Week Five

 This week concludes the Deincoccus Caeni’s susceptibility to the tetracycline test. The concentrations used during the last experiment were 0 ug/ml, 1 ug/ml, 2 ug/ml, and 6 ug/ml. These concentrations were still too high to see any growth on the plates with antibiotics so the concentrations used were 0 ug/ml, 0.25 ug/ml, 0.5 ug/ml, and 0.75 ug/ml. 

Materials:

  • Liquid culture of D. Caeni

  • Four flasks of 25 ml TGY with Agar

  • Tetracycline with a stock concentration of 2.5 mg/ml

  • Hot plate

  • Vortex

  • Micropipette and corresponding tips

Procedures:

  1. Inoculate a flask of 20 ml TGY with a colony from the D. Canei plate

  2. Place in a shaking incubator for 72 hours

  3. Autoclave four flaks of 25 ml TGY with agar to pour plates

  4. Reheat the sterilized agar on a hot plate

  5. Let cool to room temperature before adding the antibiotic 

  6. Remove TGY, add tetracycline, add bacteria then vortex for six seconds on level six, pour in labeled petri plates

    1. 0 ug/ml: remove 0 ul TGY, add 250 ul of bacteria

    2. 0.25 ug/ml: remove 2.5 ul TGY, add 2.5 ul Tet., add 250 ul of bacteria

    3. 0.50 ug/ml: remove 5 ul TGY, add 5 ul Tet., add 250 ul of bacteria

    4. 0.75 ug/ml: remove 7.5 ul TGY, add 7.5 Tet., add 250 ul of bacteria

  7. Place plates in the standard incubator at 30 degrees Celsius for 72 hours


The photo displays the growth of the D. Caeni after 72 hours in the incubator. There was no growth on the 0.25 ug/ml, 0.50 ug/ml, or 0.75 ug/ml concentration plates. The 0 ug/ml plate had growth.

It was unexpected that there was no growth on the plates containing antibiotics, even at these concentrations. Though the bacteria contains an efflux pump in its membrane, it may not be activated or effective for regulating the amount of antibiotic entering the cell. It might be possible that the conditions for the efflux pump to be effective may not be replicated in the lab. This will conclude the susceptibility test of D. Caeni to tetracycline. The next steps are to explore the capability of D. Caeni, specifically its capability of being transformed with a plasmid extracted from E coli. 

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