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Chapter Overview

• Title: Observing Microorganisms through a Microscope

• Source: Microbiology: An Introduction, Thirteenth Edition, Pearson Education, 2019

Page 1

• Introduction to Microscopy

  • Overview of observing microorganisms

  • Contains hyperlinks for additional resources, accessible via JAWS for screen readers.

Page 2

• Focus on Helicobacter Pylori

  • Discusses a specific type of bacteria important in microbiology.

Page 3

• Microscope and Magnification

  • Reference to a figure (Figure 3-2) demonstrating microscopes.

  • For details, refer to slide 78 in Appendix 1.

Page 4

• Units of Measurement

  • Learning Objective: Understand the units used to measure microorganisms.

Page 5

• Micrometer and Nanometer Measurements

  • Microorganisms are primarily measured in micrometers (μm) and nanometers (nm).

    • 1 μm = 10^{-6} m = 10^{-3} mm

    • 1 nm = 10^{-9} m = 10^{-6} mm

    • 1000 nm = 1 μm

    • 0.001 μm = 1 nm

Page 6

• Check Your Understanding

  • Question regarding conversion: How many nanometers is 10 micrometers?

Page 7

• Microscopy Instruments (Part 1)

  • Learning Objectives:

    • Diagram the path of light through a compound microscope.

    • Define total magnification and resolution.

Page 8

• Simple Microscope

  • Contains only one lens; functions similarly to a magnifying glass but has higher magnification.

Page 9

• Anton Van Leeuwenhoek

  • Reference to his observations through microscopy (Figure 1.3b).

Page 10

• Light Microscopy Types

  • Overview: Uses visible light to observe specimens. Types include:

    • Compound light microscopy

    • Darkfield microscopy

    • Phase-contrast microscopy

    • Differential interference contrast (DIC) microscopy

    • Fluorescence microscopy

    • Confocal microscopy

Page 11

• Compound Light Microscope

  • Reference to Figure 3-1a demonstrating its structure.

Page 12

• Understanding Compound Light Microscopy

  • The image is magnified by both the objective and ocular lenses.

  • Total magnification formula: objective lens × ocular lens.

Page 13

• Visual of Compound Light Microscope

  • Figure 3-1b provides visual details.

Page 14

• Resolution in Microscopy

  • Ability to distinguish between two points; importance of wavelength.

  • A microscope resolving power of 0.4 nm identifies points 0.4 nm apart.

Page 15

• Refractive Index & Immersion Oil

  • Defined as the light-bending ability of a medium.

  • Immersion oil is used to minimize refractive loss of light in microscopy.

Page 16

• Refraction in Microscopes

  • Visual reference Figure 3-3 illustrating the effects of refraction.

Page 17

• Brightfield Illumination

  • Description of the brightfield microscopy method, contrasting dark objects against light backgrounds.

Page 18

• Comparison of Microscopy Types

  • Figure 3-4a visualizes brightness in various microscopy types (brightfield).

Page 19

• Check Your Understanding

  • Questions about the lenses involved in light passage and significance of resolution values.

Page 20

• Microscopy Instruments (Part 2)

  • Learning Objectives:

    • Uses for darkfield, phase-contrast, DIC, fluorescence, confocal, two-photon, and scanning acoustic microscopy.

    • Contrast with brightfield illumination.

    • Differences between electron and light microscopy.

Page 21

• Darkfield Microscopy

  • Light objects viewed against a dark backdrop; achieved by a special condenser.

Page 22

• Darkfield View

  • Figure 3-4b shows how darkfield microscopy enhances visibility of cellular edges.

Page 23

• Phase-Contrast Microscopy

  • Allows observation of living cells and their structures by combining direct and diffracted light rays.

Page 24

• Phase-Contrast Image

  • Figure 3-4c illustrates differences in light paths.

Page 25

• Differential Interference Contrast (DIC)

  • Enhances contrast and color using two light beams and prisms.

Page 26

• Visual of DIC Microscopy

  • Reference to Figure 3-5 for illustration.

Page 27

• Fluorescence Microscopy

  • Utilizes UV light to excite fluorescent substances.

  • Cells can be stained with fluorescent dyes for visualization.

Page 28

• Immunofluorescence Technique

  • Figure 3-6b depicts immunofluorescence application.

Page 29

• Confocal Microscopy

  • Involving fluorochrome dyes and computer-aided image construction.

  • Can examine up to 100 μm depth.

Page 30

• Visual of Confocal Microscopy

  • Reference to Figure 3-7.

Page 31

• Two-Photon Microscopy

  • Uses two photons of long-wavelength light for studying living cells up to 1 mm deep.

Page 32

• Visual of Two-Photon Microscopy

  • Refers to Figure 3-8 for details.

Page 33

• Super-Resolution Light Microscopy

  • Employs two laser beams for enhanced fluorescence imaging at a nanometer scale.

Page 34

• Visual of Super-Resolution Microscopy

  • Reference to Figure 3-9 illustrating the method.

Page 35

• Scanning Acoustic Microscopy

  • Uses sound waves to analyze cells on surfaces; resolution of 1 μm.

Page 36

• Visual of Scanning Acoustic Microscopy

  • Refers to Figure 3-10 depicting a bacterial biofilm.

Page 37

• Check Your Understanding

  • Questions comparing microscopy techniques.

Page 38

• Electron Microscopy Overview

  • Utilizes electrons instead of light, offering greater resolution.

Page 39

• Transmission Electron Microscopy (Part 1)

  • Describes passage of electrons through thin specimens; potential staining.

Page 40

• Transmission Electron Microscopy Visual

  • Reference to Figure 3-11a.

Page 41

• Transmission Electron Microscopy (Part 2)

  • Magnification capabilities and resolution details.

Page 42

• Scanning Electron Microscopy (Part 1)

  • Scanning technique that generates three-dimensional images from emitted electrons.

Page 43

• Visual of SEM

  • Reference to Figure 3-11b.

Page 44

• Scanning Electron Microscopy (Part 2)

  • Magnification range and resolution description.

Page 45

• Check Your Understanding

  • Questions on why electron microscopes have higher resolution than light microscopes.

Page 46

• Scanning Tunneling Microscopy

  • High-resolution surface scanning using a tungsten probe.

Page 47

• Visual of Scanned-Probe Microscopy

  • Reference to Figure 3-12a detailing surface analysis.

Page 48

• Atomic Force Microscopy

  • Utilizes a probe to record movements, creating high-resolution 3D images.

Page 49

• Visual of Atomic Force Microscopy

  • Reference to Figure 3-12b.

Page 50

• Check Your Understanding

  • Questions addressing specific microscopy techniques (TEMeter, SEMeter).

Page 51

• Preparation of Specimens for Light Microscopy

  • Learning Objectives focus on staining techniques and differentiation methods.

Page 52

• Staining Process Explanation (Part 1)

  • Overview of the purpose and methodology of staining microorganisms.

Page 53

• Staining Process Explanation (Part 2)

  • Discussion about the contrast of live versus stained specimens.

Page 54

• Staining Composition

  • Relationship between ionic composition and dye application; distinction between basic and acidic dyes.

Page 55

• Simple Stains Overview

  • Technique using a single dye to enhance visual attributes of microorganisms.

Page 56

• Check Your Understanding

  • Questions about staining methods and procedures.

Page 57

• Differential Stains

  • Focus on distinguishing between types of bacteria (Gram stain and Acid-fast stain).

Page 58

• Gram Stain Overview

  • Differentiates bacteria based on cell wall structure:

    • Gram-positive: thick peptidoglycan walls.

    • Gram-negative: thin peptidoglycan walls with lipopolysaccharides.

Page 59 & 60

• Visuals of Gram Staining

  • Reference to Figures 3-13a and 3-13b for staining illustration.

Page 61

• Acid-Fast Stain (Part 1)

  • Targets bacteria with waxy cell wall material; key in identifying specific genera.

Page 62

• Acid-Fast Stain (Part 2)

  • Color transition explanation through the staining process.

Page 63

• Visual of Acid-Fast Bacteria

  • Reference to Figure 3-14 for visual impact.

Page 64

• Check Your Understanding

  • Questions focusing on the utility of Gram staining.

Page 65

• Special Stains Overview

  • Techniques to target specific parts of microorganisms including capsule, endospore, and flagella stains.

Page 66

• Capsule Staining

  • Capsule appears as a halo around the cell when stained properly.

Page 67

• Visual of Special Staining

  • Reference to Figure 3-15a.

Page 68

• Endospore Staining Overview

  • Steps and key colors observed during endospore staining.

Page 69

• Visual of Endospore Staining

  • Reference to Figure 3-15b.

Page 70

• Flagella Staining Method

  • Mordant application details to enhance flagella visibility.

Page 71

• Visual of Flagella Staining

  • Reference to Figure 3-15c.

Page 72

• Check Your Understanding

  • Inquiries about endospore appearance in stained and unstained conditions.

Page 73

• Copyright Notice

  • Legal implications regarding the distribution of content from the textbook.