The document discusses microscopy techniques, staining methods, and microbial classification. It describes various types of microscopes like brightfield, darkfield, phase contrast, fluorescent, confocal, transmission electron, and scanning electron microscopes. It also discusses staining to increase contrast and resolution, like simple stains, differential stains, Gram stain, and stains for electron microscopy. Finally, it covers microbial taxonomy and methods for microbial identification like physical and biochemical tests, nucleic acid analysis, and using dichotomous keys.

1. Chapter 4 Microscopy, Staining, and Classification

2. Units of Measurement Animation: Microscopy and Staining

3. Units of Measurement [INSERT TABLE 4.1]

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

5. Microscopy [INSERT FIGURE 4.1]

6. Microscopy [INSERT FIGURE 4.2]

7. Microscopy [INSERT FIGURE 4.3]

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

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

10. Microscopy Light Microscopy Bright-field microscopes Compound Use a series of lenses for magnification Light rays pass through specimen and into objective lens (one of a series of objective lenses) Oil immersion lens increases resolution because light does not refract Have one or two ocular lenses Total magnification = magnification of objective lens X magnification of ocular lens Most have condenser lens to direct light through specimen

11. Microscopy [INSERT FIGURE 4.4]

12. Microscopy [INSERT FIGURE 4.5]

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

14. Microscopy [INSERT FIGURE 4.6]

15. Microscopy Light Microscopy Phase microscopes Used to examine living organisms or specimens that would be damaged or altered by attaching them to slides or staining them Treat one set of light rays differently from another set Light rays in phase produce brighter image, while light rays out of phase produce darker image Contrast is created because light waves are 1/2 wavelength out of phase Two types Phase-contrast microscope Differential interference contrast microscope

16. Microscopy [INSERT FIGURE 4.7]

17. Microscopy [INSERT FIGURE 4.8]

18. Microscopy Light Microscopy Fluorescent microscopes Direct UV light source at specimen; causes the specimen to radiate energy back as a longer, visible wavelength UV light increases resolution and contrast Some cells and molecules are naturally fluorescent, while others must be stained Used in immunofluorescence to identify pathogens and to locate and make visible a variety of proteins

19. Microscopy [INSERT FIGURE 4.9]

20. Microscopy [INSERT FIGURE 4.10]

21. Microscopy Light Microscopy Confocal microscopes Use fluorescent dyes Use UV lasers to illuminate fluorescent chemicals in a single plane that is no thicker than 1.0  m Resolution increased by up to 40% because emitted light passes through pinhole aperture Computer constructs 3-D image from digitized images

22. Microscopy Animation: Light Microscopy

23. Microscopy Electron Microscopy Light microscopes cannot resolve structures closer than 200 nm because shortest wavelength of visible light is 400 nm Electrons have wavelengths of 0.01 nm to 0.001 nm, so electron microscopes have greater resolving power and greater magnification Magnifies objects 10,000X to 100,000X Gives detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms Two types Transmission electron microscopes Scanning electron microscopes

24. Microscopy [INSERT FIGURE 4.11]

25. Microscopy [INSERT FIGURE 4.12]

26. Microscopy [INSERT FIGURE 4.13]

27. Microscopy Animation: Electron Microscopy

28. Microscopy Probe Microscopy Uses minuscule, pointed, electronic probes to magnify more than 100,000,000 times Two types Scanning tunneling microscopes Atomic force microscopes

29. Microscopy [INSERT FIGURE 4.14]

30. Microscopy [INSERT TABLE 4.2]

31. Staining Increases contrast and resolution by coloring specimens with stains/dyes Smear of microorganisms (thin film) air dried to slide and then fixed to surface by heat or chemical fixation Microbiological stains usually salts composed of cation and anion, with one colored (chromophore) Acidic dyes stain alkaline structures; more commonly, basic dyes stain acidic structures

32. Staining [INSERT FIGURE 4.15]

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

34. Staining [INSERT FIGURE 4.16]

35. Staining [INSERT FIGURE 4.17]

36. Staining [INSERT FIGURE 4.18]

37. Staining [INSERT FIGURE 4.19]

38. Staining [INSERT FIGURE 4.20]

39. Staining [INSERT FIGURE 4.21]

40. Staining [INSERT TABLE 4.3]

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

42. Staining Animation: Staining

43. Classification & Identification of Microorganisms Taxonomy consists of classification, nomenclature, and identification Enables scientists to organize large amounts of information about organisms and make predictions based on knowledge of similar organisms

44. Classification & Identification of Microorganisms Linnaeus, Whittaker, and Taxonomic Categories Linnaeus Provided system that standardized the naming and classification of organisms based on characteristics in common Grouped similar organisms that can successfully interbreed into categories called species Used binomial nomenclature in his system

45. Classification & Identification of Microorganisms

46. Classification & Identification of Microorganisms Linnaeus, Whittaker, and Taxonomic Categories Whittaker Linnaeus proposed only two kingdoms Whittaker proposed a widely accepted taxonomic approach based on five kingdoms: Animalia, Plantae, Fungi, Protista, and Prokaryotae

47. Classification & Identification of Microorganisms [INSERT FIGURE 4.23]

48. Classification & Identification of Microorganisms Linnaeus, Whittaker, and Taxonomic Categories Linnaeus’s goal was classifying and naming organisms as a means of cataloging them More modern goal is understanding relationships among groups of organisms Major goal of modern taxonomy is to reflect phylogenetic hierarchy Greater emphasis on comparisons of organisms’ genetic material led to proposal to add domain

49. Classification & Identification of Microorganisms Domains Carl Woese compared nucleotide sequences of rRNA subunits (changes occur rarely) Proposal of three domains based on three basic types of cells as determined by ribosomal nucleotide sequences Three Domains: Eukarya, Bacteria, and Archaea Cells in the three domains also differ with respect to many other characteristics

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

51. Classification & Identification of Microorganisms [INSERT FIGURE 4.24]

52. Classification & Identification of Microorganisms [INSERT FIGURE 4.25]

53. Classification & Identification of Microorganisms [INSERT FIGURE 4.26]

54. Classification & Identification of Microorganisms [INSERT FIGURE 4.27]

55. Classification & Identification of Microorganisms Taxonomic Keys Dichotomous keys – series of paired statements worded so that 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

56. Classification & Identification of Microorganisms [INSERT FIGURE 4.28]

57. Classification & Identification of Microorganisms Animation: Dichotomous Keys