2 - Microbiology

• What antiseptic techniques can be used to avoid contamination?

  • Spray working area with disinfectant and wipe dry
  • Wash hands with antibacterial wash
  • Flame the neck of the culture bottle
  • Lift the lid of the agar plate at an angle
  • Dip spreader in ethanol and pass through bunsen flame before spreading bacteria
  • Use forceps to place antibiotic discs
• Describe how you could investigate the effect of antibiotics on bacterial growth using zones of inhibition.

  • Divide agar plate with bacteria into three segments
  • Use forceps to place filter paper disc with antiseptic in each zone
  • Loosely tape lid onto agar plate to allow oxygen to reach bacteria
  • Incubate at 25°C for 48 hours
  • Measure the diameter of clear zones (zones of inhibition) using a ruler from two opposite directions - calculate mean of measurements
  • Calculate area of clear zones
• How would you measure the zone of inhibition?

  • Use a ruler to measure from a point on one side to a point directly opposite (with lid still in place)
  • Measure again at 90° to first diameter measurement in order to calculate a mean
• Why should you not completely seal the agar plate?

  To allow oxygen to enter the agar plate, preventing the growth of harmful anaerobic bacteria.
• Why is it necessary to measure the diameter of the zone of inhibition twice?

  Clear zones are not always uniform - taking more than one measurement allows a mean diameter to be calculated.
• What equation is used to calculate the area of clear zones?

  Area = pi x radius2
• What is the method for this practical (part 1)?

  1. Spray the bench you are working on with disinfectant.
  2. Separate your agar plate into sections with a pen (not the lid).
  3. Wash hands.
  4. Place the different antiseptics onto different filter paper discs.
  5. Lift the lid of the agar plate at an angle carefully and use forceps to place each filter paper disc onto the dots. Note down the antiseptic applied to each zone.
• What is the method for this practical (part 2)?

  6. Tape the lid onto the agar plate securely, but loosely enough that oxygen can still reach the bacteria.

  7. Place the agar plate in the incubator at 25°C for 48 hours.

  8. Measure the diameter of the clear zones after 48 hours using a ruler. Take a second measurement at 90° from the first and take a mean.

  9. Record results in a table and calculate area.