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Compare and contrast brightfield, darkfield, and phase-contrast microscopes.
- Brightfield: objects dark background bright. Light reflected off specimen does not enter objective lens. For viewing colored/stained specimen
- Darkfield: objects light background dark. Only light reflected off specimen enters objective lens. For viewing unstained or live specimen.
- Phase contrast: Accentuates diffraction of light that passes through specimen. Uses 2 beams of light. Image is dark (light rays of out phase) and light (in phase). For viewing internal structures of live specimen
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Compare and contrast SEM and TEM
- EM: viewing objects smaller than 2 micrometers, short wavelength of electrons gives high resolution
- TransmissionEM: ultrathin sections of specimen. Resolves up to 2.5nm. Not 3D, artifacts present from preparation.
- ScanningEM: secondary electrons emitted from specimen produce image. 3D. Useful for looking at surface structures. Up to 20nm.
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Define basic vs. acidic dyes, explain how they work, given an example of each.
- Basic: chromophore is a cation, stains bacteria. Crystal violent, methylene blue, safranin
- Acidic: chromophore is an anion, stains background. Eosin, nigrosin, fuchsin
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Name the 3 general staining techniques, explain the mechanism and use of each
- Simple stains: single, basic dye. To look at shape, arrangement, and basic structures (negative stain)
- Differential stains: To distinguish between different types of bacteria (Gram stain, acid-fast stain)
- Special stains: To distinguish specific parts of bacterial cells (capsule stain, endospore stain, flagella stain)
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