1. PowerPoint ® Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R © 2014 Pearson Education, Inc. Microscopy, Staining, and Classification 4

2. © 2014 Pearson Education, Inc. Microscopy and Staining: Overview PLAY

3. © 2014 Pearson Education, Inc.

4. Microscopy General Principles of Microscopy Wavelength of radiation Magnification Resolution Contrast

5. © 2014 Pearson Education, Inc. Figure 4.1 The electromagnetic spectrum.

6. © 2014 Pearson Education, Inc. Figure 4.2 Light refraction and image magnification by a convex glass lens. Light Air Glass Focal point Specimen Convex lens Inverted, reversed, and Enlarged image

7. © 2014 Pearson Education, Inc. Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of various types of microscopes.

8. © 2014 Pearson Education, Inc. 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

9. © 2014 Pearson Education, Inc. Microscopy Light Microscopy Bright-field microscopes Simple Contain a single magnifying lens Similar to magnifying glass Leeuwenhoek used simple microscope to observe microorganisms

10. © 2014 Pearson Education, Inc. 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)

11. © 2014 Pearson Education, Inc. Figure 4.4 A bright-field, compound light microscope. Coarse focusing knob Moves the stage up and down to focus the image Illuminator Light source Diaphragm Controls the amount of light entering the condenser Condenser Focuses light through specimen Stage Holds the microscope slide in position Objective lenses Primary lenses that magnify the specimen Body Transmits the image from the objective lens to the ocular lens using prisms Ocular lens Remagnifies the image formed by the objective lens Line of vision Ocular lens Path of light Prism Body Objective lenses Specimen Condenser lenses Illuminator Fine focusing knob Base Arm

12. © 2014 Pearson Education, Inc. Figure 4.5 The effect of immersion oil on resolution. Glass cover slip Slide Specimen Light source Without immersion oil Lenses Immersion oil Glass cover slip Slide Light source With immersion oil Microscope objective Refracted light rays lost to lens Microscope objective More light enters lens

13. © 2014 Pearson Education, Inc. 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

14. © 2014 Pearson Education, Inc. Objective Light refracted by specimen Light unrefracted by specimen Specimen Dark-field stop Condenser Dark-field stop Figure 4.6 The light path in a dark-field microscope.

15. © 2014 Pearson Education, Inc. 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

16. © 2014 Pearson Education, Inc. 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 4.7 Principles of phase microscopy.

17. © 2014 Pearson Education, Inc. Nucleus Bacterium Bright field Dark field Phase contrast Nomarski Figure 4.8 Four kinds of light microscopy.

18. © 2014 Pearson Education, Inc. 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

19. © 2014 Pearson Education, Inc. Figure 4.9 Fluorescence microscopy.

20. © 2014 Pearson Education, Inc. Figure 4.10 Immunofluorescence. Antibodies Bacterium Cell-surface antigens Bacterial cell with bound antibodies carrying dye Fluorescent dye Antibodies carrying dye

21. © 2014 Pearson Education, Inc. 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

22. © 2014 Pearson Education, Inc. Light Microscopy PLAY

23. © 2014 Pearson Education, Inc. 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

24. © 2014 Pearson Education, Inc. Figure 4.11 A transmission electron microscope (TEM). Lamp Light microscope (upside down) Column of transmission electron microscope Lamp Condenser lens Specimen Objective lens Eyepiece Final image seen by eye Electron gun Specimen Objective lens (magnet) Projector lens (magnet) Final image on fluorescent screen

25. © 2014 Pearson Education, Inc. Electron gun Magnetic lenses Primary electrons Secondary electrons Specimen holder Vacuum system Beam deflector coil Scanning circuit Photo- multiplier Detector Monitor Figure 4.12 Scanning electron microscope (SEM).

26. © 2014 Pearson Education, Inc. Figure 4.13 SEM images.

27. © 2014 Pearson Education, Inc. Electron Microscopy PLAY

28. © 2014 Pearson Education, Inc. Microscopy Probe Microscopy Magnifies more than 100,000,000 times Two types Scanning tunneling microscopes Atomic force microscopes

29. © 2014 Pearson Education, Inc. Enzyme DNA Figure 4.14 Probe microscopy.

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32. 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

33. © 2014 Pearson Education, Inc. Figure 4.15 Preparing a specimen for staining.

34. © 2014 Pearson Education, Inc. 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

35. © 2014 Pearson Education, Inc. Staining Simple stains Differential stains Gram stain Acid-fast stain Endospore stain Special stains Negative (capsule) stain Flagellar stain

36. © 2014 Pearson Education, Inc. Figure 4.16 Simple stains.

37. © 2014 Pearson Education, Inc. Figure 4.17 The Gram staining procedure.

38. © 2014 Pearson Education, Inc. Figure 4.18 Ziehl-Neelsen acid-fast stain.

39. © 2014 Pearson Education, Inc. Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis.

40. © 2014 Pearson Education, Inc. Staining Differential Stains Histological stains Two common stains used for histological specimens Gomori methenamine silver (GMS) stain Hematoxylin and eosin (HE) stain

41. © 2014 Pearson Education, Inc. Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae. Bacterium Capsule Background stain

42. © 2014 Pearson Education, Inc. Flagella Figure 4.21 Flagellar stain of Proteus vulgaris.

43. © 2014 Pearson Education, Inc. Staining Staining for Electron Microscopy Chemicals containing heavy metals used for transmission electron microscopy Stains may bind molecules in specimens or the background

44. © 2014 Pearson Education, Inc. Staining PLAY

45. © 2014 Pearson Education, Inc. 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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47. 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

48. © 2014 Pearson Education, Inc. Figure 4.22 Levels in a Linnaean taxonomic scheme.

49. © 2014 Pearson Education, Inc. 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

50. © 2014 Pearson Education, Inc. 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

51. © 2014 Pearson Education, Inc. Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids

52. © 2014 Pearson Education, Inc. 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

53. © 2014 Pearson Education, Inc. No hydrogen sulfide Hydrogen sulfide produced Acid with gasAcid with no gasInert Gas bubbleInverted tubes to trap gas Figure 4.23 Two biochemical tests for identifying bacteria.

54. © 2014 Pearson Education, Inc. Wells Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system.

55. © 2014 Pearson Education, Inc. Figure 4.25 An agglutination test, one type of serological test.

56. © 2014 Pearson Education, Inc. Bacterial lawn Plaques Figure 4.26 Phage typing.

57. © 2014 Pearson Education, Inc. 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

58. © 2014 Pearson Education, Inc. 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

59. © 2014 Pearson Education, Inc. Figure 4.27 Use of a dichotomous taxonomic key.

60. © 2014 Pearson Education, Inc. Dichotomous Keys: Overview PLAY Dichotomous Keys: Overview