Week Three

Currently in the lab Deinococcus Canei is being sequenced. Though the genome has not yet been fully sequenced, it can be said that D. Canei is closely related to Deinococcus Aquaticus. With that, D. Canei susceptibility is tested this week to determine if it has an efflux pump, and if so, identify how useful it is against the antibiotic tetracycline. In the last test with D. Aquaticus the concentrations 0 ug/ml, 10 ug/ml, 100 ug/ml, and 1000 ug/ml. The only plate that had growth was the 0 ug/ml, the only plate that did not contain tetracycline. Since, these concentrations were bactericidal the concentrations used for this experiment were lowered to 0 ug/ml, 10 ug/ml, 50 ug/ml, and 100 ug/ml.
Materials: 
  • Liquid culture of D. Canei 
  • 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 a flask of 10 ml TGY with a colony from D. Canei plate
  2. Place in shaking incubator for 72 hours
  3. Autoclave four flaks 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 100 ul bacteria and vortex at level six for six seconds, then pour in labled petri plates
    1. 0 ug/ml: remove 0 TGY
    2. 10 ug/m: remove 40 ul TGY, add 40 ul Tetracycline 
    3. 50 ug/ml: remove 200 ul TGY, add 200 ul Tetracycline
    4. 100 ug/ml: remove 400 ul TGY, add 400 ul Tetracycline
  6. Place plates in standard incubator at 27 degree Celcius for 72 hours
The photo shown below displays the growth of bacteria after 72 hours. Once more the only plate with growth is the 0 ug/ml, which has no antibiotic. Since there is no growth on the rest of the plates, it cannot be determined whether D. Canei has an efflux pump. However, it can. Be concluded that if it did, similarly to D. Aquaticusm it is not effective in removing tetracycline from the cell. Within the next weeks, the minimum inhibitory concentration will be determined.Afterwords the utility of the efflux pump in D. Aquaticus will be analyzed, and after D. Canei has been sequenced it too will be explored more. 

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