Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

To search for possible bacteriophages in the soil, this experiment takes Soil Sample 9.5.18 and go through the Washing process to collect potential bacteriophage-containing lysate for further analysis. By using LB Broth, centrifuge and syringe filters, potential bacteriophage lysate can be collected. And by adding Arthrobacter to some of the lysate, Enrichment protocol can help with the confirmation of bacteriophage’s existence. And Soil Metadata can help us to learn more about the soil sample’s characters.

Experimental Procedure for Soil Washing, Enrichment of sample Soil 9.5.18:

Soil Washing:

1. Set up an Aseptic zone(Sprayed with Ci-Decon, wiped dry, then sprayed 70% Ethanol and let it evaporate)  on the workbench,  prepare:

1. Sample Soil 9.5.18
2. Two 15 ml Conical Tube
3. One 50 ml Conical Tube
4. LB Broth
5. Syringe Filter

2. Place sample Soil A to the 2 ml mark in the 15ml Conical Tube.

3. Pour LB Broth to the 12 ml mark in the 15 ml Conical Tube Soil 9.5.18

(1) 15 ml Conical Tube Soil 9.5.18 weight: 20.15 g

4. Vortex(5 min), Shake(5 min) & Vortex(5 min) for 15 min to mobilize the soil.

5. Centrifuge at 3000 G for 10 min.

6. Filter the supernatant from the 15 ml Conical Tube Soil 9.5.18 with syringe filter to a new 50 ml conical tube and the rest in a new 15 ml conical tube.

7. Add 0.5 ml Arthrobacter into the 50 ml conical tube with the filtered supernatant and place on shaker.

10. Stored the Supernatant filtered from the filter paper into a new 15 ml Conical Tube(Direct Isolation) and store in fridge.

Soil Metadata:

11. Soil Composition:

…..(1) Add soil to the 10 ml mark on the falcon tube.

…..(2) Add water to the 30 ml mark.

…..(3) Add 3 drops of Soil Dispersion Liquid

…..(4) Wait for 48 hrs to see the results

12. % Water:

…..(1) Weigh the empty petri dish: 7.24 g

…..(2) Weigh the petri dish with wet soil: 12.24 g

…..(3) Wait for 48 Hrs for Soil to dry and check results

13. Soil pH:

…..(1) Add a small amount of soil to the pH vial

…..(2) Add water to 80% full

…..(3) Shake the vial for 30 sec and wait for 2 min

…..(4) put in a pH paper(should not exceed 1 inch)

…..(5) Wait for 45 sec and compare the color with the pH scale to determine the pH of the soil

…..(6) The pH level for Soil Sample 9.5.18 is  pH 7.5

14. Workbench was sterilized with Ci-Decon and 70% Ethanol for clean up.

Observations, Results & Data:

During the syringe filter stage, the bubbles in the syringe cause the filter process harder due to the compressible characteristic of air.

The metadata of Soil Sample 9.5.18: pH 7.5,

other data would have to wait for 48 Hrs to get the results.

Interpretations & Conclusions:

Even with a pH scale, the results can be hard to interpret and can be subjective due to the nature of the naked eye.

It would be ideal if the lab reports next time can feature a picture of the pH paper and the scale together,

Next Step:

I will be doing spotting test and Plaque Assay on Soil Sample 9.5.18, and finishing up the data collection of the soil metadata.

In the Next Lab:

I will need (1) LB Broth

……………….(2) Arthrobacter

……………….(3) Calcium Chloride

……………….(4) Centrifuge

……………….(5) Agar Plate

……………….(6) Syringe Filter

……………….(7) Micropipette and Serological Pipette