Rationale: The purpose of this lab is to flood a webbed plate to obtain more lysate for future calculations.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
2. 5 ml of phage buffer was pipetted onto the webbed plate. The plate was shaken for one hour on the shaker.
3. The lysate was harvested from the plate with a syringe filter and filtered with a 0.22 um syringe filter into the lysate obtained from the last lab.
4. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• 5 ml of phage buffer was used to flood
•  3 ml of lysate was collected from the webbed plate, making a total of 6 ml collected.

Interpretations/Next Steps:

The procedure was complete. The next step will be to perform serial dilutions to calculate the titer of the lysate.

Control and Webbed Plate

Rationale: The purpose of this lab is to flood a plate to obtain more lysate to create a webbed plate.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
2. 5 ml of phage buffer was poured onto the plate with plaques.
3. The plate was shaken on an incubator for one hour.
4. While shaking, 4 ml of LB Broth and 45 ul of CaCl2 were added to a tube, to create top agar for two plates.
5. The lysate was harvested from the plate with a syringe and filtered with a 0.22 um syringe filter into a tube. 3 ml of lysate were obtained.
6. 10 ul of the lysate was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
7. 5 ml of 2 x TA were added to the top agar solution and pippetted up and down. 4.5 ml of the solution was added to the 0.5 ml of arthrobacter and poured onto a plate labeled LIP 11-5-18 10 ul titer test. The rest of the top agar solution was poured onto a plate labeled LIP 11-5-18 TA Control.
8. The plates were allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
9. The workstation was cleaned using aspetic technique and materials were properly stored or disposed of.

Observations:

• Plate from last lab was not completely webbed.
• Bubbles on the plates.

Interpretations/Next Steps:
The procedure was complete. The next step will be to calculate the titer and make a webbed plate.

Control, 14 ul Lysate Plate, 15 ul Lysate Plate

Rationale: The purpose of this lab is to calculate the amount of lysate needed to create a webbed plate and run a plaque assay.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
2. The plaques on the 10^-1 dilution plaque assay were counted and 92 were found.
3. The diameter of the plate was measured and found to be 86mm, and the radius was 43mm. The diameter of 10 plaques was found and the average diameter was calculated to be 2.4mm.
4. Next, the titer of the plate was found to be 9.2 x 10^4
5. The area of the plate was calculated to be 1849 pi, and the area of the plaque was found to be 1.44 pi.
6. Next, the area of the plate was divided by the area of a plaque to calculate how many plaques would be needed to web the plate. This calculation determined that 1284.03 plaques would be needed.
7. The amount of lysate needed to web a plate was then calculated by dividing the number of plaques needed by the titer, and changing the units to ul. It was determined that 14.01 ul of lysate would be needed to web the plate.
8. 14.01 ul of the lysate was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
9. Top agar solution was made for three plates. 6 ml of LB broth and 67.5 ul of CaCl2 were added to a tube.
10. 7.5 ml of 2x TA was added to the tube. 4.5 ml of top agar solution was then added to the 0.5 ml of arthrobacter and poured onto a plate labeled LIP 14.01 ul PA 10-31-18. The rest of the top agar was poured onto a plate labeled LIP LJF 10-31-18 Control.
11. The plates were then allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
12. The workstation was cleaned using aseptic technique and materials were properly stored or disposed of.

Observations/Results:

• Observations:
  • Bubbles formed on the plates
• Results:
  • Titer of lysate: 9.2 x 10^4
  • Average area of plaques: 1.44 pi
  • Area of plate:  1849 pi
  • Lysate needed to web a plate: 14.01 ul

Interpretations/Next Steps:
The procedure was complete. The next step will be to flood the webbed plate to obtain more lysate.

Rationale: The purpose of this lab is to perform serial dilutions.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
2. 90 ul of phage buffer was added to four tubes.
3. 10 ul of phage buffer lysate from the picked plaque was added to to the 10^-1 tube. 10 ul of the 10^-1 buffer was then added to the 10^-2 tube. This was done out to a dilution of 10^-4.
4. 10 ul of each tube was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
5. 10 ml of LB broth and 112.5 ul of CaCl2 were added to a tube. 12.5 ml of 2x TA were added to the tube.
6. 4.5 ml of the top agar solution was added to the 0.5 ml of arthrobacter and poured onto plates labeled LIP 10-29-18 PA 10^-1, LIP 10-29-18 PA 10^-2, LIP 10-29-18 PA 10^-3, and LIP 10-19-18 PA 10^-4. The rest of the top agar solution was poured onto a plate labeled LIP 10-29-18 control.
7. The plates were allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
8. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• Bottom top agar had not set correctly on some plates.
• Bubbles seen on plates.

Interpretations/Next Steps:
The procedure was complete. The next step will be to make a webbed plate.

Rationale: After a negative plaque assay from the flooded lysate, the purpose of this lab is to retest the flooded lysate and pick a plaque for purification. This lab will also retest enriched soil C lysate for further study.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an aseptic zone was made with an ethanol burner.
2. 100 ul of phage buffer was added to a tube. A plaque was picked and mixed in the buffer. The tube was then vortexed to mix.
3. 10 ul of phage buffer with plaque was added to 0.5 ml of arthrobacter, 100 ul of the flooded lysate was added to 0.5 ml of arthrobacter, and 10 ul of soil C lysate was added to 0.5 ml of arthrobacter, and all were allowed to sit for 10 minutes.
4. 8 ml of LB Broth and 90 ul of CaCl2 were added to a tube to create the top agar solution. 10 ml of 2x TA were added the tube.
5. 4.5 ml of the top agar solution was added to 0.5 ml of arthrobacter for each plate, and the poured directly onto plates labeled LIP 10-24-18 PA SC, LIP 10-24-18 PA-P1, and LIP 10-24-18 PA(FL). The rest of the solution was poured directly onto a plate labeled LIP 10-24-18 Control.
6. The plates were allowed to settle for 10 minutes and then inverted and stored in the incubator until the next lab.
7. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• 100 ul of flooded lysate was added instead of the usual 10 ul.
• Soil C lysate was retested.
• Some bubbles were seen on the plates.

Interpretations/Next Steps:
The procedure was complete. The next step will be to check for plaques on all the plates, and to flood the plate from the picked plaque.

Rationale: After contamination, the purpose of this lab is to sterilize the flooded lysate and to perform a plaque assay.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
2. Top agar solution was made for two plates. 4 ml of LB Broth and 45 ul of CaCl2 were added to a tube.
3. 50 ul of chloroform were added to the flooded lysate to kill any bacterial contamination. The lysate was swirled for 2 minutes.
4. 10 ul of lysate was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
5. 5 ml of 2x TA were added to the top agar solution and pipetted up and down to mix. 4.5 ml of the top agar solution were added to the arthrobacter and poured onto a plate labeled LIP 10-22-18 PA. The rest was poured directly onto a plate labeled LIP 10-22-18 Control.
6. The plates were allowed to settle for 10 minutes and then inverted and stored in the incubator until the next lab.
7. The workstation was cleaned using aseptic technique and materials were properly stored or disposed of.

Observations:

• Chloroform was used instead of a syringe filter to sterilize the lysate.
• No bubbles were seen on plates.

Interpretations/Next Steps:
The procedure was complete. The next step will be to check for plaques. If plaques are found, serial dilutions will be performed. If no plaques are found, a new plaque will need to be picked for purification.

Contaminated Plaque Assay and Control:

Rationale: After contamination, the purpose of this lab is to create a plaque assay from the flooded lysate.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an aseptic zone was created.
2. 10 ul of the flooded lysate was added to 0.5 ml of arthrobacter and was allowed to sit for 10 minutes.
3. Top agar was made for two plates. 4 ml of LB broth and 45 ul of Calcium Chloride were added to a tube. 5 ml of 2x TA was then added and pipetted up and down to mix. 4.5 ml of the top agar solution was added to the 0.5 ml of arthrobacter and poured onto a plate labeled LIP 10-17-10 PA. The rest of the top agar was added to a control plate labeled LIP 10-17-18 control.
4. The plates were allowed to sit for 10 minutes to solidify and then inverted and stored in the incubator until the next lab.
5. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• Small bubbles were seen on both plates, very few.

Interpretations/Next Steps:
The procedure was complete. The next step will be to check for plaques. If plaques are found, then serial dilutions will be performed. If no plaques are found, the purification process will need to be performed again.

Contaminated Plaque Assay and Control:

Rationale: The purpose of this lab is to create a plaque assay from the lysate collected from the flooded plate.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an aseptic zone was created.
2. Top Agar was made for three plates. 6 ml of LB Broth and 67.5 ul of CaCl2 were added to a tube.
3. 10 ul of lysate was added to 0.5 ml of arthrobacter and was allowed to sit for 10 minutes.
4. 7.5 ml of 2x TA was added to the tube and pipetted up and down to mix. 4.5 ml of the top agar solution were added to the arthrobacter and poured onto a plate labeled LIP 10-15-18 Plaque Assay. The rest of the top agar solution was poured directly onto a control plate labeled LIP SS Control 10-15-18.
5. Plates were allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
6. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• Small bubbles were seen on the control plate.

Interpretations/ Next Steps:
The procedure was complete. The next step will be to perform serial dilutions in the next lab.

Rationale: The purpose of this lab is to create a lysate from the flooded webbed plate from soil C.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
2. The top agar and phage buffer were spun for 10 minutes in the centrifuge at 5,000 x g.
3. The supernatant was filtered using a 0.22 um syringe filter and stored in the refrigerator until the next lab.
4. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• Filtration process went smoothly

Interpretations/Next Steps:
The procedure was complete. The next step will be to perform serial dilutions and a plaque assay.