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© 2012 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell North Carolina State University Chapter 4 Microscopy, Staining, and Classification.

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Presentation on theme: "© 2012 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell North Carolina State University Chapter 4 Microscopy, Staining, and Classification."— Presentation transcript:

1 © 2012 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell North Carolina State University Chapter 4 Microscopy, Staining, and Classification

2 Microscopy and Staining © 2012 Pearson Education Inc. ANIMATION Microscopy and Staining: Overview

3 Table 4.1 Metric Units of Length

4 Microscopy General Principles of Microscopy –Wavelength of radiation –Magnification –Resolution –Contrast © 2012 Pearson Education Inc.

5 Figure 4.1 The electromagnetic spectrum Visible light Micro- wave Infra- red UV light X rays Radio waves and Television One wavelength 400 nm 700 nm Gamma rays Increasing wavelength Crest 10 0 m 10 3 m 10 –4 m 10 –8 m Increasing resolving power Trough 10 –12 m

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

7 Chicken egg Human red blood cell Large protozoan (Euglena) Chloroplasts Flea Typical bacteria and archaea Diameter of DNA VirusesProteins Ribosomes Amino acids Atoms Scanning tunneling microscope (STM) 0.01 nm–10 nm Scanning electron microscope (SEM) 0.4 nm–1 mm Transmission electron microscope (TEM) nm–100 µm Atomic force microscope (AFM) 1 nm–10 nm Compound light microscope (LM) 200 nm–10 mm Unaided human eye 200 µm– Mitochondrion Figure 4.3 The limits of resolution of the human eye and of various types of microscopes

8 Microscopy General Principles of Microscopy –Contrast –Differences in intensity between two objects, or between an object and background –Important in determining resolution –Staining increases contrast –Use of light that is in phase increases contrast © 2012 Pearson Education Inc.

9 Microscopy Light Microscopy –Bright-field microscopes –Simple –Contain a single magnifying lens –Similar to magnifying glass –Leeuwenhoek used simple microscope to observe microorganisms © 2012 Pearson Education Inc.

10 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 (objective lens X ocular lens) –Most have condenser lens (direct light through specimen) © 2012 Pearson Education Inc.

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

12 Figure 4.5 The effect of immersion oil on resolution-overview Microscope objective Refracted light rays lost to lens Glass cover slip Light source Specimen Slide Without immersion oil Glass cover slip Light source Slide Microscope objective More light enters lens Lenses With immersion oil Immersion oil

13 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 © 2012 Pearson Education Inc.

14 Light refracted by specimen Light unrefracted by specimen Specimen Condenser Dark-field stop Objective Figure 4.6 The light path in a dark-field microscope

15 Microscopy Light Microscopy –Phase microscopes –Examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining –Contrast is created because light waves are out of phase –Two types –Phase-contrast microscope –Differential interference contrast microscope © 2012 Pearson Education Inc.

16 Ray deviated by specimen is 1/4 wavelength out of phase. Deviated ray is now 1/2 wavelength out of phase. Bacterium Rays in phaseRays out of phase Phase plate Figure 4.7 Principles of phase microscopy-overview

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

18 Microscopy Light Microscopy –Fluorescent microscopes –Direct UV light source at specimen –Specimen radiates energy back as a 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 © 2012 Pearson Education Inc.

19 Figure 4.9 Fluorescent microscopy-overview

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

21 Microscopy Light Microscopy –Confocal microscopes –Use fluorescent dyes –Use UV lasers to illuminate fluorescent chemicals in a single plane –Resolution increased because light passes through pinhole aperture –Computer constructs 3-D image from digitized images © 2012 Pearson Education Inc.

22 Microscopy © 2012 Pearson Education Inc. ANIMATION Light Microscopy

23 Microscopy Electron Microscopy –Light microscopes cannot resolve structures closer than 200 nm –Greater resolving power and magnification –Magnifies objects 10,000X to 100,000X –Detailed view of bacteria, viruses, ultrastructure, and large atoms –Two types –Transmission electron microscopes –Scanning electron microscopes © 2012 Pearson Education Inc.

24 Figure 4.11 A transmission electron microscope (TEM) -overview Light microscope (upside down) Column of transmission electron microscope Condenser lens (magnet) Lamp Condenser lens Objective lens Eyepiece Final image seen by eye Final image on fluorescent screen Projector lens (magnet) Objective lens (magnet) Specimen Electron gun

25 Magnetic lenses Electron gun Primary electrons Secondary electrons Specimen holder Vacuum system Specimen Photo- multiplier Detector Scanning circuit Monitor Beam deflector coil Figure 4.12 Scanning electron microscope (SEM)

26 Figure 4.13 SEM images-overview

27 Microscopy © 2012 Pearson Education Inc. ANIMATION Electron Microscopy

28 Microscopy Probe Microscopy –Magnifies more than 100,000,000X –Two types –Scanning tunneling microscopes –Atomic force microscopes © 2012 Pearson Education Inc.

29 Figure 4.14 Probe microscopy-overview Enzyme DNA

30 © 2012 Pearson Education Inc. Staining Principles of Staining –Staining increases contrast and resolution by coloring specimens with stains/dyes –Smear of microorganisms (thin film) made prior to staining –Microbiological stains contain chromophore –Acidic dyes stain alkaline structures –Basic dyes stain acidic structures

31 Spread culture in thin film over slide Pass slide through flame to fix it Air dry Figure 4.15 Preparing a specimen for staining

32 Simple Stains Differential Stains –Gram stain –Acid-fast stain –Endospore stain –Histological stain Special Stains –Negative (capsule) stain –Flagellar stain © 2012 Pearson Education Inc. Staining

33 Figure 4.16 Simple stains-overview

34 Figure 4.17 The Gram staining procedure-overview Slide is flooded with crystal violet for 1 min, then rinsed with water. Result: All cells are stained purple. Slide is flooded with solution of ethanol and acetone for 10–30 sec, then rinsed with water. Result: Smear is decolorized; Gram-positive cells remain purple, but Gram-negative cells are now colorless. Slide is flooded with safranin for 1 min, then rinsed with water and blotted dry. Result: Gram-positive cells remain purple, Gram-negative cells are pink. Slide is flooded with iodine for 1 min, then rinsed with water. Result: Iodine acts as a mordant; all cells remain purple.

35 Figure 4.18 The Ziehl-Neelsen acid-fast stain

36 Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis

37 Staining Differential Stains –Histological stain –Two popular stains for histological specimens –Gomori methenamine silver (GMS) –Hematoxylin and eosin (HE) © 2012 Pearson Education Inc.

38 Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae Background stain Bacterium Capsule

39 Figure 4.21 Flagellar stain of Proteus vulgaris Flagella

40 Staining Staining for Electron Microscopy –Transmission electron microscopy uses chemicals containing heavy metals –Absorb electrons –Stains may bind molecules in specimens or the background © 2012 Pearson Education Inc.

41 Staining © 2012 Pearson Education Inc. ANIMATION Staining

42 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 © 2012 Pearson Education Inc.

43 Classification and Identification of Microorganisms Linnaeus and Taxonomic Categories –Linnaeus –Classified organisms based on characteristics in common –Organisms that can successfully interbreed called species –Used binomial nomenclature in his system –Linnaeus proposed only two kingdoms © 2012 Pearson Education Inc.

44 l. scapularis (deer tick) l. pacificus (black-eyed tick) l. ricinus (castor bean tick) Parasitiformes (mites and ticks) Acariformes (mites) Rhipicephalus Ixodidae (hard ticks) DermacentorIxodes Argasidae (soft ticks) Araneida Scorpionida Arachnida CrustaceaInsecta Chordata (vertebrates) Arthropoda (joint-legged animals) Platyhelminthes (tapeworm) Nematoda (unsegmented roundworms) Animalia PlantaeFungi BacteriaArchaeaEukarya Family Class Kingdom Order Phylum Domain Genus Species Figure 4.22 Levels in a Linnaean taxonomic scheme-overview

45 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 © 2012 Pearson Education Inc.

46 Classification and Identification of Microorganisms Linnaeus and Taxonomic Categories –Linnaeus’s goal was to classify organisms to catalogue them –Modern goal is to understand relationships among groups of organisms –Reflect phylogenetic hierarchy –Emphasis on comparison of organisms’ genetic material –Led to proposal to add domain © 2012 Pearson Education Inc.

47 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 differ by other characteristics © 2012 Pearson Education Inc.

48 Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics –Physical characteristics –Biochemical tests –Serological tests –Phage typing –Analysis of nucleic acids © 2012 Pearson Education Inc.

49 Gas bubble Inverted tubes to trap gas Acid with gas Acid with no gasInert Hydrogen sulfide produced No hydrogen sulfide Figure 4.23 Two biochemical tests for identifying bacteria-overview

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

51 Negative result Positive result Figure 4.25 An agglutination test, one type of serological test-overview

52 Figure 4.26 Phage typing Bacterial lawn Plaques

53 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 © 2012 Pearson Education Inc.

54 Figure 4.27 Use of a dichotomous taxonomic key-overview Gram-positive cells? Rod-shaped cells? Gram-positive bacteria Obligate anaerobes Ferments lactose? Cocci and pleomorphic bacteria Can tolerate oxygen? Can use citric acid (citrate) as sole carbon source? Non-lactose- fermenters Produces gas from glucose? Produces hydrogen sulfide gas? Produces acetoin? Salmonella Enterobacter Citrobacter Escherichia Shigella Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No

55 Classification and Identification of Microorganisms © 2012 Pearson Education Inc. ANIMATION Dichotomous Key: Overview ANIMATION Dichotomous Key: Sample with Flowchart ANIMATION Dichotomous Key: Practice


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