Rationale:

The goal for today was to analyze the results of the previously performed plaque assay and either perform a serial dilution if there were plaques present or perform another plaque assay.

Results of 09/24/18

• Contamination of the group control plate yet again. Plaque assay was also negative.
• It was also discovered that the lack of plaques and contamination could be due to the apparent contamination of the Arthobacter cultures in the lab. There were no plaques present because there was no Arthrobacter present to infect. This meant that a new plaque assay had to be performed with an older culture of Arthobacter to truly determine if the soil sample is negative.
• LB Broth was clearly contaminated, contained a precipitant very similar to the past 2 contaminated plaque assays.
• 

  Contaminated LB Broth

  

  Uncontaminated LB Broth

  

  Contamination

  

  Empty Plaque Assay

   

Materials:

• 2.5-mL 2X TA
• 400-μL Arthobacter (Lab was low on uncontaminated Arthro so we had to lower amount used)
• 2.1-mL LB Broth
• 22.5-μL Calcium Chloride
• Enriched Lysate

Procedure:

• Established aseptic zone.
• Aliquoted 2.1-mL LB broth into a conical vial.
• Added 22.5-μL of calcium chloride to the LB broth.
• After, added 2.5-mL of 2X TA to control top agar and plated immediately. Left to solidify and set in the incubator.
• Combined 10-μL of lysate with the 400-μL of Arthobacter and left to infect for 15 minutes.
• After time had passed, aliquoted 2.5-mL of 2X TA to broth mixture, added infected lysate, and plated immediately.
• Left plate to solidify and put in the incubator until the next lab.

Results/Data:

• Previous plaque assay had been negative, however careful measures were taken to ensure the experiment was performed aseptically. Additional burners were added and almost all equipment was wiped with 70% ethanol before use.
• LB Broth also was examined before use and was completely clear before the experiment. If the broth is contaminated next week, there is contamination elsewhere.
• 

  Uncontaminated LB Broth

   

Conclusions:

• If the plaque assay returns negative with no contamination, we will know that there is no phage present in the soil sample taken. Recently it’s been very difficult to tell due to the influx in contamination across the lab. Still unsure as to what is causing it as extra measures are taken to ensure that the experiment is performed aseptically.
• Arthrobacter was contaminated, so using this guaranteed culture that was grown at the beginning of the year should yield legitimate results.

Next Steps:

• Analyze the plaque assay’s results and either perform a serial dilution and pick a plaque, or find a new soil sample.
• Finally find some phage in the lysate.

Rationale:

The purpose of today’s lab was to check on the metadata experiments that had been performed the week prior (water percentage and soil composition) and also perform a plaque assay with the newly enriched lysate to test for the presence of phage.

Materials:

• 2.0-mL LB Broth
• 2.5-mL 2X TA
• 22.5-μL Calcium Chloride
• 0.5-mL Arthrobacter
• 0.22 micron syringe filter
• Enriched Lysate

Procedure:

1. Established an aseptic zone.
2. Checked soil composition and weighed dry soil to calculate sand, silt, clay makeup of soil and water percentage of soil.
3. After metadata was completed, aliquoted 2.0-mL Lb broth to conical vial.
4. Filtered enriched lysate through 0.22 micron filter.
5. Added 22.5-μL of calcium chloride to LB Broth.
6. Combined 10-μL of filtered lysate with 0.5-mL of Arthobacter and left to infect for approximately 20 minutes.
7. After 20 minutes had passed, added 2.5-mL 2X TA to broth, quickly added lysate mixture, and plated immediately.
8. Let solidify and left to incubate for 48 hours.

Observations/Results/Data:

• The soil composition did not vary as drastically as last samples. It did contain 4 different layers of the soil, but the fourth dark top layer was just additional organic matter that was not poured out.  It roughly had 1.5-mL of sand, 1.0-mL of silt, and 0.5-mL of clay. Making it 50% sand, 33.33% silt, and 16.67% clay.

• 

  Soil Composition 09/24/18 Bottom Layer: Sand Dark Middle Layer: Silt Light Layer: Clay

• LB broth before use was not contaminated, no cloudiness was reported and there was no precipitate in the solution at the time of use.
• The mass of the dry soil was 6.21 grams after being left out all weekend to encourage water evaporation. Therefore making the mass of water 3.91 grams.

Analysis/Conclusions:

• Based off graphic below and the percentages of the soil composition obtained from the experiment, the best estimate for the soil type is Loam. Loam is a fertile soil with a roughly 40-20-20 mineral composition of sand-silt-clay, which is very close to the results gathered in the experiment.
• 
• By using the water percentage equation, the final calculated percentage of water in the soil was 24.80%. This is only 4% more water than the previous soil sample which was gardeners soil. Possibly this increase in water will be beneficial for the presence of phage.
  

  Work for calculating water percentage

   

Next Steps:

• Analyze the plaque assay results for the presence of phage. If there are plaques present, a serial dilution will be performed to get a high titer of phage. If no plaques are present, a second plaque assay will be performed.

Title: Plaque Assay for Original Plaque (Redo)

Date: 26 September 2018

Rationale: Due to an error with the Arthrobacter culture, the previous experiment’s results are rendered invalid. The bacteria used was not Arthrobacter, therefore an Arthrobacterphage couldn’t grow on a bacterial lawn. Therefore the experiment is repeated. The attached pictures show the previous (contaminated/invalid) plaque assays.

Procedure: Aseptic zone created by washing the lab bench with CiDecon and ethanol and a heat lamp was lit.

• The picked 10^0 lysate from the previous experiment was used for 2 more 10^0 plaque assays.

The following recipe was used to make 8 plaque assays (7 PA + 1 Top Agar Control):

• 18.9 mL LB Broth
• 180 microliters CaCl2
• 20 mL 2x TA

~ 4.6 mL pipetted into a vial containing 0.4 mL new Arthrobacter strain + 10 microliters 10^0 lysate. The mixture was plated, cooled for 10-15 minutes, and incubated. \

Conclusions: Much like the previous experiment, both plaque assays must pass in order to continue passaging. If the test fails, soil will be recollected and the washing/purification process will start over.

09/26/18

Objective:

• Make another plaque assay from the extracted phages from the plate from the first serial dilution

Pre-Lab Observations:

• After checking the positive control made by the lab instructors, it was discovered that the arthrobacter culture used on 09/24/18 was not actually arthrobacter.
• So, new plaque assays must be made from the same phage extract used on 09/24/18
• The control plate from the plaque assays prepared on 09/24/18 was not contaminated.

Procedure:

1. Cidecon was poured on the desk and wiped till the desk was dry. Then, 70% ethanol was poured and wiped until it was all over the table and then it was allowed to evaporate. After the ethanol had evaporated, an ethanol lamp was lit, setting up the aseptic zone.
2. 400 μl of arthrobacter was retrieved from the lab instructor
3. Using the micropipette, 10 microliters of the 10^0 bacteriophage mixture was transferred to the arthrobacter vial.
4. The vial was then allowed to rest on the test tube rack for 15 minutes
5. While the vial was resting, one Top Agar mixture was made for the group.
6. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
7. While in the aseptic zone, 8.4 ml of LB broth was transferred to the 50 ml conical vial.
8. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
9.  Using the micropipette, 90 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
10. The vial was then set on the rack.
11. 10 ml of the 2X TA was added to the LB broth and Cacl2 after the sample was allowed to enrich the arthrobacter for 15 minutes.
12. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to the test tube with the arthrobacter and the phage extract.
13. The contents of the test tube were then poured onto the agar plate.
14.  Part of the top agar mixture was poured into the top agar control plate for the group.
15. To let the top agar solidify, the plates were allowed to rest for 15 minutes.
16. The plates were placed upside down in the incubator, where they will remain for 48 hours

Analysis and Conclusion:

The procedures were properly performed in the aseptic zone and the chances on contamination were minimized. the control plate from 09/24/18 was not contaminated, which was a surprising outcome due to the repeated contamination of the control plates in previous spot tests and plaque assays. it may have been the plates that were contaminated when contaminated control plates were a result because more caution was used while picking plates for assays on Monday.