Rationale

10 microliter plate was flooded and had serial dilutions performed up to 10^-8. A spot titer was performed to calculate titer by Monday.

Detailed Procedure

1. Add 5 mL of PB to  10 microliters  plate from previous lab day.
2. The plates was shaken on an incubator for one hour.
3. Filtered lysate from flooded plate in two separate 50 mL 0.22 µm filtered conical vial.
4. 10 µL of 10⁰ was added with 90 µL of phage buffer to create 10^-1 dilution which was vortexed.
5. 10 µL of 10^-1 was added with 90 µL of phage buffer to create 10^-2 dilution which was vortexed.
6. 10 µL of 10^-2 was added with 90 µL of phage buffer to create 10^-3 dilution which was vortexed.
7. 10 µL of 10^-3 was added with 90 µL of phage buffer to create 10^-4 dilution which was vortexed.
8. 10 µL of 10^-4 was added with 90 µL of phage buffer to create 10^-5 dilution which was vortexed.
9. 10 µL of 10^-5 was added with 90 µL of phage buffer to create 10^-6 dilution which was vortexed.
10. 10 µL of 10^-6 was added with 90 µL of phage buffer to create 10^-7 dilution which was vortexed.
11. 10 µL of 10^-7 was added with 90 µL of phage buffer to create 10^-8 dilution which was vortexed.
12. 4 mL of LB Broth, 45 µL of CaCl₂, and 5 mL of 2X TA were combined into a 15 mL vial.
13. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into control plate labeled “SJ Control 11/30/18.”
14. Added 0.5 mL Arthro into remaining 4.5 mL TA mixture and poured onto plate labeled “SJ Spot 11/30/18” which was sectioned off into 8 parts
15. Waited 15 mins for both plates to solidify.
16. Micropipetted 10 µL of 10⁰, 10^-1, 10^-2, 10^-3 ,10^-4 , 10^-5  , 10^-6  , 10^-7  , 10^-8  in according sections of the spot test plate.
17. Rested into incubator

Conclusion

10 microliter plate was completely lysed, which gives hope that a high titer will be achieved by Monday. Only 5 mL of PB was on the plate since the graduated pipette didn’t have up to 10 mL. By next lab day, a new titer will be calculated and hope to achieve a high titer by then.

Rationale

New titer and web calculations were made since titer is not high enough. Made more webbed plates for new calculations for titer.

Detailed Procedures

1. Took 0.5 mL Arthro and added 10 microliters of 10^0. Sat for 15 mins.
2. Took 4 mL of LB Broth, 5 mL 2X TA, and 45 microliters of 1 M CaCl2 into a 15 mL vial.
3. Took 4.5 mL of top agar mixture and poured onto a plate labeled as “control.”
4. Took rest of 4.5 mL top agar mixture and poured into a 15 mL vial labeled as “10 microliters.”
5. Took mixture from step 1 and added to 15 mL vial labeled as “10 microliters.”
6. Took “10 microliter” vial and poured onto “10 microliters” plate.
7. Let both plates solidify for around 15 mins and inverted into incubator.

Results

Lab Day 31 Current titer= 1.1 x 10^8

Lab Day 31 web= 0.2627 microliter of 10^0

Conclusion

Plates from lab day 30 did not have any contamination. Plaque size compare to webbed plates from a while ago seems to get smaller. Since pipettes can’t measure 0.2627 microliters, used 10 microliters instead. Since it is the last day, flooding and serial dilutions cannot be made and continue DNA extraction, TEM, or nanodrop. This phage was from a donor, results will be looked over by the donor and compare results.

plaque size is small, used a microscope and found 11 pfu on 10^-5 spot

newly webbed plates and control

Rationale

5 microliters and 203 microliters plates were flooded and had serial dilutions performed up to 10^-12. A spot titer was performed to prevent making 13 plaque assay plates.

Detailed Procedure

1. Add 8 mL of PB to  203 microliters  plate from previous lab day.
2. Add 5 mL of PB to  5 microliters  plate from previous lab day.
3. The plates was shaken on an incubator for one hour.
4. Filtered lysate from flooded plate in two separate 50 mL 0.22 µm filtered conical vial. Labeled “SJ 11/28/18 FS 2 lysate 10⁰” and “SJ 11/28/18 FS 3 lysate 10⁰.”
5. 10 µL of “SJ 11/28/18 FS 2 lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1 dilution which was vortexed.
6. 10 µL of 10^-1 was added with 90 µL of phage buffer to create 10^-2 dilution which was vortexed.
7. 10 µL of 10^-2 was added with 90 µL of phage buffer to create 10^-3 dilution which was vortexed.
8. 10 µL of 10^-3 was added with 90 µL of phage buffer to create 10^-4 dilution which was vortexed.
9. 10 µL of 10^-4 was added with 90 µL of phage buffer to create 10^-5 dilution which was vortexed.
10. 10 µL of 10^-5 was added with 90 µL of phage buffer to create 10^-6 dilution which was vortexed.
11. 10 µL of 10^-6 was added with 90 µL of phage buffer to create 10^-7 dilution which was vortexed.
12. 10 µL of 10^-7 was added with 90 µL of phage buffer to create 10^-8 dilution which was vortexed.
13. 10 µL of 10^-8 was added with 90 µL of phage buffer to create 10^-9 dilution which was vortexed.
14. 10 µL of 10^-9 was added with 90 µL of phage buffer to create 10^-10 dilution which was vortexed.
15. 10 µL of 10^-10 was added with 90 µL of phage buffer to create 10^-11 dilution which was vortexed.
16. 10 µL of 10^-11 was added with 90 µL of phage buffer to create 10^-12 dilution which was vortexed.
17. 4 mL of LB Broth, 45 µL of CaCl₂, and 5 mL of 2X TA were combined into a 15 mL vial.
18. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into control plate labeled “SJ Control 11/28/18.”
19. Added 0.5 mL Arthro into remaining 4.5 mL TA mixture and poured onto plate labeled “SJ Spot 11/28/18” which was sectioned off into 12 parts
20. Waited 15 mins for both plates to solidify.
21. Micropipetted 10 µL of 10⁰, 10^-1, 10^-2, 10^-3 ,10^-4 , 10^-5  , 10^-6  , 10^-7  , 10^-8  , 10^-9  , 10^-10  , 10^-11   , 10^-12 in according sections of the spot test plate.
22. Rested into incubator

Conclusion/Next Steps

203 microliter plate was completely lysed, which gives hope that a high titer will be achieved by next lab day. 5 microliter plate was nearly fully webbed, but not enough, so only 5 mL of PB was poured onto the plate. Each plate, after an hour, were filtered out in separate vials. Only the 203 microliter plate was used for serial dilutions because the plate was completely lysed. By next lab day, a new titer will be calculated and hope to achieve a high titer by then.

results from lab day 29 (below)

10^-1 through 10^-12 spot titer plate

Rationale

New titer and web calculations were made since plate was not webbed at all. Made more webbed plates for flooding and serial dilutions on next lab day.

Detailed Procedures

1. Took 0.5 mL Arthro and added 203 microliters of 10^0. Sat for 15 mins.
2. Took another 0.5 mL Arthro and added 5 microliters of 10^0. Sat for 15 mins.
3. Took 6 mL of LB Broth, 7.5 mL 2X TA, and 69 microliters of 1 M CaCl2 into a 50 mL vial.
4. Took 4.5 mL of top agar mixture and poured onto a plate labeled as “control.”
5. Took another 4.5 mL top agar mixture and poured into a 15 mL vial labeled as “203 microliters.”
6. Took another 4.5 mL top agar mixture and poured into a different 15 mL vial labeled as “5 microliters.”
7. Took mixture from step 1 and added to 15 mL vial labeled as “203 microliters.”
8. Took mixture from step 2 and added to 15 mL vial labeled as “5 microliters.”
9. Took “203 microliter” vial and poured onto “203 microliters” plate.
10. Took “5 microliter” vial and poured onto “5 microliters” plate.
11. Let all three plate solidify for around 15 mins and inverted into incubator.

Results

Current titer= 1.4 x 10^7

web= 2.02 microliter of 10^0

Made a miscalculation and did 203 microliters of 10^0, so made another plate with 5 microliters of 10^0  instead of 2.02 microliters.

Conclusion/Next Steps

Plates from lab day 28 did not have any contamination. Since 10^-5 lysate was used, the plaque size was very small in size and quantity, that is why a new titer calculation was made from that. 2.02 microliters of 10^0 was small, so I was advised to used 5 microliters instead. Hopefully a webbed plate will be ready by next lab day so that flooding and serial dilutions will take place and get a high titer by Friday.

results from lab day 28

Rationale

Created 10^-5 dilution to make a newly webbed plate in hope of flooding and creating a high titer.

Detailed Procedure

1. 10 µL of “SJ 11/12/18 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1 dilution which was vortexed.
2. 10 µL of 10^-1 was added with 90 µL of phage buffer to create 10^-2 dilution which was vortexed.
3. 10 µL of 10^-2 was added with 90 µL of phage buffer to create 10^-3 dilution which was vortexed.
4. 10 µL of 10^-3 was added with 90 µL of phage buffer to create 10^-4 dilution which was vortexed.
5. 10 µL of 10^-4 was added with 90 µL of phage buffer to create 10^-5 dilution which was vortexed.
6. Took 98 µL of 10^-5 into 0.5 mL arthro. Set for 15 mins to infect.
7. Took 4 mL of LB Broth, 5 mL of 2X TA, and 45 µL of 1M CaCl2 into 15 mL vial.
8. Took 4.5 mL of top agar mixture and poured onto control plate.
9. Took rest of top agar mixture and combined with arthro and 10^-5 mixture.
10. Took resulting solution and poured onto 10^-5 plate.
11. Allowed both plates to sit for 15 mins.

Conclusion/Next Steps

Since the calculations were 97.6 µL of 10^-5 , only 98 µL were pipetted for easier pipetting. By next lab day, hopefully a webbed plate will be able to flood and make new plates to calculate the new titer. By Friday, hopefully a high titer will be calculated.

Rationale

Due to  possible miscalculations from previous titer and webbing, I had to redo the same serial dilution and plate only 10^-3 through 10^-5. From result from previous lab day, no plaque present. From the lab day before that, phage was present in the 10^4 plate. I chose to redo it because new and correct calculations are needed for a new webbed plate.

Detailed Procedure

1. 10 µL of “SJ 11/12/18 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1 dilution which was vortexed.
2. 10 µL of 10^-1 was added with 90 µL of phage buffer to create 10^-2 dilution which was vortexed.
3. 10 µL of 10^-2 was added with 90 µL of phage buffer to create 10^-3 dilution which was vortexed.
4. 10 µL of 10^-3 was added with 90 µL of phage buffer to create 10^-4 dilution which was vortexed.
5. 10 µL of 10^-4 was added with 90 µL of phage buffer to create 10^-5 dilution which was vortexed.
6. Took 50 µL of 10^-3 into 0.5 mL of Arthro. Sat for 15 mins to infect.
7. Took 50 µL of 10^-4 into 0.5 mL of Arthro. Sat for 15 mins to infect.
8. Took 50 µL of 10^-5 into 0.5 mL of Arthro. Sat for 15 mins to infect.
9. Took 8 mL of LB Broth, 10 mL of 2X TA, and 90 µL of 1M CaCl2 into a 50 mL vial.
10. Took 4.5 mL of top agar mixture onto control plate. Allowed to solidify for 15 mins.
11. Took another 4.5 mL of top agar mixture into separate 15 mL vial. Combined lysate and 10^-3 mixture into same 15 mL vial. Swirled gently to mix and poured onto 10^-3 plate.
12. Took another 4.5 mL of top agar mixture into separate 15 mL vial. Combined lysate and 10^-4 mixture into same 15 mL vial. Swirled gently to mix and poured onto 10^-4 plate.
13. Took remiaing 4.5 mL of top agar mixture and combined lysate and 10^-5 mixture into same vial. Swirled gently to mix and poured onto 10^-5 plate.
14. Allowed all plates to solidify for 15 mins.
15. Inverted all plates into incubator after 15 mins.

Conclusion

Current titer= 7.4 x 10^-6

Webbed calculations= 97.635 microliters of 10^-5 to web

Only 10^-3 through 10^-5 plates were made because the results from lab day 24 had plaque shown in the 10^-4 spot titer and made calculations based off from there. Since there might have been a miscalculation from that spot titer, individual plates were made from 10^-3 through 10^-5 to insure a more precise calculation. 10^-4 plate was used to calculate the web and current titer since 10^-5 only had 3 plaque present. In the next lab day,  I will be able to make a new webbed plate for it to be flooded.

contamination of control plate, and no phage present on 10^-4 plate from lab day 24

no contamination on control plate, plaque present in 10^-3, 10^-4, and 10^-5 plates.

Rationale

New titer and webbed plate calculation were recorded. The amount calculated (22 microliters of lysate) will be used to make a new webbed plate.

Detailed Procedure

1. Took 22 microliters of 10^0 lysate and added to 0.5 mL arthro. Sat for 15 mins.
2. Took 4 mL LB Broth, 5 mL 2X TA, and 45 microliters of 1M CaCl2 into a 15 mL vial.
3. Took 4.5 of Top Agar mixture into a plate labeled as “SJ control 11/14/18.”
4. Took remaining 4.5 mL of Top Agar mixture and added arthro+lysate mixture.
5. Poured mixture into a plate labeled as “SJ PA 22 microliters 11/14/18.”
6. Allowed plates to solidify for 15 mins and inverted into incubator.

Results

current titer= 2.4 x 10^6

need to web plate= 21.3 microliters, used 22 microliters.

Conclusion/next steps

Plates were sliding, no bubbles formed on the plate. The current titer calculation is nearly the same titer as last time. In the next lab day, hopefully, the plate will be flooded and perform serial dilutions up to 10^-8 and recalculate for a new, high titer

titer calculations were used from 10^-4 spot

Rationale

Current titer calculations from 10^-2 plate was 10⁶. The 10^-1 and 10^-2  plates from previous lab day will be flooded and filtered out. A spot test will be performed using 50 µL of 10⁰, 10^-1, 10^-2, 10^-3 ,10^-4 , and 10^-5  of the new lysate with a goal to receive a webbed plate and get a high titer.

Detailed Procedure

1. Add 8 mL of PB to 10^-1 and 10^-2  plates from previous lab day.
2. The plates was shaken on an incubator for one hour.
3. Filtered lysate from flooded plate through a 50 mL 0.22 µm filtered conical vial. Labeled “SJ 11/13/18 FS lysate 10⁰.”
4. 10 µL of “SJ 11/12/18 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1 dilution which was vortexed.
5. 10 µL of 10^-1 was added with 90 µL of phage buffer to create 10^-2 dilution which was vortexed.
6. 10 µL of 10^-2 was added with 90 µL of phage buffer to create 10^-3 dilution which was vortexed.
7. 10 µL of 10^-3 was added with 90 µL of phage buffer to create 10^-4 dilution which was vortexed.
8. 10 µL of 10^-4 was added with 90 µL of phage buffer to create 10^-5 dilution which was vortexed.
9. 4 mL of LB Broth, 45 µL of CaCl₂, and 5 mL of 2X TA were combined into a 15 mL vial.
10. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into control plate labeled “SJ Control 11/13/18.”
11. Added 0.5 mL Arthro into remaining 4.5 mL TA mixture and poured onto plate labeled “SL Spot Test 11/13/18” which was sectioned off into 6 parts.
12. Waited 15 mins for both plates to solidify.
13. Micropipetted 50 µL of 10⁰, 10^-1, 10^-2, 10^-3 ,10^-4 , and 10^-5  in according sections of the spot test plate.
14. Rested into incubator

Observations/Results/Next Step

Control plate from previous lab day had no contamination and size of plaques all seem to remain the same size. Plate seeme nearly fully webbed, it was enough to flood the plate. For the new plates, air bubbles were formed. The spot test for 10^-1 and 10^-2 collided with each other, so it might be hard to calculate the titer, but hopefully a high titer can be calculated for the rest of the spotted dilutions by next lab day.

10^-2 plate calculated a 10^6 titer

newly filtered lysate after flooding

10^0 — 10^-5 dilutions were made

results from lab day 24

spot test 11/13/18

Rationale

The two plates from previous lab day will be flooded and filtered out. Plaque assays will be performed using 10 µL of 10⁰, 10 µL of 10^-1, and 10 µL of 10^-2 of the new lysate with a goal to receive a webbed plate and get a high titer.

Detailed Procedure

1. Add 5 mL of PB to each plates from previous lab day.
2. The plates was shaken on an incubator for one hour.
3. Filtered lysate from flooded plate through a 0.22 µm syringe filter into a conical vial labeled “SJ 11/12/18 FS lysate 10⁰.”
4. 10 µL of “SJ 11/12/18 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1 dilution which was vortexed.
5. 10 µL of 10^-1 was added with 90 µL of phage buffer to create 10^-2 dilution which was vortexed.
6. 10 µL of “SJ 11/12/18 FS lysate 10⁰“, 10 µL of 10^-1, and 10 µL of 10^-2 of the new lysate were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them. Sat for 15 mins.
7. 8 mL of LB Broth, 90 µL of CaCl₂, and 10 mL of 2X TA were combined into a conical vial.
8. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
9. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the TA mixture was poured onto a plate labeled “SJ 11/2/18 Control.”
10. Waited 15 mins to solidify ad inverted into incubator for 48 hours.

Observations/Results/Next Step

Control plate from previous lab day had no contamination and size of plaques all seem to remain the same size. Although not fully webbed, it was enough to flood the plate. For the new plates, air bubbles were formed. For the next lab day, a new titer will be calculated and hope to reach a high titer by Wednesday.

results from lab day 23

new plates after flooding and serial dilutions

Rationale

Try to create a plate that is webbed enough for future flooding.

Detailed Procedures

1. Took 0.5 mL Arthro and added 524 microliters of 10^0 lysate from donor. Sat for 15 mins.
2. Took another 0.5 mL Arthro and added 550 microliters of 10^0 lysate from donor. Sat for 15 mins.
3. Took 6 mL of LB Broth, 7.5 mL 2X TA, and 69 microliters of 1 M CaCl2 into a 50 mL vial.
4. Took 4.5 mL of mixture from step 3 and poured onto a plate labeled as “control.”
5. Took another 4.5 mL of mixture from step 3 and poured into a 15 mL vial labeled as “524 microliters.”
6. Took another 4.5 mL of mixture from step 3 and poured into a different 15 mL vial labeled as “550 microliters.”
7. Took mixture from step 1 and added to 15 mL vial labeled as “524 microliters.”
8. Took mixture from step 2 and added to 15 mL vial labeled as “550 microliters.”
9. Took “524 microliter” vial and poured onto “524 microliters” plate.
10. Took “550 microliter” vial and poured onto “550 microliters” plate.
11. Let all three plate solidify for around 15 mins and inverted into incubator.

Conclusion/Results/Next Steps

Control plate was contaminated again. The plaque assay plate from previous lab day had no phage on the plate, so I had to redo 550 microliters again. Another member who had the same donated lysate also had no phage on plate as well as contamination. There was no problems in making the plates (no bubbles, good aseptic technique, no sliding.) If the plate seems to be webbed enough by next lab day, then flooding will take place. If not, then webbing will continue and will have to re-calculate.