Microscopy, Staining, & Classification

7/29/2019 Microscopy, Staining, & Classification

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© 2012 Pearson Education Inc.

Lecture prepared by Mindy Miller-Kittrell North Carolina State University

Chapter 3

Microscopy,

Staining, and

Classification



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




Figure 4.1 The electromagnetic spectrum

Visible light

Micro-waveInfra-redUVlightXrays Radio waves andTelevision

One wavelength

400 nm 700 nm

Gamma rays

Increasing wavelength

Crest

100m 103m10 –4m10 –8m

Increasing resolving power

Trough

10 –12m



Figure 4.2 Light refraction and image magnification by a convex glass lens-overview

Convexlens

Inverted,reversed, andenlarged

image

Focal point

Specimen

Glass

Light

Air



Chicken

egg

Human red

blood cell

Large

protozoan

(Euglena )Chloroplasts

FleaTypical bacteria

and archaeaDiameter

of DNA

VirusesProteins

Ribosomes

Amino

acids

Atoms

Scanning tunneling microscope(STM) 0.01 nm –10 nm

Scanning electron microscope (SEM)

0.4 nm –1 mm

Transmission electron microscope (TEM)

0.078 nm –100 µm

Atomic force

microscope (AFM)1 nm –10 nm

Compound light microscope (LM)

200 nm –

10 mm

Unaided human eye

200 µm –

Mitochondrion

Figure 4.3 The limits of resolution of the human eye and of various types of microscopes



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




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 intoobjective lens

– Oil immersion lens increases resolution

– Have one or two ocular lenses

– Total magnification (objective lens X ocular lens)

– Most have condenser lens (direct lightthrough specimen)




Figure 4.4 A bright-field, compound light microscope-overview

Line of vision

Ocular lens

Path of light

Prism

Body

Specimen

Objectivelenses

Condenser lenses

Illuminator

Ocular lens

Body

Objective lenses

Condenser

Illuminator

Remagnifies the image formed by

the objective lens

Base

Fine focusing knob

Coarse focusing knob

Diaphragm

Stage

Arm

Transmits the image from the

objective lens to the ocular lens

using prisms

Primary lenses that

magnify the specimen

Controls the amount of

light entering the condenser

Focuses light

through specimen

Holds the microscope

slide in position

Light source

Moves the stage up and

down to focus the image



Figure 4.5 The effect of immersion oil on resolution-overview

Microscopeobjective

Refracted lightrays lost to lens

Glass cover slip

Light sourceSpecimen

Slide

Without immersion oil

Glass cover slip

Light source

Slide

Microscopeobjective

More lightenters lens

Lenses

With immersion oil

Immersion oil



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




Light refractedby specimen

Light unrefractedby specimen

Specimen

Condenser

Dark-field stop Dark-field stop

ObjectiveFigure 4.6 The light path in a dark-field microscope



Microscopy


– Phase microscopes

– Examine living organisms or specimens thatwould 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


Fi 4 7 P i i l f h i i



Ray deviated byspecimen is 1/4wavelength outof phase.

Deviated rayis now 1/2wavelengthout of phase.

Bacterium

Rays in phase Rays out of phase

Phase plate

Figure 4.7 Principles of phase microscopy-overview

Fi 4 8 F ki d f li ht i i



Bright field

Bacterium

Nucleus

Phase contrast

Dark field

Nomarski

Figure 4.8 Four kinds of light microscopy-overview



Microscopy


– Fluorescent microscopes

– Direct UV light source at specimen

– Specimen radiates energy back as a visiblewavelength

– UV light increases resolution and contrast

– Some cells are naturally fluorescent; others mustbe stained

– Used in immunofluorescence to identifypathogens and to make visible a variety of proteins


Figure 4 9 Fluorescent microscopy overview



Figure 4.9 Fluorescent microscopy-overview

Figure 4 10 Immunofluorescence overview



Cell-surfaceantigens

Bacterium

Antibodies

Antibodiescarrying dye

Fluorescent dye

Bacterial cell withbound antibodiescarrying dye

Figure 4.10 Immunofluorescence-overview



Microscopy


– Confocal microscopes

– Use fluorescent dyes

– Use UV lasers to illuminate fluorescent chemicalsin a single plane

– Resolution increased because light passes

through pinhole aperture

– Computer constructs 3-D image from digitizedimages




Microscopy


ANIMATION Light Microscopy

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Microscopy

• Electron Microscopy

– Light microscopes cannot resolve structurescloser 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


Figure 4 11 A transmission electron microscope (TEM) -overview



Figure 4.11 A transmission electron microscope (TEM) overview

Light microscope

(upside down)Column of transmission

electron microscope

Condenser lens

(magnet)

Lamp

Condenser

lens

Objective lens

Eyepiece

Final image

seen by eye

Final image on

fluorescent screen

Projector lens

(magnet)

Objective lens

(magnet)

Specimen Specimen

Electron gun

Figure 4.12 Scanning electron microscope (SEM)



Magnetic

lenses

Electron gun

Primary

electrons

Secondary

electrons

Specimen

holder

Vacuum

system

SpecimenPhoto-

multiplier

Detector

Scanning

circuit

Monitor

Beam

deflector coil

Figure 4.12 Scanning electron microscope (SEM)

Figure 4 13 SEM images-overview



Figure 4.13 SEM images overview

Mi



Microscopy


ANIMATION Electron Microscopy

Mi

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Microscopy

• Probe Microscopy

– Magnifies more than 100,000,000X

– Two types

– Scanning tunneling microscopes – Atomic force microscopes


Figure 4.14 Probe microscopy-overview



gu e obe c oscopy o e e

EnzymeDNA

St i i




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

Figure 4.15 Preparing a specimen for staining



Spread culture inthin film over slide

Pass slide throughflame to fix it

Air dry

g p g p g

St i i



• Simple Stains

• Differential Stains

– Gram stain

– Acid-fast stain – Endospore stain

– Histological stain

• Special Stains

– Negative (capsule) stain – Flagellar stain


Staining

Figure 4.16 Simple stains-overview



Figure 4.17 The Gram staining procedure-overview



Slide is flooded with crystalviolet for 1 min, then rinsedwith water.

Result: All cells are stainedpurple.

Slide is flooded with solutionof ethanol and acetone for 10 –30 sec, then rinsed withwater.

Result: Smear is decolorized;Gram-positive cells remainpurple, but Gram-negativecells are now colorless.

Slide is flooded with safraninfor 1 min, then rinsed withwater and blotted dry.

Result: Gram-positive cellsremain purple, Gram-negativecells are pink.

Slide is flooded with iodinefor 1 min, then rinsed withwater.

Result: Iodine acts as amordant; all cells remainpurple.

Figure 4.18 The Ziehl-Neelsen acid-fast stain



Figure 4.19 Schaeffer-Fulton endospore stain of Baci l lus anthrac is



Staining



Staining

• Differential Stains – Histological stain

– Two popular stains for histological specimens

– Gomori methenamine silver (GMS)

– Hematoxylin and eosin (HE)


Figure 4.20 Negative (capsule) stain of Klebsiel la pneum oniae



Backgroundstain

Bacterium

Capsule

Figure 4.21 Flagellar stain of Proteus vulgar is



Flagella

Staining



Staining

• Staining for Electron Microscopy

– Transmission electron microscopy uses

chemicals containing heavy metals

– Absorb electrons – Stains may bind molecules in specimens or the

background


Staining



Staining


ANIMATION Staining

Classification and Identification of Microorganisms

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– Taxonomy consists of classification,nomenclature, and identification

– Organize large amounts of information

about organisms

– Make predictions based on knowledge of

similar organisms






• 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


Figure 4.22 Levels in a Linnaean taxonomic scheme-overview



l . scapularis (deer tick) l . paci ficus (black-eyed tick) l . ricinus (castor bean tick)

Parasitiformes(mites and ticks)

Acariformes (mites)

Rhipicephalus

Ixodidae (hard ticks)

Dermacentor Ixodes

Argasidae (soft ticks)

AraneidaScorpionida

ArachnidaCrustaceaInsecta

Chordata(vertebrates)

Arthropoda(joint-legged animals)

Platyhelminthes(tapeworm)

Nematoda(unsegmented roundworms)

Animalia Plantae Fungi

Bacteria Archaea Eukarya

Family

Class

Kingdom

Order

Phylum

Domain

Genus

Species





• Linnaeus and Taxonomic Categories

– Linnaeus proposed only two kingdoms

– Later taxonomic approach based on five

kingdoms – Animalia, Plantae, Fungi, Protista, and

Prokaryotae






• 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






• Domains

– Carl Woese compared nucleotide sequences of

rRNA subunits

– Proposal of three domains as determined byribosomal nucleotide sequences

– Eukarya, Bacteria, and Archaea

– Cells in the three domains differ by other

characteristics






• Taxonomic and Identifying Characteristics

– Physical characteristics

– Biochemical tests

– Serological tests – Phage typing

– Analysis of nucleic acids


Gas bubble Inverted tubes to trap gas

Figure 4.23 Two biochemical tests for identifying bacteria-overview



Gas bubble Inverted tubes to trap gas

Acid with gas Acid with no gas Inert

Hydrogen

sulfide

produced

No

hydrogen

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

Negative result

Positive result

Positive result

Figure 4.26 Phage typing



Bacterial lawn

Plaques




C ss c o d de c o o c oo g s s

• 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


Figure 4.27 Use of a dichotomous taxonomic key-overview



Gram-positivecells?

Rod-shapedcells? Gram-positivebacteria

Obligateanaerobes

Fermentslactose?

Cocci andpleomorphicbacteria

Cantolerateoxygen?

Can use citricacid (citrate)as sole carbonsource?

Non-lactose-fermenters

Produces gasfrom glucose?

Produces hydrogensulfide gas?

Producesacetoin?

Salmonel la

Enterobacter Citrobacter

Escher ichia Shigel la

YesNo

YesNo YesNo

YesNo

YesNo

YesNo

YesNo

YesNo




g

ANIMATION Dichotomous Key: Overview

ANIMATION Dichotomous Key: Sample with Flowchart

ANIMATION Dichotomous Key: Practice

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Microscopy, Staining, & Classification

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