Transcript Chapter 13
Chapter 4 Microscopy, Staining, and Classification © 2012 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell North Carolina State University Microscopy and Staining ANIMATION Microscopy and Staining: Overview © 2012 Pearson Education Inc. Table 4.1 Metric Units of Length Microscopy • General Principles of Microscopy – – – – Wavelength of radiation Magnification Resolution Contrast © 2012 Pearson Education Inc. Figure 4.1 The electromagnetic spectrum 400 nm 700 nm Visible light Gamma rays 10–12m X UV rays light 10–8m Infra- Microred wave Radio waves and Television Increasing wavelength 10–4m 100m 103m Crest One wavelength Trough Increasing resolving power Figure 4.2 Light refraction and image magnification by a convex glass lens-overview Light Glass Air Focal point Specimen Convex lens Inverted, reversed, and enlarged image Figure 4.3 The limits of resolution of the human eye and of various types of microscopes Diameter of DNA Ribosomes Proteins Viruses Atoms Amino acids Typical bacteria and archaea Flea Chloroplasts Mitochondrion Large protozoan (Euglena) Chicken egg Human red blood cell Scanning tunneling microscope (STM) 0.01 nm–10 nm Transmission electron microscope (TEM) 0.078 nm–100 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 Unaided human eye 200 µm– 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 © 2012 Pearson Education Inc. 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. 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. Figure 4.4 A bright-field, compound light microscope-overview Ocular lens Line of vision Remagnifies the image formed by the objective lens Body Transmits the image from the objective lens to the ocular lens using prisms Arm Objective lenses Primary lenses that magnify the specimen Stage Holds the microscope slide in position Condenser Focuses light through specimen Diaphragm 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 Ocular lens Path of light Prism Body Objective lenses Specimen Condenser lenses Illuminator Figure 4.5 The effect of immersion oil on resolution-overview Microscope objective Refracted light rays lost to lens Microscope objective Lenses More light enters lens Glass cover slip Glass cover slip Slide Slide Specimen Light source Without immersion oil Immersion oil Light source With 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 © 2012 Pearson Education Inc. 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 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. 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. Figure 4.8 Four kinds of light microscopy-overview Nucleus Bacterium Bright field Dark field Phase contrast Nomarski 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. Figure 4.9 Fluorescent microscopy-overview Figure 4.10 Immunofluorescence-overview Antibodies Bacterium Cell-surface antigens Bacterial cell with bound antibodies carrying dye Fluorescent dye Antibodies carrying dye 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. Microscopy ANIMATION Light Microscopy © 2012 Pearson Education Inc. 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. 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 Figure 4.12 Scanning electron microscope (SEM) Electron gun Magnetic lenses Primary electrons Beam deflector coil Scanning circuit Secondary electrons Specimen Specimen holder Vacuum system Photomultiplier Detector Monitor Figure 4.13 SEM images-overview Microscopy ANIMATION Electron Microscopy © 2012 Pearson Education Inc. Microscopy • Probe Microscopy – Magnifies more than 100,000,000X – Two types – Scanning tunneling microscopes – Atomic force microscopes © 2012 Pearson Education Inc. Figure 4.14 Probe microscopy-overview DNA Enzyme 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 © 2012 Pearson Education Inc. Figure 4.15 Preparing a specimen for staining Spread culture in thin film over slide Air dry Pass slide through flame to fix it Staining • Simple Stains • Differential Stains – – – – Gram stain Acid-fast stain Endospore stain Histological stain • Special Stains – Negative (capsule) stain – Flagellar stain © 2012 Pearson Education Inc. Figure 4.16 Simple stains-overview 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: Smear is decolorized; Gram-positive cells remain purple, but Gram-negative cells are now colorless. Result: Gram-positive cells remain purple, Gram-negative cells are pink. 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) © 2012 Pearson Education Inc. Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae Bacterium Capsule Background stain 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 © 2012 Pearson Education Inc. Staining ANIMATION Staining © 2012 Pearson Education Inc. 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. 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. Figure 4.22 Levels in a Linnaean taxonomic scheme-overview Domain Bacteria Archaea Eukarya Animalia Plantae Fungi Kingdom Phylum Chordata (vertebrates) Arthropoda (joint-legged animals) Platyhelminthes Nematoda (tapeworm) (unsegmented roundworms) Class Insecta Crustacea Arachnida Order Scorpionida Parasitiformes (mites and ticks) Acariformes (mites) Araneida Family Ixodidae (hard ticks) Argasidae (soft ticks) Ixodes Rhipicephalus l. pacificus (black-eyed tick) l. ricinus (castor bean tick) Genus Dermacentor Species l. scapularis (deer tick) 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. 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. 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. 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. Figure 4.23 Two biochemical tests for identifying bacteria-overview Gas bubble Acid with gas Inverted tubes to trap gas Acid with no gas Inert Hydrogen sulfide produced No hydrogen sulfide Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system Wells Figure 4.25 An agglutination test, one type of serological test-overview Negative result Positive result Negative result Positive result 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 © 2012 Pearson Education Inc. Figure 4.27 Use of a dichotomous taxonomic key-overview Gram-positive cells? No Yes Gram-positive bacteria Rod-shaped cells? No Yes Can tolerate oxygen? Cocci and pleomorphic bacteria No Yes Ferments lactose? Obligate anaerobes No Yes Non-lactosefermenters Can use citric acid (citrate) as sole carbon source? No Yes Produces gas from glucose? No Shigella Produces hydrogen sulfide gas? No Yes Escherichia Yes Produces acetoin? No Citrobacter Salmonella Yes Enterobacter Classification and Identification of Microorganisms ANIMATION Dichotomous Key: Overview ANIMATION Dichotomous Key: Sample with Flowchart ANIMATION Dichotomous Key: Practice © 2012 Pearson Education Inc.