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Microscopy, Staining, and Classification
Chapter 4 Microscopy, Staining, and Classification
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Microscopy and Staining
ANIMATION Microscopy and Staining: Overview © 2012 Pearson Education Inc. 2
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Table 4.1 Metric Units of Length
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General Principles of Microscopy
Wavelength of radiation Magnification Resolution Contrast © 2012 Pearson Education Inc. 4
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Figure 4.1 The electromagnetic spectrum
400 nm 700 nm Visible light X rays UV light Infra- red Micro- wave Radio waves and Television Gamma rays Increasing wavelength 10–12m 10–8m 10–4m 100m 103m One wavelength Crest Trough Increasing resolving power 5
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Light Air Glass Focal point Specimen Convex lens Inverted,
Figure 4.2 Light refraction and image magnification by a convex glass lens-overview Light Air Glass Focal point Figure 4.2 Light refraction and image magnification by a convex glass lens. Specimen Convex lens Inverted, reversed, and enlarged image 6
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Figure 4.3 The limits of resolution of the human eye and of various types of microscopes
Diameter of DNA Typical bacteria and archaea Ribosomes Flea Large protozoan (Euglena) Atoms Proteins Viruses Chloroplasts Chicken egg Amino acids Human red blood cell Mitochondrion Scanning tunneling microscope (STM) 0.01 nm–10 nm Transmission electron microscope (TEM) 0.078 nm–100 µm Unaided human eye 200 µm– Scanning electron microscope (SEM) 0.4 nm–1 mm Atomic force microscope (AFM) 1 nm–10 nm Compound light microscope (LM) 200 nm–10 mm 7
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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 © 2012 Pearson Education Inc. 8
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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 © 2012 Pearson Education Inc. 9
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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 (objective lens X ocular lens) Most have condenser lens (direct light through specimen) © 2012 Pearson Education Inc. 10
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Figure 4.4 A bright-field, compound light microscope-overview
Ocular lens Line of vision Remagnifies the image formed by the objective lens Body Ocular lens Transmits the image from the objective lens to the ocular lens using prisms Path of light Arm Prism Objective lenses Body Primary lenses that magnify the specimen Objective lenses Stage Holds the microscope slide in position Specimen Condenser Focuses light through specimen Condenser lenses Diaphragm Illuminator Controls the amount of light entering the condenser Illuminator Light source Coarse focusing knob Moves the stage up and down to focus the image Fine focusing knob Base 11
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Figure 4.5 The effect of immersion oil on resolution-overview
Microscope objective Microscope objective Lenses Refracted light rays lost to lens More light enters lens Immersion oil Glass cover slip Glass cover slip Slide Slide Specimen Light source Light source Without immersion oil With immersion oil 12
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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 © 2012 Pearson Education Inc. 13
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Figure 4.6 The light path in a dark-field microscope
Objective Light refracted by specimen Light unrefracted by specimen Specimen Condenser Dark-field stop Dark-field stop 14
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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 © 2012 Pearson Education Inc. 15
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Figure 4.7 Principles of phase microscopy-overview
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. 16
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Figure 4.8 Four kinds of light microscopy-overview
Nucleus Bacterium Bright field Dark field Phase contrast Nomarski 17
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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 © 2012 Pearson Education Inc. 18
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Figure 4.9 Fluorescent microscopy-overview
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Figure 4.10 Immunofluorescence-overview
Antibodies Fluorescent dye Antibodies carrying dye Bacterium Cell-surface antigens Bacterial cell with bound antibodies carrying dye 20
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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 © 2012 Pearson Education Inc. 21
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Microscopy ANIMATION Light Microscopy © 2012 Pearson Education Inc. 22
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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 © 2012 Pearson Education Inc. 23
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Figure 4.11 A transmission electron microscope (TEM) -overview
Light microscope (upside down) Column of transmission electron microscope Lamp Electron gun Condenser lens Condenser lens (magnet) Specimen Specimen Objective lens Objective lens (magnet) Eyepiece Projector lens (magnet) Final image seen by eye Final image on fluorescent screen 24
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Figure 4.12 Scanning electron microscope (SEM)
Electron gun Magnetic lenses Beam deflector coil Scanning circuit Primary electrons Secondary electrons Photo- multiplier Specimen Monitor Detector Specimen holder Vacuum system 25
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Figure 4.13 SEM images-overview
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Microscopy ANIMATION Electron Microscopy 27
© 2012 Pearson Education Inc. 27
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Microscopy Probe Microscopy Magnifies more than 100,000,000X Two types
Scanning tunneling microscopes Atomic force microscopes © 2012 Pearson Education Inc. 28
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Figure 4.14 Probe microscopy-overview
DNA Enzyme 29
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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 © 2012 Pearson Education Inc. 30
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Figure 4.15 Preparing a specimen for staining
Spread culture in thin film over slide Air dry Pass slide through flame to fix it 31
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Staining Simple Stains Differential Stains Special Stains Gram stain
Acid-fast stain Endospore stain Histological stain Special Stains Negative (capsule) stain Flagellar stain © 2012 Pearson Education Inc. 32
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Figure 4.16 Simple stains-overview
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Figure 4.17 The Gram staining procedure-overview
Slide is flooded with crystal violet for 1 min, then rinsed with water. Slide is flooded with iodine for 1 min, then rinsed with water. Result: All cells are stained purple. Result: Iodine acts as a mordant; all cells remain purple. Slide is flooded with solution of ethanol and acetone for 10–30 sec, then rinsed with water. Slide is flooded with safranin for 1 min, then rinsed with water and blotted dry. Result: Gram-positive cells remain purple, Gram-negative cells are pink. Result: Smear is decolorized; Gram-positive cells remain purple, but Gram-negative cells are now colorless. 34
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Figure 4.18 The 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 stain
Two popular stains for histological specimens Gomori methenamine silver (GMS) Hematoxylin and eosin (HE) © 2012 Pearson Education Inc. 37
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Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae
Bacterium Capsule Background stain 38
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Figure 4.21 Flagellar stain of Proteus vulgaris
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Staining Staining for Electron Microscopy
Transmission electron microscopy uses chemicals containing heavy metals Absorb electrons Stains may bind molecules in specimens or the background © 2012 Pearson Education Inc. 40
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Staining ANIMATION Staining © 2012 Pearson Education Inc. 41
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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 © 2012 Pearson Education Inc. 42
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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 Linnaeus proposed only two kingdoms © 2012 Pearson Education Inc. 43
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Figure 4.22 Levels in a Linnaean taxonomic scheme-overview
Domain Bacteria Archaea Eukarya Kingdom Animalia Plantae Fungi Phylum Chordata (vertebrates) Arthropoda (joint-legged animals) Platyhelminthes (tapeworm) Nematoda (unsegmented roundworms) Class Insecta Crustacea Arachnida Order Scorpionida Acariformes (mites) Parasitiformes (mites and ticks) Araneida Family Ixodidae (hard ticks) Argasidae (soft ticks) Genus Dermacentor Ixodes Rhipicephalus Species l. scapularis (deer tick) l. pacificus (black-eyed tick) l. ricinus (castor bean tick) 44
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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 © 2012 Pearson Education Inc. 45
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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 Emphasis on comparison of organisms’ genetic material Led to proposal to add domain © 2012 Pearson Education Inc. 46
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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 differ by other characteristics © 2012 Pearson Education Inc. 47
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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 © 2012 Pearson Education Inc. 48
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Figure 4.23 Two biochemical tests for identifying bacteria-overview
Gas bubble Inverted tubes to trap gas Hydrogen sulfide produced No hydrogen sulfide Acid with gas Acid with no gas Inert 49
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Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system
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Negative result Positive result Negative result Positive result
Figure An agglutination test, one type of serological test-overview Negative result Positive result Negative result Positive result 51
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Figure Phage typing Bacterial lawn Plaques 52
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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 © 2012 Pearson Education Inc. 53
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Figure 4.27 Use of a dichotomous taxonomic key-overview
Gram-positive cells? No Yes Rod-shaped cells? Gram-positive bacteria No Yes Cocci and pleomorphic bacteria Can tolerate oxygen? No Yes Obligate anaerobes Ferments lactose? No Yes Can use citric acid (citrate) as sole carbon source? Non-lactose- fermenters No Yes Produces gas from glucose? Produces hydrogen sulfide gas? No Yes No Yes Produces acetoin? Shigella Escherichia Salmonella No Yes Citrobacter Enterobacter 54
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Classification and Identification of Microorganisms
ANIMATION Dichotomous Key: Overview ANIMATION Dichotomous Key: Sample with Flowchart ANIMATION Dichotomous Key: Practice © 2012 Pearson Education Inc. 55
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