September
28
SEPTEMBER 21ST, 24TH, 26TH- Labs
- SEPTEMBER 21ST, 2018
- SOIL FILTRATION
- OBJECTIVE:
- Filter out new soil sample
- PROCEDURE:
- Tables were cleaned and lamps were lit
- 4 mL of soil was put into a test tube and filled to the 24mL mark with LB broth
- Tube was then shaken for 15 minutes
- Then a separate tube was filled with water to be its “mass buddy” and it was centrifuged for 7 minutes
- Next a weigh boat was massed and wet soil was added and massed
- The weigh boat its the soil was then left to dry in the fume hood
- A falcon tube was then filled with 10 ml of soil and was then filled to the 20 ml with DI water
- Then 3 drops of dispersion liquid was added to the tube, it was then shaken for 30 seconds and then left to sit over night
- Then the tube that was then spun was retrieved and then had the supernatant filtered using a top filter
- 10mL of the supernatant was left in the test tube and 5mL was put into a different test tube
- The test tube containing the 10mL had .4mL of Arthro added to it (enriched sample)
- Was then left to incubate
- The 5mL test tube was then put in the fridge (direct sample)
- RESULTS:
- Soil Meta Data
- Sand 65%
- Silt 15%
- Clay 20%
- Soil Meta Data
- Water percent
-
- Mass of plate: 7.57g
- Mass of wet soil: 13.01g
- Mass of dry soil: 12.23g
- Mass of water: .78g
- 6% water
-
- CONCLUSION:
- Metadata was collected, will now conduct plaque assay next to test for phage presence
- NEXT STEP:
- Conduct a plaque assay
- SEPTEMEBER 24TH, 2018
- PLATING PLAQUE ASSAY
- OBJECTIVE:
- Conduct a plaque assay with NO CONTAMINATION
- PROCEDURE:
- Tables were cleaned and lamps were lit
- Direct and enriched samples were filtered out using a syringe and a .22 𝝁m filter
- Next, a large test tube was filled with:
- 10 mL of LB
- 12.5 mL of 2XTA
- 112.5 CaCl
- .10 𝝁L of lysate was then added to the containing .5mL of Arthro and was left for 15 minutes
- The tube contains the 2X TA solution was then placed in a hot water bath so it would not solidify
- Next 5mL of the TA solution was added to a plate, where 10 𝝁L of the direct were added to it
- The plate was swirled then was left to solidify
- Another 5mL was pipetted onto a different plate to serve as control
- Next the tubes contain the lysate and Arthro had 5mL of the TA solution added to it
- Solution was mixed then was poured onto plates
- Plates were left to solidify and were then inverted and placed into incubator
- RESULTS:
- The results as seen in figure 13 were contaminated due to the Arthro sample being a different bacteria
- CONCLUION:
- The results seen have been deemed inconclusive due to laboratory issue with Arthro
- NEXT STEPS:
- Conduct another plaque assay
- SEPTEMBER 26TH, 2018
- PLATING ANOTHER PLAQUE ASSAY (SAME SAMPLE)
- OBJECTIVE:
- To conduct another plaque assay without contamination
- PROCEDURE:
- Test tubes containing .4mL of Arturo had 10 𝝁L of lysate added to it, and was left to sit for 10 minutes
- A large test tube was filled with
- 10mL of 2X TA
- 90 𝝁L of CaCl
- 8.4 mL LB broth
- 5mL of the TA solution was poured onto a plate and served as the control
- Then 5mL of TA solution was put into the tubes contains the Arturo and lysate
- The contents of the tubes were mixed then poured onto plates
- The plates then solidified for 10 minutes and were then inverted and placed in the incubator
- RESULTS:
- Waiting to view plates and obtain results
- CONCLUSION:
- Waiting on results
- NEXT STEPS:
- View results and determine if phage are present, if so dilutions will be carried out, and if not a new soil sample will be collected
- View results and determine if phage are present, if so dilutions will be carried out, and if not a new soil sample will be collected
