Rationale:

The purpose of todays lab was to analyze the results of the plaque assays performed prior. If plaques were present, dilutions and plaque assays would be performed from picked plaques. If no plaques were found, final soil gathering and enrichment were to take place.

Plaque Assay Results from 10/08/18:

• Plate was littered with plaques.
• Top Agar control was uncontaminated, meaning there is a high possibility this is not some other organism such as in previous trials.
• 

  Group Control Plate

  

Materials:

• Micropipette to pick plaques
• Phage Buffer for Dilutions
• 2X TA
•  Arthobacter 
• LB Broth
• Calcium Chloride
• Enriched Lysate

Procedure:

1. Established an aseptic zone
2. Began by circling 6 plaques to pick.
3. To pick a plaque, used a micropipette tip to poke the top agar and lifted out (avoid bacterial lawn beneath) and inserted tip into 100-μL of phage buffer. Repeated for each plaque in separate containers. This was the 10^0 dilution
4. Chose the first plaque picked for dilutions and added 10-μL of the 10^0 dilution to 90-μL of phage buffer, making a 10^-1 dilution.
5. Made a 10^-2 dilution by repeating the dilution process with the 10^-1 dilution and adding it to 90-μL of phage buffer.
6. Once diluted, 3 separate plaque assays were run with each dilution.
7. 2.0-mL of LB broth was added into 3 separate conical vials.
8. Next, 22.5-μL of calcium chloride was added to the conical vials.
9. 10-μL of each of the dilutions was combined with 3 separate 0.5 mL quantities of Arthrobacter and left to infect.
10. After about 15 minutes, the diluted lysates were combined with their LB Broth mixtures and 2.5-mL of 2X TA was added to the broths.
11. Swirled and plated top agars immediately.
12. Once solidified, they were added to the incubator until the next lab.

Data:

• Plaque morphology was relatively difficult to gather. The plaques were very small, seemingly circular, and some had more jagged edges than the others. For the most part they were relatively the same size except for a few outliers.
• 
• Plaque assays were performed with no complications.

Conclusions:

• There is a clear indicator of phage presence, but it is impossible to determine which type and how much are present.
• The soil sample gathered is phage positive, being the first soil sample gathered to test positive.

Next Steps:

The next steps are to check the plaque assay results and determine the titer of phage. From this, amplification and purification will be needed to single out a specific phage from the possible multiple phages that could be present.

Rationale:

The purpose of todays lab was to run a plaque assay on the newly enriched soil sample gathered previously in hopes of finding any evidence of phage presence. It was discovered that this year the Arthrobacter cultures we have been using may not have even been Arthrobacter, so for this lab we used a culture grown and kept from 2017.

Materials:

• 2.5-mL 2X TA
• 500-μL Arthobacter 
• 2.0-mL LB Broth
• 22.5-μL Calcium Chloride
• 10-μL Enriched Lysate

Procedure:

1. Established aseptic zone.
2. First began by checking soil composition results.
3. Once soil composition was recorded, began plaque assay procedure by filtering enriched lysate through 0.22 micron filter.
4. After filtration, added 2.0-mL LB Broth into a conical vial.
5. Aliquoted 22.5-μL of calcium chloride into the LB Broth.
6. Combined 10-μL of lysate with the 500-μL of Arthrobacter and left to infect.
7. Plates had to be warmed for approximately 25-30 minutes.
8. Once plates were obtained, combined lysate with the broth mixture.
9. Next added 2.5-mL of 2X TA to broth mixture and plated immediately.
10. Once solidified, plate was inserted into the incubator until next lab.
11. After incubation, grabbed a weighing plate and recorded the mass.
12. Added some of the new soil sample, recorded the mass, then left out for water to evaporate until next lab.

Results/Data:

• The soil composition had approximately 1.8-mL of sand, 1.2-mL of silt, and 0.5-mL of clay. It also had an extremely large amount of organic material floating around on the top that had to be removed. This makes a percent composition of 51.4% sand, 34.3% silt, and 14.3% clay.

• 

  Soil Composition of Sample 4 10/08/18

• The mass of the wet soil was 1.93-grams. This was gathered after subtracting the mass of the weigh plate (1.96-grams) from the total mass of the weigh plate and soil (3.89-grams).
• Plaque Assay went very smoothly and extra precautionary steps were taken to ensure an aseptic zone was maintained.

Conclusions:

• Based off the percent compositions and the soil composition chart below, the soil gathered is sandy loam. This soil is dominated by sand particles, but has enough silt and clay to allow some structure and fertility. This would make sense as the soil was sandy enough, yet was able to retain its shape and had a fair amount of water present in the soil.
• 
• Large amount of organic material present in the soil composition was due to the amount of grass that was gathered in the process of digging the soil sample as grasses were everywhere.

Next Steps:

• Analyze plaque assay results to determine presence of phage. If plaques are present, they will be picked and diluted to run plaque assays to begin purification and amplification of phage. If no plaques are present, one last soil sample will be gathered.

10/11/18

Objective:

• To extract lysate from soil sample C.
• To collect soil metadata on soil sample C

Pre-Lab Observations:

The control plate was not contaminated. there were no plaques on the plaque assay form 10/09/18. It seems that there were no phages in soil sample B and therefore a new sample will be analyzed.

Procedure:

1.  An Aseptic zone was set up.

Washing and Enrichment:

1. Using a serological pipette,  10ml of LB broth to a 15 ml conical vial containing 2 ml of soil sample C.
2. The vial was then shaken and vortexed for 15 minutes.
3. after  15 minutes, the vial was weighed and another vial of water with a similar mass ( within 0.01) was prepared and they were both centrifuged for 10 minutes.
4. using a sterile syringe and a syringe tip filter (22 microns), 10 ml of filtered lysate to a conical vial.
5.  0.5 ml of arthrobacter was added to the vial and labeled as enriched sample
6. the tube was placed in the shaking incubator for 48 hours
7.  rest of the lysate was filtered into another conical vial and labeled as direct isolation.
8. store direct isolate in the fridge

% Water

1. weighing boat was weighed on the scale ( g)
2. some soil was added onto the boat and weighed
3. the difference of the final weight ( boat and soil ) and initial weight (boat) was taken to find the weight of the soil.
4. the soil was placed in the fume hood for 48 hours, after which it will be weighed again.

Sand, Silt, Clay

1. 10 ml of soil sample was transferred to a falcon tube.
2. 20 ml of DI water was added to the tube along with soil dispersion liquid
3. the vial was shaken for 30 seconds and allowed to rest for 48 hours.

pH

1. a small amount of soil was added to the pH vial
2.  water was added to the vial until the level reached the top of the vial
3. the vial was shaken for 10 seconds and then allowed to rest for 120 seconds
4.  after 120 seconds, the pH paper was immersed in the vial for 45 seconds
5. the pH was determine using the color scale on the chart in the pH paper dispenser.

Analysis and Conclusion

due to the lack of legitimate plaques in the past few plaques, it is probable that phages were not present in soil sample B and it was time to move on. soil sample C was collected from a tree in close proximity from soil sample B, the possibility of correlation of presence of phages to the proximity of soil where phages are absent is logically nonexistent, this sample may lead to reveal a possibility if phages are absent, leading to another possible question to answer in conjecture to this research currently being performed.