Microscopy, Staining, and Classification

Transcription

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

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

3 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

4 Microscopy General Principles of Microscopy Magnification (enlarge) Resolution (tell apart 2 objects close together) Contrast (Differences in intensity between two objects) How to increase all 3 of these?

5 Figure 4.1 The electromagnetic spectrum. Smaller the object = Use smaller wavelength VIBGYOR

6 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)

7 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

8 Lenses refract (bend) the light, so that the image is magnified Light Air Glass Magnification = 50/5 = 10X Magnify using lenses Focal point 5 50 Specimen Convex lens Inverted, reversed, and Enlarged image

9 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

10 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

11 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

12 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

13 Microscopy General Principles of Microscopy Contrast Differences in intensity between two objects, or an object and its background Increase contrast by staining

14 50 µm Figure 6.3ab Brightfield (unstained specimen) Brightfield (stained specimen) Staining increases contrast

15 Figure 4.16 Simple stains.

16 Figure 4.15 Preparing a specimen for staining.

17 Microscopy Light Microscopy 1) Bright-field microscopes Simple/ Compound

18 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

19 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

20 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

21 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.

22 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

23 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

24 Figure 4.9 Fluorescence microscopy. Can help you look through clutter

25 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

26 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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28 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

29 Figure 4.11 A transmission electron microscope (TEM). needs vacuum Light microscope (upside down) Column of transmission 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

30 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

31 Figure 4.13 SEM images.

32 Microscopy Probe Microscopy Magnifies more than 100,000,000 times Two types Scanning tunneling microscopes Atomic force microscopes

33 Figure 4.14 Probe microscopy. DNA Enzyme

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

36 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

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38 Bacterium Capsule Background stain

39 Rhodospirillum rubrum

40 Figure 4.21 Flagellar stain of Proteus vulgaris. Flagella

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42 Gram Staining

43 Gram Staining Mechanism

44 Staining for Electron Microscopy Chemicals containing heavy metals used for transmission electron microscopy

45 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

46 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

47 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

48 Homo naledi (2015)

49 Microbe Naming Rules Each species have 2 names (1)Genus (2)specific epithet Genus specific epithet G. specific epithet Genus specific epithet G. specific epithet Escherichia coli Escherichia coli E. coli E. coli

50 Microbe Naming Rules X X Genus specific epithet Genus specific epithet Y Genus specific epithet Y Genus specific epithet

51 Bacteria have shapes/morphology

52 Figure 3.12 Bacterial shapes and arrangements. 3 Shapes 1) coccus 2) bacillus 3) spirillum 1) staphylo 2) strepto 2 main arrangements

53 Microbe Naming Rules Based on shape and arrangement (Arrangement+shape) (specific epithet) Staphylococcus (specific epithet) Streptobacillus (specific epithet)

54 cocci bacilli shape arrangement Streptococci Streptobacilli Staphylococci

55 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 genetic relationships among organisms Bat vs Bird

56 Figure 4.22 Levels in a Linnaean taxonomic scheme. D K P Class Our For Good So

57 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

58 Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids

59 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

60 Some bacterial colonies have distinct appearance used for identification

61 Figure 6.8 Characteristics of bacterial colonies. Shape Circular Rhizoid Irregular Filamentous Spindle Margin Entire Undulate Lobate Curled Filiform Elevation Flat Raised Convex Pulvinate Umbonate Size Punctiform Small Moderate Large Colony Texture Smooth or rough Appearance Glistening (shiny) or dull Pigmentation Nonpigmented (e.g., cream, tan, white) Pigmented (e.g., purple, red, yellow) Optical property Opaque, translucent, transparent

62 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 sulfide produced No hydrogen sulfide

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

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

65 Figure 4.26 Phage typing. Bacterial lawn Plaques

66 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

67 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

68 Figure 4.27 Use of a dichotomous taxonomic key. dichotomous taxonomic key dichotomous taxonomic key activity