Date: 8/29/2018

Plaque Assay Soil A

Objectives:

• perform the spot test

Materials Required: Filtered Enriched lysate, serological pipettes, micropipettes, micropipette tubes, LB broth, Top Agar 2X, 50 ml                                               conical vials, 1M CaCl2 stock solution

Calculations:

conversion factors:

1M= 1000mM

1 ml =1000 microliters

M1V1=M2V2

1M(V1)=(4.5mM)(10ml)

1000mM(V1)=(4.5mM)(10000 microliters)

V1=45 microliters

Procedure:

1.  First the aseptic zone was set up: pour Cidecon on the desk and wiping it till the desk dry. then pour ethanol and wipe it all over the table and let it evaporate.
2. Light the ethanol lamp to create an air current near which the samples can be opened to prevent things from getting into the samples.
3. Retrieved the LB broth, a 50 ml conical tube and a serological pipette
4. While in the aseptic zone, transfer 4.5 ml of LB broth to the 50 ml conical vial.
5. now retrive the 1 M CaCl2 stock solution.
6.  Using the micropipette, i transfered 45 microliters of the CaCl2 to the 50 ml conical tube with the LB broth.
7. retrieved 0.5 ml of arthrobacter from Lathan ( Teaching Assistant)
8. add the arthrobacter to the 50 ml conical tube.
9. add 5 ml of top agar to the 50 ml conical tube.
10. pour the contents of the 50 ml conical tube onto the agar plate.
11. to let the top agar solidify, i waited for 10 minutes.
12. collect the enriched sample tube, a syringe and a filter of 22 microns.
13. take a sample from the enriched tube using the syringe.
14. attach filter to the syringe and pour the lysate out slowly into a microcentrifuge tube
15. collect the direct isolation sample from the fridge.
16. collect a phage buffer from the instructor
17. mark the agar plate with three spots , one for the enriched, one for the direct and one for the phage buffer.
18. spot 10 microliters each of the phage buffer, enriched sample and the direct isolation sample.
19. let the sample rest for 10 minutes
20. i put the plate in the incubator, where it will remain for 48 hours.

Analysis:

there was no event that may have caused contamination to the sample. the aseptic zone was properly maintained. the procedure was properly followed.

Future notes:

read the instructions carefully and work faster so as to finish on time and prevent mistakes.

Date: 8/29/2018

Plaque Assay Soil A

Objectives:

• Analyse the results of the spot test from Monday (08/27/2018)
• Make a plaque assay with the enriched lysate of soil sample A that was filtered on Monday

Results from Spot Test:

• No plaques have formed on my Spot test for soil A. According to these results, there are no bacteriophages in my soil sample.
• I have also made a major error. I forgot my filtered enriched sample on the table on Monday and this may affect the results of the plaque test today.
• There was also some moving liquid inside my spot test dish. May be just some of the TA that had no solidifies before incubation.
• 

Adjustments:

Due to a lack of an adequate number of Agar plates, adjustments were made to the experiment for today. Everyone had to do an enriched sample plaque Assay and the following adjustments were made to the measurements of the ingredients required to make the top agar. the top agar was made for the entire group in one conical vial.

2 ml LB broth

2.5 ml 2X  Top Agar

22.5 microliters 1 M CaCl2.

These values were then multiplied by 4 to account for the fact that it was for the entire group. the measurements used for the group:

8 ml LB broth

10 ml 2X Top Agar

90 microliter 1 M Cacl2

5 ml of this mixture for the top agar was poured onto each plate.

Calculations:

conversion factors:

1M= 1000mM

1 ml =1000 microliters

M1V1=M2V2

1M(V1)=(4.5mM)(10ml)

1000mM(V1)=(4.5mM)(10000 microliters)

V1=45 microliters

Materials Required: Filtered Enriched lysate, serological pipettes, micropipettes, micropipette tubes, LB broth, Top Agar 2X, 50 ml conical vials, 15 ml conical vials

Procedure:

1.  First the aseptic zone was set up: pour Cidecon on the desk and wiping it till the desk dry. then pour ethanol and wipe it all over the table and let it evaporate.
2. Light the ethanol lamp to create an air current near which the samples can be opened to prevent things from getting into the samples.
3. Retrieved the LB broth, the same one I used on monday, a 50 ml conical tube and a serological pipette
4. While in the aseptic zone, transfer 8 ml of LB broth to the 50 ml conical vial.
5. now retrive the 1 M CaCl2 stock solution.
6.  Using the micropipette, i transfered 90 microliters of the CaCl2 to the 50 ml conical tube with the LB broth.
7. Set this vial in the rack.
8. retrieve 0.5 ml of arthrobacter.
9. using the micropipette, i transfered 10 microliters of my filtered enriched lysate to the arthrobacter.
10. i let the testtube rest for 10 minutes in its rack.
11. after the 10 minutes are up, add 10 ml of top agar to the conical vial.
12. using a serological pipette, transfer 5 ml of the top agar mixture to the test tube with the arthrobacter and the lysate.
13. My test tube cracked and spilled onto the table. i had to restart the process with the measurements for one sample.
14. i added 10 microliters of the filtered enriched lysate to another 0.5 ml culture of arthrobacter. let it rest for 10 minutes
15. Into a 15 ml conical vial in the aseptic zone, add 2 ml of LB broth, 22.5 microliters of 1M CaCl2 and 2.5 ml of 2X top agar, using the same methods as before.
16. Transfer the contents of the 15 ml conical vial to the test tube of arthrobacter and lysate after the 10 minute rest.
17. pour the contents of the test tube into the agar plate.
18. to let the top agar solidify, i waited for 10 minutes.
19. i put the plate, upside down in the incubator, where it will remain for 48 hours.
20. pour a part of the top agar mixture prepared into the top agar control plate for the 4 lab groups.

Analysis:

there was no event that may have caused contamination to the sample. the aseptic zone was properly maintained. the procedure was properly followed.

Future notes:

take extra care while handling lab equipment to prevent future damage that could lead to more delays. We have also decided on a research question : Does the presence of arthrobacter appear more dominant in the soil of one oak species than the others? Is there a correlation between the presence of Arthrobacterphage and the presence of oak wilt fungus?

8/20/2018

soil washing

Objective: todays goal is to extract the phages from the soil that was collected on 8/20/2018 , labeled soil sample A.

Supplies: sample collected on Monday, 50 ml test tube, centrifuge, LB Broth, pippetes

Procedure:1) set up an aseptic zone by cleaning your desk with Cidecon (wipe till dry) and ethanol ( 70%) ( wipe on the desk and let it evaporate) after clearing the table.

2) lit the ethanol lamp to set up an air current to help keep other microbes from getting into tube when it is open.

3) take the tube filled with the collected dirt and add LB broth into the tube to 35 ml.

4) shake the test tube for 15 minutes using hand and vortex machine

5) put the test tube into the centrifuge  at 3000g for 5 minutes.

6) ran out of time so the sample was stored in the fridge to continue the process on Friday.

Results: the end product was the large particles deposited at the bottom of the tube. the solution was still very dark and dense.

the tree from which the soil sample was extracted is located below. the mass of the test tube after the LB broth was added was 51.105g and the LB  broth was poured into the solution at 3:09 pm.

Analysis: due to the dense nature of the supernatant  in the test tube, it seemed that it was going to take a long time to filter the lysate from the supernatant. the aseptic zone was properly created and maintained.

Future notes: manage time more effectively and work faster so as to finish this process at the same time. Keeping the supernatant solution idle for a few days might affect the time it takes to separate the lysate from the supernatant because some of the solid might mix again  with the part of the tube containing the phages.

8/24/2018

enrichment

Objective: todays goal is to extract the phages from the soil that was collected on 8/20/2018, soil sample A.

Supplies: sample collected on Monday in 50 ml test tube, pipettes, tube top vacuum filter, shaking incubator, vacuum pump, 15 ml vials, 0.5 ml Arthrobacter host

Procedure:

1) set up an aseptic zone by cleaning your desk with Cidecon (wipe till dry) and ethanol ( 70%)( wipe on the desk and let it evaporate) after clearing the table.

2) lit the ethanol lamp to set up an air current to help keep other microbes from getting into tube when it is open.

3) divide the supernatant solution into two 15 ml vials and put it into the desk centrifuge for 17 minutes.

3) set up the tube top vacuum filter, put in the vacuum tube and put in the supernatant from the two vials using a pipette .

4) turn on the vacuum pump and let the supernatant filter.

5) After filtration, take out the lysate(solution filtered) and dispose of the waste appropriately .

6) Keep 10 ml of lysate in the tube and move the rest into an 15 ml vial in the aseptic zone.

7) put the lysate in the 15 ml vial in the fridge for direct isolation.

8) add the Arthrobacter host to the 10 ml of filtered lysate for enrichment.

9) put the enriched sample into the shaking incubator at 38 degrees Celsius for 76 hours

Results: the end product was the large particles deposited at the bottom of the tube. the solution was still very dark and dense.

Analysis: the lysate was filtered faster than initially predicted due to the second time use of the centrifuge. the aseptic zone was properly maintained and so far no apparent events that could have caused contamination. the filtered lysate that was acquired was 15.5 ml. the diagram for the process is below

enriched sample is labelled: ADP 08/24/2018 enriched ; and direct isolation sample is labelled: ADP 08/24/2018

Future notes: dividing the supernatant into the two vials and putting them into the centrifuge helps a lot with the washing process. next lab we will be to do the spot test and observe the plaque assay in the petri dish, which will be made from the enriched sample to test the presence of phages in the soil. we will know if there are phages in the soil if there are blank spots on the dish because that would represent dead bacteria, which will be the bacteria that were infected by bacteriophages.