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Plasmid Curing
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 Materials required

 

  • Laminar air flow
  • Petri plate
  • Bunsen burner
  • Test tube rack
  • Loop
  • Micropipette
  • Incubator
  • Shaker incubator
  • Swabs
  • Penicillin discs bottle
  • Ciprofloxacin discs bottle
  • Forceps
  • Marker pen

 

 Reagents required

 

  • Acridine orange
  • LB medium
  • Muller-Hinton Agar

 

 Procedure



1. Add 50┬Ál of Acridine orange (0.10 mg/ml) to 5ml of LB broth.


2. Antibiotic resistant S.aureus culture is inoculated to LB broth having acridine orange.


3. Keep it for incubation (24 hrs) in a shaker incubator.


4. After incubation, this culture is swabbed in to the MHA Plates.


5. Put the Penicillin and Ciprofloxacin discs in to the plate.


6. Plates are incubated for 24 hrs at 37oC.

 

 

Differences Encountered in Real Laboratory

 

In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:

 

  1. Before starting the experiment in the laminar air flow, sterilize it by wiping with ethanol or other disinfectants.
  2. While inoculating agar media (plates and agar slants), make sure not to gouge or tear  the surface of the agar with the loop.
  3. Always label all tubes and plates with:

                   a. Name of the organism
                   b. Name of the media
                   c. The date
     
  4. Make sure that all the media used are sterilized.
  5. Make sure that the Microfuge tube containing Acridine orange is covered with aluminium foil because Acridine orange is light sensitive.
  6. Make sure to place agar plates in the incubator upside down.  This prevents water condensation from dripping onto the agar surface and spreading over the colonies. This will also prevent contamination and aid in the formation of isolated colonies.
  7. Properly adjust the flame of the Bunsen burner. The proper flame is a small blue cone; it is not a large plume, nor is it orange.
  8. While flaming the loop (or needle), be sure that each segment of metal glows orange/red-hot before you move the next segment into the flame. Be careful; the metal will get extremely hot.
  9. Once you have flamed your loop (or needle), do not lay it down, blow on it,touch it with your fingers,or touch it to any surface other than your inoculum or the sterile media. If you do touch the tip to another surface or blow on it, you will have to re-flame the loop before you proceed with your inoculation.
  10. Allow your loop or needle to cool before you try to pick up your organism. If you pick up organism with a hot tool, your cells will be killed.To cool your loop or needle quickly, place it on a section of agar that is uninoculated or is at least different from the area from which you will remove cells.
  11. Ensure that you are transferring the correct organism into your labeled tube by double-checking the name of the organism on the stock culture from which you are collecting your inoculum.
  12. When removing the caps from tubes, always keep the caps in your hand.  Never put them on the table, as they could pick up contaminants.
  13. Always handle open tubes at an angle; never let them point directly up, since airborne or other environmental organisms could fall into the tube and cause contamination.  Also, keep the lid over a plate when removing inoculum, as this will help prevent environmental contamination.
  14. Always flame the mouth of a culture tube when you open it and before you replace the cap.
  15. As soon as you are done inoculating, flame your loop or needle. Never place a contaminated tool on your workbench.
  16. Discard all contaminated materials properly and return your supplies to the proper storage locations, and clean up your working area.
  17. Always disinfect your work area when you are finished.

 



 

 

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