microscopy chapter 4 microscopy, staining and classification general principles of microscopy...
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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
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