4 Microscopy, Staining, and Classification

Microscopy and Staining: Overview PLAY Microscopy and Staining: Overview

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Microscopy General Principles of Microscopy Wavelength of radiation Magnification Resolution Contrast

Figure 4.1 The electromagnetic spectrum. 5

Light Air Glass Focal point Specimen Figure 4.2 Light refraction and image magnification by a convex glass lens. Light Air Glass Focal point Inverted, reversed, and Enlarged image Specimen Convex lens 6

Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of various types of microscopes. 7

Microscopy General Principles of Microscopy Contrast Differences in intensity between two objects, or an object and its background Important in determining resolution Staining increases contrast Use of light that is in phase increases contrast

Microscopy Light Microscopy Bright-field microscopes Simple Contain a single magnifying lens Similar to magnifying glass Leeuwenhoek used simple microscope to observe microorganisms

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)

Figure 4.4 A bright-field, compound light microscope. Line of vision Ocular lens Remagnifies the image formed by the objective lens Body Transmits the image from the objective lens to the ocular lens using prisms Ocular lens Path of light Arm Prism Objective lenses Primary lenses that magnify the specimen Body Objective lenses Stage Holds the microscope slide in position Specimen Condenser Focuses light through specimen Condenser lenses Diaphragm Controls the amount of light entering the condenser Illuminator Illuminator Light source Coarse focusing knob Moves the stage up and down to focus the image Fine focusing knob Base 11

Figure 4.5 The effect of immersion oil on resolution. 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

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

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

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, while 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

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. 16

Figure 4.8 Four kinds of light microscopy. Nucleus Bacterium Bright field Dark field Phase contrast Nomarski 17

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. 19

Figure 4.10 Immunofluorescence. Antibodies Fluorescent dye Antibodies carrying dye Bacterium Cell-surface antigens Bacterial cell with bound antibodies carrying dye 20

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

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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). Light microscope (upside down) Column of transmission electron microscope Lamp 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 24

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

Figure 4.13 SEM images. 26

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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 29

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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

Figure 4.15 Preparing a specimen for staining. 33

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 since most cells are negatively charged

Staining Simple stains Differential stains Special stains Gram stain Acid-fast stain Endospore stain Special stains Negative (capsule) stain Flagellar stain

Figure 4.16 Simple stains. 36

Figure 4.17 The Gram staining procedure. 37

Figure 4.18 Ziehl-Neelsen acid-fast stain. 38

Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis. 39

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 41

Figure 4.21 Flagellar stain of Proteus vulgaris. 42

Staining Staining for Electron Microscopy Chemicals containing heavy metals used for transmission electron microscopy Stains may bind molecules in specimens or the background

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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

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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. 48

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

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 Hydrogen sulfide produced No hydrogen sulfide Acid with gas Acid with no gas Inert 53

Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system. Wells 54

Figure 4.25 An agglutination test, one type of serological test. 55

Bacterial lawn Plaques Figure 4.26 Phage typing. Bacterial lawn Plaques 56

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. 59

Dichotomous Keys: Overview PLAY Dichotomous Keys: Overview