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nester03a - Important Point Part 2 will cover prokarotic...

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Unformatted text preview: Important Point: 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). Compound Scope: More than one lens Advantage = Potential for greater magnification In a simple microscope the light passes through only a single lens. Advantage: Less optical distortion, simpler to build. Disadvantage: Less magnification. Additional Scope Classifications Part 1 will cover microscopy, through ~p. 53. Simple Scope We will cover this chapter in two parts. Compound Scope Chapter 3 Notes Chapter 3: Microscopy and Cell Structure 1 Additional Scope Classifications Anatomy of a Microscope Bright background, most common, what you will be using in lab. 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 Anatomy of a Microscope The Eyepiece is Commonly Described as the Ocular lens. 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. Which image is least resolved? What is Resolution? What are the two names we give to the two lenses that together make up a compound scope? Higher Resolution; Note the “Sharpness” of the Image. Lower Resolution; Note that the otherwise similar image with otherwise similar magnification nevertheless is less sharp. 2 Air has a different Index of Refraction from water (so light bends). Oil Immersion Increases Resolution Anatomy of a Microscope “High and Dry,” generally = 40x, must not touch oil. 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). Only the “Oil Immersion” Objective (generally 100x) is to come in contact with Immersion Oil! Blue Light Increases Resolution Light Interactions Oil Immersion Increases Resolution Blue Light Increases Resolution All but transmission reduces resolution. Blue light has shorter wavelength than other visible regions of the electromagnetic spectrum. Shorter wavelength results in higher resolution. Blue filter is inserted between light source and condenser. 3 Freeze Fracturing (TEM) Transmission Electron Microscopy (TEM): electrons are transmitted through substance. Scanning Electron Microscopy (SEM): electrons bounce off the surface of specimen resulting in a more 3-D image. Electron Microscopy (EM) Transmission Electron Microscopy EM = Increased Resolution Typically the clearest image of the specimen will also be realized with the Condenser raised as far as it can be raised. Shadow Casting (TEM) Anatomy of a Microscope 4 False Coloration Scanning Electron Microscopy Contrast is Really Important 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. 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. Differential Stain: The Gram Stain Gram Staining Bacillus anthracis Escherichia coli 5 Escherichia coli Note that the background is stained as well as the bacteria, plus there is a “halo” around the bacteria. The halo represents the capsule. Acid-Fast Staining Bacillus anthracis Negative Stain (note capsule) Gram Staining Special Stain: Capsule Staining “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 violetiodine complex during prolonged decolorization.” Note that the acidfast bacteria are Mycobacterium avium complex (MAC) with acid fast stain found as red often has the characteristic appearance shown here of clumps with numerous mycobacteria filling macrophages. Such cells. filamentous macrophages may be distributed diffusely or in clusters. Capsules, made by and surrounding bacteria, are simply difficult to stain. Acidic dye fails to adhere to acidic surface of most bacteria. Link to Next Presentation 6 Additional Scope Classifications Bright background, most common, what you will be using in lab. For viewing unstained cells. You will use this as part of demonstrations. We will also consider electron microscopy: High magnification, resolution, and contrast. 3a ...
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