Rationale:

To wash Soil Sample 3 using a syringe and 0.22 µm filter and to also perform experiments to collect Metadata on the soil sample.

Procedure:

Due to the negative results from the second plaque assay, the newest soil collection was washed. To determine the next step to be taken, the spot test, plaque assay, and negative control were checked from 2 mL of the sample and 10mL of LB broth were combined in a 50 mL conical vial, then vortexed for 15 minutes. The vial was then weighed to be placed in a centrifuge for 10 minutes at 10,000 g. While it was spinning, procedures were done to determine the soil composition, pH, and % water of the soil. For the pH, a small amount of soil was added to a pH vial in which was filled to the brim with DI water. After shaking for 45 seconds, the vial was left alone on the table for 2 minutes. A strip of pH paper was inserted into the vial for 45 seconds which was then used to determine the pH. The soil composition consisted of mixing 4 mL of soil, 8 mL of DI water, and 3 drops of soil dispersion liquid. Then, the tube was shaken for 30 seconds and left alone for 30 seconds. To measure the % water, the weight of a weigh boat was taken (initial) along with the soil (3 g-5 g)in the weigh boat (final). After the allotted time in the centrifuge, the samples were taken out and filtered using the serological pipette, syringes, and some filters. Out of the lysate, 2 mL became the Direct Isolation and the rest became the Enriched. 0.5 mL of Arthrobacter was added to the Enriched vial and shaken for 5 seconds.

Results and Analysis:

Plaque Assay 3 and Negative Control 

It was later found out that the reason for the contamination was because the LB broth was the contaminated.

The LB broth on the left was contaminated and the one that was used.

% Water

Weigh boat- 2.32 g

Weight boat and soil- 6.11 g

Soil- 3.79 g

pH

pH of 6.3

Soil Composition

A little more than 4mL of soil was added due to the large spaces at the bottom despite the many tries to break the clumps down.

Soil Sample 3

Weight: 19.74 g

Direct Isolation and Enriched (without Arthrobacter)

5 mL Enriched

2.5 mL Direct Isolation

Conclusion and Future Plans:

The 3rd plaque assay that was conducted on 9/17/18 came back negative and the control was contaminated. It was soon found out that the reason for the contamination was due to the contamination of the LB broth. Today (9/19/18), Soil Sample 3 was washed using a syringe and filter and metadata for % water, soil composition, and pH was collected. There was a total of 7.5 mL of lysate after centrifuging the sample. The procedures to collect the soil metadata was successfully completed.

In the future (9/21/18), the enriched lysate will be filtered again using the same method and the metadata will be collected. If there is enough time, a plaque assay will be performed.