Required Practical 6: Aseptic Techniques and Culturing Microorganisms
Laura Armstrong & Joe Wolfensohn
Teachers
Contents
Recall Questions
This topic requires prior knowledge of bacteria, antibiotics and natural selection. You can test your knowledge on these below.
How do bacteria reproduce?
Bacteria reproduce asexually by binary fission, where one cell divides into two identical daughter cells.
What is an antibiotic and how does it work?
An antibiotic is a substance that kills or inhibits the growth of bacteria, often by preventing cell wall formation or disrupting protein synthesis.
Why are some bacteria resistant to antibiotics?
Some bacteria have mutations that make them resistant to antibiotics. These bacteria survive, reproduce, and pass on the resistance allele, leading to antibiotic resistance.
Topic Explainer Videos
Check out this @JoeDoesBiology video that explains investigating microbial growth, or read the full notes below. Once you've gone through the whole note, try out the practice questions!
Required Practical 6
What is Required Practical 6?
This practical investigates how to safely culture microorganisms and test the effect of antibiotics or antiseptics on bacterial growth using aseptic techniques.
Aseptic Techniques: Why Are They Important?
Aseptic techniques prevent:
Contamination of bacterial cultures by unwanted microorganisms.
Contamination of the environment by the bacteria being cultured.
Aseptic Techniques – Step by Step
1. Sterilise Equipment:
- Work near a Bunsen burner to create an upward air current, preventing contamination with bacteria in the air that may fall onto the agar plate.
- Flame the inoculating loop in a bunsen burner until red hot before and after use. This kills any bacteria present on the inoculating loop.
- Disinfect surfaces before and after the experiment.
- Boil the agar before pouring into the petri dish to destroy any bacteria that will contaminate your culture.
2. Prepare the Bacterial Culture:
- Sterilise the neck of the bottle in which the bacteria you wish to culture is being kept. You can do this by passing the neck of the bottle through a bunsen burner flame.
- Use a sterile pipette or a sterile inoculating loop to transfer bacteria onto an agar plate.
- Spread the bacteria evenly with a sterile glass spreader. Glass spreaders can be sterilised by flaming in a Bunsen burner or dipping in ethanol to kill any bacteria.
3. Introduce Antibiotic Discs:
- Soak sterile paper discs in different antibiotics or antiseptics.
- Use sterile forceps to place the discs onto the agar plate.
- Keep the lid of the petri dish over the plate and at an angle whilst doing this to prevent any contamination with airborne bacteria.
- Close the plate and tape shut (but do not fully seal to allow oxygen in for bacterial respiration).
4. Incubate at 25°C for 24-48 Hours:
- Why 25°C? – This temperature is safe and prevents growth of human pathogens (which thrive at 37°C).
- Store the plate upside down to prevent condensation from dripping onto the bacteria.
5. Measure the Zone of Inhibition:
- After incubation, measure the zone of inhibition around each antibiotic disc.
- A larger zone of inhibition indicates a more effective antibiotic as it shows more bacteria have been killed by the antibiotic.
Example Calculation:
- Measure the diameter of the clear zone (zone of inhibition).
- Calculate the area: Area = πr2 (where r is the radius of the clear zone).
Expected Results
Antibiotics that inhibit bacterial growth will produce large clear zones.
Resistant bacteria will show no or small zones of inhibition.
A control disc soaked in water shows that bacteria do not die from handling or disc placement. This proves it is the antibiotic that has killed the bacteria.
Example Interpretation:
If bacteria grow up to the edge of an antibiotic disc, they are resistant.
If a large zone of inhibition forms, the bacteria are susceptible to the antibiotic.
Key Terms
Aseptic Technique: Laboratory methods used to prevent contamination of microbial cultures.
Sterilisation: The process of removing or killing all microorganisms from equipment.
Agar Plate: A petri dish containing nutrient agar used for culturing bacteria.
Antibiotic: A substance that kills or inhibits the growth of bacteria.
Zone of Inhibition: The clear area around an antibiotic disc where bacteria have not grown.
Binary Fission: The process by which bacteria reproduce asexually.
Antibiotic Resistance: When bacteria evolve to survive exposure to antibiotics.
No answer provided.
Exam Tips
When answering questions on aseptic techniques, always be specific with the techniques used:
Flaming the loop.
Flaming the neck of the bottle.
Disinfecting surfaces.
Keeping the lid partially closed.
If a question asks how to compare antibiotic effectiveness, refer to the size of the zone of inhibition and use the equation πr² to calculate its area.
No answer provided.
A student investigated the effect of three types of disinfectant on the growth of Lactobacillus bacteria.
During the investigation, the student:
Boiled the agar before pouring the agar plates.
Transferred of a diluted liquid culture of Lactobacillus onto each agar plate.
Left some agar plates as controls.
Added to other agar plates different concentrations of the disinfectants as shown in the table. After 2 days, she counted the number of colonies of bacteria on each agar plate.
Explain the purpose of:
Boiling the agar. (1 mark)
Transferring the same volume of liquid culture onto each agar plate. (1 mark)
The liquid culture the student transferred was diluted by 1 in 10,000 . Use information in this question to calculate how many bacteria were present in of undiluted liquid culture. (2 marks)
So no contamination / no other bacteria present.
b) So same number of bacteria transferred to allow for comparison.
c) 6,000,000 OR 6 × 10 6.
Explanation: There were 300 bacteria present in 0.5 cm3 of bacterial culture.
This had been diluted 10,000 times.
So, 300 x 2 gives us the number of bacteria present in of diluted culture = 600 bacteria.
Then multiply 600 x 10,000 to find the number present if it was undiluted= 6,000,000 bacteria.
Practice Question
Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!