2. Methods

First, we will grow the bacteria and separate the individual colonies by streaking and incubating the plate for 24 hours at 37 degrees Celsius.
   
    After that, we will prepare the bacteria culture by selecting one or two colonies and mixing them in liquid LB broth. We will divide the petri dish that will be use for disinfecting into three thirds, and swab the whole plate with the bacteria culture prepared.

    For testing of disinfection agent, we have come up with a method inspired by the Kirby-Bauer method. Instead of antibiotic disks, we use diffusion disks and soak them with the disinfectant, and placing them in the centre of the third that was divided earlier and incubating the plate for 24 hours at 37 degrees Celsius.

    Below are the detailed step-by-step procedures:

  • Part I - Isolating Bacterial Colonies
    First, we need to grow the bacteria and separate the individual colonies. To do that:

  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 4 lines 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 a to i FOR Staphylococcus aureus


    Diagram of Streaking Method (Fig 2.2.1)
  • Part II - Preparing bacteria culture
    After isolating the individual colonies, we have to prepare the bacteria culture. To do that:
  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 a to d FOR Staphylococcus aureus
  • Part III - Disinfection Process
    With the bacteria culture, we can start the disinfection process. To do that:
  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 a to i FOR ALL OTHER PETRI DISHES
Diagram of Kirby-Bauer Method

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