Microscopy, Staining, and Classification. ~10 um. Red Blood Cells = mm 1500 um. Width of penny

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1 PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 4 Microscopy, Staining, and Classification Figure 3.4 Approximate size of various types of cells. ~10 um Red Blood Cells 1.5mm 1500 um = 1500 Width of penny Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of various types of microscopes. 1

2 Official definitions Magnification, the ratio of an object s image size to its real size Resolution, the measure of the clarity of the image, or the minimum distance of two distinguishable points Contrast, visible differences in brightness between parts of the sample Microscopy General Principles of Microscopy Magnification (enlarge) Resolution (tell apart 2 objects close together) Contrast (Differences in intensity between two objects) General rule for any microscopy/detector Smaller the wavelength smaller the object you can see (small objects need small hands) Think about giant fingers and texting 2

3 Figure 4.1 The electromagnetic spectrum. Smaller the object = Use smaller wavelength VIBGYOR Microscopy Microscopes are used to visualize cells In a light microscope (LM), visible light is passed through a specimen and then through glass lenses Glass lenses focuses light and enlarges and resolves objects Lenses refract (bend) the light, so that the image is magnified Light Air Glass Magnification Magnify using = 50/5 lenses = 10X Focal point 5 50 Specimen Convex lens Inverted, reversed, and Enlarged image 3

4 Microscopy Light Microscopy Bright-field microscopes Simple single lense Contain a single magnifying lens Similar to magnifying glass Leeuwenhoek used simple microscope to observe microorganisms Microscopy Light Microscopy Bright-field microscopes Compound multiple lenses More than one - Series of lenses for magnification Light passes through specimen into objective lens Have one or two ocular lenses Total magnification = magnification of objective lens X magnification of ocular lens E.g. 10 times X 10 times = 100 times Figure 4.4 A bright-field, compound light microscope. occular lens 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 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 Ocular lens Path of light Prism Body Objective lenses Specimen Condenser lenses Illuminator Coarse focusing knob Moves the stage up and down to focus the image Fine focusing knob Base objective lens 4

5 50 µm Figure 4.5 The effect of immersion oil on resolution. Special type of objective lens = oil immersion lens Microscope objective Lenses Microscope objective Refracted light rays lost to lens Glass cover slip More light enters lens Glass cover slip Immersion oil Slide Slide Specimen Light source Light source Without immersion oil With immersion oil Increases magnification and resolution Microscopy General Principles of Microscopy Contrast Differences in intensity between two objects, or an object and its background Increase contrast by staining Figure 6.3ab Brightfield (unstained specimen) Brightfield (stained specimen) Staining increases contrast 5

6 Figure 4.16 Simple stains. Figure 4.15 Preparing a specimen for staining. Microbe Naming Rules XGenus specific epithet XGenus specific epithet XGenus specific epithet Genus specific epithet G. specific epithet Genus specific epithet G. specific epithet Escherichia coli Escherichia coli E. coli E. coli 6

7 Figure 3.12 Bacterial shapes and arrangements. 3 Shapes 1) coccus 2) bacillus 3) spirillum 1) staphylo 2) strepto 2 main arrangements Microbe Naming Rules Based on shape and arrangement Arrangement+shape species Staphylococcus species Streptobacillus species 7

8 cocci bacilli shape arrangement Streptococci Streptobacilli Staphylococci Microscopy Light Microscopy 1) Bright-field microscopes Simple/ Compound Figure 4.8 Four kinds of light microscopy. Nucleus Bacterium Bright field Dark field 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 8

9 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 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. 9

10 Figure 4.8 Four kinds of light microscopy. Nucleus Two types of Phase microscopes 1) Phase-contrast microscope 2) Differential interference contrast microscope aka Nomarski 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 10

11 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 11

12 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 Column of transmission (upside down) electron microscope Live? 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 Vacuum system Photomultiplier Detector Monitor 12

13 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 13

14 Staining Principles of Staining Dyes used as stains are usually salts Chromophore is the colored portion of the dye Acidic dyes (negatively charged) stain alkaline structures Basic dyes (positively charged) stain acidic structures Basic dyes are more commonly used since inside of most cells is negatively charged Staining Simple stains Differential stains Gram stain Acid-fast stain Endospore stain Special stains Negative (capsule) stain Flagellar stain 14

15 Bacterium Capsule Background stain Rhodospirillum rubrum 15

16 Figure 4.21 Flagellar stain of Proteus vulgaris. Flagella Gram Staining 16

17 Gram Staining Mechanism Staining for Electron Microscopy Chemicals containing heavy metals used for transmission electron microscopy 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 17

18 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 Classification and Identification of Microorganisms Binomial nomenclature naming species of living things by giving each species a name composed of two parts humans belong to the genus Homo and within this genus to the species Homo sapiens. noun-genus adjective-specific epithet homo naledi (2015) 18

19 Classification and Identification of Microorganisms Linnaeus and Taxonomic Categories Linnaeus's goal was classifying organisms to catalog them Linnaeus - 2 kingdoms Later on - 5 kingdoms Later on 3 domains (genetic material) Goal of modern taxonomy is to reflect phylogenetic hierarchy understanding relationships among organisms Figure 4.22 Levels in a Linnaean taxonomic scheme. D K P Class Our For Good So 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 19

20 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 Biochemical tests Acid with gas Acid with no gas Inert Hydrogen No sulfide hydrogen produced sulfide 20

21 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 21

22 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 dichotomous taxonomic key activity 22