Microbiology Ch 04 lecture_presentation

PowerPoint® Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University

C H A P T E R

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Microscopy, Staining, and Classification

4

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Microscopy and Staining: Overview

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Units of Measurement

• Tell Me Why• Why do scientists use metric rather than English units?

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Microscopy

• Microscopy: the use of light or electrons to

magnify objects

• Science of microscopy begun by Antoni van

Leeuwenhoek

• Various types of light and electron microscopes

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Microscopy

• General Principles of Microscopy• Wavelength of radiation

• Magnification

• Resolution

• Contrast

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Figure 4.1 The electromagnetic spectrum.

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

Light

Air Glass

Focal point

Specimen Convexlens

Inverted,reversed, andenlargedimage

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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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Microscopy

• General Principles of Microscopy• Contrast

• Differences in intensity between two objects or between

an object and its 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 = magnification of objective lens x magnification of ocular lens

• Most have condenser lens (direct light through specimen)

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Figure 4.4 A bright-field, compound light microscope.

Coarse focusing knobMoves the stage up anddown to focus the image

IlluminatorLight source

DiaphragmControls the amount oflight entering the condenser

CondenserFocuses lightthrough specimen

StageHolds the microscopeslide in position

Objective lensesPrimary lenses thatmagnify the specimen

BodyTransmits the image from theobjective lens to the ocular lensusing prisms

Ocular lensRemagnifies the image formed bythe objective lens

Line of vision

Ocular lens

Path of light

Prism

Body

Objectivelenses

Specimen

Condenserlenses

Illuminator

Fine focusing knobBase

Arm

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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

Microscopeobjective

Refracted lightrays lost to lens

Microscopeobjective

More lightenters lens

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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 refractedby specimen

Light unrefractedby specimen

Specimen

Dark-field stop

Condenser

Dark-field stop

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Microscopy

• Light Microscopy• 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, whereas light rays out of phase produce darker image

• Contrast is created because light waves are out of phase

• Two types

• Phase-contrast microscope

• Differential interference contrast microscope

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Rays in phase Rays out of phase

Phase plate

Bacterium Ray deviated byspecimen is 1/4wavelength outof phase.

Deviated rayis now 1/2wavelengthout of phase.

Figure 4.7 Principles of phase microscopy.

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Figure 4.8 Four kinds of light microscopy.

Nucleus

Bacterium

Bright field Dark field

Phase contrast Nomarski

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Microscopy

• Light 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

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Figure 4.9 Fluorescence microscopy.

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Antibodies

BacteriumCell-surfaceantigens

Bacterial cell withbound antibodiescarrying dye

Fluorescent dye

Antibodiescarrying dye

Figure 4.10 Immunofluorescence.

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Microscopy

• Light Microscopy• Confocal microscopes

• Use UV lasers to illuminate fluorescent chemicals in a

single plane

• Resolution increased because emitted light passes

through pinhole aperture

• Each image is "optical slice" through specimen

• Computer constructs 3-D image from digitized images

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Light Microscopy

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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

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Figure 4.11 A transmission electron microscope (TEM).

Lamp

Light microscope(upside down)

Column of transmissionelectron microscope

Condenserlens

Specimen

Objective lens

Eyepiece

Final imageseen by eye

Electron gun

Specimen

Objective lens(magnet)

Projector lens(magnet)

Final image onfluorescent screen

Condenser lens(magnet)

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Figure 4.12 Scanning electron microscope (SEM).

Electron gun

Magneticlenses

Primaryelectrons

Secondaryelectrons

Specimen

Specimenholder

Vacuumsystem

Beamdeflector coil

Scanningcircuit

Photo-multiplierDetector

Monitor

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Figure 4.13 SEM images.

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Electron Microscopy

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Microscopy

• Probe Microscopy• Magnifies more than 100 million times

• Two types

• Scanning tunneling microscopes

• Atomic force microscopes

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Microscopy

• Probe Microscopy• Scanning tunneling microscopes

• Passes metallic probe above specimen surface

• Measures the electron flow (tunneling current) to and from

the probe and the specimen's surface

• Atomic force microscopes

• Passes probe lightly on the specimen surface

• Deflection of laser beam translated into atomic

topography

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Figure 4.14 Probe microscopy.EnzymeDNA

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Microscopy

• Tell Me Why• Why is magnification high but color absent in an

unretouched electron micrograph?

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Staining

• Most microorganisms are difficult to view by bright-

field microscopy

• Coloring specimen with stain increases contrast

and resolution

• Specimens must be prepared for staining

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Figure 4.15 Preparing a specimen for staining.

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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 stain alkaline structures

• Basic dyes stain acidic structures

• More common because most cells are negatively charged

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Staining

• Simple stains—composed of single dye

• Differential stains—use more than one dye • Gram stain

• Acid-fast stain

• Endospore stain

• Histological stains

• Special stains—reveal specific structures• Negative (capsule) stain

• Flagellar stain

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Figure 4.16 Simple stains.

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Figure 4.17 The Gram staining procedure.

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Figure 4.18 Ziehl-Neelsen acid-fast stain.

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Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis.

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Staining

• Differential Stains• Histological stains

• Two common stains used for histological specimens

• Gomori methenamine silver (GMS) stain

• Hematoxylin and eosin (HE) stain

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Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae.

Bacterium

Capsule

Backgroundstain

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Flagella

Figure 4.21 Flagellar stain of Proteus vulgaris.

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Staining

• Staining for Electron Microscopy• Chemicals containing heavy metals are used for

transmission electron microscopy

• Stains may bind molecules in specimens or the

background

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Staining

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Microscopy

• Tell Me Why• Why is a Gram-negative bacterium colorless but a

Gram-positive bacterium purple after it is rinsed with

decolorizer?

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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• Current taxonomy system began with Carolus Linnaeus

• His system classified organisms based on characteristics

in common

• Grouped organisms that can successfully interbreed into

categories called species

• Used binomial nomenclature

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Figure 4.22 Levels in a Linnaean taxonomic scheme.

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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

• Linnaeus's goal was to classify organisms in order to catalog

them

• Modern goal is to understand relationships among organisms

• Goal of modern taxonomy is to reflect phylogenetic hierarchy

• Greater emphasis on comparisons 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 also differ with respect to

many 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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Classification and Identification of Microorganisms• Taxonomic and Identifying Characteristics• 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

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Figure 4.23 Two biochemical tests for identifying bacteria.

No hydrogen

sulfide

Hydrogen sulfide

producedAcid with gas Acid with no gas Inert

Gas bubble Inverted tubes to trap gas

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

Wells

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Classification and Identification of Microorganisms• Taxonomic and Identifying Characteristics• Serological tests

• Serology—study of serum (liquid portion of blood after

clotting factors removed)

• Many microorganisms are antigenic

• Trigger immune response that produces antibodies

• Serum is an important source of antibodies

• Antibodies can be isolated and bind to the antigens that

triggered their production

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

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Classification and Identification of Microorganisms• Taxonomic and Identifying Characteristics• Phage typing

• Bacteriophage (phage)—virus that infects bacteria

• Phages are specific for the host they infect

• Phage typing is based on this specificity

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Bacterial lawn

Plaques

Figure 4.26 Phage typing.

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Classification and Identification of Microorganisms• Taxonomic and Identifying Characteristics• 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

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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.

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Dichotomous Keys: Overview

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Classification and Identification of Microorganisms• Tell Me Why• Why didn't Linnaeus create taxonomic groups for

viruses?