SEA Phage Lab Journal

September 10th, 2018

Metadata and Enrichment

Objectives: Collect Metadata on soil sample 2, filter lysate and separate into direct and enriched portions.

Rationale: The metadata for each tree our unit collects from can be used to better understand the way the presence of Arthrobacter phage correlates with the health of different trees. Once we have discovered correlation, we could test causation. Filtering lysate allows me to test for phage effectively.

Results from last Lab: I collected soil from a large Live Oak tree right outside of the SLC, near a very popular walkway. I have the soil sample in a 15 mL vial and a plastic bag, as well as several leaves.

Procedure: Added 2 mL of soil sample 2 into a 15 mL tube, for enrichment. The other roughly 10 mL of soil was saved in the same plastic bag it was stored in, for metadata collection. Added 10 mL of LB Broth to the 15 mL enrichment tube. Then, I shook the tube for 15 minutes to mix the soil and LB Broth together. After shaking, I massed my tube, found it to be 18.72 grams, and ended up adding several drops of DI water to match a lab partner for centrifuging at 19.22 grams.

While the tubes were in the centrifuge, I began to collect metadata. Because I didn’t collect as much soil as I should have, I couldn’t complete the proportions as instructed. Instead of filling a tube to 10 mL of soil, I filled one to 4 mL, saving roughly half of the remaining soil for later. I then added DI water to 12 mL, and 3 drops of soil dispersion liquid. Shook for 30 seconds, and let the soil settle out. Afterwards, I massed a weigh boat at 2.45 grams, and my remaining wet soil in the same boat at 8.10 grams. This means the wet soil was 5.65 grams.

The soil and LB Broth finished the 10 min in the centrifuge. I set up my filter apparatus and used a bubble pipette to move the supernatant to the filter on the top. I was only able to collect around 9 mL of lysate, so I opted to stick with just an enriched sample rather than enriched and a direct. I added .5 mL of arthrobacter and set the enriched lysate in a shaking incubator at 26 degrees Celsius for the next 48 hours.

Finally, I took a few drops of the liquid from my dispersed soil sample and added DI water until it filled a small vial. I took pH paper and tested for 45 seconds. The pH was 6.0.

Analysis and Interpretation: The soil was light colored, especially when compared with other Live Oak tree soil in the area. The pH seemed to be acidic, but I don’t know what other trees are like.

Future Plans: Next lab I will complete a spot test with my enriched lysate, and determine the need for a plaque assay the class afterwards. I will also find the percent composition of my soil, and find the proper classification based on Lathan’s chart.

SEA Phage Lab Journal

September 12th, 2018

Spot Testing and Metadata

Objectives: Complete a Spot test with my second enriched lysate, and finish soil metadata.

Rationale: Testing for plaques and a basic understanding of the environment around my tree.

Procedure: Massed enriched lysate after incubation from the last lab. Found mass buddy (21.55g), and gave the 50 mL tube to Dr. Adair to centrifuge for 5 minutes at 3,000 g. I found the mass of my dry soil in the weigh boat, now 7.38g, and found the percent water loss, at 12.7% of the original mass.

I found the dispersed soil from the last lab to be 3.5 mL inside the tube. 1.75 mL was clay, (50%),, 1 mL was silt, (28.6%), and .75 mL was sand, (21.4%). When plotted, the readout was firmly in the Clay section of the sand/silt/clay chart.

I began to filter my lysate into a microcentrifuge tube with a syringe, and a 22 micrometer filter attached to it, but as my lab partners moved around me, I drifted out of the aseptic zone and needed to repeat. I got a new syringe and filter, as well as a microcentrifuge tube. I pulled slightly less than 2 mL of enriched lysate from my 15 mL tube, attached my new filter, and pushed the plunger slowly until I had filtered everything into the microcentrifuge tube. While I was doing this, my lab partners had made an Agar plate and separated it into quadrants. We used a pipette and transferred 10 microliters of each lysate into our respective quadrants, saving one quadrant as a control. It occurred to me after lab that I didn’t notice Henry switching pipette tips, but I likely just didn’t see it. Thought it was important to note, however.

Once that was done, we let the plate sit for around 10 minutes, then set it in the incubator.

Analysis and Interpretations: I suspect my soil has a different composition from others’, due to the high amount of clay and the distinct coloration as compared to others’. I will be interested to see where other people’s soil falls on the graph.

Future Plans: Depending on how my spot test goes, I will either complete a plaque assay next lab or reevaluate my soil and determine a new place to search for samples, based on our question.