RPQ 6 - testing effects of antibiotics - investigating selec

• antibiotics are medicines that are designed to kill bacteria - this makes them an antimicrobial substance type - can investigate the effects of different antibiotics on bacterial growth - whole experiment must be carried out using aseptic techniques
• Liquid broth


  Mixture of distilled water, bacterial culture and nutrients
• Transferring bacteria from broth to agar plate

  1. Use sterile pipette to transfer bacteria

  2. Spread bacteria over plate using sterile plastic spreader
• Placing antibiotic discs on agar plate


  1. Use sterile forceps to place paper discs soaked with different antibiotics spread apart on the plate

  2. Use various concentrations of antibiotic

  3. Add a negative control disc soaked only in sterile water
• Incubating agar plate


  1. Tape a lid onto the petri dish (without completely sealing it)

  2. Invert and incubate the plate at about 25 degrees for 48 hours
• Incubation of agar plate

  • Allows bacteria to grow forming a 'lawn'
  • Anywhere bacteria can't grow can be seen as a clear patch in the lawn of bacteria - an inhibition zone
• Inhibition zone


  Size of the inhibition zone tells you how well an antibiotic works - the larger the zone the more the bacteria were inhibited from growing
• no inhibition zone = resistant
• control shows that the other results must be due to the presence of antibiotics - not the paper disc
• similar method can be used to test the effects of antiseptics or disinfectants on microbial growth - just replace the paper discs soaked in antibiotics with discs soaked in antiseptics or disinfectants
• aseptic techniques are used to prevent contamination of cultures by unwanted microorganisms - this is important bc contamination can affect the growth of the microorganism you are interested in

  also important to avoid contamination with disease-causing microbes that could make you ill
• Minimise contamination


  1. Regularly disinfect work surfaces

  2. Don't put any utensils on the work surface

  3. Place contaminated utensils in a beaker of disinfectant
• Use sterile equipment


  1. Use glassware sterilised before and after use in an autoclave

  2. Use pre-sterilised plastic instruments once, then safely discard
• Work near a Bunsen flame


  Hot air rises, so any microbes in the air should be drawn away from the plate
• Minimise time with lid off agar plate

  Reduce chance of airborne microorganisms contaminating the culture
• Flame the neck of the glass container of broth

  1. Briefly flame just after it is opened and just before it is closed

  2. Causes air to move out of the container, preventing unwanted microorganisms from falling in
• Method for flaming


  1. Loosen cap of the bottle so it is easy to remove

  2. Lift bottle/tube with left hand

  3. Remove cap/cotton wool plug with little finger curled towards palm of right hand by turning bottle not the cap

  4. Do not put cap/cotton wool down

  5. Flame the neck of the bottle by passing forward and backwards through the blue cone of bunsen flame

  6. Replace cap/ cotton wool plug with little finger again turning bottle not cap taking care as bottle will be hot

  7. If cotton wool has partially lost its shape it will be easier to guide back into the neck of the bottle by slowly twisting mouth of the vessel whilst pushing plug down
• lift lid of gar plate away from you towards the flame of the Bunsen as little as possible placing sample onto petri dish and immediately close lid and replace lid of bottle of culture - place outside of immediate work area
• using a glass spreader which should be flamed before and after use, spread the culture sample out evenly around the agar plate to ensure an even distribution of the sample and therefore even growth - providing uniform layer of bacteria growth on a solid medium - this is done by flooding the surface of the solid medium with a liquid culture, pipetting of the excess inoculum, spreading the culture and incubating at 25 degrees
• tape the lid on at 3,6,9,12 o'clock but do not fully seal to ensure that anaerobic bateria are not being produced
• invert the plate during storage to prevent a build-up of condensation disrupting the bacteria production
• to calculate inhibition zone measure 2 diameters of the clear zone around each chad