Discovery of Eppendorf

 Direct Isolation of Sample 1

Date: 08/27/2024

Redo:  No

Purpose: To isolate a bacteriophage from an environmental sample and use the isolated phage to infect host bacteria using plaque assay.

Notes:

1. 1. Dress in appropriate PPE and sterilize the lab bench, light bunsen burner to maintain a sterile field to work under
   2. Grab the refrigerated soil sample in a 15 ml conical tube and 5ml of PYCa liquid media. Soil sample #1 filled half conical tube.
   3. Using a 5 ml sterile pipette, add the PYCa liquid media to the 15 ml conical tube until the soil is submerged under 2-3 ml.
   4. Once the liquid media has been added, cap the sample and invert several times to mix
   5. Place the sample in a shaking incubator operating at 250 rpm for 1-2 hours
   6. Allow the sample to sit for 10 minutes to settle. Our sample had been divided into 3 layers, the bottom consisting of soil, the middle a murky liquid, and the top floating debris
   7. Using a .22 μm syringe filter and a 5 ml sterile syringe we extracted the middle liquid layer from the 15 ml conical tube 
   8. Placing the .22 μm filter above a microcentrifuge tube allowed us to capture the filtered drainage 
   9. Once at least .5 ml had been filtered into the microcentrifuge tube, we immediately capped it. Now begin preparing for plaque assay
   1.  Obtain 250 μl host bacteria
   2. Using a 5 ml serological pipette, transfer the liquid in the microcentrifuge tube to the host bacteria and rotate/flick the test tube to mix
   3. Let the test tube sit for 10 minutes to allow attachment
   4. Grab molten top agar stored at 55-60C, using a 5ml serological pipette we transferred 3ml of top molten agar into the test tube and immediately aspirated it back into the pipette.
   5. We quickly dispersed the molten agar and host bacteria mixture onto the agar plate leaving a tiny amount in the pipette to prevent the formation of bubbles.
   6. Once the mixture made contact with the agar plate, we gently tilted to ensure all areas of the agar plate were evenly covered
   7. The agar plate was left to sit for 20 minutes to allow the agar to solidify, then placed in an incubator at 29C f to sit for 24-48 hours.

Results: 9/6/24

The plate showed phage growth.

Conclusions and Next Steps: We have concluded that Sample 1 was a successful example of our goal.  The next step is to isolate a single phage using plaque assay and serial dilution. 

Serial Dilution #1

Date: 09/9/2024

Redo:  No

Purpose: To use serial dilution and plaque assay to isolate a single phage 

Notes:

1. 1. Sterilize bench and begin aseptic technique by lighting bunsen burner to maintain a sterile environment.
   2. Prepare for serial dilution by gathering an appropriate number of microcentrifuge tubes, today we used six (10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6)
   3. Label each microcentrifuge tube with its corresponding dilution 10^-1, 10^-2… 
   4. Distribute 90 ul of phage buffer into each of the microcentrifuge tubes
   5. Using a 100 microliter micropipette select one phage from the sample 1 plate
   6. Circle the phage that was used
   7. Using the micropipette deposit the phage into your first (10^-1) dilution.
   8. Transfer 10 ul from 10^-1 t0 10^-2, making sure to gently mix the samples together
   9. Continue to add 10 ul to each of the 1o fold dilutions, switching the tip of the micropipette after every dilution
   10. Add plates to the incubator set at 29C while you perform other steps to ensure they will be ready
   11. Gather six tubes of host bacteria M. Foluriom and label them the same as the microcentrifuge tubes
   12. Add 10 ul of the microcentrifuge tubes to each of the corresponding host bacteria tubes
   13. Gently swirl the tubes to mix and let them sit for 10 minutes to allow attachment
   14. Remove PYCa plates from the incubator and label date, initals, and which dilution each plates will be (10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6).
   15.  Draw up 3 ml of top agar stored at 55C using a 5 ml serological pipette and add to the host bacteria
   16. Immediately aspirate and dispense the mixture into the corresponding PYCa plate
   17. Do this for each of the dilutions
   18. Let plates sit for 20 minutes to allow them to solidify
   19. Place plates in incubator at 29C for 24-48 hours

Results: 9/11/24

10^-1: Uncountable number of phages

10^-2: Uncountable number of phages

10^-3: Around 60 phages

10^-4: Around 30 phages

10^-5: 17 Phages

10^-6: 11 Phages

 

Conclusions and Next Steps:

 The 10-fold dilution did show bacterial growth that decreased with each concentration. We will use the 10^-6 dilution to continue our second serial dilution. The plates were sealed with parafilm and refrigerated. 

 

           

 Second Serial Dilution

Date: 9/11/2024

Redo:  No

Purpose: To dilute and plate phage sample until a single isolated phage remains 

Sample: 1, 10^-6

Notes:

1. 1. Steralize bench and begin aseptic technique by a lightning bunsen burner to maintain sterile environment
   2. Prepare for serial dilution by gathering an appropriate number of microcentrifuge tubes, today we used four (10^-7, 10^-8, 10^-9, 10^-9)
   3. Label each microcentrifuge tube with the corresponding dilution
   4. Distribute 90 ul of phage buffer into each of the microcentrifuge tubes
   5. Using a 100 microliter micropipette set the volume to 10 ul
   6. Select one phage from 10^-6 and mark the circle sample used on the plate
   7. Deposit 10 ul of the phage into the first dilution (10^-7)
   8. Transfer 10 ul from 10^-7 into the 10^-8 tube, making sure to gently mix together
   9. Continue this process through the remaining samples changing the tip after every dilution
   10. Gather PYCa plates and place them in an incubator at 29C to prepare for plaque assay
   11. Gather four tubes of  host bacteria (M. Foluriom) tube for every microcentrifuge sample and label them 10^-7, 10^-8, 10^-9, 10^-10
   12. Add 10 ul of the microcentrifuge tubes to the corresponding host bacteria tubes
   13. Gently swirl the tubes to mix and let them sit for 10 minutes to allow attachment
   14. While waiting, label each PYCa plate with the correct dilutions: 10^-7, 10^-8, 10^-9, 10^-10
   15. Draw up 3 ml of top agar stored at 55C , working quickly using a 5 ml serological pipette, and add to the host bacteria
   16. Immediately aspirate and dispense into the appropriate plate, do this for each of the dilutions
   17. Let the plates sit for 20 minutes to allow them to solidify
   18. Place in an incubator at 29C for 24-48 hours

Results: 9/13/24

10^7: Uncountable number of phage

10^-8:Uncountable number of phage

10^-9:Uncountable number of phage

10^-10: About 50 phages

Conclusions and Next Steps:

Using the 10^-10 diluted plates we will repeat this process of 10-fold dilutions to further isolate the phages to narrow it down to a single phage. The plates were sealed with parafilm and refrigerated. 

 

Third Serial Dilution

Date: 9/18/2024

Redo: No

Purpose: To dilute and plate phage sample until a single isolated phage remains

Notes:

1. 1. Steralize bench and begin aseptic technique by a lightning bunsen burner to maintain sterile environment
   2. Prepare for serial dilution by gathering an appropriate number of microcentrifuge tubes, today we used 4 (10^-11, 10^-12, 10^-13, 10^-14)
   3. Label each microcentrifuge tube with the corresponding dilution
   4. Distribute 90 ul of phage buffer into the microcentrifuge tube
   5. Using a 100 microliter micropipette select one phage from 10^-10 and mark the circle sample used on the plate
   6. Deposit the phage into the first dilution (10^-11)
   7. Transfer 10 ul from 10^-11 into the 10^-12 tube, making sure to gently mix together
   8. Continue this process through the remaining samples
   9. Gather agar plates and place them in an incubator at 29C to prepare for plaque assay
   10. Gather a host bacteria (M. Foluriom) tube for every microcentrifuge sample and label them 10^-11, 10^-12, 10^-13, 10^-14
   11. Add 10 ul of the microcentrifuge tubes to the corresponding host bacteria tubes
   12. Gently swirl the tubes to mix and let them sit for 10 minutes to allow attachment
   13. While waiting, label each plate with the correct dilutions: 10^-11, 10^-12, 10^-13, 10^-14
   14. Draw up 3 ml of top agar stored at 55C , working quickly using a 5 ml serological pipette, and add to the host bacteria
   15. Immediately aspirate and dispense into the appropriate plate
   16. Do this for each of the 10-fold dilutions, then place in an incubator at 29C for 24-48 hour

Results:

 

Conclusions and Next Step

 Harvesting Low-Volume Lysate

Date: 09/23/2024

Redo:  No

Purpose: To make a low-volume lysate to determine titer.

Notes:

1. 1. 1. Sterilize bench and light Bunsen burner to maintain a sterile field 
      2. Using the 10^-13 dilution from the third serial dilution to make low-volume lysate
      3. Flood the plate using 8 ml of phage buffer 
      4. Leave the plate to sit (room temperature) for 2 hours with the lid on 
      5. To collect the lysate, gently remove the plate lid and place it on the lab bench 
      6. Carefully prop the plate pooled with lysate under its lid to create a tilt
      7. Gather .22um filter and a 5ml syringe filter 
      8. Use the 5 ml syringe to gather the pooled lysate 
      9. Attach the .22um filter to the 5 ml syringe with lysate and dispense into a 15 ml sterile conical tube 
      10. Label the conical tube low volume lysate, date, and initials 
      11. Record the volume and place in refrigerator until use 

Results:

Conclusions and Next Step:

We now have a low volume lysate so now we can calculate titer and use that when performng serital dilutions to create webbed plate.

 Spot Titer

Date: 09/25/2024

Redo:  No

Purpose: Xxxxx.

Notes:

• Sterilize lab bench and light bunsen burner to maintain sterile field 
• Place a single PYCa plate into the incubator to ensure it is ready by the time it is needed 
• Once the plate is ready, mark it into a 3×3 grid.
• Label each square in the grid with the appropriate dilution (-1, -2, -3, -4, -5, -6, -7, -8)
• Have one tube of 250 ul culture bacteria ready 
• Using a 5ml sterile pipette transfer 2ml of top molten agar into the pipette and draw back into the 250 culture bacteria tube 
• Immediately aspirate back up and dispense onto the plate (making sure to work quickly with the top agar)   
• Let this sit to cool for 15 minutes (or until solid)
• Using our low-volume lysate, perform 8 dilutions
• Grab 8 microcentrifuge tubes (one for every dilution, for the spot titer we did eight)
• Add 90 ul of phage buffer into each microcentrifuge tube 
• Label each tube with its corresponding dilution (-1, -2, -3, -4, -5, -6, -7, -8)
• Using a micropipette set the volume adjustment to 10 ul 
• Collect the 10 ul in the micropipette from the low-volume lysate 
• Dispense into the first (10^-1) diluted microcentrifuge tube 
• Changing the tip after each dilution, work your way down dilutions using 10 ul from each microcentrifuge tube into the next one.
• Switching the volume adjuster on the micropipette to 3 ul takes three microliters from each dilution in the microcentrifuge tubes and dispenses them into their corresponding dilution on the PYCa plate 
• Leave the dilutions to set on the PYCa plate, after about 15 minutes place in the incubator at 29 C for 48 hours 

Results:

 Success, there was phage growth.

Conclusions and Next Step:

We will use our spot titer to calculate our phage titer and perform a full plate titer.

 Full Plate Titer 

Date: 09/30/2024

Redo:  No

Purpose: Make webbed plates 

Notes:

• •  Using the spot titer, calculate the titer to use to make a webbed plate 
  • We used the equation Titer(pfu/ml)= (#pfu/volume used in ul)(10^3 ul/ml)(dilution factor)
  • We observed 2 plaques in our 10^-8 dilution using 3 ul 
  • (⅔) (10^3)(10^*)
  • Our titer is .66×10^11
  1. Sterilize bench and light bunsen burner to maintain sterile field 
  2. Place one PYCa plate in the incubator at 29C 
  3.  Gather 11 microcentrifuge tubes 
  4. Fill each microcentrifuge tube with 90 ul of phage buffer
  5. Repeating the steps from the low-volume serial dilution 
  6. Using a micropipette, draw up 10 ul of low-volume lysate and add it to the 10^-1 dilution the fold 
  7. Switching tips after every dilution, add 10 uls of the lysate/buffer solution down each of the dilutions
  8. Knowing our titer, we are now mainly focused on the 10^-11 dilution 
  9. Remove the plate from the incubator 
  10. Gather 250 ul culture of host bacteria (M. Folorium) and add 10 ul of the 10^-11 microcentrifuge tube into the host bacteria tube
  11. Gently swirl to mix and leave it to sit for 10 minutes to allow attachment 
  12. Using top molten agar stored at 55C, draw up 3 ml of agar and dispense into the host bacteria tube 
  13. Immediately draw this back up and add to the PYCa plate 
  14. Let the plate sit for 20 minutes or until solid and incubate for 48 hours at 29C 

Results:

 The results were negative, there was no phage growth

Conclusions and Next Step:

We will repeat the full plate titer in hopes to make a webbed plate

Full Plate Titer #2

Date: 10/02/2024

Redo:  Yes (2nd attempt)

Purpose: To make webbed plate 

Notes:

1. Sterilze bench and light bunsen burner to maintain sterile field 
2. Place a Pyca plate in the incubator for ready-use
3.  Gather 11 microcentrifuge tubes 
4. Set the volume of the micropipette to 90 uls
5. Fill each microcentrifuge tube with 90 ul of phage buffer
6. Repeating the steps from the low-volume serial dilution 
7. Set the volume of the micropipette to 10 ul and draw up 10 uls of low-volume lysate
8. Add the 10 ul of low volume lysate to the 10^-1 microcentrifuge tube
9. Switching tips after every dilution, add 10 uls of the lysate/buffer solution down each of the dilutions
10. Knowing our titer, we are only plating the 10^-11 diltuion  
11. Remove the PYCa plate from the incubator and label the plate 
12. Gather 250 ul of host bacteria (M. Folorium) and add 10 ul of the 10^-11 microcentrifuge tube into the host bacteria tube
13. Gently swirl to mix and leave it to sit for 10 minutes to allow attachment 
14. Use top molten agar stored at 55C, draw up 3 ml of agar and dispense it into the host bacteria tube 
15. Immediately draw this back up and add to the Pyca plate 
16. Let the plate sit for 20 minutes or until solid and incubate for 48 hours at 29C 

 

Results:

 Results were negetive, there was no phage growth 

Conclusions and Next Step:

We will perfom another full plate titer 

 Full Plate Titer #3

Date: 10/07/2024

Redo:  Yes (3rd attempt)

Purpose: To make webbed plate 

Notes:

1. Sterilize bench and light bunsen burner to maintain sterile field 
2. Place two Pyca plates in the incubator for ready-use
3.  Gather 11 microcentrifuge tubes 
4. Set micropipette volume to 90 ul and fill each microcentrifuge tube with 90 ul of phage buffer
5. Repeating the steps from the low-volume serial dilution 
6. Set micropipette volume to 10 ul 
7. Draw up 10 ul of low-volume lysate and add it to the 10^-1 dilution the fold 
8. Switching tips after every dilution, add 10 uls of the lysate/buffer solution down each of the dilutions
9. We know we have to use 10^-11 dilution from our titer, we will also plate our 10^-10 dilution 
10. Remove the plates from the incubator and label them appropriately 
11. Gather two tubes of 250 ul of host bacteria (M. Folorium)
12. Using 10 ul from the 10^-10 from the microcentrifuge tube, add to the host bacteria
13. Do the same for the 10^-11 dilution 
14. Gently swirl to mix and leave it to sit for 10 minutes to allow attachment 
15. Using top molten agar stored at 55C, draw up 3 ml of agar and dispense it into the host bacteria tube 
16. Immediately draw this back up and add it to the Pyca plate 
17. Repeat this step for both the 10^-10 and 10^-11 dilutions
18. Let the plate sit for 20 minutes or until sold and incubate for 48 hours at 29C 

Results:

 The results were negative, there was no phage growth

Conclusions and Next Step:

We will repeat the full plate titer, but plate more and lower dilutions 

 Full Plate Titer #4

Date: 10/09/2024

Redo:  Yes (4th attempt)

Purpose: To make webbed plate 

Notes:

1. Sterilize the lab bench and light bunsen burner to maintain sterile field to work under
2. Place five PYCa plates in the incubator (29C) for ready-use
3.  Gather 11 microcentrifuge tubes and label them 10^-1 through 10^-11
4. Using 100 microliter pipette set the volume to 90 ul
5. Fill each microcentrifuge tube with 90 ul of phage buffer and label them
6. Repeating the steps from the low-volume serial dilution 
7. Set the micropipette to 10 ul
8. Add 10 ul of low-volume lysate and add it to the 10^-1 dilution 
9. Switching tips after every dilution, add 10 uls of the lysate/buffer solution down each of the dilutions
10. Remove the plates from the incubator and label 
11. Gather five tubes 250 ul of host bacteria (M. Folorium)
12. We are plating dilutions 10^-5 through 10^-9
13. Add 10 ul from each of the microcenrifuge tubes into their corresponding host bacteria tube 
14. Wait 10 minutes to allow attachment 
15. Using top molten agar stored at 55C, draw up 3 ml of agar and dispense into the host bacteria tube 
16. Immediately draw this back up and add to the Pyca plate 
17. Plate all 5 tubes onto the corresponding plates 
18. Let the plate sit for 20 minutes or until sold and incubate for 48 hours at 29C 

Results:

Conclusions and Next Step: 

The 10^-2 plate has the best optimal phage growth to make out high titer lysate. We will use the 10^-2 dilution to get to make a webbed plate and then a high titer lysate.

 Harvesting Low Volume Lysate 

Date: 10/21/2024

Redo:  Yes

Purpose:  To be to able to determine titer 

Notes:

1. Sterilize bench and light Bunsen burner to maintain a sterile field 
2. Using the 10^-14 dilution from the third serial dilution to make low-volume lysate
3. To flood the plate, use 8 ml of phage buffer 
4. Leave the plate to sit (room temperature) for 2 hours with the lid on 
5. To collect the lysate, gently remove the plates lid and place it on the lab bench 
6. Carefully prop the plate pooled with lysate under its lid to create a tilt
7. Gather .22um filter and a 5ml syringe filter 
8. Use the 5 ml syringe to gather the pooled lysate 
9. Attach the .22um filter to the 5 ml syringe with lysate and dispense into a 15 ml sterile conical tube 
10. Label the conical tube low volume lysate, date, and initials 
11. Place in refrigerator until use

Results:

 5 ml of low volume lysate 

Conclusions and Next Step:

Now that low-volume lysate has been made it will be used to determine titer in a serial dilution.

Full Plate Titer 

Date: 10/23/2024

Redo:  Yes

Purpose: To make webbed plate 

Notes:

• Sterilize lab bench and light bunsen burner to maintain sterile field to work under 
• Place eight PYCa plates in the incubator at 29C for ready use 
• Gather eight microcentrifuge tubes and label -1, -2, -3, -4, -5, -6, -7, -8
• Using 100 microliter micropipette set the volume to 90 ul 
• Add 90 ul to each of the eight microcentrifuge tubes 
• Set the volume of micropipette to 10 ul 
• Add 10 ul of low-volume lysate to the 10^-1 microcentrifuge tube
• Further dilute this by adding 10 ul down the fold, changing the tip of the micropipette after every dilution 
• Gather eight tubes of 250 ul of host bacteria (M. Folorium) and label them to match the microcentrifuge tubes 
• Add 10 ul from each of the microcentrifuge tubes into its corresponding host bacteria tube 
• Let the tubes sit for 10 minutes to allow attachment 
• Remove the PYCa plates from the incubator and label initials, date, and dilution (10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7, 10^-8)
• Using top agar stored at 55C draw up 3 ml and add to each bacteria tube 
• Immediately draw back up from the host bacteria tube and add onto the right PYCa plate 
• Do this for all 8 dilutions 
• Let the plates sit for 20 minutes or until solidified and incubate for 24-48 hours at 29C 

Results:

 

Conclusions and Next Step

 Phage did grow, using the 10^-2

 

Title: Making Webbed Plates from Known Titer

Date: 10/16/2024

Redo:  No

Purpose: Xxxxx.

Notes:

1. 1.  Sterilize the lab bench and light Bunsen burner to maintain a sterile field
   2. Place 6 pyca plates from the fridge into the incubator
   3. Select plate from the previous round of dilutions that best made webbed plate (we used our 10^-2)
   4. Place phage buffer (90 ul) into two microcentrifuge tubes to get to your 10^-2 dilution
   5. Only using the 10^-2 serial fold dilution now
   6. Have 6 different host bacteria tubes ready, and place 10 ul from the microcentrifuge tubes into each of the tubes containing host bacteria
   7. Label each of the host bacteria tubes with the appropriate dilution (-1, -2, -3, -4, -5, -6)
   8. Let this sit for 10 minutes after swirling gently
   9. Remove the pyca plates from the incubator and label them with the correct dilution that will be plated
   10. Using a serological pipette draw up 2 ml of top agar and dispense into the first (10^-1) dilution in the bacteria tube
   11. Immediately draw back up and dispense onto the pyca plate
   12. Repeat this step for the remaining five host bacteria tubes
   13. Once all six dilutions have been plated, let them sit for 20 minutes or until they solidify.

Results:

 

Conclusions and Next Steps:

 

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