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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
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 Compound Scope: More than one lens Advantage = Potential for greater magnification
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 Microscope The Eyepiece is Commonly Described as the Ocular lens.
Anatomy of a Microscope What are the two names we give to the two lenses that together make up a compound scope?
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?
Higher Resolution; Note the “Sharpness” of the Image. Which image is least resolved? Lower Resolution; Note that the otherwise similar image with otherwise similar magnification nevertheless is less sharp.
All but transmission reduces resolution. Light Interactions
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 Microscope “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 has shorter wavelengththan 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 Microscope Typically the clearest image of the specimen will also be realized with the Condenser raised as far as it can be raised.
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.
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 Microscope 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.
Gram Staining Bacillus anthracis Escherichia coli
Gram Staining 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 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 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) Capsules, made by and surrounding bacteria, are simply difficult to stain. Acidic dye fails to adhere to acidic surface of most bacteria.