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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 © 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 • Light Microscopy – Bright-field microscopes – uses light – 1. – – – © 2012 Pearson Education Inc. Simple Contain a single magnifying lens Similar to magnifying glass Leeuwenhoek used simple microscope to observe microorganisms 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) © 2012 Pearson Education Inc. 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 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 • 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 • 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 – Acidic dyes stain alkaline (base) structures – Nigrosin, India Ink – Basic dyes stain acidic structures – Crystal violet, methylene blue, safranin, malachite green © 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 – 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 © 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 Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae Bacterium Capsule Background stain 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 © 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 – genus and species © 2012 Pearson Education Inc. 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 – grouping organisms reflecting their evolution from common ancestors – Emphasis on comparison of organisms’ genetic material – Led to proposal to add domain © 2012 Pearson Education Inc. 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 © 2012 Pearson Education Inc. 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 © 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