Investigating Antibiotic Resistance GCSE Biology
Investigate the effect of antiseptics or antibiotics on bacterial growth using agar plates and measuring zones of inhibition.
Students are required to prepare a bacterial lawn plate on which to test the effectiveness of antibiotics. Aseptic technique must be followed during this practical and care must always be taken when handling bacterial cultures. Pre-impregnated antibiotic discs could be used as an alternative to students producing their own. If using Penicillin discs of different concentrations, it is recommended that Micrococcus Luteus cultures are used to produce the lawn plate.
Equipment (per pair of participants)
• Bunsen burner
• Safety goggles
• 1 x nutrient agar plate
• 1 x bacterial culture (M. Luteus or E. Coli)
• 1 x sterile L-shaped spreader
• 1 x sterile 1ml syringe
• 1 x permanent marker or chinagraph pencil
• Blank filter paper discs and a selection of antiseptics to test (or pre-impregnated antibiotic discs)
• 1 x discard beaker of Virkon® disinfectant
• 1 x forceps
• 1 x roll of sellotape and scissors to cut
Investigating the Effects of Antiseptics on Bacteria Growth
Method
- Preparation and Safety
- Safety Precautions
- Put on safety goggles and tie back long hair.
- Disinfect the working area using an appropriate disinfectant, then wipe dry with paper towels.
- Set Up Equipment
- Place a Bunsen burner at your station and light it, leaving it on the safety flame.
- Personal Hygiene
- Wash hands thoroughly with anti-bacterial hand wash (avoid alcohol-based hand sanitizers to prevent fire hazards).
- Preparing the Agar Plate
- Label the Plate
- Turn over the petri dish containing nutrient agar and label the underside with:
- Your name/initials
- The date
- Your class
- The name of the bacterial culture being used.
- Divide the Plate
- Draw three equal sections on the base of the plate and number them 1-3.
- Place a dot in the centre of each section to indicate where the antiseptic discs will go.
- Adding the Bacterial Culture
- Flame the Bottle
- Turn the Bunsen burner to a blue flame.
- Unscrew the bacterial culture bottle lid, keeping the lid in your hand. Pass the bottle neck through the blue flame, twisting it as you go.
- Collect the Bacteria
- Use a 1ml syringe to collect 1ml of bacterial culture.
- Flame Again
- Pass the bottle neck through the flame once more, then replace the lid securely.
- Place the bottle near the Bunsen burner.
- Apply the Culture
- Slightly lift the lid of the agar plate and dispense the 1ml of bacteria onto the agar surface.
- Close the lid immediately.
- Place the used syringe into a Virkon® disinfectant pot, ensuring it is filled with the liquid to fully disinfect it.
- Spread the Bacteria
- Use a sterile L-shaped spreader to evenly distribute the bacteria over the surface of the agar.
- Discard the spreader into the Virkon® disinfectant pot after use.
- Adding Antiseptic Discs
- Prepare Antiseptic Discs
- If making your own, soak three filter paper discs in different antiseptics.
- Alternatively, use pre-impregnated discs.
- Place the Discs
- Slightly lift the petri dish lid again and, using sterile forceps, place one disc on the dot in each section.
- Gently press each disc to ensure proper contact with the agar.
- Record Placement
- Make a note of which antiseptic/antibiotic corresponds to each numbered section for accurate results.
- Securing and Incubating the Plate
- Secure the Lid
- Use two small pieces of tape to secure the lid, placing them on opposite sides of the dish.
- This ensures the plate does not become anaerobic during incubation.
- Incubation
- Hand the plate to the Technician to incubate for 48 hours at 25°C.
- Clean Up
- Tidy the Area
- Turn off the Bunsen burner and ensure it has cooled before handling.
- Clean and disinfect the working area thoroughly.
- Personal Hygiene
- Wash hands thoroughly with hot water and anti-bacterial soap.
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Technician tips
Advance preparation
• Nutrient agar is prepared by adding 28g to a litre of water.
• Any microbiology practical session requires meticulous advanced planning, regardless of the size of your school and number of classes.
The technician needs to be well prepared by making sure that:
• Ample sterile nutrient agar plates are available for the number of students;
• Viable cultures of bacteria are sub-cultured in advance and are ready for students to use; and
• There is enough space in the prep room or incubator to allow the plates to incubate.
• If multiple classes are conducting the required practical at the same time, the technician may have to keep track of a large number of student bacterial plates. Counting these, keeping them grouped by class and noting their location in the
incubator helps.
• In the absence of an incubator, the plates will still develop at room temperature, but may take longer than 48 hours.
• All discard beakers, syringes and L-Shaped spreaders will need to be autoclaved after use.
• All incubated agar plates must be put into an autoclave bag and be autoclaved and destroyed once measurements have been taken by the class.
• Strict hand hygiene should be followed whenever bacteria, or bacterial plates are handled. Always be sure to wash your hands thoroughly before and afterwards with hot water and anti-bacterial soap.
• Using deionized water in your autoclave will help to reduce limescale and prolong its life.
To do this practical, you will need
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