August 31

8/27/18 Spot Test

8/27/18 Spot Test

Objective:

The goal of this procedure is to determine weather or not bacteria phages were present in the soil collected last week. This will be determined by using small amounts of lysate on auger plates to look for plaques that would indicate phage particles. By performing this test

Procedures and Protocols:

Materials:

  • CiDecon
  • 70% Ethanol
  • Ethanol Burner
  • .5 ml Arthrobacter
  • refrigerator
  • Pipette
  • Test tube stand
  • 50 ml tubes
  • LB Broth
  • 2X TA
  • 1M Calcium Chloride
  • Agar plate
  • Micropipette

In order to complete the procedure an aseptic zone was created.

  1. Clean off the work space (lab table) with CiDecon applied with a squeeze bottle and wiped away with a paper towel
  2. Apply 70% Ethanol with a squeeze bottle, spread with a paper towel, and allow to evaporate
  3. Light an ethanol burner in order to use the rising heat from the flame to form the aseptic zone

Then the spot test could be preformed.

  1. Divide the bottom of an Agar plate into three sections and label them E for enriched isolation, D for direct isolation, and B for phage buffer *note that this was a collaborative effort to some degree so three agar plates were created*
  2. Create a separate Agar plate for a top agar (TA) control, label and set aside
  3. Gather previously created enriched isolation and direct isolation lysate
  4. Use a syringe to aseptically draw 3 ml of the enriched lysate out of the 50 ml tube
  5. Reseal tube
  6. Attach a filter to end of syringe and gently push 1.5 ml of lysate through the filter and into a pipette tip
  7. Cap the tip and set aside
  8. Set aside two 50 ml tubes and label them as TA control and Arthrobacter
  9. In the Arthrobacter tube add 13.ml of LB broth *The Arthrobacter tube was originally overfilled so some of the LB broth was pipette into the TA control tube*
  10. In the TA control tube add 4.5 ml of LB broth *Note that while adding LB broth to the TA tube the broth was spilled and the tube contents had to be emptied and new broth added*
  11. Set aside
  12. Add 42.75 μl Calcium Chloride and 5.0 ml of 2X TA to the TA control tube
    1. *The amount of calcium chloride to be added was determined based on the following calculations:
  13. Mix the contents of the TA control by shaking the tube vigorously *note that a pipette should have been used*
  14. Pipette 10 ml of the contents in the TA control tube into the agar plate labeled TA control
  15. cap plate and set aside for agar to solidify
  16. In the tube labeled Arthrobacter add 135 μl Calcium Chloride, 1.5 ml Arthrobacter, and 15.0 ml of 2X TA
  17. Use a pipette to mix solution by pulling it into the pipette and then expelling it several times
  18. Pipette 10 ml of solution from the Arthrobacter tube into each of the Agar plates
  19. On the Agar plate pour the remaining contents of the tube onto the plate because slightly less than 10 ml are left
  20. Allow Agar to solidify
  21. Using a Micropipette pipette 10 μl of filtered enriched lysate onto the section of plate labeled E, then pipette 10 μl of direct isolation lysate onto the section of plate labeled D, finally pipette 10 μl of phage buffer onto the section of plate labeled B
  22. Allow the plate to sit for about 10 minutes before being placed into the incubator
  23. Leave to incubate until next class (approximately 46 hours)
Results:

The results of this procedure will not be immediately clear until Wednesday’s lab, but once they are available they will be included here. Based on visible observations from lab today, the results of this procedure were four agar plates that appear to have been created correctly.

Update: There are visible plaques!!

Analysis:

Based on the Wednesday update, it can be inferred that there are bacteria phage present in my enriched and direct lysates. I will confirm this will a plaque update.

Future:

My future actions will heavily depend on the results of these spot tests. Regardless of weather or not plaques form though I will be doing plaque assay. So my next lab will detail that.

Category: Lucy FIsher | LEAVE A COMMENT
August 30

Spot Test 8/27/18

Rationale: Today’s goal was to correctly perform a spot test to determine the presence of phage in the soil samples that we had extracted in the week prior to this one. Hopefully, if done correctly with no contamination, spots will appear in our plaque after 48 hours of incubation.

Materials:

  • Lysate
  • PY Broth
  • PY 2X TA
  • 40% Dextrose
  • 1M Calcium Chloride
  • 0.5 mL of Arthrobacter host
  • Agar plate
  • Pipettes (Micropipettes and serological pipettes)

Procedure:

  1. This procedure started with establishing an aseptic zone to decontaminate the work bench. The bench was sprayed and wiped down with CiDecon, then followed up with 70% Ethanol, which was spread on the table allowed to evaporate. Following this, I grabbed our burner to finish setting up the aseptic zone.
  2. Next, I gathered the materials necessary to create the top agar that is needed for the spot test. I grabbed 4 plates (one for each group member and one for control), 2 serological pipette tips,a 50 mL conical tube, and I also set out four micro centrifuge tubes which will be used later in the lab. I then marked my plate with a sharpie to show where I would spot my direct isolation, my enriched isolation, and my phage buffer.
  3. Next, I began to create the top agar, I measured out 4.5 milliliters of LB broth to add to the conical tube using a serological pipette. My pipette was having issues, and could not go past 4 milliliters of liquid, so I had to use the serological pipette for the 4 milliliters, then I had to use a micropipette to gather 500 micro-liters to add to my broth to equal 4.5 milliliters to add to my 50 mL conical tube.
  4. After the LB broth was added, I needed to add calcium chloride to our top agar. Initially, the quantity of calcium chloride needed was unknown, but what was known was that we needed the molarity of calcium chloride to equate to 4.5 mM or millimolar. To solve for the volume needed, there is a simple equation that I used to get a final value of 45 microliters of calcium chloride
  6. Following the calculation, the calcium chloride was added to our broth mixture using a 20-200 microliter pipette.
  7. Instead of adding arthrobacter next, I filtered my enrichment from the previous lab through a 22 micron filter by syringe. I pulled out approximately 2 milliliters and pushed it gently through the filter into a micro-centrifuge tube.
  8. After the filtration process, 0.5 ml of arthrobacter was added to the broth mixture.
  9. Followed up immediately by pipetting 5.0 milliliters of PY 2x Top Agar with serological pipette and allowed the top agar to mix.
  10. Then I very quickly plated my top agar and the group’s control plate, swirled the plate around, and waited 15 minutes for the plate to solidify.
  11. After 15 minutes, I added 10 microliters of my direct, enriched, and phage buffer to their designated spots and put the tray to incubate for approximately 45 hours.
  12. After 45 hours I removed the tray to analyze my results.

Observations/Results/Data:

  • After examining the plate, it was concluded that there was no phage present in the lysate that I had extracted from my soil sample. The plate looked the same as it did when it was put in the incubator, no spotting whatsoever. This indicates a lack of phage as the arthobacter had not been cleared from the plate. There was no contamination as the agar looked exactly like the control.
  • Plate had the same color, but had a liquid forming on the top. Unsure what exactly caused that. 

Interpretations/Conclusions:

  • The results of the spot test indicate that there is no phage present in the soil sample that I collected the week before. This can be deduced by the lack of any clearing or any indication of bacterial death on the plate. If phage were to be present, there should be minor bacterial clearing in any part of the plate.

Next Steps:

  • There may still be hope for the soil sample. The next step is to perform a plaque assay to view the results of that experiment, as plaque assays and spot test have the ability to yield different results. The plaque assay will be performed the next time I am in lab, and if the results are negative that means I have to collect more soil samples to test for phage.
Category: Gabriel Andino, Uncategorized | LEAVE A COMMENT
August 30

Spot Test Soil A Results and Plaque Assay Soil A

8/29/18

Spot Test Soil A Results and Plaque Assay Soil A

Rational:

To confirm the results of the spot test on soil A with a plaque assay of soil A. This will give results if the sample was contaminted and will confirm results found in the spot test.

Procedure:

  • Cleaned the lab desk with CiDecon and 70% ethanol
  • Labeled the plate plaque assay soil A with enriched
  • Labeled conical vial team 2 TA
  • Added 22.5 ML CaCl2 using a micropipette
  • LB broth splashed out preventing us from knowing how much was added- conical vial was thrown out
  • Labeled new vial team 2 TA
  • Added 22.5 ML CaCl2 using micropipette
  • Added 2.0 mL LB broth using a serological pipette
  • In order to have 5 mL for each team member from the vial and 1 mL for the control plate we added 67.5 ML more CaCl2 with a micropipette and 6 mL LB broth with 10 mL pipette
  • Labeled tube with arthrobacter (0.5 mL arthrobacter) arthrobacter with enriched soil A
  • Added 10 ML lysate to the tube with a micropipette
  • Set a timer for 15 min
  • Added 10 mL of TA
  • Added 5 mLmof TA mixture to the tube with the lysate (some spilled) (KEA and my tubes were mixed up so the plates were re-labeled)
  • Added 1 mL of TA mixture without lysate to the control plate
  • Put plates in the incubator at 26 C at 4:00 8/29
  • Cleaned lab desk with CiDecon and ethanol

Results:

*results from spot test on 8/27                                                                                                                                   Contamination was found on the control plate as well as my plate so plate                           

fig.4 – shows where contamination and bubbles                  fig.7 – shows the plate after incubating for 46 hrs.               are found on the plate

                          

fig.5 – shows where contamination was found                     fig.6 -show the dots of contamination on the control plate  on the control plate

Analysis and Interpretation:

The results from the spot test indicated that there was no bacteriophage present in the soil A, however the contamination observed prevents this from being confirmed in the spot test.

Future Plans:

I plan to check the plaque assay for plaques during the next lab. If no plaques are present then a new soil sample will be collected. The new soil samplewill then have LB broth added and filtered. Then a new spot test and plaque test will be done (not all in one lab). If a plaque is observed the plate will be saved and a new test to confirm the presence will be done. If there is contamination the test will be done over trying to prevent contamination again.

Category: Emily Balint | LEAVE A COMMENT
August 30

Lab Day 3: Plaque Assay

After two days, my plate looks cloudy compared to the control. The control plate was very clear with no sign of contamination. My plate looks like it had been contaminated (probably from my errors in Lab Day 2) and looked like some parts of TA didn’t settle properly.

Detailed Procedure

  1. Took 0.5 Arthro vial from the back and added 10 microliters of enriched lysate with a pipette in the aseptic zone. Let it sit for 15 mins.
  2. With rest of group, add 2.0 mL of LB Broth and 2.5 microliters of 1M CaCl2 with a pipette
  3. As soon as each group members waited 15 mins, we added 2.5 mL of 2X TA.
  4. Spilt above mixture equally into 3 vials.
  5. Took one of the vials and added the mixture from step 1 and used pipette to mix it thoroughly.
  6. My vial tilted over, which resulted in a small spill.
  7. Poured the rest of the vial onto a separate plate labeled as “Plaque Assay”
  8. Wait 10 mins for it to solidfy

Thoughts

  • Due to a small spill in step 6, I was unable to get exactly 4.5 mL onto my plate.
  • This might have caused some contamination.

Questions

  • Can certain oak trees be more prone to bacteria
  • Turns out the soil from the tree my group and I took was from a red oak. If we can get soil from a white oak in our area, we might be able to find plaque from tests. We would need to find another red oak and white oak to test this.
  • Does the present of Arthro appear more dominant in one oak tree species than the others? If so, in this species, is there a correlation between the presence of Arthro and the present of oak wilt fungus growth?

  • plaque assay plate

    control plate from Lab Day 2. Shows no sign of contamination

    signs of contamination. No sign of plaque

    second pic of spot test results

Category: Soo-Un Jeong | LEAVE A COMMENT
August 30

Spot Test Soil A

8/27/18

Spot Test Soil A

Rational:

This experiment will test for the presence of bacteriophage the specifically infect arthrobacter in the soil sample A. A spot test will be used in order to test this.

Procedure:

  • Cleaned lab table with CiDecon and ethanol
  • Used a 2 Mm filter on the enriched soil A to filter it out in order to get 1.5 mL filter sterile (FS) enriched lysate soil A in a microcentrifuge tube
  • Labeled agar plate with FS lysate soil A, direct isolation soil A, and negative control
  • Top agar- 0.5 mL arthrobacter sp. , 4.5 mL LB broth, 5.0 mL 2X TA, and 45.0 ML CaCl2 in a tube (CaCl2 calculations- (1,000 mM)*V1=(4.5 mM)*(10,ooo ML) V1= 45.0 ML CaCl2)
  • For control CaCl2 is 42.75 ML (rounded to 42.8 ML) (calculations- (1,000 mM)*V1=(4.5 mM)*(9,500 ML) V1= 42.75 ML)
  • 45.0 ML of CaCl2 was added to TA mixture
  • Added 4.5 LB broth to TA mixture using serological pipette using 5 mL tip
  • Added 0.5 mL arthrobacter under the clean hood using a micropipette
  • Added 5.0 mL TA using serological pipette with a 10 mL tip
  • Added TA mixture to plate and waited 10 min for agar to solidify
  • Added 10 mL of direct isolation to plate
  • Added 10 mL of FS lysate
  • Added PB (phage buffer) to negative control quadrant on the plate
  • Incubated plate at 26 C from 4:30 8/27 – 2:30 8/29 (46 hrs)
  • Cleaned lab desk with CiDecon and ethanol

*see results on next entry

Future Plans:

During the next lab I will check the results of the spot test. If I observe a plaque I will save the plate and do a plaque assay for the direct isolation and direct lysate for soil A. If there is no plaque I will throw away the plate after recording data and do a plaque assay with enriched lysate soil A to confirm this. If the plate was contaminated then I will do a plaque assay with enriched lysate soil A.

*Note: there were a few bubbles between the negative control and the direct lysate (see fig 3 and 4       

fig.3 – shows where the bubbles are located       fig.2 – the plate after TA was added ( you can see where some of the      on the plate                                                                bubbles are located)

Category: Emily Balint | LEAVE A COMMENT
August 30

Lab Day 2: Spot Test

Detailed Procedure

  1. Add 5.0 mL LB Broth to 50 mL vial in the aseptic zone. Labeled vial as control.
  2. On a different 50 mL vial, labeled it as “E” and put 13.5 mL of LB Broth in aseptic zone.
  3. Add 45 microliters of CaCl2 in control vial.
  4. Add 135 microliters of CaCl2 in E vial.
  5. Took my plate and labeled it into  sections (D,E,B)
  6. Add 5.0 mL of 2X TA into control vial and used pipette to mix it.
  7. Poured control vial onto control plate and let it sit for 15 mins.
  8. While it dries, filter 2 mL of enriched lysate from Lab Day 1 using a syringe filter, and add it into a small cap in the aseptic zone.
  9. Add 1.5 mL of Arthro in E vial
  10. Add 5.0 mL of 2X TA (by mistake, therefore it did not work)
  11. Took 4.5 mL of LB Broth into a separate 50 mL vial and add 4.5 microliters of CaCl2.
  12. Went to the back and got 0.5 mL of Arthro into the vial with a pipette.
  13. Add 5.0 mL of 2X TA and swirled vial to mix (did not use pipette to mix)
  14. Steps 11-13 done in aseptic zone.
  15. Poured separate vial onto plate that was labeled in step 5.
  16. Wait 15 mins for it to dry.
  17. Add 10 microliters of direct, enriched, and buffer onto correct labeled areas of the plate.
  18. Try to prevent bubbles to appear on the plate.

Thoughts

  • There were times where I wasn’t as careful such as spilling some TA on the side of my vial.
  • Bubbles did appear onto the plate and tried to “pop” the bubbles with the pipette.
  • Due to human errors above, plate may result in contamination or inaccurate measurement of spotting each solution.

    Plate for spot test

    control

    process of spot testing in aseptic zone

Category: Soo-Un Jeong | LEAVE A COMMENT
August 30

Lab Day 1: Enriched Lysate

Detailed Procedure

  1. Shake the bag of soil into vial Soil A. It was around 15 mL of lumpy soil.
  2. Add the LB Broth to my vial at the 35 mL mark. (I had to use two bottles of LB Broth since the first one did not fill up to the 35 mL mark.)
  3. Shake the vial by hand and vortex for 15 mins.
  4. Weighed the tube= 52.21 grams
  5. Centrifuge for 5 mins.
  6. Tube is now separated  from soil pellet and supernatant. Looks very cloudy.
  7. After two days, I filtered it out.
  8. To filter, use a dropper to put the supernatant into the tube top filter.
  9. After 23 mins, I poured 12.5 mL of filter supernatant into a 50 mL vial.
  10. Went to aseptic zone to pour arthro into the 50 mL vial labeled as Enriched A
  11. For the rest of supernatant that didn’t filter, put it in a centrifuge for 5 mins  in a 15 mL vial.
  12. Used a syringe filter for 30 mins and got 4.5 mL of Direct Isolation A into a 15 mL vial.

Thoughts

  • As I put the soil in the tube, it came out lumpy and slightly wet. Perhaps this might have affected the result of a cloudy appearance when put in the centrifuge
  • Some large particles in the soil may have clogged up the filtering process.
  • If the soil was dry, the supernatant filtering might have been easier.
Category: Soo-Un Jeong | LEAVE A COMMENT
August 30

Filtration Soil A

8/22/18

Filtration Soil A

Rational:

The soil sample will be filtered out in order to get a sample containing only bacteriophages in the broth. This will allow me to test for the presence of bacteriophage that infects arthrobacter since the bacteriophage will eventually be placed in and environment containing only arthrobacter (no other bacteria).

Procedure:

  • Cleaned the lab desk with CiDecon and 70% ethanol
  • Used burner to set up an aceptic zone and added 40 mL of broth to the soil sample A (weight 60.07 mg)
  • Shook the tube for 15 min to break up the soil sample A
  • The soil sample was then matched with another sample with a similar weight and centrifuged for 5 min at 3,000 G
  • The liquid at the top of the sample was then filtered through a top filter
  • The filtered lysate was then separated into a direct isolation sample of 8.3 mL and an enriched sample of 10 mL (with arthrobacter added)
  • Lab desk was cleaned with CiDecon and ethanol

Future Steps:

I will do a spot test and later a plaque assay using the direct isolationa and enriched lysate in order to test for the presence of bacteriophage that infect arthrobacter in the soil sample A

fig.1 – shows the layers seen in the soil sample                                                                                                                          after the sample was centrifuged

Category: Emily Balint | LEAVE A COMMENT
August 30

Plaque Assay 8.29.18

 Plaque Assay 8.29.18

Rationale)

To conduct a plaque assay using an enriched lysate derived from Soil Sample A in the effort to find and isolate a phage via the presence of a plaque, and if no plaque is formed the plaque assay will determine the necessity of collecting another soil sample to try again.

Results from 8.27.18)

I was able to check my Spot Test from 8.27.18 which based upon careful visual inspection did not have any plaques that had formed in the top agar. Therefore my results were negative, however, there was no evidence of contamination present in the dish. Subsequently, chances are high that another soil sample will have to be collected in order to isolate a lysate to be tested.

       

Procedures)

1. Setup an aseptic zone using CiDecon, 70% ethanol, and an ethanol flame.

2. Retrieve a 50mL conical vial and label “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay”

3. Retrieve micro test tube labeled “Enriched Lysate for Spot Test NMN 8.27.18”, a red-capped vial of arthrobacter, which is then labeled as “Top Agar for Plaque Assay 8.29.18 NMN”, and a plate with base agar, labeled as “NMN 8.29.18 Plaque Assay”.

4. Add 10μL of “Enriched Lysate for Spot Test NMN 8.27.18” to the red-capped vial labeled “Top Agar for Plaque Assay 8.29.18 NMN” and let the arthrobacter and lysate interact for 13 minutes.

5. Add 8mL of LB Broth to “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” under aseptic conditions using a 10mL serological pipette.

6. Add 90μL of 1M CaCl2 to “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” using a 20-200 micropipette under aseptic conditions.

7. Add 10mL of 2xTop Agar to “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” using a 10ml serological pipette under aseptic conditions.

8. Mix, by swirling for 10 seconds, “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” then using a 1000μL micropipette add about 1mL of  “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” to the group 3 section of the Top Agar control plate, put the remaining top agar in the micropipette back into “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” all under aseptic conditions.

9. Distribute 5mL of the remaining top agar in “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” into the 3 separate vials for group 3, including “Top Agar for Plaque Assay 8.29.18 NMN” all under aseptic conditions using a 10mL serological pipette.

10. Mix carefully, by swirling for 15 seconds, “Top Agar for Plaque Assay 8.29.18 NMN” then add the vial’s top agar solution, under aseptic conditions, to the plate “NMN 8.29.18 Plaque Assay” under aseptic conditions, shake the plate gently to evenly distribute the top agar across the surface of the plate.

11. Let the plate solidify for 5 minutes then invert the plate and place it in the incubator for a total of 48 hours with the intent to check on Friday for the development of plaques.

Observations)

I observed minor bubbles when pouring the top agar into my plate which could potentially be mistaken to be plaques if care is not taken when looking for them. I also observed that there were string-like structures forming in the top agar as I was mixing it right before I was about to pour it into the plate. Ultimately we perceived little to no error in the performing of these procedures.

Conclusion)

From the negative result from the spot test on 8.27.18 I can conclude that if the plaque assay produces negative results as well another soil sample will need to be acquired. As Soil Sample A will be shown to not contain a phage that can affect arthrobacteria, however, if the plaque assay produces positive results it can be seen that Soil Sample A may contain arthrobacter phage and the spot test or the manner in which it was conducted was ineffective in allowing the formation of plaques.

Next Steps)

The next step in conducting this plaque would be to wait about 48 hours or until Friday to check and see if the plaque assay produces positive or negative results. This would be determined, respectively, by the presence or absence of a plaque in the plaque assay that I conducted. If a plaque was formed then the plaque could be picked and a phage could potentially be isolated from that, however, if the results are negative another soil sample will have to be acquired.

Update from 8.31.18)

As of checking Friday, August 8th, 2018 the plaque assay, “NMN 8.29.18 Plaque Assay”, produced negative results in terms of the formation of plaques, however, the bacteria growth was very grainy in appearance potentially illustrating an issue with the top agar mixing or setting. Either way, based upon the two negative results I have observed in both the previous spot test and this plaque assay it can be concluded that lysate from Soil Sample A did not contain any phage that infected Arthrobacteria. Subsequently, I will have to collect another soil sample, Soil Sample LH, in the hopes of isolating a phage.

Category: Nathan Newton | LEAVE A COMMENT
August 30

8/27 – Spot Test Procedure

Rationale: After obtaining my lysate, I will be performing a spot test to see if there are any bacteriophage present in my lysate that will infect arthrobacter.

 

Procedure:

  • First, we cleaned our lab area/station with CiDecon and 70% ethanol to create an aseptic area where we can work with our samples, minimizing the chance of contamination
  • I then isolated my lysate via a 3 mL syringe and filtering; I ran the lysate through the filter and into a microcentrifugetube (Labled JY 8/27/18 FS lysate) [FS stands for filtered sterile]
    • The filter was 22μL in size
  • My group then got 4 petri dishes; one singular dish for each member, as well as a dish for our team that we will use as our top agar control
    • Throughout this blog entry (and later entries), I will abbreviate top agar as TA
    • For my personal plate, I drew on the bottom and split the plate into three separate thirds and labeled them as “Negative Control, FS Lysate Soil A (Enriched), and FS Lysate Soil A (Enriched)
      • When I created my enriched lysate, I was only able to filter out 10 mL, so I wasn’t able to create an direct isolation
      • The plate was also labeled Spot Test Soil A
    • Our team plate was labeled “Team 2 8/27/18 Soil A TA Control”
  • We then did calculations to see how much of the following materials we needed for our TA
    • For my personal TA, I need 0.5mL Arthrobacter, 4.5 mL LB Broth, 5mL 2x TA, and 45μL 1M CaCl2
    • For our teams TA, we needed 4.5mL LB Broth, 5mL 2x TA, and 42.8μL 1M CaCl2
  • We used a blue pipette w/ a yellow sticker (The P200 pipette) [Min. 20μL and Max 200μL] to transfer 42.5μL of 1M CaCL2 to a 50 mL conical vial
    • This vial became our Team 2 TA vial
  • I used the same pipette to transfer 45μL of 1M CaCL2 to a 50 mL conical vial
    • This vial became my personal TA vial, and was labeled accordingly with my initials, date and TA description
  • We then used a cartwheel pipette with a 5mL tip to transfer 4.5 mL of LB Broth into both our team vial and my personal TA 50 mL vial
  • I then took my vial to Lathan (Our TA) to have 0.5mL Arthrobacter added into my 50 mL TA vial
  • We then added 5mL of 2X TA (Via a cartwheel pipette with a 10 mL tip) into both our team vial and my personal TA 50 m vial
  • The contents of my vial was then immediately poured into my petri dish, and allowed to sit for 10 minutes for the TA to solidify
    • The contents of the team’s control TA vial was also poured into the team’s petri dish
  • I then used a white pipette (The P10 pipette) [Min. 1μL and Max 10μL] to drop 10μL of my FS Lysate to both sections on my plate
  • I also dropped 10μL of phage buffer into the Negative Control area of my petri dish
  • All our dishes were placed into incubators at room temperature
  • We cleaned and sprayed down our work area with CiDecon and 70% ethanol

 

Observations:

  • In one of my thirds labeled “FS Lysate”, there were bubbles from when the TA was added
  • Using the cartwheel pipette proved harder than anticipated, since it is based on how fast you release the liquid

Image of my plate after the drop of lysate was added

The circled areas are where we noticed bubbles in the TA

 

Next Steps:

  • The next steps would be to see if the drop test turned out positive or negatively, and regardless of the outcome, proceed with a plaque assay, which will also give us information if there is bacteriophage in my lysate
Category: Justin Yu | LEAVE A COMMENT