PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 4 Microscopy, Staining, and Classification
Microscopy Light Microscopy 1) Bright-field microscopes Simple/ Compound
Microscopy Light Microscopy 2) 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
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
Figure 4.8 Four kinds of light microscopy. Nucleus Bacterium Bright field Dark field
Microscopy Light Microscopy 3) 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, while light rays out of phase produce darker image Contrast is created because light waves are out of phase
Figure 4.7 Principles of phase microscopy. 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. Nucleus Two types of Phase microscopes 1) Phase-contrast microscope 2) Differential interference contrast microscope Phase contrast Nomarski
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
Figure 4.9 Fluorescence microscopy. Can help you look through clutter
Figure 4.10 Immunofluorescence. Can help you target specific structures Antibodies Fluorescent dye Bacterium Cell-surface antigens Antibodies carrying dye Bacterial cell with bound 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 emitted light passes through pinhole aperture Computer constructs 3-D image from digitized images
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
Figure 4.11 A transmission electron microscope (TEM). needs vacuum Light microscope (upside down) Column of transmission electron microscope Lamp Electron gun Condenser lens Specimen Specimen Objective lens Objective lens (magnet) Eyepiece Projector lens (magnet) Final image seen by eye Final image on fluorescent screen need sections
Figure 4.12 Scanning electron microscope (SEM). Electron gun Magnetic lenses Beam deflector coil Primary electrons Scanning circuit Secondary electrons Specimen Specimen holder Photomultiplier Detector Monitor Vacuum system
Figure 4.13 SEM images.
Microscopy Probe Microscopy Magnifies more than 100,000,000 times Two types Scanning tunneling microscopes Atomic force microscopes
Figure 4.14 Probe microscopy. DNA Enzyme
Staining Simple stains Differential stains Gram stain Acid-fast stain Endospore stain Special stains Negative (capsule) stain Flagellar stain
Figure 4.16 Simple stains.
Figure 4.17 The Gram staining procedure.
Figure 4.18 Ziehl-Neelsen acid-fast stain.
Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis.
Staining Differential Stains Histological stains Two common stains used for histological specimens Gomori methenamine silver (GMS) stain Hematoxylin and eosin (HE) stain
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 Chemicals containing heavy metals used for transmission electron microscopy Stains may bind molecules in specimens or the background
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
Classification and Identification of Microorganisms Linnaeus and Taxonomic Categories Linnaeus His system classified organisms based on characteristics in common Grouped organisms that can successfully interbreed into categories called species Used binomial nomenclature
Figure 4.22 Levels in a Linnaean taxonomic scheme. D K P Class Our For Good So
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 classifying organisms to catalog them Modern goal is understanding 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
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
Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids Relate it to Linnaeus and Woese Methods
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
Figure 4.23 Two biochemical tests for identifying bacteria. Gas bubble Inverted tubes to trap gas Acid with 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.
Figure 4.26 Phage typing. Bacterial lawn Plaques
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
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
Figure 4.27 Use of a dichotomous taxonomic key. dichotomous taxonomic key