9.12.18: Metadata Collection and Spot Test

Rationale: Metadata tests that needed additional time to settle had been given enough, so results of the tests could be processed and analyzed. Then, a Spot Test could be performed with Enriched Isolation that was obtained on Monday, 9/10/18.

Metadata Results/Data:

• % Water: 2.390g measured after 48 hours of evaporation/3.145g initial soil added= 75.99% Soil. 1-0.7599= 24.01% Water
• After observing tube that contained soil being analyzed for percentages of sand, silt, and clay, it was found to contain 3.25mL of layered soil. The bottom layer was 1.5mL of sand, the middle layer was 1mL of silt, and the top layer appeared to be 0.75mL of clay. The corresponding percentages were: 46.15% Sand, 30.77% Silt, and 23.08% Clay. The category of soil this fits under is Sandy Clay Loam.

Procedure:

1. Lab bench was cleaned with Cidecon and 70% ethanol.
2. Ethanol burner was lit to establish an aseptic zone surrounding the flame.
3. Obtained mass of 50mL Conical tube labelled “HMB Enriched Lysate 9/10/18 S.S.B.”
4. Paired tube with another of similar mass and sent for centrifuging at 3000g for 5 minutes in order to pellet out Arthrobacter.
5. While the tube was being centrifuged, metadata results were collected.
   1. Weigh boat containing original soil sample B labelled “HMB Water” was re-massed and found to contain 2.390g of soil. The corresponding calculations resulted in discovering the % water of the soil was 24.01%. Weigh boat containing soil was disposed of under the instruction of Leo.
   2. The tube with soil in layers was taken up from lab bench and examined. After examination and documentation, the tube was cleaned and returned properly.        
6. LB Broth, plate, and 50mL conical tube were obtained. Plate was labelled with each of Group Three’s initials on one section of the plate and “Spot Test 9.12.18” on the bottom.
7. Placed 2mL of LB Broth in the 50mL conical tube.
8. 0.5mL of Arthrobacter was added to same 50mL conical tube.
9. 22.5 microliters of 1M CaCl2 was pipetted into same 50mL conical tube.
10. Finally, 2.5mL of 2x Top Agar solution was added and swished in same 50mL conical tube. After mixing, solution was rapidly poured onto plate to avoid cooling and hardening in tube rather than on plate.
11. Plate with freshly poured Top Agar was given 15 minutes to settle and harden.
12. While plate was setting, a 3mL syringe was obtained along with the enriched lysate from Monday (9/10/18).
13. 2mL of enriched lysate were drawn up into syringe. Filter was screwed onto end, and the 2mL were pushed through the filter and into a microcentrifuge tube.
14. 10 microliters of filtered sterilized enriched lysate was added to the section of the plate labelled “HB”.
15. Plate was allowed to set for 15 minutes before being placed into the incubator to be observed on Friday.

Observations:

• Soil in tube was not evenly layered. For example, the silt on one side appeared to encompass over 1mL of the total sample, but on the other side it made up closer to 0.5mL. To get values, averages of these were taken, leading to possible inaccuracies in final values.
• While the top agar was being formed, some residual bubbles remained on the outside of the agar. They were unable to be popped, so they remained around the ring. However, there was more than enough space on the plate to avoid them.
• When samples were applied to the plate during the initial spot test, they seemed to sit on the surface momentarily before settling into the agar. However, the same phenomena was not observed during this Spot Test. It will be interesting to see whether or not the results are different, but any discrepancies would not be able to be formally attributed to that due to the number of variables changed.

Next Steps:

• On Friday during open lab, the spot test sample will be analyzed. If there is a plaque, it will need to be picked and a Plaque Assay will be run to ensure that the results obtained were accurate and repeatable. If there is no plaque, another soil sample will need to be obtained and examined.