2. Observing and Counting Bacteria

• small
• 1-10 um long, 0.3-1 um wide
• transport by diffusion, surface/volume ratio large

• resolution limit 0.2 um
• poor contrast without staining
• can visualize whole cells

magnification by objective lens (10-100X) x magnification by oculars (10X)

• smallest distance between two objects that allows them to be seen as distinct
• R= λ/2NA

light-gathering ability of the objective

oil has refractive index similar to glass so more light rays pass into objective, increases NA

• 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

• 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 +

• special lens with phase ring amplifies small phase differences
• can observe living specimen without altering
• poor resolution

• 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

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

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

• 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

• 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

• 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