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Chapter 3: Microscopy and Cell Structure. Important Point:. If you are having trouble understanding lecture material: Try reading your text before attending lectures. And take the time to read it well!. We will cover this chapter in two parts.

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Chapter 3 microscopy and cell structure l.jpg
Chapter 3:Microscopy andCell Structure


Important point l.jpg
Important Point:

If you are having trouble

understanding lecture material:

Try reading your text

before attending lectures.

And take the time to read it well!


Chapter 3 notes l.jpg

  • We will cover this chapter in two parts.

  • Part 1 will cover microscopy, through ~p. 53.

  • Part 2 will cover prokarotic cell structure, through ~p. 72.

  • Part 3 we will not cover nor will you be held responsible for it (this section covers eukaryotic cell structure).

Chapter 3 Notes


Simple scope l.jpg

In a simple microscope the light passes through only a single lens.

Simple Scope

Advantage: Less optical distortion, simpler to build.

Disadvantage: Less magnification.


Compound scope l.jpg
Compound Scope single lens.

Compound Scope: More than one lens

Advantage = Potential for greater magnification



Additional scope classifications7 l.jpg

Bright background, most common, what you will be using in lab.

Additional Scope Classifications

For viewing unstained cells.You will use this as part of demonstrations.

We will also consider electron microscopy:High magnification, resolution, and contrast.



Anatomy of a microscope9 l.jpg
Anatomy of a Microscope lab.

The Eyepiece is Commonly Described as the Ocular lens.


Anatomy of a microscope10 l.jpg
Anatomy of a Microscope lab.

What are the two names we give to the two lenses that together make up a compound scope?


What is resolution l.jpg

  • The object of microscopy is not just to increase magnification, but to do so while retaining sufficient resolution.

  • Resolution is the ability to see two items as two separate things, i.e., two dots as two separate dots.

  • The resolution a microscope is capable of achieving is the smallest distance between two dots such that the two dots may be observed (resolved) as separate entities.

  • In less technical terms, lower resolution means an increased degree of fuzziness, i.e., less focusable specimens.

  • Greater resolution can be achieved by using oil immersion, by filtering out not-blue light, and by replacing light with electrons.

What is Resolution?


Which image is least resolved l.jpg

Higher Resolution; Note the “Sharpness” of the Image. magnification, but to do so while retaining sufficient resolution.

Which image is least resolved?

Lower Resolution; Note that the otherwise similar image with otherwise similar magnification nevertheless is less sharp.


Light interactions l.jpg

All but transmission reduces resolution. magnification, but to do so while retaining sufficient resolution.

Light Interactions


Oil immersion increases resolution l.jpg
Oil Immersion Increases Resolution magnification, but to do so while retaining sufficient resolution.


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Air has a different Index of Refraction from water (so light bends).

Oil Immersion Increases Resolution

Air has a different Index of Refraction from glass (so light bends).

The Mineral Oil has the same Index of Refraction as glass (so light does not bend).


Anatomy of a microscope16 l.jpg
Anatomy of a Microscope bends).

“High and Dry,” generally = 40x, must not touch oil.

Only the “Oil Immersion” Objective (generally 100x) is to come in contact with Immersion Oil!



Blue light increases resolution18 l.jpg

Blue light has shorter wavelength bends).than other visible regions of the electromagnetic spectrum.

Blue Light Increases Resolution

Shorter wavelength results in higher resolution.

Blue filter is inserted between light source and condenser.


Anatomy of a microscope19 l.jpg
Anatomy of a Microscope bends).

Typically the clearest image of the specimen will also be realized with the Condenser raised as far as it can be raised.



Em increased resolution l.jpg

Transmission Electron Microscopy (TEM): electrons are transmitted through substance.

EM = Increased Resolution

Scanning Electron Microscopy (SEM): electrons bounce off the surface of specimen resulting in a more 3-D image.


Transmission electron microscopy l.jpg
Transmission Electron Microscopy transmitted through substance.


Freeze fracturing tem l.jpg
Freeze Fracturing (TEM) transmitted through substance.


Shadow casting tem l.jpg
Shadow Casting (TEM) transmitted through substance.


Scanning electron microscopy l.jpg
Scanning Electron Microscopy transmitted through substance.


False coloration l.jpg
False Coloration transmitted through substance.


Contrast is really important l.jpg

  • Contrast results from differences in the index of refractive between specimen and background, or within specimen = # visible shades in specimen.

  • Black vs. White = high contrast!

  • Unfortunately, unstained bacteria under bright-field microscopy are nearly transparent.

  • If you can’t contrast what you are looking at from the background then you can’t distinguish what you are looking at from the background.

  • Closing Iris Diaphragm increases contrast.

  • Contrast can also be increased by employing optical “tricks” such as with phase contrast.

  • “Stains provide contrast between bacteria and surrounding media.”

  • Types of staining:Simple staining, Differential staining, Special stains: capsule, endospore.

Contrast is Really Important


Anatomy of a microscope28 l.jpg
Anatomy of a Microscope between specimen and background, or within specimen = # visible shades in specimen.

The Iris Diaphragm controls contrast with greater contrast achieved by letting less light through to the specimen.

Generally you want to close (= less light) the Iris Diaphragm as far as you can get away with while still allowing sufficient illumination of the specimen.


Differential stain the gram stain l.jpg
Differential Stain between specimen and background, or within specimen = # visible shades in specimen.: The Gram Stain


Gram staining l.jpg
Gram Staining between specimen and background, or within specimen = # visible shades in specimen.

Bacillus anthracis

Escherichia coli


Gram staining31 l.jpg
Gram Staining between specimen and background, or within specimen = # visible shades in specimen.

Bacillus anthracis

“One of the most common mistakes is to decolorize a smear for too long a time period. Even Gram-positive cells can lose the crystal violet-iodine complex during prolonged decolorization.”

Escherichia coli


Acid fast staining l.jpg
Acid-Fast Staining between specimen and background, or within specimen = # visible shades in specimen.

Note that the acid-fast bacteria are found as red clumps of filamentous cells.

Mycobacterium avium complex (MAC) with acid fast stain often has the characteristic appearance shown here with numerous mycobacteria filling macrophages. Such macrophages may be distributed diffusely or in clusters.


Special stain capsule staining l.jpg
Special Stain: between specimen and background, or within specimen = # visible shades in specimen.Capsule Staining

Note that the background is stained as well as the bacteria, plus there is a “halo” around the bacteria. The halo represents the capsule.


Negative stain note capsule l.jpg
Negative Stain between specimen and background, or within specimen = # visible shades in specimen. (note capsule)

Capsules, made by and surrounding bacteria, are simply difficult to stain.

Acidic dye fails to adhere to acidic surface of most bacteria.


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Link to Next Presentation between specimen and background, or within specimen = # visible shades in specimen.


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