chapter 4 microscopy, staining, and classification azeem ahmad, ph.d des moines area community...

Chapter 4

Microscopy, Staining, and Classification

Azeem Ahmad, Ph.DDes Moines Area Community College

(DMACC-URBAN)

Microscopy and Staining

© 2012 Pearson Education Inc.

ANIMATION Microscopy and Staining: Overview

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- wave

Infra- red

UV light

X rays

Radio waves and Television

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, andenlargedimage

Focal point

Specimen

Glass

Light

Air

Chickenegg

Human redblood cell

Largeprotozoan(Euglena)Chloroplasts

Flea Typical bacteriaand archaea

Diameterof DNA

Viruses Proteins

Ribosomes

Aminoacids

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 eye200 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

• 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)


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 bythe objective lens

BaseFine focusing knob

Coarse focusing knob

Diaphragm

Stage

Arm

Transmits the image from theobjective lens to the ocular lensusing prisms

Primary lenses thatmagnify the specimen

Controls the amount of light entering the condenser

Focuses lightthrough specimen

Holds the microscopeslide in position

Light source

Moves the stage up anddown 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

• 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


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

• 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


Ray deviated byspecimen is 1/4wavelength outof phase.

Deviated rayis now 1/2wavelength out of phase.

Bacterium

Rays in phase Rays out of phase

Phase plate

Figure 4.7 Principles of phase microscopy-overview

Bright field

Bacterium

Nucleus

Phase contrast

Dark field

Nomarski

Figure 4.8 Four kinds of light microscopy-overview

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


Figure 4.9 Fluorescent microscopy-overview

Cell-surfaceantigens

Bacterium

Antibodies

Antibodiescarrying dye

Fluorescent dye

Bacterial cell withbound antibodiescarrying dye

Figure 4.10 Immunofluorescence-overview

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


Microscopy


ANIMATION Light Microscopy

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


Figure 4.11 A transmission electron microscope (TEM) -overview

Light microscope(upside down)

Column of transmissionelectron microscope

Condenser lens(magnet)

Lamp

Condenserlens

Objective lens

Eyepiece

Final imageseen by eye

Final image onfluorescent screen

Projector lens(magnet)

Objective lens(magnet)

Specimen Specimen

Electron gun

Magneticlenses

Electron gun

Primaryelectrons

Secondaryelectrons

Specimenholder

Vacuumsystem

SpecimenPhoto-multiplier

Detector

Scanningcircuit

Monitor

Beamdeflector coil

Figure 4.12 Scanning electron microscope (SEM)

Figure 4.13 SEM images-overview

Microscopy


ANIMATION Electron Microscopy

Microscopy

• Probe Microscopy– Magnifies more than 100,000,000X

– Two types– Scanning tunneling microscopes– Atomic force microscopes


Figure 4.14 Probe microscopy-overview

EnzymeDNA

BREAK

5 min


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

Spread culture inthin film over slide

Pass slide throughflame to fix it

Air dry

Figure 4.15 Preparing a specimen for staining

• 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 for10–30 sec, then rinsed with water.

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

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

Result: Gram-positive cells remain purple, Gram-negativecells are pink.

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

Result: Iodine acts as amordant; all cells remain purple.

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)


Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae

Backgroundstain

Bacterium

Capsule

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


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 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 by ribosomal 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

Acid with gas Acid with no gas Inert

Hydrogensulfide

produced

No hydrogen

sulfide

Figure 4.23 Two biochemical tests for identifying bacteria-overview

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

Wells

Negative result

Negative result

Positive result

Positive result

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

Figure 4.26 Phage typing

Bacterial lawn

Plaques


• 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?

Produces acetoin? Salmonella

EnterobacterCitrobacter

EscherichiaShigella

YesNo

YesNo YesNo

YesNo

YesNo

YesNo

YesNo

YesNo



ANIMATION Dichotomous Key: Overview

ANIMATION Dichotomous Key: Sample with Flowchart

ANIMATION Dichotomous Key: Practice

chapter 4 microscopy, staining, and classification azeem ahmad, ph.d des moines area community...

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