Chapter 2 Flashcards
Microscopy (Light Microscopy)
Uses light to illuminate cells.
Types: bright field, phase contrast, dark field, fluorescence.
Bright field microscope
Specimens are visualized due to differences in contrast between specimen and surroundings.
Two sets of lens form the image
-Ocular and objective lens
-Total magnification= ocular mag x objective mag
Max magnification is 2000x
Resolution
The ability to distinguish between two adjacent objects as separate and distinct.
Determined by wavelength of light and numerical aperture of lens.
Staining
Improving contrast results in a better image.
Staining improves contrast.
-dyes are organic compounds that bind to specific cellular materials
Stains can be very specific and tell us something about bacteria.
Simple Staining
- Prepare the smear.
- Heat fixing and staining
- microscopy
The gram stain
Differential stain.
Used widely in microbiology.
-Bacteria can be divided into two major groups: gram negative and gram positive.
-Gram positive bacteria appear purple, gram negative bacteria appear red after staining.
Steps in Gram staining
- flood heat fixed smear with crystal violet (cells purple)
- Add iodine solution (cells purple)
- decolorize w/ alcohol (gram+ remain purple; gram- are colorless)
- counterstain with safranin (gram+ are purple, gram- are red)
Increased contrast 2: phase contrast
Specimens stand out against background.
- improves contrast of sample without stain
- allows for visualization of live samples
- dark cells on a light background
Increases contrast 3: dark field
Light reaches specimen from side.
Light reaches lens scattered by specimen.
Light specimen against a dark background.
Excellent for observing motility.
Increased contrast 4: fluorescence
Visualize specimens that fluoresce.
-emit light of one color when illuminated with another color of light.
Cells fluoresce naturally or after being stained with DAPI
Used in microbial ecology for enumerating bacteria
Fish 1
Fluorescence In Situ Hybridization
DNA probe attached to fluorescent dye
Identify and count cells
Atomic Force Microscopy (AFM)
Stylus is placed close to specimen.
Measures weak repulsive forces between it and the specimen.
Computer generates image based on data received from stylus.
Confocal Scanning Laser Microscopy (CSLM)
Computerized microscope coupled with laser source to generate a 3D image.
Can focus on a single layer.
Electron Microscopy
Uses electrons instead of photons to image structures.
Two types: transmission electron microscope (TEM) and scanning electron microscope (SEM)
Transmission Electron Microscope (TEM)
Uses electrons.
Uses electromagnets instead of a lens.
Operates in a vacuum.
High magnification and resolution.
Structures can be visualized at the molecular level.
-Proteins and nucleic acids can be seen.
Specimen must be very thin and stained with a heavy metal.
Scanning Electron Microscope (SEM)
Specimen coated with a thin layer of heavy metal.
Electron beams scan the specimen.
Scattered electrons are collected by a detector and an image is produced.
Large specimens can be observed.
Magnification from 15x-100,000x
Basic cell types
Two major classifications
- Prokaryotes: bacteria and archaea spindle shaped)
- Eukaryotes: Everything else (round shaped)
Common to all cells
cytoplasmic membrane
cytoplasm
ribosomes
Cell membrane
Fluid mosaic structure
-phospholipid bilayer
-proteins interspersed in mosaic
Regulates movement of materials into and out of cells
-nutrients enter and wastes exit
Determines boundary of cell
Assists in capturing ATP and capturing proteins