4️⃣PAG 7: Microbiolical techniques Flashcards
Safety
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 25C and sealed with a few strips of sticky tape. An airtight seal must not be formed.
Exercise caution around blue bunsen flame - tie back all long hair
Make sure to wash your hands thoroughly before and after the experiment.
Make sure to disinfect all work surfaces after the experiment.
Make sure to safely dispose of any equipment that had come into contact with bacteria
Equipment
- disinfectant solution/alcohol to sterilise benches
- Bunsen burner
- sterile Petri dish containing 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
- antiseptics (e.g., antibacterial liquid soap, mouthwash, liquid toothpaste) or pre-soaked antibiotic discs
- distilled water
What are antimicrobial agents + 2 examples
Antimicrobial agents are chemicals that kill microorganisms or inhibit their growth. They include antiseptics and antibiotics.
Antiseptics
Antiseptics are used to try to prevent the spread of potentially harmful bacteria. These chemicals kill or neutralise the bacteria but do not damage human tissue.
Antibiotics
Antibiotics are medical drugs that kill bacteria without damaging your cells. Different types of antibiotic kill a different range of bacteria.
Describe how to investigate the effectiveness of an anti specific or antibiotic
You can investigate the effectiveness of an antiseptic or an antibiotic by setting up an agar plate containing a bacterial culture. You then place paper discs soaked in different antiseptics or antibiotics on top of your agar. An area of clear agar gel indicates that the bacteria have been killed or cannot grow. This zone of inhibition can be measured and used to compare the effectiveness of different chemicals on bacterial growth
Method - part 1 - preparation of the work area and Preparing the agar plates for growth of a colony of bacteria
Preparation of the work area
Clear the work space of all non-essential items.
Clean the desk with disinfectant.
Reason - this kills all unwanted bacteria and so decreases the chance of the agar plate becoming contaminated.
Preparing the agar plates for growth of a colony of bacteria
Glass petri dishes and agar gel must be sterilised before use by using an autoclave, or pre-sterilised plastic petri dishes can be bought.
Reason - this will kill any unwanted bacteria that are present in the solution or on the petri dishes.
Pour the agar into the sterile petri dishes and allow to set fully.
Reason – this provides the selected bacterium with all the nutrients needed for them to grow.
Method par 2
- plating bacteria
Plating the bacteria
The following should be done beside a blue Bunsen flame.
Reason - to create an updraft to stop the agar media getting contaminated with unwanted bacteria from the air.
Swirl (do not shake) the bacterial suspension to make sure that the bacterial culture is well mixed.
Reason - to make sure that the bacteria aren’t all at the bottom of the container
Sterilise the inoculating loop, by heating it in the Bunsen burner flame. Leave it to cool. Alternatively, sterilise it by placing it in pure alcohol for a few seconds.
Reason - kills any unwanted bacteria that are present on the loop.
Remove the lid from the bacterial bottle and put the mouth of the bottle in the Bunsen flame.
Reason - to kill off any unwanted bacteria that could be on the bacterial bottle.
Dip the inoculation loop into the microorganism solution and make streaks on the surface of the agar plate.
Reason - this allows the bacteria to spread out and to grow in individual colonies on the agar plate. A lawn of bacteria can be produced by using a sterile spreader to evenly spread the bacteria across the whole of the plate.
Replace the lid of the petri dish as soon as possible and secure with tape. Allow the plate to dry then label the half of the petri dish containing the media (do not label the top). Invert the plate and store it upside down.
Reason - The lid stops additional unwanted bacteria in the air contaminating the plate. Do not fully seal the lid, as this will stop oxygen reaching the bacterium, and this may encourage harmful anaerobic bacteria to grow. Labels are important, as this identifies the growing bacterium. If the lid is separated from the petri dish for some reason, the label will stay with the part that has the bacteria on it, so it can be identified.
Incubate at a maximum temperature of 25°C in schools and colleges.
Reason - this reduces the chance of growing harmful pathogens, which would grow at 37°C in a human body. Hospital laboratories would incubate plates at 37°C (body temperature) to allow quick growth and identification.
Method part 3 - clearing up
Clearing up following the activity
All contaminated materials need to be disposed of either in autoclave bags (for disposable materials that need to be sterilised, eg spreaders/Petri dishes) or pots (for items that are to be washed, sterilised and then reused).
It is essential that all work surfaces need to be thoroughly disinfected at the end of the activity. Ensure that hands are washed with soap and water at the end of the activity.
PAG method
Method
Soak filter paper discs in a variety of solutions. For example, use different concentrations of the same antibiotic solution, or a variety of different antibiotic or antiseptic solutions.
Reason - the effectiveness of the solutions at killing the bacteria can be tested experimentally.
Place the discs onto the surface of the pre-prepared agar plate, in contact with the bacteria. A control disc must be also included which has no anti-microbial substance on it. Leave for at least 24 hours at room temperature.
Reason - the antibiotic or antiseptic will diffuse through the agar and come into contact with the bacteria.
Measure the diameter of the clear area around the soaked filter paper discs using a ruler.
Reason – size of zone of inhibition indicates the effect of the substance tested on the growth of the specific bacterium. The larger the clear zone, the more bacteria were killed so the higher the effectiveness of the substance.
Sources of error
Sources of Error
Contamination of the petri dish may occur.
The shape of the clear zones may not be regular so it would be difficult to find a suitable diameter.
