2. Methods
2.1 Equipment
Equipment
Equipment
|
Quantity
|
Purpose
|
Fruits (Nectarine, Peach, Avocado, Raspberry)
|
2 sets of each except Raspberry
|
Juice in the fruits that is needed to be extracted
|
Knife
|
1
|
To cut the fruits
|
Blender
|
1
|
To blend the fruits for the juice
|
Thermo Bag
|
1
|
To keep the fruit juices cooled and prevent it from spoiling
|
Used iPhone
|
1
|
Bacteria collection site
|
Paper Towels
|
12
|
To clean work area after sanitizing with 70% ethanol
|
Apparatus
Apparatus
|
Quantity
|
Purpose
|
Gloves
|
48
|
To keep the bacteria form hands from contaminating the results
|
Micropipette 10-100ul
|
1
|
To extract the juices into the microtube
|
Micropipette 100-1000ul
|
1
|
To extract the juices into the microtube
|
Microtip
|
30
|
Attached to the micropipette for extracting mixture
|
Microtube
|
30
|
Used to store the extracted mixture
|
Agar plates
|
30
|
To grow the bacteria in so that results can be seen
|
L-Shaped spreaders
|
30
|
To help spread the mixture in the agar plates evenly
|
Parafilm strips
|
30
|
To secure the agar plates to prevent air-borne bacteria from entering and contaminating it
|
Bottle of 70% ethanol
|
1
|
To cleanse the table and get rid of the bacteria
|
Sterile swabs
|
30
|
To swab the bacteria from the iPhone
|
2.2 Diagram
Diagram 1
Use the sterile swabs to swipe across the screen to collect bacteria.
Diagram 2
Spreading of bacteria onto agar plate.
Diagram 3
Wrapping parafilm onto the edge of the agar plate which already has the juice and the bacteria inside
Diagram 4
Agar plate ready for incubation
Diagram 5
Putting the mixture into a microtube using a micropipette
2.3
Procedures
2.3 Procedures
Preparation
1. Wash hands with antibacterial handwash to kill all bacteria on hands so that it is clean and will not affect any results.
2. Wear gloves to prevent excess bacteria on hands from contaminating the agar plates.
3. Use 70% Ethanol to clean the table to ensure that the table is cleansed and that there are no bacteria left on the table.
Collection of fruit juices from the 4 different fruits
1. Refrigerate the fruits to down slow the the process of rotting
2. Cut the fruits into smaller pieces for the ease of blending and collecting the juices.
3. Pour the juice into different containers so it is much easier when bringing it to school for the experiment.
Gathering the bacteria from the black screen area of the iPhone
1. Use sterile cotton swabs to swab the black screen area of the iPhone in a zigzag pattern for the gathering of bacteria so that data can be collected.
2. Repeat the step 3 times to ensure that sufficient amount of bacteria is collected.
3. Use the sterile swabs used from gathering the bacteria to apply bacteria onto the agar plate using a zig-zag motion to allow the bacteria collected from the speaker area of the iPhone to start cultivating.
4. Use parafilm to seal up all the agar plates so that air-borne bacteria will not contaminate the results.
5. Leave the bacteria collected in the agar plates for incubation in order to allow the bacteria to start growing so that the experiment can be initiated during the next lab session.
Growing bacteria from the black screen area of the iPhone
1. Take a micropipette and attach a microtip to it so that the juices can be extracted. Also, after using a microtip, dispose it so that there will not be any mixture of fruit juices or contamination.
2. Then use the micropipette to extract the 1000 microlitres of the different fruit juices from the containers and put them into one microtube per fruit. This is to ease the collection of the mixture which is very important for the later part of the experiment.
3. Use a micropipette to fill a microtube with 1000 microlitres of LB Broth. Those are for the to-be-collected bacteria to be mixed with the LB Broth.
4. Use an Inoculating Loop to scrape up a bacteria colony from the agar plate of every fruit for the collection of the cultivated bacteria from the agar plate.
5. Mix the bacteria collected into the LB Broth and stir the mixture properly with the Inoculating Loop till the bacteria colony completely dissolves into the LB Broth for every fruit. Doing this is helpful as LB Broth is a medium for growth so mixing the bacteria with it actually helps the bacteria to grow faster.
6. Incubate the microtube containing the mixture(LB Broth+Bacteria) until the continuation of the experiment during the next lesson. This allows the bacteria to grow to a suitable number of bacteria colonies to grow for the experiment to take place.
7. Use the micropipette and add 30 microlitres of the mixture (LB Broth+Bacteria) into another microtube containing 1000 microlitres of sterile water. The new mixture will then act as the control when testing for results.
8. Again, use the micropipette and add 1000 microlitres of each of the different fruit juices into the microtubes containing the mixture(LB Broth+Bacteria).
9. Leave the new mixture and the control for incubation till the next lab session. This will allow the bacteria to continue interacting with the acids in the fruit juice so that the effects can be seen during further experimentation.
10. Using a micropipette, extract 30 microlitres of the new mixture (LB Broth+Bacteria/Sterile water+Fruit Juice) from all the different microtubes and put each of the new mixture onto a fresh agar plate. By doing this, it allows the bacteria in the 30 microlitres of new mixture to grow into individual bacterial colonies so that it is countable.
11. Use L-Shaped spreaders to spread the mixtures evenly around the agar plates to ensure that the bacteria is spread to every part of the agar dish and not cramped on one spot only. Every time after using the spreaders, change it to prevent contamination. Make sure to not talk when doing this step to ensure that the saliva will not enter the mixture and contaminate it.
12. Use parafilm to seal up the agar plates so as to prevent air-borne bacteria from contaminating the mixture spreaded on the agar plate.
13. Incubate the agar plates to allow the bacteria to start growing into colonies.
Gathering the bacteria from the speaker area of the iPhone
1. Use sterile cotton swabs to swab the speaker area of the iPhone in a zigzag pattern for the gathering of bacteria so that data can be collected.
2. Repeat the step 3 times to ensure that sufficient amount of bacteria is collected.
3. Use the sterile swabs used from gathering the bacteria to apply bacteria onto the agar plate using a zig-zag motion to allow the bacteria collected from the speaker area of the iPhone to start cultivating.
4. Use parafilm to seal up all the agar plates so that air-borne bacteria will not contaminate the results.
5. Leave the bacteria collected in the agar plates for incubation in order to allow the bacteria to start growing so that the experiment can be initiated the next lab session.
Growing bacteria from the speaker area of the iPhone
1. Take a micropipette and attach a microtip to it so that the juices can be extracted. Also after using a microtip, dispose it so that there will be no mixture of fruit juices or contamination.
2. Use the micropipette to extract the 1000 microlitres of the different fruit juices from the containers and put them into one microtube per fruit. This is to ease the collection of the mixture which is very important for the later part of the experiment.
3. Use a micropipette to fill a microtube with 1000 microlitres of LB Broth. Those are for the to-be-collected bacteria to be mixed with the LB Broth.
4. Use an Inoculating Loop to scrape up a bacteria colony from the agar plate of every fruit for the collection of the cultivated bacteria from the agar plate.
5. Mix the bacteria collected into the LB Broth and stir the mixture properly with the Inoculating Loop till the bacteria colony completely dissolves into the LB Broth for every fruit. Doing this is helpful as LB Broth is a medium for growth so mixing the bacteria with it actually helps the bacteria to grow faster.
6. Incubate the microtube containing the mixture(LB Broth+bacteria) until the continuation of the experiment during the next lesson. This allows the bacteria to grow to a suitable number of bacteria colonies to grow for the experiment to take place.
7. Use the micropipette and add 30 microlitres of the mixture (LB Broth+Bacteria) into another microtube containing 1000 microlitres of sterile water. The new mixture will then act as the control when testing for results.
8. Use the micropipette and add 1000 microlitres of each of the different fruit juices into the microtubes containing the mixture(LB Broth+Bacteria).
9. Leave the new mixture and the control for incubation till the next lab session. This will allow the bacteria to continue interacting with the acids in the fruit juice so that the effects can be seen during further experimentation.
10. Using a micropipette, extract 30 microlitres of the new mixture (LB Broth+Bacteria/Sterile water+Fruit Juice) from all the different microtubes and put each of the new mixture onto a fresh agar plate. By doing this, it allows the bacteria in the 30 microlitres of new mixture to grow into individual bacteria colonies so that it is countable.
11. Use L-Shaped spreaders to spread the mixtures evenly around the agar plates to ensure that the bacteria is spread to every part of the agar dish and not cramped on one spot only. Every time after using the spreaders, change it to prevent contamination. Make sure to not talk when doing this step to ensure that the saliva will not enter the mixture and contaminate it.
12. Use parafilm to seal up the agar plates so as to prevent air-borne bacteria from contaminating the mixture spreaded on the agar plate.
13. Incubate the agar plates to allow the bacteria to start growing into colonies.
Dilution of the mixture
Dilution only needs to be done if the bacteria colonies found on the agar plates are not countable. If they are countable, dilution is not needed in the experiment. These are the following steps for proper dilution of the mixture:
1. Use the micropipette to extract 100 microlitres of sterile water and put it into 8 different microtubes for the dilution of the soon-to-be-added fruit juices.
2. Using the micropipette again, extract only 900 microlitres of the fruit juices and put them into the different microtubes filled with water.
3. Stir the mixture containing the sterile water and fruit juice to ensure that it is properly mixed.
4. Next, use the micropipette to extract 30 microlitres of the diluted mixture and put it onto a fresh agar plate so that the bacteria can now be seen more clearly and are countable.
5. Use a L-shaped spreader to spread the diluted mixture evenly around the agar plate to ensure that the bacteria is spread evenly around the agar plate and not cramped on one spot only.
6. Use parafilm to seal up the agar plate so that air-borne bacteria will not enter and contaminate the mixture.
7. Incubate the agar plates to allow the bacteria to start growing into colonies.
*IMPORTANT NOTE: (Due to the different concentration levels of acid in the different fruits, the number of dilutions needed to be done for visible results may vary.The mixture of diluted three different times with three different dilution levels, each using the previous diluted sample.)
Method of analysis of results
1. Place the agar plate with the cover facing downwards (so that it will be easier and more exact to count) on the counting plate of the colony counter and use the plastic block provided to hold the agar plate in the centre so that the magnifying glass is positioned just above the agar plate for easier counting as some of the colony counters’ counting plate are larger in size.
The counting plate is illuminated so that the individual colonies would be more visible.
2. Using a black marker, dot on the top of bacteria colonies on the agar plate so as to not recount a counted colony. Try to use a hard and thin tipped marker as it will be easier for identifying the bacteria and will not smudge when the tip becomes soft after pressing too many times on the agar plate.
With every press, there will be a “beep” noise and the number shown will increase.
2.4 Risk Assessment and Management
Potential Risk
-Bacteria from hands may contaminate the agar dishes when handling them
-Excess bacteria from the table may contaminate the agar dishes when not cleansed properly before the experiment
-When extracting fruit juices, it might get contaminated with other fruit juices or bacteria
-Saliva may contaminate the agar dishes when spreading the mixture due to talking
-Equipments such as Microtubes might get infected with bacteria if opened and not used immediately
-Agar plates may get contaminated with the L-shaped spreader if it is being reused
-Fruit juices might get contaminated if the microtips is being reused
-Mixing up of microtubes after extracting the fruit juices and putting them into it because there was no labeling
-Mixing up of microtubes after extracting the fruit juices and putting them into it because there was no labeling
-Air borne bacteria might enter and contaminate the agar plates
Safety Precautions
-Wash hands before handling any equipments so any bacteria found can be killed
-Wear gloves when conducting the experiment so that any excess bacteria from hands cannot contaminate the agar dishes
-Use 70% Ethanol to cleanse the table so that no bacteria are left when initiating the experiment
-Do not talk or open mouth when spreading the mixture on the agar dish or when extracting the fruit juices into the microtubes
-Make sure that microtubes are covered instantly after fruit juices are put into them
-Use an equipment instantly after taking it out to prevent it from being infected with bacteria
-Ensure that the microtubes, microtips and L-shaped spreader are not being reused to prevent contamination
-Label every microtubes after putting the fruit juices in so as to avoid confusion
-Use parafilm strips to secure the agar plates which will prevent bacteria from entering.
2.5 Data Analysis
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