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

Process:

1. Wash table and set up aseptic zone
2. Added ~10 mL LB broth to ~2 mL soil
3. Shook and vortexed for 15 minutes
4. Centrifuged for 10 minutes
5. 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
6. Added 0.5 mL arthro to enriched isolation
7. Incubated enriched for 48 hours
8. 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 = 6.0
• sand/silt/clay
  • added to falcon tube:
    • 4 mL soil
    • 8 mL  deionized water
    • 3 drops dispersion oil
  • shook for 45 seconds
  • let sit under hood for 48 hours

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

Rationale: The spot test from the previous class showed no plaques, so group 6 collectively decided to make one last push in soil collection.

Spot test from 10/8/2018 that did not have any plaques

Control plate from 10/8/2018 that shows the agar used was not contaminated

Process:

• Dug dirt from under a red oak tree off campus, approximately 2 feet away from the trunk
• Recorded tree measurements in survey
  • trunk circumference
  • avg crown width
  • tree height
  • tree conditions
  • tree location

Observations:

• had to dig past a lot of mulch to get to actual tree soil

Next Steps:

Wash soil to get direct and enriched isolations. In addition, start collecting soil metadata (%H2O, pH, and sand/silt/clay tests).

Rationale: Complete spot test with new sample isolations to check for plaques.

Process:

1. Set up aseptic system
2. Made LB agar media for 1 control plate and 2 sample plates

   1. Reagents	Control	Sample
      LB Broth	2 mL	4 mL
      2x TA	2.5 mL	5 mL
      CaCl2	23 µL	45 µL
      arthro	NA	1 mL

3. poured 5 mL of LB agar media into each plate and let sit ~5 min
   1. plates must have been too cold because the agar kept sliding around
4. filtered enriches sample with syringe filter, keeping tip clean with ethanol
5. pipetted 5 µL of direct, enriched, and phage buffer (negative control) into designated areas of sample plate and let sit ~10 min
6. incubated for 48 hours
   1. did not invert because of the slippery agar

Next steps: If the spot test is positive, perform a plaque assay to check for plaques again. If the spot test, is negative, dig more dirt!

Rationale: Plates from last week were plaque-less yet again, so we decided to find new soil from another red oak.

Spot test from 9/26/2018. No plaques found.

Process:

• Dug dirt from under a red oak tree, approximately 2 feet away from the trunk
• Recorded tree measurements in survey
  • trunk circumference
  • avg crown width
  • tree height
  • tree conditions
  • tree location
• In the lab, started soil metadata tests
  • % H2O
    • mass of weigh boat = 2.32 g
    • mass of wet soil = 4.45 g
  • sand/silt/clay
    • filled falcon tube with ~4 mL soil
    • added DI to 12 mL mark
    • added 3 drops soil dispersion
    • shook for 30 seconds
    • let sit for 48 hours

Next Steps:

Wash soil to get enriched and direct sample isolations.  Measure pH of soil and calculate the % H2O and sand/silt/clay.

Rationale: the arthro from the last class was probably contaminated because the positive controls did not yield any plaques. The results from the previous spot test are inconclusive so the experiment had to be repeated

Invalid spot test results from previous class

Process:

1. Set up aseptic system
2. Made LB agar media for 1 control plate and 1 sample plate
   1. We only had 400 µL of arthro available so 5 samples were spotted on the sample plate

   2. Reagents	Control	Sample
      LB Broth	2.1 mL	4.2 mL
      2x TA	2.5 mL	5 mL
      CaCl2	23 µL	45 µL
      arthro	NA	400 µL

3. poured 5 mL of LB agar media into each plate and let sit ~5 min
4. pipetted 5 µL of direct (RSM), direct (LCG), enriched (RSM), enriched (LCG), and phage buffer (negative control) into designated areas of sample plate and let sit ~10 min
5. incubated for 48 hours

Next steps: If the spot test is positive, perform a plaque assay to check for plaques again. If the spot test, is negative, dig more dirt!

Rationale: Perform spot test with new direct and enriched isolations to check for the presence of phages

Process:

1. set up aseptic system
2. made LB agar broth for sample plates and a control plate

   1. Reagents	Control	Sample
      LB Broth	2 mL	4 mL
      2x TA	2.5 mL	5 mL
      CaCl2	23 µL	45 µL
      arthro	NA	1 mL

3. poured 5 mL of LB agar broth into each plate and let sit for ~10 min
4. used syringe filter to filter enriched sample
5. pipetted 5 µL of direct isolation, enriched isolation, and phage buffer (negative control) into designated areas of sample plate and let sit for ~10 min
6. put plates in incubator for 48 hours

soil metadata:

1. % H2O:
   1. mass of water = 0.49 g
   2. mass of wet soil = 6.01 g
   3. 0.49 g / 6.01 g * 100% = 8.15% H2O
2. sand/silt/clay
   1. total volume = 2.75 mL appr.
   2. sand = 1 mL appr.
   3. silt = 1 mL appr.
   4. clay = 0.75 mL apprx.

1 / 2.75 * 100% = 36.4% sand

1 / 2.75 * 100% = 36.4% silt

0.75 / 2.75 * 100% = 27.2% sand

soil metadata: sand, silt, and clay layers

Next steps: If the spot test is positive, perform a plaques assay with enriched isolation to check for plaques again. If the spot test is negative, look for a new soil sample outside.

Rationale: wash soil samples and produce a direct isolated sample and an enriched isolated sample.

Process:

Soil Washing

1. Cleaned lab bench with CiDecon and 70% ethanol
2. Using aseptic techniques, added LB broth to the 15 mL mark of the tube containing ~ 2 mL of soil
3. shook and vortexed the tube for 10 minutes
4. Added small amount of water to get a final weight of 19.21g
5. Centrifuged the tube with classmates
6. Used a syringe and filter to filter supernatant into fresh tubes
   1. Used ethanol to keep the filter tip clean
   2. Ended with ~9 mL for enriched and ~1 mL for direct samples
7. Added 0.5 mL of artho to the enriched sample
8. Stored it in the fridge
9. Left enriched isolated sample in the shaking incubator for 48 hours

Soil Metadata

1. mass of dry soil and sample = 7.89g
2. measured pH of soil
   1. added a pinch of dirt and DI to tube
   2. shook for 45 seconds
   3. held pH paper in tube for 45 seconds
   4. pH = 6.0
3. sand/silt/clay soil dispersion
   1. Added ~8mL DI to ~4mL soil
   2. Added 3 drops of soil dispersion liquid
   3. shook for 30 seconds
   4. let sit for 48 hours

Next Steps:

Perform spot test with enriched and direct isolations. Measure sand/silt/clay dispersion and calculate % H2O.

Rationale: perform spot test on direct and enriched isolations from the previous lab to check for presence of phages

Steps:

1. labeled plate with spots for enriched, direct, and negative control (phage buffer)
2. found enriched sample mass = 21.22g
3. centrifuged samples as a class
4. made LB agar for mine and Lilly’s plates, and a control plate

   1. Reagents	control plate	sample plates
      Arthro	NA	0.5 mL
      LB Broth	~2 mL	4 mL
      2x TA	2.5 mL	5 mL
      1M CaCl2	23 µL	45 µL

5. pipetted both solutions to mix
6. pipetted 5 mL control agar onto control plate
7. pipetted 5 mL of the arthro agar onto mine and Lily’s plates
8. Let plates sit for 10 minutes
9. Used a syringe and filter to filter enriched isolation into a new 15 mL conical tube
   1. filter had very little resistance – I tried two different filters and nothing changed
10. pipetted 5 µL of the enriched and direct isolations as well as the negative control onto the pre-marked places on the plates
11. let plates sit for 15 minutes
12. put plates into the incubator (NOT inverted)

Soil Metadata Note:

weight of dry sample and weigh boat = 6.31g

the soil level experiment that I had put into two different tubes had to be redone because it did not show anything. To improve the experiment I didn’t pour out the supernatant because that part ended up having a substantial amount of soil that shouldn’t have been separated from the rest of the sample.

Next Steps:

Check plates for sign of phages.

Perform plaque assay with enriched sample.

analyze new metadata results

Rationale: wash a new soil sample to get new direct and enriched isolations. In addition, prepare the soil for analysis to get soil metadata.

Soil Washing:

1. Filled 15 mL conical tube with ~2mL of soil
2. Added LB the 12 mL mark of the tube
3. Started 10 minutes of shaking and vortex-ing
   1. the tube started leaking so I tried to switch the mixture into another tube but then I switched it back when not all of the soil came out of the original tube. I ended up losing ~1mL of my LB and soil mixture
4. After shaking I weighed the tube and got 19.83 g
5. The tubes were centrifuged as a class
6. I used a syringe and filter to filter the supernatant of the centrifuges mixture into a new 15 mL tube
   1. I used aseptic technique, and consistently washed the tip of the filter with 70% ethanol
   2. I lost a lot of solution to the table trying to get the last of the supernatant into the syringe
7. I ended up getting ~7 mL for my enriched isolation and ~0.75 mL for my direct isolation, both in 15 mL conical tubes
8. I added 0.5 mL of arthro to my enriched isolation and left it to incubate for 48 hours at 28 °C

Soil Metadata:

1. Filled a falcon tube with ~4 mL soil
2. Added DI water to the 12 mL mark of the tube
3. Added 3 drops of soil dispersion
4. shook tube with glove over the opening for 30 seconds
5. poured supernatant into 50 mL conical tube and let both tubes sit under the hood for 48 hours
6. measured the mass of wet soil sample in a weigh boat
   1. mass of weigh boat = 2.37g
   2. mas of wet soil = 4.62 g
7. Took the pH of soil sample
   1. added pinch of soil to pH tube
   2. filled tube with DI
   3. shook for 10 seconds
   4. let sit for 2 min
   5. put 1 in long pH paper into tube for 45 sec
      1. dropped first paper into sample so had to use a new one
   6. pH = 6.5

Next Steps: Perform a spot test to check for phages and continue analyzing soil metadata.