❌ 2. Required Practical: Microbiology - Analysing Bacterial Growth RP (B5)

Question 1

Health and Safety Measures

Answer 1

• Bench tops need to be wiped with disinfectant or alcohol before starting your experiment. Allow alcohol fumes to diffuse before lighting a Bunsen burner.
• Use a Bunsen burner to flame any apparatus used to transfer bacteria between media
• Benches must be cleaned with disinfectant or alcohol after the experiment. During this part of the procedure no naked flames must be used
• Equipment must be sterilised in an autoclave before and after the experiment.
• Agar plates must be incubated at a maximum temperature of 25°C and sealed with a few strips of sticky tape. An air tight seal must not be formed.

Question 2

Equipment List

Answer 2

• Disinfectant solution/alcohol to sterilise benches/worktops
• Bunsen Burner
• Sterile Petri dish containng nutrient agar or sterile Petri dish and cooled molten nutrient agar
• Inoculating Loop
• Sticky tape
• Marker pen or chinagraph pencil
• Culture of a bacterium (such as Bacillus subtilis) in a small capped bottle
• Tweezers
• Small pieces of filter paper
• Ruler
• Examples of antiseptics (e.g., antibacterial liquid soap, mouthwash, liquid toothpaste) or pre-soaked antibiotic discs

Question 3

Method for Microbiology RP

Answer 3

1. Prepare your work space by santising the area using the disinfectant.
   (a. You should wash your hands during this procedure as well, to minimise the risk of contamination.)
2. Ignite the bunsen burner, setting it to a safety flame. Ensure that windows and doors are open.
   (a. This is to help regulate a consistent airflow to remove any airborne pathogens that could affect the reliability of the results.
   b. It also helps remove any carbon monoxide from the air cycle that is produced from the flame.)
3. Open the vial, and using the syringe, put the required amount of the pathogen on the petri dish and spread it around evenly using the spreader. Be careful not to damage the agar jelly as this might affect the results!
4. Add your antipathogenic solution/substance to the plate in different amounts, in different areas of the petri dish, using filter paper.
   (a. Make sure you label the lid of the petri dish so you know which area is which, for example, you might label the area with the least amount of the antipathogenic solution/substance as (1).)
5. Once the pathogen has been spread around the agar and you’ve added your antipathogenic substance, put the lid on the petri dish and seal it. Turn the plate so the pathogen faces down towards the ground.
   (a. Sticky tape often does the job - make sure you only add small bits and they don’t cover the entire gap, otherwise you’ll suffocate your pathogen.)
6. Place the plate into the incubator. Ensure that the incubator is below 25°C to avoid dangerous pathogens from maturing.
7. Wait a day or two for the pathogens to culture.

Question 4

How Do You Successfully Put Your Sterile Paper Discs?

Answer 4

How Do You Successfully Put Your Sterile Paper Discs:

Adding antibiotic or antiseptic soaked patches to pre-prepared agar plates.
By adding filter paper soaked in a variety of anti-microbial solutions to the pre-prepared agar plate (method A), the effective of the solutions can be tested experimentally. A clear area (zone of inhibition) indicates that the bacteria have been killed by the solution or have not been able to reproduce.

    1. Soak filter paper disks in a variety of solutions, use either different concentrations of the same solution, or a variety of different solutions.
	(This will kill any bacteria that are present in the solution or on the petri dishes.)
	2. Pour the sterile agar plates and allow to set fully.
	(The effectiveness of the solutions at killing the bacteria can be tested.)
	3. Measure the clear area around the soaked filter paper disks. A control disk must be also included.
	(Size of zone indicates the effect of the substance tested on the growth of the specific bacterium.)

Question 5

Where is the Penicillin, the Exclusion Zone, and the Bacterial Growth on a typical Petri Dish

Answer 5

Penicillin - This will be an impregnanted paper disc on penicillin in the middle.

Exclusion Zone - The clear circle around the penicillin disc is the exclusion zone where no bacteria has got into.

Bcaterial Growth - This is the area outside of the exclusion zone where bacteria has been allowed to grow and culture.

Question 6

What is the Independant Variable in this Experiment

Answer 6

The Independant Variable in this experiment is the type of antispetic/antibiotic that is applied to agar plate.

Question 7

What is the Dependant Variable in this Experiment

Answer 7

The Dependant Variable is the diameter of the exclusion zone.

Question 8

What are the Control Variables in this Experiment

Answer 8

The Control Variables are the type of amount of each antiseptic/antibiotic used, the type of agar jelly, the size of the agar plate, the temperature of the incubator, the spreading technique, the type of pathogen, the heat of the bunser burner, and the amount of the pathogen applied.

Question 9

How To Test The Effect Of Other Disinfectants And Antibiotics Too?

Answer 9

You can add circles of filter paper soaked in different types or concentrations of disinfectant or antibiotic when you set up your culture plate. An area of clear agar gel indictaes that the bacteria have been killed or cannot grow in that area. This zone of inhibition / exclusion zone can be measured and used to investigate the effect of idfferent anyiseptics or antibiotics on the growth of the bacteria.

Question 10

How To Calculate The Effect Of Disinfectnats And Antibiotic On Bacterial Growth (Area of The Exclusion Zone)

Answer 10

To measure the effectiveness of each chemical tested, you will need to calculate the area of each zone of inhibition surrounding the paper disc. To do this, you:

• Measure the diameter of any circles of agar gel around your filter paper circles.
• Divide the diameter by 2 to find out the radius of the circle.
• Now you can work out the area of the clear circles of clear agar gel using πr^2 and compare the effectiveness of the different chemicals in a table.

Question 11

How To Create A Table To Show The Results?

Answer 11

1. Design a suitable results table and record your findings. Remember to give the
   units of your measurements.
2. List your antiseptics/antibiotics from the most effective to the least effective.
3. To measure the effectiveness of each chemical tested you will need to calculate the area of each zone of inhibition surrounding the paper disc and put that in the table.

Question 12

How do you lay out the Results Table for comparing the Effectiveness of 4 different Antibiotics/Antiseptics?

Answer 12

|——————————————————|
| Type of Antiseptic | Exlcusion Zone | Exclusion Zone |
| | Diameter (cm) | Area (cm^2) |
|——————-|—————-|——————|
| Distilled Water | | |
|——————-|—————-|——————|
| Streptomycin | | |
|——————-|—————-|——————|
| Penicillin | | |
|——————-|—————-|——————|
| TCP | | |
|——————————————————|

Question 13

How do you lay out the Graph for comparing the Efficveness of 4 different Antibiotics/Antiseptics?

Answer 13

I would use a bar graph, with the Type of Antisepctic/Antibiotic on the X-Axis, and I would have the Exclusion Zone Area (cm^2) on the Y-Axis.

Then you plot the average results for the area of each Antisepctic/Antibiotic that you have collected from your table, and then the graph is complete

Question 14

What is the difference betwen antibiotics, antispetics, and disinfectants?

Answer 14

An antibiotic is a type of antimicrobial substance that is ‘active’ against bacteria but does not affect human cells. An antispetic is a antimicrobial substance, like an antibiotic, but is instead applied to the tissue/skin of a living thing to reduce the chance of future infection. However, a disinfectnat is slightly different in the fact that they are ‘chemical agents’ that destroy microorganisms on certain surfaces.