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
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.
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
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)