[Text on screen: NSW Department of Education
Microbiology investigation
Testing the antiseptic properties of substances against a bacteria]

[Image on screen: teacher holding petri dishes, in front of testing equipment]

Speaker

In this video, we will walk you through some microbiology techniques for testing the antiseptic properties of substances against bacteria.

[Text on screen: 1 Aseptic technique
The set of procedures used to prevent contamination of cultures, equipment, and us]

Aseptic technique refers to the set of procedures used to prevent contamination of cultures, equipment and us. It protects the integrity of an investigation and models the techniques real scientists use for students.

In microbiology aseptic technique is used when preparing agar plates, preparing the antiseptic discs with their treatments, transferring bacteria to the agar plates and when handling bacterial cultures.

To start, we need to close windows and doors to reduce drafts and prevent sudden movements which might disturb the air. We need to tie back long hair and wash our hands thoroughly to remove any surface microbes. If there are any cuts or wounds on the hands, they must be covered with a waterproof dressing to prevent contamination.

[Text on screen: Prepare equipment and materials

• Sterilise equipment that will be used (for example: forceps, Schott bottles for storing agar, nutrient agar)
• Prepare space and self
• Close windows and doors
• Turn off AC and fans
• Tie hair back
• Wash hands thoroughly]

[Video shows: teacher in front of science lab table. On the table is equipment required for experiment]

Once hands are clean and protected, we then put on sterile disposable gloves to create a final barrier against contamination. These steps reduce the risk of introducing unwanted microbes into the experiment, and prevent contact between our hands and the bacteria.

Next, wipe down the bench with 70% ethanol and a paper towel. Allow the ethanol to evaporate to ensure the surface is fully sterilised before you start working. This process will reduce the likelihood that your equipment and materials will become contaminated.

[Video shows: teacher throwing paper towel in bin then bringing the Bunsen burner into the centre of the table, lighting it with a match]

Then we need to set up a Bunsen burner. It is important to work close to a Bunsen burner, roughly 30cm from the flame [Visual graphic overlaid on the teacher shows the upward direction of the updraft created by the Bunsen burner], as it provides an updraft that reduces contamination from the surrounding air, maintaining a sterile area.

All of our equipment should be located within the sterile area we have created. [Visual graphic overlaid on the screen shows a blue circle around the Bunsen burner, depicting the sterile area].

Always work within this area. Opening culture plates and tubes and handling sterile tools close to the flame to help maintain the sterile environment. We use a blue flame for microbiology work.

Note that in the classroom, you can choose not to use the Bunsen burner when working with plates. This may reduce any risks associated with using the Bunsen burner.

This will increase potential contamination on the plates. However, it should not pose a risk since the plates are not opened after sealing. If removing the Bunsen, ensure you have enough sterile equipment such as forceps for each use during the investigation.

[Text on screen: Summary

• Personal protective equipment
• Tie back long hair and wash hands thoroughly
• Cover any cuts with waterproof dressings
• Wear sterile disposable gloves as a barrier against contamination.

Reducing contaminations sources

• Close windows and doors to minimise draughts
• Use sterile equipment (for example Schott bottles and forceps)
• Disinfect the bench with 70% ethanol]

[Text on screen: Summary

• Maintaining the integrity of the investigation
• Keep all equipment within the sterile zone
• Handle culture and tools near the flame to ensure sterility
• Sterilise equipment such as forceps between uses (OR use a new set of sterile equipment each time) Work quickly so agar is not exposed to the environment longer than necessary.]

[Text on screen: 2 Conducting the investigation
Creating a bacterial lawn, placing antiseptic discs on the agar plates and waste disposal procedures]

Now that we have established a sterile environment, we can set up for the microbiology investigation.

In this next part of the video, we will outline how to conduct the investigation, to test the antiseptic properties of different substances. You will learn how to prepare a bacterial lawn, place the antiseptic discs on the agar plate and follow waste disposal procedures, once the investigation is complete.

First we set up our sterile work area, as previously shown.

[Video shows: teacher cleaning table and sterilising the workspace]

Next, we organise all our equipment. You'll need a lit Bunsen burner, nutrient agar plates, your pre-prepared antiseptic discs, bacterial culture, sterile cotton swabs, a waste beaker with bleach, 70% ethanol in a beaker and a pair of sterile forceps.

[Text on screen: Safety tip
Only use a small volume of ethanol if flaming the forceps. Sit the ethanol away from the Bunsen burner as it is flammable.]

[Visuals on the screen show teacher holding up items for the experiment]

Now let's make our bacterial lawn. Place nutrient agar plate on the sterilised bench close to the Bunsen flame. The plates should be labelled with a permanent marker. The label should include the abbreviated name of the bacteria used, the date the plate was used and initials of the person or persons conducting the investigation the specific treatment shown using numbers in each quadrant, in this example, and where relevant, the trial or repeat number.

[Visual graphic on screen shows two plates, the one on the right labelled along its edge ‘Control blank disc 4/9/25 LK & TL’ the left side labelled along its edge ‘S. epidermidis 4/9/25 LK & TL Rpt1. It has two lines that intersect in the middle to show four quadrants. Top left quadrant has a 1 and moving clockwise the next quadrant is 2, then 3 and 4. A text box pointing to the left plate has the following text ‘The numbers tell us which antiseptic treatment is in each area of the plate.].

[Video shows the teacher writing on the top of the agar plate]

[Text on screen: Labelling the agar plate
Plates should be labelled with the name of the bacteria, date, initials of the person, and treatment on the plate]

Carefully open a sterile cotton swab from its packaging, avoiding contamination.

Hold the cotton swab and bacterial culture in one hand, and use your other hand to open the lid of the bacterial culture. Briefly flame the neck in the Bunsen flame to ensure that it remains uncontaminated.

[Text on screen: Flaming the bacterial culture tube
A quick pass through the flame is sufficient. Extended flaming may melt the vial.]

Moisten the swab by gently dipping it into the bacterial broth. The bacterial culture is then passed back through the Bunsen flame before the lid is replaced.

Now carefully lift the lid of the agar plate and pass the swab over the entire surface.

[Text on screen: Creating a bacterial lawn
Pass the swab over the entire surface in a gentle rolling motion to maximise coverage and prevent breakages in the agar surface.]

Once we have swabbed the plate in one direction, we rotate it 45 degrees and swab again. We swab the entire plate three times to ensure an even bacterial lawn.

Place the used swab in the waste beaker containing bleach. Now we can set these aside until we are ready to apply our antiseptic disc treatments.

Some equipment, such as forceps are sterilised by carefully dipping them in 70% ethanol, then lighting them in the Bunsen flame to burn off the alcohol. You need to hold the forceps at an angle facing the end down, this prevents ethanol from running up the forceps and causing burns.

[Text on screen: Mitigate risk
You can avoid the need to sterilise forceps with the ethanol and the flame, by using a new set of sterile forceps for each disk application.]

Once the flame has gone out, the forceps can be used to carefully pick up an antiseptic disc and then place it onto the agar plate in its pre labelled section.

[Text on screen: Antiseptic disks
Antiseptic disks are prepared under aseptic techniques prior to the investigation. They can be stored in a sterile petri dish until required.]

We now re-sterilise the forceps before placing the next antiseptic disc, this reduces the chance of any contamination. This is repeated until all the antiseptic discs have been placed in the pre labelled sections on the agar.

To seal the plate we can use parafilm. Use a strip of parafilm to wrap around the entire plate once to seal it.

Alternatively, you can use four pieces of sticky tape placed at the twelve, three, six, and nine o’clock positions to seal the agar plate.

Now we are ready to place our plates in the incubator at no more than 30°C for 24 to 48 hours. The agar plates should be stacked upside down. This stops condensation from forming on the agar surface. The incubator should not be set at or near 37°C, due to the potential of growing human pathogens. By setting the incubator at 30°C or lower, these organisms, if present, will grow more slowly because it's not their optimal temperature.

[Text on screen: Incubating the plates

• Sealed plates are stored upside down
• Plates are incubated at <30°C for 24–48 hours
• Incubation time varies depending on the type of bacteria used]

Now it's time to clean up. Pack away equipment to clear your bench. Decontaminate work surfaces with 70% ethanol after working with microorganisms.

[Video shows teacher wiping down table with disinfectant and paper towel]

Remove gloves by pinching near the wrist, pulling them off inside out and holding them in the gloved hand, use your other hand to slide a finger under the wrist and peel off the glove inside out over the first glove, then dispose of both gloves.

Finally, wash your hands thoroughly to remove any microbes you may have picked up while taking the gloves off. Then dry your hands thoroughly. This reduces the risk of spreading microbes.

Once the agar plates have been incubated, they can be stored in a lab refrigerated tray or in a bench in the prep room until required for class. There should be a zone of inhibition around the antiseptic disc, if it was successful preventing bacterial growth.

To measure the zone of inhibition, we use a rule up to measure the diameter.

[Graphic on screen shows the plate with its four quadrants and a different sized circle in each. On the lower right number 4 quadrant a ruler sits across the circle (the largest of the four circle on the plate) showing a width of approximately 34mm.]

Here we can see that the zone of inhibition for treatment four is approximately 34mm. The larger zone of inhibition indicates that this antiseptic substance is more effective than the other treatments tested.

Once the results are collected, all agar plates, including cultures, must be sterilised or autoclaved before disposal. The agar plates and any cultures should be placed in an autoclavable waste disposal bag or an oven cooking bag.

Place the bag in an autoclave or pressure cooker. Heat to 121°C and maintain a pressure of 110 kilopascals for 25 minutes. Let the items cool. Then place them in a garbage bag and put the bag in the regular waste bin.

[Text on screen: Summary
Organisation

• Organise equipment: agar plates, forceps, antiseptic discs, and swabs
• Label the agar plates
• Creating a bacterial lawn and applying antiseptic discs
• Use aseptic techniques to ensure that unwanted microbes are not transferred to the plates
• Swab agar plates methodically to ensure even distribution of bacteria
• Apply antiseptic discs using sterilised forceps.]

[Text on screen: Summary

• Sealing and incubating
• Seal plates with parafilm or sticky tape
• Incubate at <30°C for 24–48 hours
• Observing the results
• Handle plates with gloves
• Never open cultured agar plates
• Measure the zone of inhibition with a plastic ruler
• Wash hands thoroughly after inspecting plates]

[Text on screen: Post experiment cleanup

• Decontaminate surfaces with 70% ethanol
• Wash hands thoroughly
• Autoclave all equipment, cultures, and used agar plates
• Dispose autoclaved cultures and plates in a waste bag in the general waste.]

[Text on screen: 3 More information
Finding more information about microbiology in schools]

[Text on screen: More information

• Chemical safety in schools 3.2.6: Safe use of biological materials/organism/tissues (https://education.nsw.gov.au/inside-the-department/health-and-safety/risk-management/compliance-and-environment/chemical-safety-in-schools/section-3--curriculum-support-documents/3-2-6--safe-use-of-biological-materials-organism-tissues). This page outlines procedures related to microbiology in schools.
• Information related to risk management procedures can be found on the department’s website (https://education.nsw.gov.au/policy-library/policies/staff-only/pd-2013-0454-04).
• The Science 7–10 Disease assessment task contains more detailed instructions for completing this investigation: Disease — Stage 5, Science (https://curriculum.education.nsw.gov.au/learning-area/103/stage/3371/stageYear/24/unit/1270).
• Contact the Science curriculum team at science7-12@det.nsw.edu.au.]

[Text on screen: NSW government logo
copyright State of New South Wales (Department of Education) 2026.]

[End of transcript]