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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.