10.10.18: Spot Test and Plaque Assay for Soil Sample E

Rationale: Since soil E was washed during Monday (10/08/18), it is possible to test the lysate obtained to determine whether or not phage was present in the sample. Thus, it is necessary to determine whether or not it is possible to progress with purification of phage or use an alternate procedure.

Procedure:

1. Aseptic zone established
2. A syringe and filter were used to obtain 2mL of Filter Sterilized Enriched Lysate (FSEL) – placed in tube “HMB FSEL 10/10/18”.
3. 10µL FSEL was added to 0.5mL of Arthrobacter. Let sit to mix for 15 minutes.
4. 2mL of LB Broth was added to both control and experimental (“HMB Plaque Assay 10/10/18 Sample E”) tubes.
5. 22.5µL CaCl2 was added to control and experimental tubes
6. 2.5mL 2X Top Agar was added to both tubes
7. Overlay solutions were plated on respective plates (“HMB Plaque Assay Soil E 10/10/18” and “NMN HMB Top Agar Control”) after brief swishing.
8. 6mL LB Broth was added to new tube
9. 67.5µL CaCl2 was added to tube
10. 2mL of LB Broth and CaCl2 solution was added to separate tube that contained 0.5mL arthrobacter
11. 2.5mL 2X Top Agar was added to arthrobacter tube and original tube (control tube).
12. Both tubes were swished, then plated as an experimental (“HMB ST 10/10/18 Sample E”) and control plate. Let sit for 10 minutes to solidify overlay solution
13. Experimental Plate: 10µL FSEL, Direct Lysate, and Phage buffer were added to respective spots indicated on plate
14. Both plates let sit for 15 minutes, then incubated.

Observations:

• Plaque assay plate solidified quickly and showed signs of very few air bubbles on the plate. This should lead to little confusion between plaque and air bubble.
• Spot Test plate did not absorb the drops of FSEL, Direct Lysate, and Phage buffer well. Therefore, the plate was left unflipped in the incubator to allow for more absorption time.
• Plates for spot test were created using a different method than normal. There appeared to be no difference between using separate tubes and using one main tube, but strange results may be attributed to this.

Conclusions/Next Steps:

• The next step in the process will be to check the plates to see whether or not there is plaque present on either plate. If there is, there would be phage present in the sample, and it would be possible to move forth with the purification process.

Repeat Enrichment due to bad LB Broth

10/1/2018

Objectives: Repeat Enrichment Procedure to separate a lysate, collect metadata

Rationale: Must have a lysate to isolate phage, if present, and must have metadata to better analyze potential patterns in phages.

Procedure:

• Isolated 2 mL of the same soil from last lab
• Added 10 mL LB Broth
• Shook 15 Min
• Centrifuged tube for 10 min at 5000 rpm

When centrifuging, completed metadata

• Massed dry weigh boat
• Found water to make up 40.2% mass
• Determined soil makeup to be comfortably clay

Removed mixture from Centrifuge

• Filtered through vacuum apparatus
• Left lysate in shaking incubator

Cleaned lab station and left.

Nothing to analyze or interpret.

Future Plans: Complete a Plaque Assay on new Lysate.

Plaque Assay 3

10/5/18

Results from last lab: Last Lab I was busy and couldn’t make it in for long enough to call it an actual day. I moved the lysate from the shaking incubator to the freezer, and left.

Objectives: Complete Plaque Assay on Lysate 3

Rationale: In order to check for the presence of phages, a spot test or plaque assay needs to be performed. Most groups have found a plaque assay to be more effective and valuable time wise.

Procedure:

• Filtered lysate into micropipette tube through a 22 nm filter
• Added 2 mL LB Broth, .5 mL Arthrobacter, 22.5 microliters Calcium Chloride, and 2.5 mL 2X Top Agar to 15mL conical tube
• Added mixture to plate to solidify
• Found that Arthro had condensed and gotten stringy- rather than throwing it out, it was labeled and added to the incubator
• Added 2 mL LB Broth 2.5 mL 2X Top Agar to a 15mL conical tube
• Added 2 mL LB Broth, .5 mL Arthrobacter, 22.5 microliters Calcium Chloride, and 2.5 mL 2X Top Agar to 15mL conical tube
• Arthro dispersed properly this time, so both the control and plaque assay plates sat for 15 minutes, and were placed in the incubator

Analysis and Interpretation: Verify date of arthro before use in the future. Investigate how the arthro turns out next lab period.

Future Plans: Analyze Plaque Assay for the presence of phages, and continue inputting soil metadata into the survey on canvas.

Rationale: Wash newly collected soil in order to get both a direct and enriched sample isolation. In addition, calculate soil metadata.

Process:

1. Wash table and set up aseptic zone
2. Added ~10 mL LB broth to ~2 mL soil
   1. accidentally poured to much and used a bulb pipette to remove ~2 mL LB broth
3. Shook and vortexed for 15 minutes
4. Mass of tube = 20.33 g
5. Centrifuged for 10 minutes
6. Filtered with syringe filter
   1. used ethanol to constantly clean tip
   2. ended with ~7 mL for enriched isolation and ~1 mL for direct isolation
7. Added 0.5 mL arthro to enriched isolation
8. Incubated enriched for 48 hours
9. Stored direct in fridge

Soil Metadata

• % H2O
  • mass of dry soil and weigh boat = 6.66 g
  • mass of wet soil and weigh boat = 6.77 g
  • mass of H2O = 0.11 g
  • mass of wet soil = 4.45 g
  • % H2O = 0.11 g / 4.45 g * 100% = 5.27%
• pH
  • added pinch of dirt to pH tube
  • filled with DI
  • shook for 10 seconds
  • held pH paper in for 45 seconds
  • pH = 5.5
• sand/silt/clay
  • total ≈ 4 mL
  • sand ≈ 2 mL
  • silt ≈ 1 mL
  • clay ≈ 1 mL
  • % sand = 2 mL / 4 mL * 100% = 50%
  • % silt = 1 mL / 4 mL * 100% = 25%
  • % clay = 1 mL / 4 mL * 100% = 25%

sand/silt/clay dispersion in falcon tube

Next steps: Perform spot test to check for any plaques.

Rationale:

The goal in the lab today was to analyze the results of the plaque assay performed on 10/01/18 and perform metadata experiments. If plaques are present, a serial dilution would be performed to get a high titer of phage. If the results were negative, the new soil sample will be cleaned and enriched for future plaque assays.

Results From 10/01/18:

• Plaque assay from 10/01/18 was negative, with contamination on the group control plate.
• The LB broth used for the plaque assay was contaminated again as well.

Materials:

• 10-mL LB broth
• DI water
• .22 micron syringe filter
• Conical Vials
• 0.5-mL Arthrobacter

Procedure for Enrichment:

• Established aseptic zone.
• Added soil up to 2-mL mark of conical vial and filled with LB broth to 12-mL mark.
• Vortex soil and broth mixture for approximately 15 minutes.
• Once vortex was completed, conical vial was weighed and DI water was added to balance out for centrifuge.
• Soil was centrifuged on table top centrifuge for 10 minutes.
• Soil was removed after 10 minutes and lysate was transferred to a new conical vial to separate from pellet.
• Filtered separated lysate through 0.22 micron syringe filter. Repeated until all lysate was filtered into a conical vial.
• After filtration, 0.5-mL of Arthrobacter was added to lysate to create an enrichment.
• Lysate was left to grow in shake incubator.

Procedure for pH and Soil Dispersion:

• Soil was added to the 4-mL line of a falcon tube and DI water was added to the 12-mL line.
• 3 drops of soil dispersion liquid was added to the soil and the tube was shaken for approximately 30 seconds.
• Soil was then put on a rack to settle for 48 hours.
• For pH testing, a small amount of soil was added to a vial and then filled with DI water.
• Soil was then shaken for 10 seconds and left to rest for 2 minutes.
• pH paper was inserted into the soil for 45 seconds and removed for examination.

Results/Observations:

• Lysate at the end of enrichment contained no particles, no apparent signs of error during procedure. Also LB broth used was clear and was kept in aseptic zone with caution to prevent any possible contamination.
• pH paper was removed and kept a yellow color relatively similar to the original just slightly dimmer.
• 
• Soil seemed to contain a lot of sand resting at the bottom right after the initial shaking. Could possibly even out after the settling for 48 hours.

Conclusions:

• From the metadata taken, the pH of the soil seems to be slightly acidic with a pH of around 5.5. This is the first soil sample taken that has had any pH below 7.0. Could possibly provide a more optimal environment for Arthobacter and phage, seeing as the previous basic samples have yielded no phage.

Next Steps:

• The next steps for this experiment are to perform plaque assays with the new enrichment to test for the presence of phage. Also additional metadata experiments need to be performed to gather water percentage and soil composition.

Title: Soil Collection and Washing

Date: 3 October 2018

Rationale: The previous dilutions all failed, therefore the conclusion was made that there was not a phage present in the previous sample. A new soil was collected to restart the phage discovery process.

Procedure: New soil was collected from a tree on the North side of Teal Residential hall. The tree was closer to the building, on the other side of the sidewalk. After soil was collected, it was washed and enriched.

Aseptic zone created by washing lab bench with CiDecon and Ethanol, and an Ethanol heat lamp was lit.

• 2 mL of fresh soil sample added to 15 mL conical vial
• LB Broth added to ~12 mL mark
• Vortex-ed for 15 minutes
• Centrifuged for 15 minutes at 3000 G
• Supernatant decanted into top-filter and vacuum filtered to yield ~9.5 mL direct lysate
• 0.5 mL Arthrobacter added to lysate to enrich
• Placed into shake incubator to complete enrichment process

Conclusions: The selected tree appeared slightly wilted with various dead branches, perhaps from a disease caused by a bacteriophage. The tree fit the criteria for the research question as it was likely planted by humans, not naturally grown like the large Oaks across campus. The metadata for the soil will be taken in the following lab session and the lysate tested for phage presence.