Soil Metadata and Dilution Plaque Assays (10/17/18)
Rationale:
Soil metadata will be completed for future correlations. Also, plaque assays will be run with 100, 10-1, and 10-2 dilutions. From the results of these plates further calculations will be made to determine what steps need to find a high titer.
Procedure:
- To complete the soil metadata, the “KEA 10/15 soil E” weigh boat’s weight and the “KEA 10/15 soil E” falcon tube different layers were recorded.
- Once an aseptic zone was established, 10 µL of 100, 10-1, and 10-2dilutions were added to correlation labeled test tubes which already had 0.5 mL of Arthrobacter in them.
- 8 mL of LB Broth, 90 µL of CaCl2, and 10 mL of 2X TA were combined into a conical vial.
- Transferred and mixed 4.5 mL of the Top Agar mixture from the conical vial into each test tube.
- Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the Top Agar mixture was poured onto a plate labeled “KEA 10/17 Control TA.”
- These plates were placed in the incubator at room temperature.
Observations:
- The following calculations were performed to determine the amount of LB Broth, 2X TA, and CaCl2needed for 4 plates.
|
Original Recipe |
X4 |
|
2 mL LB Broth |
8 mL LB Broth |
|
2.5 mL 2X TA |
10 mL 2X TA |
|
22.5 μL CaCl2 |
90 μL CaCl2 |
- The falcon tube, as shown below, had a layer of dark brown sand with a layer of cinnamon-brown sand 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 thin tannish-brown color which was clay.
- When pouring the TA mixture, bubbles and lumps formed on the plates.
Metadata:
Percent Water
Data Table
| mass of empty weigh boat (mi) |
2.39 grams |
| mass of weigh boat with wet soil sample (mt) |
6.15 grams |
| mass of wet soil sample (mwet soil)
mt – mi= mwet soil |
3.76 grams |
| mass of weigh boat with dry soil sample (mf) |
5.45 grams |
| mass of dry soil sample (mdry soil)
mf – mi= mdry soil |
3.06 grams |
| mass of water in the soil sample (mwater)
mwet soil – mdry soil = mwater |
0.70 grams |
Equation for Percent Water
Percent Water = 18.61%
Percent Sand, Percent Silt, and Percent Clay
Data and Calculation Table
|
Texture |
Approximate Amount | Calculations | Percent |
|
Clay |
1.0 mL | 1.0 mL / 11 mL | 9.09% |
|
Silt |
1.0 mL | 1.0 mL / 11 mL |
9.09% |
| Sand | 9.0 mL | 9.0 mL / 11 mL |
81.82% |
| Total | 11 mL | 11 mL / 11 mL |
100.00% |
Percent Clay = 9.09%
Percent Silt = 9.09%
Percent Sand = 81.82%
Classifying Soil Texture
Based off the percent sand, percent silt, and percent clay, calculations using a soil texture triangle classified the soil sample’s texture to be loamy fine sand. 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:
If the plates are contaminated, the plaque assays will be run again with the 100, 10-1, and 10-2 dilutions. If the plates are positive, another plaque will be picked, serial dilutions, and more plaque assays will be run.
