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:

A Plaque Assay helps us determine if there is the presence of bacteriophages by adding Arthrobacter directly to the lysate. We can tell the existence of bacteriophages by checking the presence of plaques on the agar plate. A spotting test can help determine if bacteriophages are present in the lysate or not. And Soil Metadata can help us to learn more about the soil sample’s characters.

Experimental Procedure for Spotting Test for Soil 9.5.18:

……………………. 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) Direct Isolation & Enrichment Lysate

…………………………………………………….(2) LB Broth

…………………………………………………….(3) 2x TA

…………………………………………………….(4) 1M CaCl2

…………………………………………………….(5) 0.5 ml Arthrobacter

…………………………………………………….(6) 50ml conical tubes

…………………………………………………….(7) Micropipettes & Serological Tubes

…………………………………………………….(8) Phage Buffer

 …………………….2. Centrifuge the enrichment Lysate at 3000x g for 5 min

……………………..3. Add 2 ml LB Broth, 0.5 ml Arthrobacter & 22.5 ul CaCl2 (aq) to a 50 ml conical …………………………tube.

……………………..4. Section a new Agar Plate into 3 sections, each label  PB (Phage Buffer as control), ………………………..(D) Direct Isolation & (E) Enrichment. (Plate labeled: JY5 Spotting test 9.5.18 …………………………9/12/18)

……………………..5. Add 2.5 ml of 2x TA to 50 ml conical tube and pipette with serological tube …………………………before adding the solution on to a new Agar plate, slightly swirl the plate to let it …………………………cover the whole plate evenly and wait for 10 min.

……………………..6. Filter 2 ml Soil 9.5.18 Enrichment Lysate with filter paper (0.22um) to filter out ………………………….bacteria into an Eppendorf. (labeled: JY5 9.5.18)

……………………..7. Use micropipette to drop 1o ul of Phage Buffer, Direct Isolation Lysate & ………………………….Enrichment Lysate to the middle of each section and set still for 10 min before ………………………….placing in the incubator.

Experimental Procedure for Plaque Assay for Soil 9.5.18:

…………………….1. Add 0.5 ml Arthrobacter and 10 ul Filtered Enriched Lysate to an Eppendorf 10

……………………….min for infection.

…………………….2. Add 8 ml of LB Broth, 90 ul Calcium Chloride (aq) to a 50 ml conical tube

…………………….3. Add 10 ml of 2x Top Agar to the 50 ml conical tube, quickly distribute among four

………………………..new 15 ml conical tubes for the whole group.

…………………….4. Take one tube and add the infected lysate, pipette and carefully pour on to a new

………………………..agar plate

…………………….5. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Soil Metadata:

…………………….1. Remove the supernatant in the falcon tube

…………………….2. Record the results and discard the soil in the biohazard bin, wash up the tube and

………………………..place on rack

…………………….3. Weigh the petri dish, record the mass, discard the petri dish in the biohazard bin

…………………….4. Calculate the Soil Composition and % Water

Observations, Results & Data:

.Soil Metadata for Soil Sample 9.5.18:

…………………………….(1) % Water: 21.6% ( (12.24 – 11.16) / (12.24 – 7.24 ) = 0.216 )

 ……………………………(2) Sand: 57.1% (4ml / 7 ml)

 ……………………………(3) Slit: 28.6% (2 ml / 7 ml)

 ……………………………(4) Clay: 14.3% (1 ml / 7 ml)

The raw data from the falcon tube is extremely hard to identify since the differentiation of layers isn’t obvious.

From the data collected, the soil type seems to be sandy loam, which is generally the soil type for gardening.

Spotting Test for Soil Sample 9.5.18:

The plate appears to have an unknown white dot on the plate, its effects on the experiment are yet to be seen.

Interpretations & Conclusions:

By spinning down the Enrichment lysate, the bacteria are mostly at the bottom of the 50 ml conical tube, which made the syringe filter step extremely easy due to the lack of blockage on the filter paper.

When mixing the top agar for Plaque Assay, the time passed since the 2x Top Agar was added to the mixed solution to the mixture was poured onto the plate could potentially affect the quality of the test, since the agar starts to solidify as soon as it was added to the mixture.

The white dot is a giant variable in the spotting test, if the dot is a contamination on the agar plate the test would lose its accuracy and reliability,  after further investigation the dot seemed to be embedded in the bottom agar so it was not removable.

The result of the soil composition metadata corresponds to the environment it was sampled in since the sample was originated from an artificial garden, it is not surprising that the soil analysis indicated that it was sandy loam. However, the high percentage of water contained in the soil was astounding and the effects of the % water in the soil needs more background research.

Next Step:

I will be checking the results of both the Spotting Test and Plaque Assay on the Soil Sample 9.5.18