SCHOOL OF SCIENCE AND TECHNOLOGY, SINGAPORE

INVESTIGATIVE SKILLS IN SCIENCE

Names: Md Danish Karlin (16), Md Danish B Mass. (17), Tan Zhi En (23)

Class: S2-01   

Group Reference: A / B / C / D / E / F / G / H

1. Indicate the type of research that you are adopting:

[    ] Test a hypothesis: Hypothesis-driven research

e.g. Investigation of the antibacterial effect of chrysanthemum

[ X ] Measure a value: Experimental research (I)

e.g. Determination of the mass of Jupiter using planetary photography

[    ] Measure a function or relationship: Experimental research (II)

e.g. Investigation of the effect of temperature on the growth of crystals

[    ] Construct a model: Theoretical sciences and applied mathematics

e.g. Modeling of the cooling curve of naphthalene  

[    ] Observational and exploratory research

e.g. Investigation of the soil quality in School of Science and Technology, Singapore  

[    ] Improve a product or process: Industrial and applied research

e.g. Development of a SMART and GREEN energy system for households  

2. Write a research proposal of your interested topic in the following format:

Title: An analysis of the most effective disinfectant agent available to common household against Escherichia coli and Staphylococcus aureus.

2A. Question or Problem being addressed

Infection is defined to be the invasion of a host’s body by disease-causing agents such as viruses, prions, bacteria, parasites and fungi. For infection to occur, each link of the chain of infection have to be present and be in sequential order. The chain of infection is defined to be: Infectious Agent > Reservoir > Portal of Exit > Mode of Transportation > Portal of Entry > Susceptible Host. By disinfection, we eliminate the first ring of the chain, Infectious Agent, which eliminates any risk of infection. Infections caused by bacteria and virus such as Methicillin Resistant Staphylococcus Aureus and Human Immunodeficiency Virus are usually very pernicious and even deadly. Therefore, it is imperative to prevent the spread of infection. Disinfectants like Hydrogen Peroxide (H2O2) are often used to disinfect surfaces. Our research is to determine which disinfectant agent (see list below for disinfectant agents used in this research) is the most effective at killing bacteria at its naturally gotten concentration.

2B. Goals / Expected Outcomes / Hypotheses

Hypothesis: The disinfectant agent, Chlorhexidine Gluconate will lyse the most amount of bacteria for both E. coli and S. aureus.

The independent variable is the type of disinfection agent

The dependent variable is the the lysis of the bacteria.

The constants are:

2C. Description in detail of method or procedures

  1. Remove the sterile inoculating loop from it package.
  2. Remove the lid from bottle containing the bacteria culture of E.coli.
  3. Dip the sterile inoculating loop into the bacteria culture of E.coli.
  4. Using a new agar plate, lift the lid just enough to insert the loop.
  5. Spread the loop containing the bacteria at the top end of the agar plate moving in a random circular pattern at one end of the plate.
  6. Rotate the plate about 60 degrees and streak straight, separate but close lines from the previous pattern.
  7. Rotate the plate about 60 degrees and spread the bacteria from the end of the second streak into a new area in the same pattern until you have 16 lines.
  8. Replace the lid, invert the plate and seal it with parafilm.
  9. Incubate the plate for 24 hours at 37 degrees Celsius.
  10. REPEAT STEPS 1-9 FOR Staphylococcus aureus

Refer to Fig 1.2 for streaking diagram

  1. Prepare liquid LB of E.coli and transfer into sterile flip-top dilution bottle.
  2. Using a sterile inoculating loop, gently scrape off a single colony of E.coli from agar plate.
  3. Drop the inoculating loop into the liquid LB and swirl.
  4. Using a sterile swab and a new agar plate, cover the entire surface of agar plate with the liquid LB of E.coli.
  5. REPEAT STEPS 1-4 FOR Staphylococcus aureus
  1. Prepare all disinfection agents.
  2. Divide all the petri dishes into 3 equal parts by writing on the side of the petri dish with the agar.
  3. Using a pair of forceps sterilized by the glass bead steriliser, gently pick up a diffusion disk.
  4. Soak the diffusion disk in the disinfectant and shake the diffusion disk until no more disinfectant is dripping off the diffusion disk
  5. Open the petri dish lid and place the soaked diffusion disk into the centre of a divided part of the petri dish.
  6. Repeat steps 4-6 for the all other diffusion disks, placing on different divided parts of the dish.
  7. After placing 3 diffusion disks, close the lid and invert the petri dish.
  8. Seal the petri dish using parafilm.
  9. Incubate the plate for 24 hours at 37 degrees Celsius.
  10. REPEAT STEPS 1-9 FOR ALL OTHER PETRI DISHES

    Equipment required:
  1. Incubator (1)
  2. Petri dish with agar culture (20)
  3. Glass bead sterilizer (1)
  4.  Methanol (50 ml)
  5. Chlorhexidine Gluconate/Soap (50ml)
  6. Chloroxylenol/Dettol (50 ml)
  7. Hydrogen Peroxide (50 ml)
  8. Bleach (50 ml)
  9. 70% Ethanol (500 ml)

      10. Ethyl Alcohol, chlorhexidine hand rub (50ml)

      11. Isopropyl alcohol (120ml)

      12. LB broth (5ml)

      13. Sterile cotton swab (20)

      14. Sterile disk (100)

      15. Forceps (6)

      17. Inoculating loop (5)

      18. E. coli culture in a bottle (10ml)

      19. S. aureus culture in a bottle (10ml)

      20. Sterile flip-top dilution bottle (2)

      21. Parafilm (2 rolls)

Diagrams: 

Diagram of Kirby Bauer Method (Fig 1.1)

(CDC, 2013)

Diagram of streaking method (Fig 1.2)

Procedures:

Detail all procedures and experimental design to be used for data collection

  1. We will find out which disinfectant agent can lyse the most colonies of E.coli and S.aureus by measuring the clearing of the lawn using a ruler. (Refer to Figure 1.1)
  2. From there we can determine the most effective disinfectant available to the common household in killing the bacteria.

Potential Risks

Due to the fact that we are working with bacteria and high heat, there is a serious risk of:

- Possible risk of infection due to handling of E.Coli or Staphylococcus aureus

- Risk of chemical/heat burns, due to use of glass bead steriliser and disinfection agents.

Safety Precautions

  1. Keep mouth and nose away from any bacteria culture or any equipment that has come into contact with bacteria used in experiment to avoid inhaling or ingesting the bacteria.
  2. Dispose all disposable equipment in a biohazard waste bin after use to avoid bacteria from spreading to other unintended areas.
  3. Wear gloves at all times throughout the duration of the experiment.
  4. Sterilise work area with 70% Ethanol solution or any other disinfectant before and after use.
  5. Handle glass bead steriliser and forceps sterilised by the glass bead steriliser with care, as it might get extremely hot.
  6. Sterilise any other non-disposable equipment after experiment to avoid bacteria from spreading to other unintended areas.

  1. Lay the petri dish with the agar face up against a black piece of paper.
  2. Using a ruler, measure the radius of the bacteria that was killed around the diffusion disk (the radius of the bacteria that was killed is marked by clear agar).
  3. Plot a table of the size of the radius of bacteria killed and the type of disinfectant used.
  4. Find the average radius of the bacteria killed by adding all 3 measurements from the same agar plate and divide by 3.
  5. We will then order the disinfectants by order of the biggest to smallest radius through a table. The best disinfectant is the one with the largest radius.

2D. Bibliography: List at least five (5) major references (e.g. science journal articles, books, internet sites) from your literature review. If you plan to use vertebrate animals, one of these references must be an animal care reference. Choose the APA format and use it consistently to reference the literature used in the research plan. List your entries in alphabetical order.