Discovery of BlueMoth

Sample 1 Isolation

Date: 09/06/2024

Redo:  Yes

Purpose: The isolation of sample 1 is to determine if we had a viable phage to further purify.

Notes:

1. 1. Using an aseptic technique and once the environmental sample was collected in the 15 mL tubes, we started a direct isolation by using a buffer (PYCa) to mix our dirt sample in.
   2. We added the media until it was submerged beneath 2-3 mL of liquid.
   3. The sample was mixed and placed in the shaking incubator at 250 rpm for 1 hour.
   4. We let the sample sit for 20 minutes.
   5. Next we opened a 0.22 μm syringe filter, leaving the filter in the packaging.
   6. The filter was attached to the syringe first then the plunger was removed from the syringe.
   7. The sample was tipped into the barrel of the syringe.
   8. The plunger was attached and 0.5 mL filtrate was dispensed into a microcentrifuge tube.
   9. It was incubated at 4°C for seven days.
   10. After seven days, we proceeded with the plaque assay.
   11. Using an aseptic technique, we used a micropipettor to dispense our isolated sample into a culture tube of 250 µL of M. Foliorum.
   12. The culture tube was flicked a couple of times to mix everything and let it sit for 10 minutes.
   13. We retrieved a container of molten agar and retrieved 3 mL using a micropipettor.
   14. We dispensed the molten agar into our culture tube of phage sample and M. Foliorum. Then it was retrieved back into the micropipettor and dispensed the liquid onto the agar plate.
   15. We rotated the agar plate to evenly coat the surface and let it solidify for 20 minutes.
   16. At 10:17 AM, we incubated the sample for 48 hours at 29°C.

Results:

After about 48 hours of incubating in 29°C, there is a very small plaque starting to grow on the plate. It’s a little too early to tell its morphology. So we let it incubate for another 4 days. After additional incubation, we did not find any viable plaque growing.

Conclusions and Next Steps:

We had to restart the experiment with sample 2.

 

Sample 2 Isolation

Date: 09/06/2024

Redo:  yes

Purpose: The isolation of sample 2 is to determine if we had a viable phage to further purify.

Notes:

1. 1. 1. Using an aseptic technique and once the environmental sample was collected in the 15 mL tubes, we started a direct isolation by using a buffer (PYCa) to mix our dirt sample in.
      2. We added the media until it was submerged beneath 2-3 mL of liquid.
      3. The sample was mixed and placed in the shaking incubator at 250 rpm for 1 hour.
      4. We let the sample sit for 20 minutes.
      5. Next we opened a 0.22 μm syringe filter, leaving the filter in the packaging.
      6. The filter was attached to the syringe first then the plunger was removed from the syringe.
      7. The sample was tipped into the barrel of the syringe.
      8. The plunger was attached and 0.3 mL filtrate was dispensed into a microcentrifuge tube.
      9. We noticed the filtrate was colored differently; a clear brownish red.
      10. It was incubated at 4°C for five days.
      11. After five days, we proceeded with the plaque assay.
      12. Using an aseptic technique, we used a micropipettor to dispense our isolated sample into a culture tube of 250 µL of M. Foliorum.
      13. The culture tube was flicked a couple of times to mix everything and let it sit for 15 minutes.
      14. We retrieved a container of molten agar and retrieved 3 mL using a micropipettor.
      15. We dispensed the molten agar into our culture tube of phage sample and M. Foliorum. Then it was retrieved back into the micropipettor and dispensed the liquid onto the agar plate.
      16. We rotated the agar plate to evenly coat the surface and let it solidify for 20 minutes.
      17. At 9:43 AM, we incubated the sample at 29°C.

Results:

 On 9/11/24 Unfortunately, no plaque was growing. 

Conclusions and Next Steps: 

 The plate was discarded and we restarted the experiment with sample 3, during this time we will be collecting a 4th sample. 

Sample 3 Isolation

Date: 09/09/2024

Redo:  yes

Purpose: The isolation of sample 3 is to determine if we had a viable phage to further purify.

 

Notes:

• Using an aseptic technique and once the environmental sample was collected in the 15 mL tubes, we started a direct isolation by using a buffer (PYCa) to mix our dirt sample in.
• We added the media until it was submerged beneath 2-3 mL of liquid.
• The sample was mixed and placed in the shaking incubator at 250 rpm for 1 hours
• We let the sample sit for 20 minutes.
• Next we opened a 0.22 μm syringe filter, leaving the filter in the packaging.
• The filter was attached to the syringe first then the plunger was removed from the syringe.
• The sample was tipped into the barrel of the syringe.
• The plunger was attached and 0.3 mL filtrate was dispensed into a microcentrifuge tube.
• It was incubated at 4°C for two days
• After two days, we proceeded with the plaque assay.
• Using an aseptic technique, we used a micropipettor to dispense our isolated sample into a culture tube of 250 µL of M. Foliorum.
• The culture tube was flicked a couple of times to mix everything and let it sit for 2 hr 
• We retrieved a molten agar container and 3 mL using a micropipettor.
• We dispensed the molten agar into our culture tube of phage sample and M. Foliorum. Then it was retrieved back into the micropipettor and dispensed the liquid onto the agar plate.
• We rotated the agar plate to evenly coat the surface and let it solidify for 20 minutes.
• At 12:40 PM, on 09/11/24 we incubated the sample at 29°C. 

 

Results:

Waiting on results of plate

Conclusions and Next Steps: 

 

Sample 4 Isolation

Date: 09/16/2024

Redo:  No

Purpose: The isolation of sample 4 is to determine if we had a viable phage to further purify.

 

Notes:

• Using an aseptic technique and once the environmental sample was collected in the 15 mL tubes, we started a direct isolation by using a buffer (PYCa) to mix our dirt sample in.
• We added the media until it was submerged beneath 2-3 mL of liquid.
• The sample was mixed and placed in the shaking incubator at 250 rpm for 2 hours
• We let the sample sit for 20 minutes.
• Next we opened a 0.22 μm syringe filter, leaving the filter in the packaging.
• The filter was attached to the syringe first then the plunger was removed from the syringe.
• The sample was tipped into the barrel of the syringe.
• The plunger was attached and 0.3 mL filtrate was dispensed into a microcentrifuge tube.
• It was incubated at 4°C for 5 days
• After 5 days, we proceeded with the plaque assay.
• Using an aseptic technique, we used a micropipettor to dispense our isolated sample into a culture tube of 250 µL of M. Foliorum.
• The culture tube was flicked a couple of times to mix everything and let it sit for 2 hr 
• We retrieved a molten agar container and 3 mL using a micropipettor.
• We dispensed the molten agar into our culture tube of phage sample and M. Foliorum. Then it was retrieved back into the micropipettor and dispensed the liquid onto the agar plate.
• We rotated the agar plate to evenly coat the surface and let it solidify for 20 minutes.
• At 9:53 AM, on 09/16/24 we incubated the sample at 29°C. 

 

Results:

We have a plaque!

Conclusions and Next Steps: 

We proceeded to our first serial dilution with sample 4.

 

 

 

Sample 4 Serial Dilution #1

Date: 09/23/2024

Redo:  yes

Purpose: The first serial solution of sample 4 is to dilute our phage sample further to isolate plaques on a plate

 

Notes:

• Using an aseptic technique, we used a micropipettor to pick a plaque from the agar plate.
• The plaque picked was then transferred to a microcentrifuge tube, labeled OG and containing 90 microliters of PYCa buffer, by carefully tapping the micropipette tip on the inside of the microcentrifuge tube.
• Another eight microcentrifuge tubes were gathered and labeled 10^-1, 10^-2, and so on.
• Then, using a micropipette, 10 microliters were taken from the OG sample and dispensed into the microcentrifuge tube labeled 10^-1.
• The microcentrifuge labeled 10^-1 was vortexed for about 3 seconds.
• Then 10 microliters were taken from the microcentrifuge tube labeled 10^-1 and dispensed into the microcentrifuge tube labeled 10^-2.
• The microcentrifuge tube labeled 10^-2 is vortexed for about 3 seconds.
• The previous two steps are repeated for all remaining microcentrifuge tubes.
• Using a micropipette, 10 microliters are taken from each microcentrifuge and dispensed into separate culture tubes of 250 µL of M. Foliorum.
• The culture tube was flicked a couple of times to mix everything and let it sit for 10-20 minutes.
• We retrieved a molten agar container and 3 mL using a micropipettor.
• We dispensed the molten agar into our culture tube, retrieved it back into the micropipettor, and dispensed the liquid onto the agar plate. This was done for all culture tubes.
• We rotated the agar plate to evenly coat the surface and let it solidify for 20 minutes.
• At 9:53 AM, on 09/16/24 we incubated the sample at 29°C. 

 

Results:

Dilution was unsuccessful

Conclusions and Next Steps: 

We will redo the dilution procedure

 

Sample 4 Serial Dilution #2

Date: 09/25/2024

Redo:  no

Purpose: The second serial solution of sample 4 is to dilute our phage sample further to isolate 1-3 plaques on a plate.

 

Notes:

• Using an aseptic technique, we used a micropipettor to pick a plaque from the agar plate.
• The plaque picked was then transferred to a microcentrifuge tube, labeled OG and containing 90 microliters of PYCa buffer, by carefully tapping the micropipette tip on the inside of the microcentrifuge tube.
• Another eight microcentrifuge tubes were gathered and labeled 10^-1, 10^-2, and so on.
• Then, using a micropipette, 10 microliters were taken from the OG sample and dispensed into the microcentrifuge tube labeled 10^-1.
• The microcentrifuge labeled 10^-1 was vortexed for about 3 seconds.
• Then 10 microliters were taken from the microcentrifuge tube labeled 10^-1 and dispensed into the microcentrifuge tube labeled 10^-2.
• The microcentrifuge tube labeled 10^-2 is vortexed for about 3 seconds.
• The previous two steps are repeated for all remaining microcentrifuge tubes.
• Using a micropipette, 10 microliters are taken from each microcentrifuge and dispensed into separate culture tubes of 250 µL of M. Foliorum.
• The culture tube was flicked a couple of times to mix everything and let it sit for 10-20 minutes.
• We retrieved a molten agar container and 3 mL using a micropipettor.
• We dispensed the molten agar into our culture tube, retrieved it back into the micropipettor, and dispensed the liquid onto the agar plate. This was done for all culture tubes.
• We rotated the agar plate to evenly coat the surface and let it solidify for 20 minutes.
• At 10:16 AM, on 09/25/24 we incubated the sample at 29°C. 

 

Results:

Dilution was successful

Conclusions and Next Steps: 

We now proceed with creating a webbed plate

 

Sample 4 Webbed Plate Serial Dilution #3

Date: 09/30/2024

Redo:  no

Purpose: This 3rd dilution was to see if we can create a webbed plate to collect a lysate from 

Notes:

• Using an aseptic technique, we used a micropipettor to pick a plaque from the agar plate.
• The plaque picked was then transferred to a microcentrifuge tube, labeled OG (10^0) and containing 90 microliters of PYCa buffer, by carefully tapping the tip of the micropipette on the inside of the microcentrifuge tube.
• Another 2 microcentrifuge tubes were gathered and labeled 10^-1, and 10^-2,
• Then, using a micropipette, 10 microliters were taken from the 10^0 sample and dispensed into the microcentrifuge tube labeled 10^-1.
• The microcentrifuge labeled 10^-1 was vortexed for about 3 seconds.
• Then 10 microliters were taken from the microcentrifuge tube labeled 10^-1 and dispensed into the microcentrifuge tube labeled 10^-2.
• The microcentrifuge tube labeled 10^-2 is vortexed for about 3 seconds.
• Using a micropipette, 10 microliters are taken from each microcentrifuge tube and dispensed into separate culture tubes of 250 µL of M. Foliorum.
• The culture tube was flicked a couple of times to mix everything and let it sit for 20 minutes.
• We retrieved a molten agar container and 3 mL using a micropipettor.
• We dispensed the molten agar into our culture tube, retrieved it back into the micropipettor, and dispensed the liquid onto the agar plate. This was done for all culture tubes with respective labeled plates.
• We rotated the agar plate to evenly coat the surface and let it solidify for 20 minutes.
• At 10:07 AM, on 09/30/24 we incubated the sample at 29°C. 

Results:

We have a webbed plate ready to flood

Conclusions and Next Steps: 

We will now collect a phage lysate by flooding the plate.

1.  

    

Sample 4 Collecting Low-Volume Lysate

Date: 10/2/2024

Redo:  n/a

Purpose: The purpose of this experiment is to collect a vial of low-volume phage lysate.

Notes:

1. Using an aseptic technique, we used a pipettor to collect 8 mLs of phage buffer
2. We dispensed the PYCa buffer onto the webbed plate at let it sit at room temperature for 3 hrs
3. After allowing the plate to sit, the plate was tilted by placing the lid underneath the plate on one side. This allows the lysate to pool.
4. Using a 5mL syringe, the lysate was aspirated.
5. Then a freshly opened 0.22 μm filter was attached to the syringe.
6. The lysate was dispensed into a 15 mL conical tube.
7. The conical tube containing the lysate was labeled and stored at 4°C for five days.

Conclusions and Next Steps: 

We will plate the low-volume lysate to obtain a viable plate for a high-volume lysate.

 

Sample 4 Webbed Plate Serial Dilution 

Date: 10/07/2024

Redo:  no

Purpose: This dilution was to see if we could create a webbed plate to collect a high-volume lysate from

Notes:

• Using an aseptic technique, we used a micropipettor to pick a plaque from the agar plate.
• The plaque picked was then transferred to a microcentrifuge tube, labeled OG (10^0) and containing 90 microliters of PYCa buffer, by carefully tapping the micropipette tip on the inside of the microcentrifuge tube.
• Another 5 microcentrifuge tubes were gathered and labeled 10^-1, and 10^-2,
• Then, using a micropipette, 10 microliters were taken from the 10^0 sample and dispensed into the microcentrifuge tube labeled 10^-1.
• The microcentrifuge labeled 10^-1 was vortexed for about 3 seconds.
• Then 10 microliters were taken from the microcentrifuge tube labeled 10^-1 and dispensed into the microcentrifuge tube labeled 10^-2.
• The microcentrifuge tube labeled 10^-2 is vortexed for about 3 seconds.
• Using a micropipette, 10 microliters are taken from each microcentrifuge tube and dispensed into separate culture tubes of 250 µL of M. Foliorum.
• The culture tube was flicked a couple of times to mix everything and let it sit for 20 minutes.
• We retrieved a molten agar container and 3 mL using a micropipettor.
• We dispensed the molten agar into our culture tube, retrieved it back into the micropipettor, and dispensed the liquid onto the agar plate. This was done for all culture tubes with respective labeled plates.
• We rotated the agar plate to evenly coat the surface and let it solidify for 20 minutes.
• At 10:07 AM, on 10/7/24 we incubated the sample at 29°C. 

Results: Positive results

Conclusions and Next Steps: The 10^1 dilution was our best. We will use it to make multiple plates to obtain our high lysate.

Collecting high-volume Lysate #1

Date: 10/09/2024

Redo:  no

Purpose: The purpose of this experiment was to make multiple plates of the same dilution to collect our high-volume lysate

Notes:

Plating the LTL plates to collect HTL

1. 1. Using an aseptic technique, we set up 7 microcentrifuge tubes and prefilled them with PYCa buffer.
   2. we collected 10 microliters of our liquid LTL (low titer lysate) and dispensed it into the first microcentrifuge tube.
   3. This was our 10^0 tube, from previous knowledge in plating our LTL, we determined 10^-1 was the best dilution for creating webbed plates.
   4. 10 microliters were collected from the 10^0 tube and placed in each of the 6 prefilled tubes.
   5. We now had a “clone” of the 10^-1 dilution 6 times
   6. Using a micropipette, 10 microliters are taken from each microcentrifuge tube and dispensed into separate culture tubes of 250 µL of M. Foliorum.
   7. The culture tube was flicked a couple of times to mix everything and let it sit for 20 minutes.
   8. We retrieved a molten agar container and 3 mL using a micropipettor.
   9. We dispensed the molten agar into our culture tube, retrieved it back into the micropipettor, and dispensed the liquid onto the agar plate. This was done for all culture tubes with respective labeled plates.
   10. We rotated the agar plate to evenly coat the surface and let it solidify for 20 minutes.
   11. At 10:19 AM, on 10/9/24 we incubated the sample at 29°C for 5 days. 

Collecting HTL

1. 1. On 10/14/24, the plates were flooded with 8mL of phage buffer.

Results: 

We have a high volume lysate of about 6mL

 

Conclusions and Next Steps:

We will proceed to create TEM grids for electron microscopy 

Mounting Phage Samples for TEM and Staining with Uranyl Acetate (Classic Protocol Using Pelco Tabs)

Date: 10/21/2024

Redo:  no

Purpose: The purpose of this experiment was to make TEM grids for electron microscopy to view our phage

Notes:

Preparing phage samples.

1. 1. Using an aseptic technique, 1 ml of our high-titer lysate was transferred into a sterile microcentrifuge tube.
   2. We centrifuged the tube for 1 hour at 4 °C at 15,000 rpm to concentrate the phage particles at the bottom of the tube.
   3. Using a micropipettor, we carefully remove as much supernatant as possible without disrupting the concentrated phage at the bottom of the tube.
   4. 100 μl of phage buffer was added and the sample was resuspended at 4 °C for 30 minutes.

Staining the Sample

1. Using a micropipettor, we placed 10 μl of lysate onto the grid without touching the tip to the grid itself.
2. The phage was allowed to settle and attach onto the grid for at least 2 minutes, this was based on our calculated titer of 2.89 x 10^9
3. Using a small wedge of filter paper, the rest of the fluid was wicked off
4. The grid was washed with 10 μl of sterile water onto the grid, wicked off, and repeated twice
5. 10 μl of 1 % uranyl acetate was added to the grid, allowed to sit for 2 minutes, and wicked off.
6. The surface of the grid looked like a rainbow oil slick, the grid air dried before putting it safely back into the grid box, labelled A1

Results:

 

Conclusions and Next Steps:

 

Title

Date: ##/##/2024

Redo:  Yes/No

Purpose: Xxxxx.

Notes:

Results:

 

Conclusions and Next Steps:

DNA Extraction of DNA Sample 1

Date: 10/23/2024

Redo:  Yes

Purpose: The purpose of the experiment was to see if we could get a pure and concentrated DNA sample

Notes:

Day 1

Results:

Our sample had too much salt and was not pure enough, we did have a large concentration though.

 

Conclusions and Next Steps:

We will redo this procedure and save this sample for possible precipitation. 

 

DNA Extraction of DNA Sample 2

Date: 10/30/2024

Redo:  Yes

Purpose: The purpose of the experiment was to see if we could get a pure and concentrated DNA sample

Notes:

Day 1

Results:

 This sample was slightly better but still had too much salt.

Conclusions and Next Steps:

We will now reprecipitate DNA sample 1 to see if we can get it to be more pure.

Reprecipitation of DNA Sample 1

Date: 10/…/2024

Redo:  Yes

Purpose: The purpose of the experiment was to see if we could get a pure and concentrated DNA sample

Notes: