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Chapter 2 How We See the Invisible World

The document discusses the principles and techniques of microscopy, including the properties of light, types of microscopes, and staining methods. It covers the historical development of microscopes, the differences between simple and compound microscopes, and various microscopy techniques such as fluorescence and electron microscopy. Additionally, it explains specimen preparation and staining procedures, highlighting their clinical relevance.

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jmsronline2013
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16 views

Chapter 2 How We See the Invisible World

The document discusses the principles and techniques of microscopy, including the properties of light, types of microscopes, and staining methods. It covers the historical development of microscopes, the differences between simple and compound microscopes, and various microscopy techniques such as fluorescence and electron microscopy. Additionally, it explains specimen preparation and staining procedures, highlighting their clinical relevance.

Uploaded by

jmsronline2013
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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How We See the Invisible World

OpenStax Microbiology Chapter 2


Identify and define the
characteristics of
The electromagnetic radiation
used in microscopy
Properties of
Light: Explain how lenses are
Objectives used in microscopy to
manipulate visible and
ultraviolet light
Light Basics

• Lets review basic


terminology
Light Basics

• Reflection

• Absorbance

• Interference

• Diffraction
Lenses and Refraction

• Refractive index
Lenses and Refraction

• Image point

• Focal point

• Focal length
Electromagnetic Spectrum

• Dispersion

• Fluorescent dyes

• Phosphorescence

• Magnification

• Resolution
Peering Into the Invisible World: Objectives

Describe historical developments and individual


contributions that led to the invention and development of
the microscope

Compare and contrast the features of simple and


compound microscopes
Early Microscopes

• Simple vs. Compound


• Robert Hooke
Identify and describe the parts of a
brightfield microscope

Instruments of
Microscopy: Calculate total magnification for a
compound microscope

Objectives
Describe the distinguishing
features and typical uses for
various types of light microscopes,
electron microscopes, and
scanning probe microscopes
Light Microscopy

• Lets go through the basics


• Calculate total magnification
• Obj mag. X ocular mag.

• Produces bright image


• Stains increase contrast
Oil Immersion

• Bends light
Darkfield Microscopy

• Opaque disk between illuminator


and condenser
• Blocks most light
• Produces high contrast, high
resolution images
• No Stain needed
• Live specimens

• Treponema pallidum
Phase-Contrast Microscopy

• Uses refraction and interference by structures in


specimen.
• High resolution and contrast
• No stains
• Annular stop
• Produces cone of light
• Phase plate
• Waves become out of phase
Differential Interference
Contrast Microscopy

• Use interference patterns


• Two beams of light
• Polarization differs
• High contrast images
• 3D
• Fonsecaea pedrosoi
• Chromoblastomycosis
Fluorescence Microscopy

• Fluorochromes
• Absorb light
• Emits light
• Immunofluorescence
• Identify disease causers
• Living vs. dead cells
• Locations of molecules
Confocal
Microscopy

• Uses laser
• Scans z-planes
• 2-d images
• Put together into 3D
images
• Thick specimens
Electron
Microscopy

• Transmission electron
microscope
• TEM
• Scanning electron microscope
• SEM
TEM vs. SEM

• TEM requires thin


specimens
• 20-100nm
• Specimens enhanced with
heavy metal stains
• 2-d images
• 500,000X
• SEM
• Sputter-coated with gold
• 3D external image
• 30,000X
Electron Microscopy Images
Scanning Probe Microscopy

• Scanning tunneling microscope


• Probe passes above specimen
• Voltage bias causes electric
current
• Intensity is based on distance
between probe and specimen
• Atomic force microscope
• Thin probe passing above
specimen
• Measures variations in a constant
current
• Atomic level resolution
Microscopy Summary
Microscopy Summary
Microscopy Summary
Differentiate between simple
and differential stains

Staining
Microscopic Describe the unique features of
Specimens: commonly used stains

Objectives
Explain the procedures and
name clinical applications for
Gram, endospore, acid-fast,
negative capsule, and flagella
staining
Preparing Specimens

• Wet mounts
• Specimen placed in liquid
• Stains often added
• Coverslips
• Fixation
• Heating or Chemical
• Attaches specimen to slide
• Kills microbes
Staining

• Basic dye
• Chromophore has positive charge
• Acidic dye
• Chromophore has negative charge
• Positive stain
• Absorbed by the cells
• Negative stain
• Absorbed by background
• Simple vs. Differential
Gram Staining

• Primary stain
• Gives all cells color
• Purple
• Mordant
• Stabilizes stains or dyes
• Iodine
• Decolorizing agent
• Removes color from peptidoglycan
• 95% ethanol
• Counterstain
• Stains decolorized cells
• Safranin
Gram Stain Examples

• Clinical Relevance?

This Photo by Unknown Author is licensed under CC BY-SA

This Photo by Unknown Author is licensed under CC BY-SA

This Photo by Unknown Author is licensed under CC BY-SA


Acid-Fast stains

• Zihel-Neelsen technique
• Carbolfuchsin
• Methylene blue
• heat
• Kinyoun technique
• Carbolfuchsin
• Methylene blue
• No heat
• Clinical Relevance?
• Differentiates acid fast and non acid fast
Capsule Staining

• Highlights cell structure


• Negative staining
• Positive and Negative staining
• Clinical relevance?
Endospore Staining

• Schaeffer-Fulton
• Heat
• Malachite Green
• Safranin

• Clinical relevance?
• Clostridium difficile
Flagellar staining

• Highlights cell structure


• No clinical relevance
Staining Summary

• Simple stains
Staining Summary

• Differential stains
Preparing for Electron
Microscopy: TEM

• Embed cells in plastic


• Dehydrate
• Series of ethanol solutions
• Ultramicrotome
• Cuts extremely thin sections
• Fix samples to copper wire
• Stained
• Uranyl acetate
• Osmium tetroxide
Preparation for Electron
Microscopy: SEM

• Dehydrated samples
• Ethanol series
• Dry with liquid carbon
dioxide
• Sputter-coated with gold
• Specimens can’t be charged
by electron beam

This Photo by Unknown Author is licensed under CC BY-ND

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