Week Four

During this week Deinococcus Caeni’s susceptibility to tetracycline was still being tested. It has been confirmed that this bacteria strain does have an efflux pump just as Deinococcus Aquaticus did. However, the concentrations used last week were 0 ug/ml, 10 ug/ml, 50 ug/ml, and 100 ug/ml. We expected growth on at least the 10 ug/ml plate, but that concentration was bactericidal. The concentrations were reduced to 0 ug/ml, 1 ug/ml, 2 ug/ml, and 6 ug/ml. 

Materials:

  • Liquid culture of D. Caeni
  • Four flasks of 25 ml TGY with Agar
  • Tetracycline with stock concentrations of 2.5 mg/ml
  • Hot plate
  • Vortex
  • Micropipette and corresponding tips

Procedures:

  1. Inoculate flask of 10 ml TGY with colony from D. Canei plate
  2. Place in shaking incubator for 72 hours
  3. Autoclave four flask of 25 ml TGY with 1.5% agar to pour plates
  4. Reheat the sterilized agar on a hot plate
    1. Let cool to room temperature before adding antibiotic 
  5. Remove TGY, add tetracycline, add 250 ul of bacteria and vortex at level six for six seconds, then pour in labeled petri plates
    1. 0 ug/ml: remove no TGY, add 250 ul of bacteria
    2. 1 ug/ml: remove 10 ul TGY, add 10 ul of tetracycline, add 250 ul of bacteria
    3. 2 ug/ml: remove 20 ul TGY, add 20 ul of tetracycline, add 250 ul of bacteria
    4. 6 ug/ml: remove 60 ul TGY, add 60 ul of tetracycline, add 250 ul of bacteria
  6. Place plates in standard incubator at 27 degrees Celcius for 72 hours

The photo below shows the growth of the D. Caeni after 72 hours in the incubator. Even at these calculations there was only growth on the 0 ug/ml, the only plate without antibiotic.

There will be a final experiment with concentrations lower than 1 ug/ml. It was surprising to see that there was no growth at these concentrations, since the efflux pump is supposed to prevent the tetracycline from causing the bacteria to die. The thought that the tetracycline prevented the tetR from being read and therefore does not release the repression on the tetA is not the reason. This is because the pump should be removing the antibiotic from the cell before it is able to bind to anything. Thus, the reason as to why this pump is ineffective is still not know. 

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