1. Microscopy, Staining, and Classification
Chapter 4
Microscopy, Staining, and Classification

2. Microscopy and Staining
Animation: Microscopy and Staining: Overview

3. Units of Measurement
Table 4.1

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

5. The electromagnetic spectrum
Figure 4.1

6. Light refraction and image magnification
Figure 4.2

7. The limits of resolution
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
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. A bright-field, compound light microscope
Figure 4.4

12. The effects of immersion oil on resolution
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. The light path in a dark-field microscope
Figure 4.6

15. 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. Principles of phase microscopy
Figure 4.7

17. Four kinds of light microscopy
Figure 4.8

18. 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. Fluorescent microscopy
Figure 4.9

20. Immunofluorescence
Figure 4.10

21. 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. Animation: Light Microscopy

23. 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. A transmission electron microscope (TEM)
Figure 4.11

25. Scanning electron microscope (SEM)
Figure 4.12

26. SEM images
Figure 4.13

27. Animation: Electron Microscopy

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

29. Probe microscopy
Figure 4.14

30. 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; more commonly, basic dyes stain acidic structures

31. Preparing a specimen for staining
Figure 4.15

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

33. Simple stains
Figure 4.16

34. The Gram staining procedure
Figure 4.17

35. Ziehl-Neelsen acid-fast stain
Figure 4.18

36. Schaeffer-Fulton endospore stain
Figure 4.19

37. Negative (capsule) stain
Figure 4.20

38. Flagellar stain
Figure 4.21

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

40. Staining
Animation: Staining

41. 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

42. Classification and Identification of Microorganisms
Linnaeus, Whittaker, and Taxonomic Categories
Linnaeus
System classified organisms based on characteristics in common
Grouped organisms that can successfully interbreed into categories called species
Used binomial nomenclature in his system

43. Levels in Linnaean taxonomic scheme
Figure 4.22

44. Classification and Identification of Microorganisms
Linnaeus, Whittaker, and Taxonomic Categories
Whittaker
Linnaeus proposed only two kingdoms
Whittaker proposed taxonomic approach based on five kingdoms
Animalia, Plantae, Fungi, Protista, and Prokaryotae

45. Whittaker’s five-kingdom taxonomic scheme
Figure 4.23

46. Classification and Identification of Microorganisms
Linnaeus, Whittaker, and Taxonomic Categories
Linnaeus’s goal was classifying organisms to catalogue them
Modern goal is understanding relationships among groups of organisms
Goal of modern taxonomy is to reflect phylogenetic hierarchy
Greater emphasis on comparisons of organisms’ genetic material led to proposal to add domain

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 also differ with respect to many other characteristics

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

49. Two biochemical tests for identifying bacteria
Figure 4.24

50. One tool for the rapid identification of bacteria
Figure 4.25

51. An agglutination test, one type of serological test
Figure 4.26

52. Phage typing
Figure 4.27

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

54. Use of dichotomous taxonomic key
Figure 4.28

55. Classification and Identification of Microorganisms
Animation: Dichotomous Keys: Overview