CP 5: Microbial Cultures

• What will you prepare to investigate the effect of antibiotics on bacterial growth?

  Bacterial lawn plates
• Why is it essential to use aseptic techniques during the experiment?

  To prevent contamination of the bacterial cultures

  
• What are the steps to set up your sterile work environment?

  • Disinfect work surfaces
  • Set up and light a Bunsen burner
  • Collect a pre-prepared sterile Petri dish
• What should you do with the pipette after using it to take broth culture?

  Place the pipette in disinfectant
• How should you spread the drops of broth culture across the agar?

  Using a side-to-side motion with a sterile spreader
• What should you do after placing the antibiotic discs in the Petri dish?

  Tape the lid onto the dish and turn it upside down

  
• How do you calculate the cross-sectional area of each clear space around the antibiotic discs?

  Using the formula $A =$$\pi r^2$

  
• What does the clear area around the antibiotic discs indicate after incubation?

  It indicates the effectiveness of the antibiotic in inhibiting bacterial growth

  
• What is the purpose of the control disc in the experiment?

  To provide a baseline for comparison with the antibiotic discs

  
• What can you conclude from measuring the diameter of the clear spaces around the antibiotic discs?

  The larger the clear space, the more effective the antibiotic is

  
• What should you do after measuring the clear spaces around the antibiotic discs?

  • Work out the radius of each clear space
  • Calculate the cross-sectional area using $A =$$\pi r^2$
  • Draw a graph showing cross-sectional area against concentration of antibiotic

  
• What should you do after completing the experiment?

  • Wash hands thoroughly
  • Clean up the work station using disinfectant
• What should you describe when comparing results between different groups?

  • The differences in results
  • Possible reasons for these differences
• What is the first step in preparing bacterial lawn plates?

  Collect a sterile pipette
• Why is it important to flame the mouth of the broth culture bottle?

  To sterilize the opening and prevent contamination
• What is the significance of incubating the Petri dish upside down?

  To prevent condensation from dripping onto the agar surface

  
• What should you do with the forceps after placing the antibiotic discs?

  • Sterilize the forceps again
  • Use them to place the antibiotic discs in the correct sections
• What is the final step after completing the experiment?

  Give the dish to your teacher for incubation
• What is the aim of the microbiology experiment described?

  To investigate the effect of different antiseptics or antibiotics on bacterial growth using agar plates.
• What equipment is needed for the microbiology experiment?

  • Agar plate
  • Heatproof mat
  • Filter paper discs
  • Three antiseptics (e.g., mouthwash, TCP, antiseptic cream)
  • Disinfectant bench spray
  • 1% Virkon disinfectant
  • Antibacterial handwash
  • Forceps
  • Clear tape
  • Hand wash
  • Wax pencil or permanent marker
  • Incubator set to 25°C
• What should be done to the bench before starting the experiment?

  Spray the bench with disinfectant and wipe dry with paper towels.
• How should the agar plate be marked?

  Mark with a wax pencil: three segments, a dot in the middle of each segment, and your initials, date, and name of bacteria.
• What should be done after marking the agar plate?

  Wash your hands with antibacterial handwash.
• How should the antiseptics be applied in the experiment?

  Place the different antiseptics onto different filter paper discs.
• What is the correct way to place the filter paper discs on the agar plate?

  Lift the lid of the agar plate at an angle and use forceps to place each filter paper disc onto the dots.
• How should the lid of the agar plate be secured?

  Tape the lid onto the agar plate securely, but loosely enough for oxygen to reach the bacteria.
• At what temperature should the agar plate be incubated?

  25°C
• How long should the agar plate be incubated?

  48 hours
• How should the diameter of the clear zones be measured?

  Use a ruler to measure the diameter and take a second measurement at 90 degrees, then calculate the mean.
• What formula is used to calculate the area of the clear zones?

  The area is calculated using the formula $\pi r^2$.
• What should be done with the results after measuring the clear zones?

  Record the results in a table.
• What are the controlled variables in the experiment?

  • Species of bacteria
  • Area of filter paper disc
• What is the extension of the experiment regarding antiseptics?

  • Repeat the procedure with different concentrations of the same antiseptic.
  • Investigate the optimum concentration of antiseptic used to kill bacteria.
• What are some sources of error in the experiment?

  The area of the clear zone may be irregular, and contamination may have occurred.
• What safety precautions should be taken during the experiment?

  • Wear safety goggles when handling Virkon disinfectant.
  • Wash hands before and after handling bacteria.