Microscopy, Staining, and Classification

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PowerPoint® Lecture

Presentations prepared by

Mindy Miller-Kittrell,

North Carolina

State University

C H A P T E R

© 2014 Pearson Education, Inc.

Microscopy,

Staining, and

Classification

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Figure 3.4 Approximate size of various types of cells.

Red Blood Cells

~10 um

15001.5mm

1500 um

Width of penny

=


Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of

various types of microscopes.

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• Magnification, the ratio of an object’s image

size to its real size

• Resolution, the measure of the clarity of the image, or

the minimum distance of two distinguishable points

• Contrast, visible differences in brightness between parts

of the sample

Official definitions


Microscopy

• General Principles of Microscopy

• Magnification (enlarge)

• Resolution (tell apart 2 objects close together)

• Contrast (Differences in intensity between two objects)


Smaller the wavelength

smaller the object you can see

(small objects need small hands)

Think about giant fingers and texting

General rule for any microscopy/detector

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Figure 4.1 The electromagnetic spectrum.

VIBGYOR

Smaller the object = Use smaller wavelength


Microscopy

• Microscopes are used to visualize cells

• In a light microscope (LM), visible light is passed

through a specimen and then through glass

lenses

• Glass lenses focuses light and enlarges and

resolves objects


Light

Air Glass

Focal point

Specimen Convexlens

Inverted,

reversed, and

Enlarged

image

5 50

Magnification = 50/5 = 10X

Lenses refract (bend) the light, so that the image is magnified

Magnify using lenses

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Microscopy

• Light Microscopy

• Bright-field microscopes

• Simple – single lense

• Contain a single magnifying lens

• Similar to magnifying glass

• Leeuwenhoek used simple microscope to observe

microorganisms


Microscopy


• Bright-field microscopes

• Compound – multiple lenses

• More than one - Series of lenses for magnification

• Light passes through specimen into objective lens

• Have one or two ocular lenses

• Total magnification = magnification of objective lens X

magnification of ocular lens

• E.g. 10 times X 10 times = 100 times


Figure 4.4 A bright-field, compound light microscope.

Coarse focusing knob

Moves the stage up and

down to focus the image

Illuminator

Light source

Diaphragm

Controls the amount of

light entering the condenser

Condenser

Focuses light

through specimen

Stage

Holds the microscope

slide in position

Objective lenses

Primary lenses that

magnify the specimen

Body

Transmits the image from the

objective lens to the ocular lens

using prisms

Ocular lens

Remagnifies the image formed by

the objective lens

Line of vision

Ocular lens

Path of light

Prism

Body

Objective

lenses

Specimen

Condenser

lenses

Illuminator

Fine focusing knob

Base

Arm

objective lens

occular lens

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Figure 4.5 The effect of immersion oil on resolution.

Glass cover slip

Slide

Specimen Light source

Without immersion oil

Lenses

Immersion oil

Glass cover slip

Slide

Light source

With immersion oil

Microscope

objective

Refracted light

rays lost to lens

Microscope

objective

More light

enters lens

Special type of objective lens = oil immersion lens

Increases magnification and resolution


Microscopy

• General Principles of Microscopy

• Contrast

• Differences in intensity between two objects, or an object

and its background

Increase contrast by staining


Figure 6.3ab

Brightfield(stained specimen)

50 µ

m

Staining increases

contrast

Brightfield(unstained specimen)

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Figure 4.16 Simple stains.


Figure 4.15 Preparing a specimen for staining.


Microbe Naming Rules

Genus specific epithetGenus specific epithet

Genus specific epithet

Genus specific epithet

X

X

Escherichia coli

Escherichia coli

E. coli

E. coli

G. specific epithet

G. specific epithet

Genus specific epithetX

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Figure 3.12 Bacterial shapes and arrangements.

2) strepto

1) coccus 2) bacillus

3 Shapes

3) spirillum

2 main arrangements

1) staphylo


Microbe Naming Rules

Based on shape and arrangement

Arrangement+shape species

Staphylococcus species

Streptobacillus species

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cocci bacilli

arrangement

shape

Streptococci

Staphylococci

Streptobacilli


Microscopy


• 1) Bright-field microscopes

• Simple/ Compound


Nucleus

Bacterium

Bright field Dark field

Figure 4.8 Four kinds of 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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Objective

Light refracted

by specimen

Light unrefracted

by specimen

Specimen

Dark-field stop

Condenser

Dark-field stop

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


Microscopy


• 3) Phase microscopes

• Used to examine living organisms or specimens that would

be damaged/altered by attaching them to slides or staining

• Light rays in phase produce brighter image, while light rays

out of phase produce darker image

• Contrast is created because light waves are out of phase


Rays in phase Rays out of phase

Phase plate

Bacterium Ray deviated by

specimen is 1/4

wavelength out

of phase.

Deviated ray

is now 1/2

Wavelength

out of phase.

Figure 4.7 Principles of phase microscopy.

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Nucleus

Phase contrast Nomarski

Figure 4.8 Four kinds of light microscopy.

Two types of Phase microscopes

1) Phase-contrast microscope

2) Differential interference contrast microscope

aka Nomarski


Microscopy


• Fluorescent microscopes

• Direct UV light source at specimen

• Specimen radiates energy back as a longer, 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


Figure 4.9 Fluorescence microscopy.

Can help you look through clutter

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Figure 4.10 Immunofluorescence.

Antibodies

Bacterium

Cell-surfaceantigens

Bacterial cell withbound antibodiescarrying dye

Fluorescent dye

Antibodiescarrying dye

Can help you target specific structures


Microscopy


• Confocal microscopes

• Use fluorescent dyes

• Use UV lasers to illuminate fluorescent chemicals in a

single plane

• Resolution increased because emitted light passes

through pinhole aperture

• Computer constructs 3-D image from digitized images


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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


Figure 4.11 A transmission electron microscope (TEM).

Lamp

Light microscope(upside down)

Column of transmissionelectron microscope

Lamp

Condenser

lens

Specimen

Objective lens

Eyepiece

Final image

seen by eye

Electron gun

Specimen

Objective lens

(magnet)

Projector lens

(magnet)

Final image on

fluorescent screen

needs vacuum

need sections

Live ?


Electron gun

Magneticlenses

Primaryelectrons

Secondaryelectrons

Specimen

Specimenholder

Vacuumsystem

Beamdeflector coil

Scanningcircuit

Photo-multiplier

Detector

Monitor

Figure 4.12 Scanning electron microscope (SEM).

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Figure 4.13 SEM images.


Microscopy

• Probe Microscopy

• Magnifies more than 100,000,000 times

• Two types

• Scanning tunneling microscopes

• Atomic force microscopes


EnzymeDNAFigure 4.14 Probe microscopy.

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Staining

• Principles of Staining

• Dyes used as stains are usually salts

• Chromophore is the colored portion of the dye

• Acidic dyes (negatively charged) stain alkaline

structures

• Basic dyes (positively charged) stain acidic structures

• Basic dyes are more commonly used since inside of most

cells is negatively charged


Staining

• Simple stains

• Differential stains

• Gram stain

• Acid-fast stain

• Endospore stain

• Special stains

• Negative (capsule) stain

• Flagellar stain

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Bacterium

Capsule

Background

stain


Rhodospirillum rubrum

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Flagella

Figure 4.21 Flagellar stain of Proteus vulgaris.



Gram Staining

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Gram Staining Mechanism


Staining for Electron Microscopy

• Chemicals containing heavy metals used for

transmission electron microscopy


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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Microorganisms

• Linnaeus and Taxonomic Categories

• Linnaeus

• His system classified organisms based on characteristics

in common

• Grouped organisms that can successfully interbreed into

categories called species

• Used binomial nomenclature



Microorganisms

• Binomial nomenclature

humans belong to the genus Homo and within

this genus to the species Homo sapiens.

naming species of living things by giving

each species a name composed of two

parts

noun-genus adjective-specific epithet



Microorganisms

• Linnaeus and Taxonomic Categories

• Linnaeus's goal was classifying organisms to catalog them

• Linnaeus - 2 kingdoms

• Later on - 5 kingdoms

• Later on 3 domains (genetic material)

• Goal of modern taxonomy is to reflect phylogenetic hierarchy

• understanding relationships among organisms

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Figure 4.22 Levels in a Linnaean taxonomic scheme.

So

Good

For

Our

Class

P

K

D



Microorganisms

• Domains

• Carl Woese compared nucleotide sequences of rRNA

subunits

• Proposal of three domains as determined by ribosomal

nucleotide sequences

• Eukarya, Bacteria, and Archaea



Microorganisms

• Taxonomic and Identifying Characteristics

• Physical characteristics

• Biochemical tests

• Serological tests

• Phage typing

• Analysis of nucleic acids

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Microorganisms


• Physical characteristics

• Can often be used to identify microorganisms

• Protozoa, fungi, algae, and parasitic worms can often

be identified based only on their morphology

• Some bacterial colonies have distinct appearance

used for identification – fig 6.8 pg 173


Shape

Circular Rhizoid Irregular Filamentous Spindle

Margin

Elevation

Size

Texture

Appearance

Pigmenta-

tion

Optical

property

Entire Undulate Lobate Curled Filiform

Flat Raised Convex Pulvinate Umbonate

Punctiform Small Moderate Large

Glistening (shiny) or dull

Nonpigmented (e.g., cream, tan, white)

Pigmented (e.g., purple, red, yellow)

Opaque, translucent, transparent

Smooth or rough

Colony

Figure 6.8 Characteristics of bacterial colonies.


No

hydrogen

sulfide

Hydrogen

sulfide

producedAcid with gas Acid with no gas Inert

Gas bubble Inverted tubes to trap gas

Figure 4.23 Two biochemical tests for identifying bacteria.

Biochemical tests

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Wells

Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system.


Figure 4.25 An agglutination test, one type of serological test.


Bacterial lawn

Plaques

Figure 4.26 Phage typing.

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Virus

Bacterium

Virusesassemblinginside cell

Figure 1.9 A colorized electron microscope image of viruses infecting a bacterium.

Viruses (acellular)



Microorganisms


• Analysis of nucleic acids

• Nucleic acid sequence can be used to classify and

identify microbes

• Prokaryotic taxonomy now includes the G + C content of

an organism's DNA



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.

dichotomous taxonomic key

dichotomous taxonomic key activity

Microscopy, Staining, and Classification

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