cpac 12

• Aseptic technique


  Used to avoid contamination of the sample from outside substances, such as microorganisms
• Aseptic technique


  • Important to get reliable and repeatable data
• Colorimetry


  A quick and relatively simple way of measuring bacterial growth
• Colorimetry


  • Doesn't provide a direct count and also counts non-viable bacteria
  • Colorimetry readings have to be related to bacterial count via a calibration curve produced using a haemocytometer
• Aseptic Technique


  1. Wipe down surfaces with antibacterial cleaner both before and after experiment

  2. Use a Bunsen burner in the work space so that convection currents draw microbes away from the culture

  3. Flame the wire hoop before using to transfer bacteria

  4. Flame the neck of any bottles before use to prevent any bacteria entering the vessel

  5. Keep all vessels containing bacteria open for the minimum amount of time

  6. Close windows and doors to limit air currents
• Equipment


  • Bench disinfectant
  • Paper towels
  • Bunsen burner
  • Colorimeter
  • Cuvettes
  • Measuring cylinders
  • Yeast suspension
  • Microscope
  • Slides and coverslip
  • Pipette
  • Graph paper photocopied to acetate
• Method


  1. Use a cuvette filled with glucose culture medium to set the absorbance of the colorimeter to zero

  2. Fill a measuring cylinder with yeast suspension. Transfer it into a cuvette. Measure the absorbance

  3. Repeat steps 1 and 2 at least five times over twelve hours

  4. To relate colorimetry readings to a direct cell count, use a haemocytometer, or determine your microscope's field of view with a piece of graph paper photocopied onto acetate. Stain the yeast suspension with methylene blue, add a drop to the slide and count the visible yeast cells

  5. Calculate the volume of one drop by measuring the volume of ten drops. This information will enable you to calculate an overall density and therefore cell count
• Hazards


  • Disinfectant
  • Biohazard
  • Naked flame
• Biohazard


  Contamination; infection
• Naked flame


  Fire hazard; burns
• Safety Precautions


  1. Keep disinfectant away from naked flame

  2. Use disinfectant; wash hands with soap after dissection; do not incubate at human body temperature; do not open agar plate post incubation

  3. Keep away from flammable materials; tie up long hair
• In emergency


  1. Put out fire; seek assistance

  2. Seek assistance

  3. Put out fire; seek assistance; run burns under cold water immediately
• Risk Level


  • Low
  • Low/medium (depends on likeliness of bacteria sample used to cause infection)
  • Low
• Graph


  Plot a graph of absorbance against time
• Absorbance increases when number of bacteria increases. This makes the suspension more opaque, transmission decreases and absorbance increases.
• Bacterial growth curve


  1. Lag (bacteria don't grow at maximum rate as they adapt to new environmental conditions)

  2. Log (exponential growth - bacteria grow at theoretical maximum rate)

  3. Stationary (as resources begin to be used up, bacterial growth is equal to bacterial death so number doesn't change)

  4. Death (many more bacteria are dying and so number decreases)