2. Methods

2.1 Equipment List
  1. 10 Petri Dishes with agar
  2. Different Cleaning Agents acquired from the hospital such as:
  • EC Oticide Cleaner
  • Glutasept LA
  • Dettol 4-in-1 Multi Action Cleaner Green Apple
  • Clorox Clean-Up All Purpose Cleaner
  • Floor Shine Floor Cleaner
  1. 3 mechanical Micropipette (1,000 μml, 100 μml and 10 μml)
  2. Bore tips for the 3 Micropipette and 1 Manual Pipette
  3. 1 manual pipette (10ml)
  4. 1 Tweezer
  5. 1 packet of 6mm Filter Paper disks
  6. 15 15ml Centrifuge Tubes
  7. Spreader (packets of 5)(2 packets)
  8. Tube of E. Coli
  9. Tube of B. Subtilis
  10. Deionised Water ( 1 Bottle )
  11. Oil Candle (Flame)
  12. Lighter
  13. Ethanol 70% (Used for Sterilization of work space)
  14. 3 pairs Rubber Gloves  
  15. A piece of A3 Black Paper

2.2 Diagrams of experimental setup  

Caption: All the equipment that is needed in the experiment (except pipette)


Caption: Mixing the Deionized Water with the 1 ml of Cleaning Agent
Caption : Mixing the cleaning agents with the deionized water

Figure 1.1 : Experimental setup used to calculated the concentration of the cleaning agent







Figure 1.2 : Dripping an equal amount of dilution onto each respective section of the agar plate.


Figure 1.3
Caption : Petri Dishes with Filter Paper on it.

2.3 Procedures


Procedure to cultivate the bacteria

  1. Using a black permanent marker, label a petri dish “E.Coli with EC Oticide Cleaner concentrations” and draw lines to “split” the petri dish into 4 different sections and label the areas: Control, (Cleaning agent name)10-1, (Cleaning agent name)10-2 and (Cleaning agent name) Standard.
    Example: Control, EC Oticide 10-1, EC Oticide 10-2 and EC Oticide Standard
 
Example of the petri dish shown below:
       


  1. Plate E. Coli Bacteria onto the agar in the petri dish.
  2. Repeat steps 1-2 for 4 other petri dishes for for Clorox Clean-Up All Purpose Cleaner, Floor Shine Floor Cleaner, Glutasept LA and Dettol 4-in-1 Multi Action Cleaner Green Apple.
  3. Repeat step 1- 3 for B. Subtilis Bacteria.


Procedure to prepare the cleaning agent


  1. Prepare 3 separate centrifuge tubes
  2. Prepare the standardized dilution of EC Oticide Cleaner in the centrifuge tube labelled “EC Oticide standard”.
  3. Prepare 10-1 the dilution of the standardized dilution of the EC Oticide Cleaner in the centrifuge tube labelled “EC Oticide 10-1”.
  4. Prepare 10-2 the dilution of the standardized dilution of the EC Oticide Cleaner in the centrifuge tube labelled “EC Oticide 10-2”.
  5. Repeat steps 1-4 for Clorox Clean-Up All Purpose Cleaner, Floor Shine Floor Cleaner, Glutasept LA and Dettol 4-in-1 Multi Action Cleaner Green Apple.


Procedure to place the filter paper with the cleaning agents on the plated agar.


  1. Take the petri dish plated with E. Coli Bacteria labelled with standard, 10-1, 10-2 and Control.
  2. Take a filter paper disk with a tweezer place a filter paper and place it in the centre of the section in the petri dish labelled “Standard”.
  3. Repeat step 2 for 10-1, 10-2 and Control.
  4. Drip 10 μm drops of the different concentrations of EC Oticide Cleaner from the centrifuge tubes onto the filter papers in the respective sections of the petri dish with a 10 μml pipette.
  5. Sterilize the tweezer over an oil candle flame and once cooled, place it back into the container containing ethanol.  
  6. Repeat step 1-5 for B. Subtilis bacteria and Clorox Clean-Up All Purpose Cleaner, Floor Shine Floor Cleaner, Glutasept LA and Dettol 4-in-1 Multi Action Cleaner Green Apple.


Procedure for incubating bacteria


  1. After plating the bacteria, use parafilm to seal the petri dishes.
  2. Place the sealed petri dishes into the incubator and cultivate the bacteria overnight.


Procedure for data collection


  1. Take the petri dish out of the incubator.
  2. Place the the petri dish onto a piece of black paper.
  3. Record the area around the filter paper that has no visible bacteria.
  4. Place the recorded area into the data collection table.
      
Example of the data collection table


Procedure to dispose of bacteria/cleaning agent


Cleaning Agent


  1. Pour the unused liquid down the sink drain and wash the centrifuge tubes


Bacteria

  1. In terms of wanting to keep the petri dishes for further reference, place the petri dish in the refrigerator.
  2. If not, seal each petri dish properly and dispose them in the “Biohazard” bin found in the lab.



2.4 Risk Assessment and Management  


Risk
Assessment
Management
As this experiment involves liquids, there may be spillages which may cause people to
slip and fall.
Low / Medium / High
If any spillages occur, one must immediately clean it up.
Cleaning Agents may be corrosive to the skin and hands as they may contain certain chemicals that may poisonous.
These cleaning agents may have fumes that also may cause harm if breathed in.
Low / Medium / High
Safety goggles and protective gloves are worn and maybe the inclusion of safety masks.
Disposal of bacteria, If not disposed properly may result in sudden growth of bacteria and may cause some health hazards
(Example: Flu, fever and cough)
Low / Medium / High
Disposal of bacteria into the Biohazard bin to ensure the bacteria does not grow in the wrong place and disposed of correctly.
The breakage of the petri dishes may result in sharp pieces that may injure one’s hand.
Low / Medium / High
Use rubber gloves or try not to hold it in such a way that the chance of dropping the petri dish would increase.

2.5 Data Analysis


As shown, when we plated E. Coli with the agar, Dettol works the best at the concentration of 10-1 and EC Oticide works best at the concentration of 10-1 when we plated B Subtilis with agar. Through this, we could determine that 10-1 concentration is the most cost effective dilution.

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