2. Observing and Counting Bacteria

  1. bacteria size
    • small
    • 1-10 um long, 0.3-1 um wide
    • transport by diffusion, surface/volume ratio large
  2. bright-field microscopy
    • resolution limit 0.2 um
    • poor contrast without staining
    • can visualize whole cells
  3. total magnification
    magnification by objective lens (10-100X) x magnification by oculars (10X)
  4. resolving power (resolution)
    • smallest distance between two objects that allows them to be seen as distinct
    • R= λ/2NA
  5. numerical aperture
    light-gathering ability of the objective
  6. oil-immersion lens
    oil has refractive index similar to glass so more light rays pass into objective, increases NA
  7. contrast
    • need contrast to see object - objects absorb or scatter light more than surrounding medium, most do not scatter enough
    • create contrast by staining - have affinity for specific cell materials, can use for detection
  8. gram stain
    • differential stain
    • used in diagnostic microbiology, can't observe living specimen
    • 1) stain with crystal violet - stains outermost surface
    • 2) iodine - mordent makes color stick
    • 3) alcohol decolorizes - dissolves outer membrane of gram -
    • 4) counterstain with safranin - purple stuck in peptidoglycan in gram +
  9. phase contrast
    • special lens with phase ring amplifies small phase differences
    • can observe living specimen without altering
    • poor resolution
  10. transmission electron microscopy
    • shorter wavelength, small NA
    • resolution is 0.2-0.3 nm
    • whole-mounted you can see surface details, not interior
    • thin sections allow us to see interior details, must kill
    • chemical treatments create contrast
  11. fluorescence
    immunofluorescence - antibody attached to fluor, treat fixed cells with antibody, use specific wavelength light

    advantages - locate proteins in cell, can use wild-type cells, uses light microscope

    disadvantages - cells fixed, antibodies can be not specific enough, fixation can destroy protein
  12. green fluorescent protein
    protein with a corresponding gene, fuses genes to express fusion protein in living bacteria

    advantages - don't need antibody, living cells- can see protein movement

    disadvantages - have to genetically manipulate, GFP can interfere with normal function, signal is weaker
  13. direct microscopic count
    • counting chamber with cover slip
    • see all cells and can tell if more than one shape, rapid
    • can't tell living or dead, have to immobilize, sampling error
  14. viable count or colony count
    • count number of cells capable of forming colony on solid medium, only living cells counted
    • different morphologies can be observed
    • medium may be suitable for one species not others, can take long time to grow
  15. turbidity
    • indirect, optical density is proportional to number of cells present, must generate standard curve of OD to cell number by another method
    • rapid, no sample destroyed
    • doesn't distinguish between living and dead cells or different kinds of cells
Card Set
2. Observing and Counting Bacteria
general microbiology midterm 1