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.
If you are having trouble
understanding lecture material:
Try reading your text
before attending lectures.
And take the time to read it well!
Advantage: Less optical distortion, simpler to build.
Disadvantage: Less magnification.
Compound Scope: More than one lens
Advantage = Potential for greater magnification
For viewing unstained cells.You will use this as part of demonstrations.
We will also consider electron microscopy:High magnification, resolution, and contrast.
The Eyepiece is Commonly Described as the Ocular lens.
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. 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.
All but transmission reduces resolution. magnification, but to do so while retaining sufficient resolution.Light Interactions
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).
“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 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.
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.
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.
“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.”
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.
Note that the background is stained as well as the bacteria, plus there is a “halo” around the bacteria. The halo represents the capsule.
Capsules, made by and surrounding bacteria, are simply difficult to stain.
Acidic dye fails to adhere to acidic surface of most bacteria.