Title: Soils Metadata and Plaque Testing

Date: 8 October 2018

Rationale: In the previous lab session, the soil was washed, enriched and the lysate incubated. The metadata needs to be taken and the initial plaque tests need to be run.

Procedure:

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

• Soil Metadata taken
  • % Water: 14.381g of dirt massed in weigh boat and set to evaporate under fume hood for 48 hours
  • % Sand, Silt, Clay: ~4mL dirt added to falcon tube, DI water added to 12 mL mark. 3 drops Soil Dispersion Liquid added, and shaken for 30 seconds. Tube placed in fume hood to disperse.
  • pH: small amount of soil placed in pH vial. DI water added to rest of vial. The tube was shaken for 10 seconds and a pH strip used to test the pH. The found pH was 6.0
• Incubated lysate retrieved from shake incubator and filtered through 0.2 micron filter to prepare a filtered lysate for a spot test and plaque assay.

The following recipe was used to make a spot test:

• 2 mL LB Broth
• 2.5 mL 2X Top Agar
• 22.5 microliters CaCl2
• 0.5 mL Arthrobacter

The mixture was plated and left to solidify for 15 minutes. 10 microliters of lysate was then spotted into a designated section of the plate along with a Phage Buffer negative control. The plate was then put unto the incubator for 48 hours.

The following recipe was used to make 1 plaque assay:

• 2 mL LB Broth
• 2.5 mL 2X Top Agar
• 22.5 microliters CaCl2

~4.5 mL added to 0.5 mL tube containing Arthrobacter + 10 microliters of filtered lysate. The plate was left to cool for 15 minutes and then incubated for 48 hours.

Conclusions: This soil sample constitutes the last attempt at finding a phage. The plaque assay and spot test can collectively indicate the presence of a phage that, if positive, will be picked in a following lab.

Title: Plaque Dilution

Date: 1 October 2018

Rationale: Possible plaques were found on both plaque assays (with split top agar). A suspected plaque was picked and diluted to be passaged and purified.

Procedure: Aseptic zone created by washing bench with CiDecon and Ethanol, and lighting an ethanol flame.

• Previous plaque assay picked for a 10^0 lysate and placed into 100 microliters phage buffer. 10 microliters of the 10^0 lysate was transferred to another tube with 90 microliters phage buffer to create a 10^-1 lysate.
• The above process was repeated to create a 10^-2 lysate, generating 3 different lysates for a plaque assay.

The following recipe was used for 10 plaque assays (9 + 1 Top Agar control)

• 20 mL LB Broth
• 225 microliters CaCl2
• 25 mL 2X Top Agar

~ 4.5 mL pipetted into tube containing 0.5 mL Arthrobacter culture + 10 micro liters diluted lysate (one plaque assay for each dilution), cooled for 15 minutes, and incubated.

Conclusions: The suspected plaques are not confirmed, and if the dilutions fail, then new soil will be collected. If the plaques pass, then the passaging sequence will continue to purify the phage.

Title: Plaque Assay for Original Plaque

Date: 24 September 2018

Rationale: The previous 10^0 plaque assays failed, so 2 more plaque assays of 10^0 will be done to determine a phage presence that must pas to continue passaging. Otherwise, soil must be collected again.

Procedure: An aseptic zone was created by washing the lab bench with CiDecon, Ethanol, and lighting a lamp.

– The original plaque assay (9/19/18) was picked to create a new 10^0 lysate for plating. This test will determine if the soil is still viable for passaging. After the new 10^0 was picked, the following recipe was used to make 9 plaque assays (8 PA, 1 Top Agar Control):

• 18 mL LB Broth
• 22.5 mL 2x TA
• 202.5 microliters CaCl2

~ 4.5 mL pipetted into vial containing 0.5 mL Arthrobacter + 10 microliters 10^0 lysate. The vial was then plated, cooled for 10-15 min, and incubated. A Top Agar control was also plated and incubated

Conclusions: If the 2 plaque assays do not grow, soil will be collected again to re-passage a collected phage. If the plates pass, the dilution and passaging process will resume for the present phage to eventually isolate.

Title: New Soil Enrichment + Metadata

Date: September 10, 2018

Rationale: The new soil collected in the previous week is to be washed and enriched for further testing to make progress on our research question

Procedure: Aseptic zone created by the following procedure:

• Counter washed and wiped with CiDecon
• Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
• Ethanol lamp lit to create rising heat and a current that protests samples from falling contamination.

1. ~2mL of soil was added to a 15mL vial
2. ~10mL of LB Broth was added to bring the vial up to the 12mL mark
3. Vial was shaken for 10 minutes

Meanwhile, soil metadata was taken

% Water: An empty weigh boat was massed at 2.39 grams, soil was added to bring the mass of the weigh boat to 7.76 grams. The weigh boat was left to evaporate for 48 hours. The mass of the dry dirt will then be used to calculate the % water of the soil.

Sand, Silt, Clay: ~4mL of dirt added to Falcon tube, DI water was added to cover the dirt, and soil dispersion liquid was added to separate the soil. The entire mixture was shaken for 30 seconds and left to disperse over a 48-hour period.

pH: A small amount of dirt was added to a pH testing vial and was filled the rest of the way with DI water. The mixture was shaken for 10 seconds and the pH was recorded on a pH testing strip. The recorded pH was 6.0

A 22 micrometer Top filter was then used to generate ~7.5 mL of lysate. 0.5mL of Arthrobacter was added to enrich the lysate and incubated at 28 degrees Celsius for 48 hours.

Cooper Johnson

Title: Soil Collection 2

Date: September 5, 2018

Rationale: With a new group and a refined question, new soil was collected to begin the process for discovering a phage to answer the proposed question.

Materials: 15-mL vial, bag

Question: Is there a difference in Arthrobacterphage in natural, live oak trees at Baylor versus planted trees in developed areas (surrounded by concrete, etc.)?

Procedure: Groups 3 and 4 worked together to formulate the new question and then split up to acquire the soil. Group 3 sampled dirt from natural, live oak trees while Group 4 sampled from various planted trees. My selected tree was on the northern side of Teal Residential College. It is the second tree from the corner and has a “no parking” sign near it. I observed the tree to make sure it would make for a fitting sample, dug approximately 5 inches into the dirt, and collected the sample for future washing. I then picked leaves from the tree for identification and further analyzation.

Observations/Conclusions: The tree appeared to be healthy and appropriately aged; no anomalies stood out that would discount the tree or soil from being a suitable sample. The patch of dirt that the tree was rooted in was surrounded by a concrete sidewalk. There were occasional patches of brown leaves, but nothing significant. However, browning leaves were found almost exclusively on the street side at the bottom of the canopy. The dirt will be washed and tested for a phage next week. Attached are pictures of the tree and its surroundings.

Previous Results- The direct plaque assay returned negative. The spots pictured were determined to be a top agar split, and not a plaque. The newly collected dirt will be tested for a new phage on Monday.

Date: August 29, 2018

Title: Plaque Assay for Phage Isolation

Rationale: Regardless of spot test result, a plaque assay is done to obtain another measure of phage presence.

Materials: Arthrobacter, LB Broth, 2X Top Agar, 1M CaCl₂, Enriched lysate, pipet, micropipette, plate, vials

Procedure: To create an aseptic zone, the following was done:

• Counter washed and wiped with CiDecon
• Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
• Ethanol lamp lit to create rising heat and a current that protests samples from falling contamina

• mL Arthrobacter and 10 microliters of filtered enriched lysate (filtered at spot test)

In 50 mL tube the following was added (for 3 plaque assays and 1 group control):

• 8 mL LB
• 10 mL 2X Top Agar
• 90 microliters CaCl₂

~18 mL of solution

• Once the solution was finished, 4.5 mL was added to the small vial of Arthrobacter + lysate (enriched, filtered) and poured onto the plate. (NOTE: One vial was broken in the transition to the plate. The counter was re-sanitized, and another vial was prepared)

Results/Observations: The plate and the solution were yellowish and was put into the incubator to grow over the weekend. If the result is positive, I would expect to see growth on the plaque assay, and a negative result on the control. A negative result would constitute contamination on the control, or no growth on the plaque assay. Assuming a positive result, the next step would be to begin isolating the discovered phage. (See attached picture of resulting plaque assay plate before incubation)

Plaque Assay (Pre-incubation)

Date: Monday, August 27, 2018

Title: Spot Test for Lysate

Rationale: By conducting a spot test, the phage (if present) will be given an opportunity to grow on a bacterial lawn and establish a plaque for further research.

Materials: Arthrobacter culture, LB Broth, CaCl₂, 2X Top Agar, 50 mL test tubes, micro centrifuge tubes, pipettes, micropipettes, 22-micron filter, plates, phage buffer

Preparation: To create an aseptic zone, the following was done:

• Counter washed and wiped with CiDecon
• Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
• Ethanol lamp lit to create rising heat and a current that protests samples from falling contamina

Procedure:

• Enriched lysate filtered through 22-micron filter into micro centrifuge tube to prepare for spotting.
• With a new 50 mL vial, the following was added:
  • 5 mL ArthrobacterI (pipetted by TA)
  • 5 mL LB Broth
  • 0 mL 2X Top Agar
  • 45 microliters CaCl₂ (via micropipette)

~ 10 mL added to plate to solidify for 15 minutes

• After the plate solidified, 10 micro liters of filtered lysate was spotted onto the section labelled “E” for enriched. 10 microliters of direct lysate were spotted onto the section labelled “D” for direct. There was a third section labelled “C” that is designated as a negative control area. The plate was incubated for 48 hours to discover if a phage was present.

Results/Observations: After 48 hours, the spot test showed a potential phage growth from the direct isolation spot, and was put into a walk-in fridge to further incubate in case of a negative plaque assay. (See pictures)

Potential Plaque from Spot Test

Potential Plaque from Spot Test

Potential Plaque from Spot Test (View from Bottom)

Date: Wednesday, August 22, 2018

Title: Soil Washing and Lysate Preparation

Rationale: By washing the collected soil and preparing two different kinds of lysate from a centrifuged supernatant, the process of isolating and discovering a phage will begin.

Materials Used: CiDecon, 70% EtOH, 50 mL test tubes, 15 mL test tubes, top filter, LB Broth, Arthrobacter culture, centrifuge

Preparation: To create an aseptic zone, the following was done:

• Counter washed and wiped with CiDecon
• Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
• Ethanol lamp lit to create rising heat and a current that protests samples from falling contamination.

Procedure: With the soil that was collected from an oak tree, the following was added to a test tube:

• LB Broth (poured to 35mL mark on tube) (Broth creates a favorable environment for bacteriophages to grow in)

Then, the tube was shaken for 15 minutes

Observation – The shaken tube was much lighter with the addition of LB Broth, turning the dark brown dirt to a lighter, coffee color uniform throughout the whole tube.

The tube was then inserted into a centrifuge and spun for 15 minutes to separate the supernatant and the solid soil.

After the tube was centrifuged for 15 minutes, the supernatant and the soil were separated clearly, with a dark solid at the bottom of the tube and a yellow supernatant filling the rest of the tube.

The newly acquired supernatant was then poured through a 22-micrometer top filter to filter out any bacteria (phages are just barely small enough to pass through.)

The supernatant was poured into the top of the filter and turned on via a vacuum to activate the filter.

The filtered supernatant, now lysate, was split into 2 different isolations

• A direct isolation, where pure lysate was poured into a 15 mL tube and refrigerated until Monday
• An enriched isolation, where lysate and 0.5 mL of Arthrobacter ATCC 21022 were combined and incubated until the following Monday

Conclusion/Observations: With two different isolations, different experiments and tests can be run to try and discover a phage. In the case of no growth, the process can be repeated until a phage is found. Within the enriched lysate, Arthrobacter, LB Broth, and the supernatant are all in one suspension, providing a favorable environment for growth and, hopefully, infection.