Microscopy PDF - Microbiology

PowerPoint® Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University CHAPTER 4 Microscopy, Staining, and Classification © 2015 Pearson Education, Inc.  Microscopy and Staining: Overview © 2015 Pearson Education, Inc. © 2015 Pearson Edu...

PowerPoint® Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University CHAPTER 4 Microscopy, Staining, and Classification © 2015 Pearson Education, Inc.  Microscopy and Staining: Overview © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc.  Units of Measurement Tell Me Why Why do scientists use metric rather than English units? © 2015 Pearson Education, Inc.  Microscopy Microscopy: the use of light or electrons to magnify objects Science of microscopy begun by Antoni van Leeuwenhoek Various types of light and electron microscopes © 2015 Pearson Education, Inc.  Microscopy General Principles of Microscopy Wavelength of radiation Magnification Resolution Contrast © 2015 Pearson Education, Inc.  Figure 4.1 The electromagnetic spectrum. © 2015 Pearson Education, Inc.  Figure 4.2 Light refraction and image magnification by a convex glass lens. Light Air Glass Focal point Specimen Convex Inverted, lens reversed, and enlarged image © 2015 Pearson Education, Inc.  Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of various types of microscopes. © 2015 Pearson Education, Inc.  Microscopy General Principles of Microscopy Contrast Differences in intensity between two objects or between an object and its background Important in determining resolution Staining increases contrast Use of light that is in phase increases contrast © 2015 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 © 2015 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 = magnification of objective lens x magnification of ocular lens Most have condenser lens (direct light through specimen) © 2015 Pearson Education, Inc.  Figure 4.4 A bright-field, compound light microscope. Line of vision Ocular lens Remagnifies the image formed by the objective lens Body Ocular lens Transmits the image from the objective lens to the ocular lens Path of light using prisms Prism Arm Objective lenses Body Primary lenses that magnify the specimen Objective Stage lenses Holds the microscope slide in position Specimen Condenser Focuses light Condenser through specimen lenses Diaphragm Controls the amount of Illuminator light entering the condenser Illuminator Light source Coarse focusing knob Moves the stage up and down to focus the image Fine focusing knob Base © 2015 Pearson Education, Inc.  Figure 4.5 The effect of immersion oil on resolution. Microscope Microscope objective Lenses objective Refracted light More light rays lost to lens enters lens Immersion oil Glass cover slip Glass cover slip Slide Slide Specimen Light source Light source Without immersion oil With immersion oil © 2015 Pearson Education, Inc.  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 © 2015 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 © 2015 Pearson Education, Inc. Dark-field stop Dark-field stop  Microscopy Light Microscopy Phase microscopes Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining Light rays in phase produce brighter image, whereas light rays out of phase produce darker image Contrast is created because light waves are out of phase Two types Phase-contrast microscope Differential interference contrast microscope © 2015 Pearson Education, Inc.  Figure 4.7 Principles of phase microscopy. Rays in phase Rays out of phase Phase plate Bacterium Ray deviated by Deviated ray specimen is 1/4 is now 1/2 wavelength out wavelength of phase. out of phase. © 2015 Pearson Education, Inc.  Figure 4.8 Four kinds of light microscopy. Nucleus Bacterium Bright field Dark field Phase contrast Nomarski © 2015 Pearson Education, Inc.  Microscopy Light Microscopy Fluorescent microscopes Direct UV light source at specimen Specimen radiates energy back as a longer, 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 © 2015 Pearson Education, Inc.  Figure 4.9 Fluorescence microscopy. © 2015 Pearson Education, Inc.  Figure 4.10 Immunofluorescence. Antibodies Fluorescent dye Antibodies Bacterium carrying dye Cell-surface antigens Bacterial cell with bound antibodies carrying dye © 2015 Pearson Education, Inc.  Microscopy Light Microscopy Confocal microscopes Use UV lasers to illuminate fluorescent chemicals in a single plane Resolution increased because emitted light passes through pinhole aperture Each image is "optical slice" through specimen Computer constructs 3-D image from digitized images © 2015 Pearson Education, Inc.  Light Microscopy © 2015 Pearson Education, Inc.  Microscopy Electron Microscopy Light microscopes cannot resolve structures closer than 200 nm Electron microscopes have greater resolving power and magnification Magnifies objects 10,000x to 100,000x Detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms Two types Transmission electron microscopes Scanning electron microscopes © 2015 Pearson Education, Inc.  Figure 4.11 A transmission electron microscope (TEM). Light microscope Column of transmission (upside down) electron microscope Lamp Electron gun Condenser Condenser lens lens (magnet) Specimen Specimen Objective lens Objective lens (magnet) Eyepiece Projector lens (magnet) Final image Final image on seen by eye fluorescent screen © 2015 Pearson Education, Inc.  Figure 4.12 Scanning electron microscope (SEM). Electron gun Magnetic Beam lenses deflector coil Scanning Primary circuit electrons Secondary electrons Photo- Specimen multiplier Monitor Specimen Detector holder Vacuum system © 2015 Pearson Education, Inc.  Figure 4.13 SEM images. © 2015 Pearson Education, Inc.  Electron Microscopy © 2015 Pearson Education, Inc.  Microscopy Probe Microscopy Magnifies more than 100 million times Two types Scanning tunneling microscopes Atomic force microscopes © 2015 Pearson Education, Inc.  Microscopy Probe Microscopy Scanning tunneling microscopes Passes metallic probe above specimen surface Measures the electron flow (tunneling current) to and from the probe and the specimen's surface Atomic force microscopes Passes probe lightly on the specimen surface Deflection of laser beam translated into atomic topography © 2015 Pearson Education, Inc.  Figure 4.14 Probe microscopy. DNA Enzyme © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc.  Microscopy Tell Me Why Why is magnification high but color absent in an unretouched electron micrograph? © 2015 Pearson Education, Inc.  Staining Most microorganisms are difficult to view by bright- field microscopy Coloring specimen with stain increases contrast and resolution Specimens must be prepared for staining © 2015 Pearson Education, Inc.  Figure 4.15 Preparing a specimen for staining. © 2015 Pearson Education, Inc.  Staining Principles of Staining Dyes used as stains are usually salts Chromophore is the colored portion of the dye Acidic dyes stain alkaline structures Basic dyes stain acidic structures More common because most cells are negatively charged © 2015 Pearson Education, Inc.  Staining Simple stains—composed of single dye Differential stains—use more than one dye Gram stain Acid-fast stain Endospore stain Histological stains Special stains—reveal specific structures Negative (capsule) stain Flagellar stain © 2015 Pearson Education, Inc.  Figure 4.16 Simple stains. © 2015 Pearson Education, Inc.  Figure 4.17 The Gram staining procedure. © 2015 Pearson Education, Inc.  Figure 4.18 Ziehl-Neelsen acid-fast stain. © 2015 Pearson Education, Inc.  Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis. © 2015 Pearson Education, Inc.  Staining Differential Stains Histological stains Two common stains used for histological specimens Gomori methenamine silver (GMS) stain Hematoxylin and eosin (HE) stain © 2015 Pearson Education, Inc.  Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae. Bacterium Capsule Background stain © 2015 Pearson Education, Inc.  Figure 4.21 Flagellar stain of Proteus vulgaris. Flagella © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc.  Staining Staining for Electron Microscopy Chemicals containing heavy metals are used for transmission electron microscopy Stains may bind molecules in specimens or the background © 2015 Pearson Education, Inc.  Staining © 2015 Pearson Education, Inc.  Microscopy Tell Me Why Why is a Gram-negative bacterium colorless but a Gram-positive bacterium purple after it is rinsed with decolorizer? © 2015 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 © 2015 Pearson Education, Inc.  Classification and Identification of Microorganisms Linnaeus and Taxonomic Categories Current taxonomy system began with Carolus Linnaeus His system classified organisms based on characteristics in common Grouped organisms that can successfully interbreed into categories called species Used binomial nomenclature © 2015 Pearson Education, Inc.  Figure 4.22 Levels in a Linnaean taxonomic scheme. © 2015 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 Linnaeus's goal was to classify organisms in order to catalog them Modern goal is to understand relationships among organisms Goal of modern taxonomy is to reflect phylogenetic hierarchy Greater emphasis on comparisons of organisms' genetic material led to proposal to add domain © 2015 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 also differ with respect to many other characteristics © 2015 Pearson Education, Inc.  Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids © 2015 Pearson Education, Inc.  Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics Physical characteristics Can often be used to identify microorganisms Protozoa, fungi, algae, and parasitic worms can often be identified based only on their morphology Some bacterial colonies have distinct appearance used for identification © 2015 Pearson Education, Inc.  Figure 4.23 Two biochemical tests for identifying bacteria. Gas bubble Inverted tubes to trap gas Hydrogen No sulfide hydrogen Acid with gas Acid with no gas Inert produced sulfide © 2015 Pearson Education, Inc.  Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system. Wells © 2015 Pearson Education, Inc.  Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics Serological tests Serology—study of serum (liquid portion of blood after clotting factors removed) Many microorganisms are antigenic Trigger immune response that produces antibodies Serum is an important source of antibodies Antibodies can be isolated and bind to the antigens that triggered their production © 2015 Pearson Education, Inc.  Figure 4.25 An agglutination test, one type of serological test. © 2015 Pearson Education, Inc.  Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics Phage typing Bacteriophage (phage)—virus that infects bacteria Phages are specific for the host they infect Phage typing is based on this specificity © 2015 Pearson Education, Inc.  Figure 4.26 Phage typing. Bacterial lawn Plaques © 2015 Pearson Education, Inc.  Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics Analysis of nucleic acids Nucleic acid sequence can be used to classify and identify microbes Prokaryotic taxonomy now includes the G + C content of an organism's DNA © 2015 Pearson Education, Inc.  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 © 2015 Pearson Education, Inc.  Figure 4.27 Use of a dichotomous taxonomic key. © 2015 Pearson Education, Inc.  Dichotomous Keys: Overview © 2015 Pearson Education, Inc.  Classification and Identification of Microorganisms Tell Me Why Why didn't Linnaeus create taxonomic groups for viruses? © 2015 Pearson Education, Inc.