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Summary of Lab 2 for BIOL 112 (Winter 2011). Week of Jan 17, 2011.

Microbiology I: bacteria.

1Introduction to bacteria

  • Pathogens - disease-causing bacteria
  • Morphology: three main shapes
    • Cocci (singular: coccus); spheres
    • Bacilli (singular: bacillus); rods
    • Spirilla (singular: spirillum); helical (curved rods with flagella)
  • Division: usually by binary fission, form two functionally distinct cells
  • Staining: bacteria are usually colourless, so we use dyes that have special affinities for particular components
    • Gram staining: procedure to classify bacteria based on their cell wall
    • Thick peptidoglycan cell wall - gram positive (dark blue)
    • Thin peptidoglycan layer - gram negative (pink)
    • More later
  • Bacterial cultures
    • Medium - fluid or solid surface they grow on
    • Pure culture: a population of bacteria, all the progeny of a single ancestor
  • Bacterial growth
    • Defined in terms of population size
    • When a bacterial cell has almost doubled in size, it will divide by binary fission to form two identical daughter cells
    • Time required for one cell to divide into two: doubling or generation time
    • Best measured by the time it takes for the colony to double in size (number of bacteria)
  • Sterile techniques
    • Put shit in "biohazardous materials" bags
    • If you spill, disinfect with 5% bleach
    • When pipetting, do not let any solution draw up into the pipetting aid
    • Try to keep containers open only briefly
    • Inoculating loop: partly insulated wire, for transferring microorganisms
      • First heat it over a flame until red-hot, then cool it and use to transfer bacteria
    • Spreader: can be plastic or glass; if plastic, don't heat it; if glass, sterilise briefly over a flame
      • Return it to the alcohol beaker afterwards (it's the ethanol that kills bacteria or something)
      • Use it to spread bacteria that has been transferred to a medium
    • Test tube culture: hold the test tube at an angle to reduce bacteria falling in from the air when transferring
      • Flame the mouth of the tube before and after transferring
    • Petri plate cultures
      • Solid agar media (melt at 100C, solidify at 40C); useful for obtaining isolated colonies

2Bacterial colonies

  • Observe colonies, describe shapes - e.g. round or irregular, convex or flat, small or large, etc
  • Serratia marcescens:
    • Small, diseased-looking (reddish), convex, pigmented, hard-looking (but not actually)
  • Bacillus subtilis:
    • Yellow, buttery, flat, round, large
  • Staphylococcus epidermidis:
    • Pinpoint, yellowish, mucoid, flat

3Bacterial morphology

  • Gram-staining each of the types of bacteria mentioned above, viewing on slides
  • Gram staining method:
    • Using sterile techniques, put a bit of water, then a bit of the bacteria on a slide
    • Allow the smear to dry in air, then pass it over the flame a bit, gently warming it
    • Lay it on the rack
    • Flood the slide with gentian violet for one minute
    • Rinse it off with Gram's iodine (mordant), leave flooded for one minute
    • Rinse with tap water, flood with ethanol until dye is all gone (45-60 seconds max)
    • Flood with dilute carbol fuchsin for 30 seconds
    • Rinse with tap water, blot dry
  • Gram positive bacteria are NOT decolorised by the water/ethanol, so they keep the purple colour (iodine)
  • Gram negative bacteria ARE decolourised, so they take up the carbol fuchsin (pink) counterstain
  • Observe under the microscope with oil immersion:
    • Serratia marcescens:
      • Rods, magenta, many of them everywhere, forming small clusters
      • Gram-negative bacilli
    • Bacillus subtilis:
      • Rods, blue border, purple-magenta inside, random scattering
      • Gram-positive bacilli
    • Staphylococcus epidermidis:
      • Clusters and sometimes chains of cocci, dark blue/purple
      • Gram-positive cocci
  • Note that if you don't follow the time guidelines exactly, you could screw up the gram stain
    • For instance, if you didn't rinse the serratia enough, it could still appear blue (from the gentian violet)
    • Or, if you rinsed the bacillus or staphylococcus TOO much, they could be decolourised and thus take up the counter-stain (pink)
  • Spirillum volutans: gram negative, spirilli

4Bacterial growth

  • Generation time - time it takes a population to double, should stay constant in exponential phase
  • Phases:
    • Lag phase: population is growing, adjusting to new environment
      • Cells growing in volume or mass, synthesising proteins, increasing in metabolic activity
      • Dependent on many factors including size of inoculum, and time required to do shit
    • Exponential phase: growing exponentially, like bacteria are wont to (binary fission)
      • Cells dividing at a pretty much constant rate
      • Formula: $\displaystyle 2^n = \frac{\text{number of cells now}}{\text{number of cells initially}}$ where n is the number of generations
    • Stationary phase: growth becomes limited (not enough space or food), bacteria stop dividing
      • Or, equal numbers of bacteria are dying and being produced
      • Either way, population numbers stay constant
    • Death phase: start dying et
      • Population numbers go down, theoretically exponentially but not really
  • Plating technique, experiment with serratia
    • Vortex the bacterial culture before each time we plate so we get a homogeneous solution (each time = representative sample)
  • Count the number of colonies, plot a graph, etc