Microscopy

Chapter 4

Microscopy, Staining and Classification•General Principles of Microscopy

– Wavelength of radiation – uses light or electrons

– Magnification – increase in size of object

– Resolution - clarity

– Contrast – difference between 2 objects or object and background; stain to increase contrast

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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

• Light Microscopy– Bright-field microscopes – uses light

– 1. Simple– Contain a single magnifying lens– Similar to magnifying glass– Leeuwenhoek used simple microscope to

observe microorganisms

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Microscopy

– 2. Compound– Series of lenses for magnification– Light passes through specimen into

objective lens – Have one or two ocular lenses– Total magnification (objective lens X ocular

lens – 10X) – Low – red – 4X – total 40X– medium - yellow – 10X – total100X– high – blue – 40X – total 400X– Oil immersion – white -100x – total

1000X - Oil immersion lens increases resolution

- Most have condenser lens (direct light through specimen)

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Microscopy

- 3. Darkfield microscope – background dark – used for unstained organisms

- 4. Fluorescent microscope – specimen stained with fluorescent dyes

- 5. Phase-contrast microscope – allows observation of dense structures in living organisms – no staining needed – sharp contrast

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

• 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(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

• 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

EnzymeDNA

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Staining

• Principles of Staining– Staining increases contrast and resolution by

coloring specimens with stains/dyes

– Smear of microorganisms (thin film) made prior to staining

– Acidic dyes stain alkaline (base) structures– Nigrosin, India Ink

– Basic dyes stain acidic structures– Crystal violet, methylene blue, safranin,

malachite green

Spread culture inthin film over slide

Pass slide throughflame to fix it

Air dry

Figure 4.15 Preparing a specimen for staining

• Simple Stains – single basic dye• Differential Stains – use more than 1 dye

– Gram stain – for positive or negative– Acid-fast stain – for waxy cell walls– Endospore stain – uses heat to force stain – Histological stain – for tissue (cancer or fungi)

• Special Stains – Negative (capsule) stain – stains background– Flagellar stain

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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

Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae

Backgroundstain

Bacterium

Capsule

Figure 4.21 Flagellar stain of Proteus vulgaris

Flagella

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 – genus and species

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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

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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 – grouping

organisms reflecting their evolution from common ancestors

– Emphasis on comparison of organisms’ genetic material – Led to proposal to add domain

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Classification and Identification of Microorganisms

• Domains– 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 – microbes ability to utilize or produce certain chemicals

– Serological tests – use antiserum (serum containing antibodies); clumping of antigen with antibodies (agglutination) indicates presence of targeted cells

– Phage typing – reveals if 1 bacterial strain is/is not susceptible to a particular phage – plaques (clear area) form where bacteria killed by phage

– Analysis of nucleic acids

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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

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-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