October
12
OCTOBER 5TH, 8TH, 10TH- Labs
- OCTOBER 5TH, 2018
- RESULTS: (META DATA RESULTS WERE NOT VIEWED UNTIL 10/5)
- Soil meta data Results:
- Sand: 65%
- Silt: 25%
- Clay: 10%
- Soil meta data Results:
- CONCLUSION:
- The soil composition has been determined, now the percent water must be calculated in order to have more data on the soil sample.
- OBJECTIVE:
- Plate plaque assay with no contamination
- PROCEDURE:
- Tables were cleaned and lamps were lit
- The sample was filtered using a syringe to push the solution through a .22𝝁m filter into a micro centrifuge tube
- A weigh boat was massed and then had wet soil added which was then massed
- 10 𝝁L of the lysate was then pipetted into the Arthro, and was left to sit for 10 minutes
- During the 10 minutes a test tube was filled with: (TA is added last)
- 6mL LB broth
- 7.5mL 2X TA
- 67.5 𝝁L CaCl2
- 4.5mL of the 2X TA solution was added to the test tube with the lysate and Arthro
- Before the tubes were able to be poured into plates, the 2X TA solidified in the test tubes, so the tubes were held over an open flame to put it back into a liquid form
- It was then poured onto plates and were inverted and placed into the incubator
- RESULTS:
- Percent Water:
- Plate: 7.575g
- Wet soil + plate: 9.940g
- Dry soil + plate: 9.420g
- % water: 5.23%
- Plaque Assay Results:
- As seen in Figure 15, the control plate has no contamination, while the plaque assay has been contaminated
- Percent Water:
- CONCLUSION:
- It was discovered that the arthro used in the experiment was contaminated, hence why the control, which has no arthro on it, was not contaminated while the plaque assay, which contains arthro, was contaminated. This has rendered the results to be inconclusive.
- NEXT STEPS:
- Conduct the plaque assay with the same soil sample, due to contamination rendering the results inconclusive
- RESULTS: (META DATA RESULTS WERE NOT VIEWED UNTIL 10/5)
- OCTOBER 8TH, 2018
- OBJECTIVE:
- Conduct another plaque assay with the same sample
- PROCEDURE:
- Tables were cleaned, lamps were lit
- A test tube was filled with: (2X TA was added last to prevent solidifying)
- 8mL LB broth
- 90 𝝁L CaCl2
- 10mL 2X TA
- 10 𝝁L of lysate was pipetted into test tube containing arthro and was left to sit for 10 minutes
- After the 10 minutes 4.5mL of the 2X TA solution was mixed into the test tube with the lysate and arthro
- The test tubes were then poured into the plates, which then solidified, and were then inverted and placed in the incubator
- RESULTS:
- As seen in Figure 16, the control is not contaminated while the plaque assay is contaminated
- CONCLUSION:
- The source of the contamination is unknown, it could potentially be arthro or a contamination issue that is occurring during the pouring of the TA 2X solution on to the plate.
- NEXT STEPS:
- A new soil sample will be collected, and a plaque assay will be conducted one more time with the same sample, and an arthro control will also be conducted to help determine the source of contamination
- OBJECTIVE:
- OCTOBER 10TH, 2018
- OBJECTIVE:
- Wash new soil sample, and conduct another plaque assay with the old sample and have no contamination
- PROCEDURE:
- Tables were cleaned and lamps were lit
- A test tube was filled to the 2mL mark with soil, which then had 10mL of LB broth added to it
- The tube was then shaken for 15 minutes
- The tube was then centrifuged for 10 minutes at 3,000 G
- During this time the 10 𝝁L lysate (from the previous sample) was put into a test tube with arthro and was also left to sit for 10 minutes
- A separate test tube containing arthro had 10 𝝁L of phage buffer added (this will serve as arthro control)
- A large test tube was then filled with: (2X TA was added last to prevent solidifying)
- 10mL LB broth
- 112.5 𝝁L CaCl2
- 12.5mL 2X TA
- After the 10 minutes 4.5mL of the 2X TA solution was mixed into the test tube with the lysate and arthro, and the test tube with phage buffer and arthro
- The test tubes were then poured into the plates, which then solidified, and were then inverted and placed in the incubator
- After the soil sample was done being centrifuged, a top filter was used to filter the supernatant
- .45mL of arthro was then added to the filtered supernatant, which was then placed into the incubator
- RESULTS:
- Waiting for results
- CONCLUSION:
- Waiting for results
- NEST STEPS:
- Conduct a plaque assay with new sample
- Conduct a plaque assay with new sample
- OBJECTIVE:
