Finish Metadata and Plaque Assay of Soil D (10/05/18)
Rationale:
Finished collecting soil metadata that could possibly be used later to find correlations. A plaque assay will be run with the enriched lysate from soil D to determine whether or not there are bacteriophages in the sample that specifically target Arthrobacter.
Procedure:
- To complete the soil metadata, the “KEA 10/3/18 Soil D” weigh boat’s weight and the “KEA 10/3 Soil D” falcon tube different layers were recorded.
- Once an aseptic zone was established, enriched filtered lysate from soil D was filtered with a 0.22 μL tipped syringe into a microcentrifuged tube labeled “KEA 10/5 FS enriched.”
- 10μL of “KEA 10/3 FS enriched” was added and mixed into a test tube which already had 0.5 mL of Arthrobacter.
- 6 mL of LB Broth, 67.5 μL of 1 M CaCl2, and 7.5 mL of 2X TA were combined into a conical vial.
- Transferred 4.5 mL of the Top Agar mixture from the conical vial onto a plate labeled “KEA 10/5/18 PA.”
- The test tube mixture was poured into the plate.
- The “KEA 10/5/18 PA” plate was placed in incubator at room temperature.
Observations:
- The following calculations were performed to determine enough LB Broth, TA, and CaCl2needed for 3 plates.
|
Original Recipe |
X3 |
|
2 mL LB Broth |
6 mL LB Broth |
| 2.5 mL 2X TA |
7.5 mL 2X TA |
| 22.5 μL CaCl2 |
67.5 μL CaCl2 |
- Dry soil appeared lighter than the wet soil. The pictures below show the soil from before and after; however, it is hard to tell color differences from the photos.
- The falcon tube, as shown below, had a layer of dark brown clay with a layer of cinnamon-brown clay on top of that. The silt layer on top of these two layers appeared a whitish-gray. The remaining layer on top of this was a tannish-brown color.
- The TA started to solidified when pouring the control TA into the TA plate. The picture below shows the control plate. The TA solidified in the Arthrobacter test tubes before it was poured into the plates. The test tubes were held over an EtOH (100%) flame to reverse this.
Metadata:
Percent Water
Data Table
| mass of empty weigh boat with lid (mi) |
3.40 grams |
| mass of weigh boat with wet soil sample (mt) |
6.30 grams |
| mass of wet soil sample (mwet soil)
mt – mi= mwet soil |
2.90 grams |
| mass of weigh boat with dry soil sample (mf) |
5.77 grams |
| mass of dry soil sample (mdry soil)
mf – mi= mdry soil |
2.37 grams |
| mass of water in the soil sample (mwater)
mwet soil – mdry soil = mwater |
0.53 grams |
Equation for Percent Water
Percent Water = 18.28%
Percent Sand, Percent Silt, and Percent Clay
Data and Calculation Table
|
Texture |
Approximate Amount | Calculations | Percent |
|
Clay |
1.5 mL |
1.5 mL / 8 mL |
18.75% |
|
Silt |
1.5 mL |
1.5 mL / 8 mL |
18.75% |
|
Sand |
5 mL |
5 mL / 8 mL |
62.5% |
| Total | 8 mL | 8 mL / 8 mL |
100.00% |
Percent Clay = 18.75%
Percent Silt = 18.75%
Percent Sand = 62.5%
Classifying Soil Texture
Based off the percent sand, percent silt, and percent clay, calculations using a soil texture triangle classifies the soil sample’s texture to be sandy loam. The point is plotted on the soil texture triangle below.
-This image was retrieved from the Natural Resources Conservation Service website at https://www.nrcs.usda.gov/wps/portal/nrcs/detail/soils/survey/?cid=nrcs142p2_054167.
Next Steps:
On Monday, if the plaque assay is negative, new soil will be collected. If the plaque assay is contaminated, the plaque assay will be run again with the enriched soil D. If the plaque assay is positive, the plaques will be selected.
