick) (black-eyed tick) (castor bean tick)
Classification and Identification of Microorganisms

• Linnaeus and Taxonomic Categories

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

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Classification and Identification of Microorganisms

• Linnaeus and Taxonomic Categories

– Linnaeus’s goal was to classify organisms to
catalogue them
– Modern goal is to understand relationships
among groups of organisms
– Reflect phylogenetic hierarchy
– Emphasis on comparison of organisms’
genetic material
– Led to proposal to add domain

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Classification and Identification of Microorganisms

• 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 differ by other
characteristics

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Classification and Identification of Microorganisms

• Taxonomic and Identifying Characteristics

– Physical characteristics
– Biochemical tests
– Serological tests
– Phage typing
– Analysis of nucleic acids

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Figure 4.23 Two biochemical tests for identifying bacteria-overview
Gas bubble Inverted tubes to trap gas

Hydrogen No
sulfide hydrogen
Acid with gas Acid with no gas Inert produced sulfide
Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system

Wells
Figure 4.25 An agglutination test, one type of serological test-overview

Negative result Positive result

Negative result Positive result

Figure 4.26 Phage typing

Bacterial lawn

Plaques
Classification and Identification of Microorganisms

• 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

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Figure 4.27 Use of a dichotomous taxonomic key-overview

Gram-positive
cells?
No Yes
Rod-shaped Gram-positive
cells? bacteria
No Yes
Cocci and Can
pleomorphic tolerate
bacteria oxygen?
No Yes
Obligate Ferments
anaerobes lactose?
No Yes
Can use citric
Non-lactose- acid (citrate)
fermenters as sole carbon
source?
No Yes
Produces gas Produces hydrogen
from glucose? sulfide gas?
No Yes No Yes
Produces
Shigella Escherichia Salmonella
acetoin?
No Yes
Citrobacter Enterobacter
Classification and Identification of Microorganisms

ANIMATION Dichotomous Key: Overview

ANIMATION Dichotomous Key: Sample with Flowchart

ANIMATION Dichotomous Key: Practice

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