September 20

9/19/18 Round Two of Plaque Picking/Plaque Assays

Rationale: Test to see if the bacteriophage is present by performing multiple plaque tests.

Question: Why did 10^0 serial dilution not have any plaque, yet both 10^-1 and 10^-2 have plaques?

  • From group four, only Justin had successful plaques in all three serial dilutions. Cooper had only one successful serial dilution, and Michael had two successful serial dilutions.
  • Rule out the possibility of contaminations since Group 4 used the same formula for the plating solution.
  • Control was contaminated, but all groups had a contaminated solution,
  • One reason why plaques are not showing up in the 10^o serial dilution is that the bacteria could be in a lytic cycle, whereas Justin’s bacteria is clearly in a lysogenic phase. The bacteria in both Michael’s and Cooper’s experiment could both be in the lytic cycle, meaning the bacteria are “temperate” and not as expressive as a lysogenic cycle.

Procedure:

Before starting we had to create an aseptic zone to ensure that all bacteria were killed, and the working space would not contaminate our experiments.

  1. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
  2. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.
  3. We then got an ethanol burner, and our aseptic zone was created.
  • Picked Plaque from Plaque Assay from the 10^-1, which was performed on 9/17/18.
    • 10^0 did not have any plaque.
    • 10^-1 had three plaques.
    • 10^-2 had one plaque.
      • Side note: Since 10^0 did not have plaque, this experiment performed on 9/19/18 was done to test whether or not phage is present by only performing two 10^0 plaque assays. No serial dilution was done since the purpose of this experiment was to simply see whether or not phage presence from the 10^o.
  • Added Plaque to the 100 microliter PB, and pipetted/mixed well through the microcentrifuge. Labeled this solution as 10^0 solution on the microcentrifuge.
  • Once this was done, went to get a 50mL vial to make the solution needed for the plaque assay.
    • This formula was used to make our solution for 7 plates (two for each of the 10^0 solutions and one for the control).
      • 20mL LB Booth (x7)
      • 22.5 microliters of Calcium Chloride (x7)
      • 25mL 2X TA (x7)
  • Added the TA last to each of the vials, shook the vial, and quickly poured the solution onto the plates.
  • Sat each plate for about 15mins to the solution solidify.
  • The remaining solution that was left in the 50mL vial was used for the control.
    • Added TA and poured that solution onto the last plate.

10^0 Plaque Assays

Control

Observations:

  • Group four had successful plaque assays from the experiment performed on 9/17/18.
  • Justin had plaques on all three serial dilutions, whereas Cooper and Michael did not. Michael had plaques on his 10^-1 and 10^-2, and Cooper had only one plaque on his 10^-2 dilution.

 10^0 Plaque Assay

10^-1 Plaque Assay

10^-2 Plaque Assay

  • control was contaminated.
    • Possible reason: LB broth contamination.

Next Steps/Conclusions:

On Monday, check all two plates to see if any plaques appear. If plaques do appear, perform the experiment again by picking the plaque of one of the plates. If no plaque, simply redo the experiment with the plaques assay that does have plaque (experiment performed on 9/12/18). Overall, the experiment was very easy since the experiment was done on Monday. The hard part was to determine where the plaques were, and whether if or not plaques were actually there, which they were.

 

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
September 20

9.19.18 Plaque Assay and Spot Test

Rationale)

To conduct a spot test and a plaque assay in order to test, via the formation of plaques, for the presence of phage in lysate produced from Soil C and to finish collecting metadata on Soil C.

Procedures)

  1. Setup an aseptic zone by wiping the work area with CiDecon and 70% ethanol, light an ethanol flame.
  2. Labeled two 50mL conical vials “NMN 9.19.18 Top Agar for Plaque Assay” and “NMN, HMB 9.19.18 Top Agar for Plaque Assay Control” respectively.
  3. Added 2mL of LB broth to both conical vials.
  4. Added 22.5 microliters of 1M CaCl2 to both vials.
  5. Retrieved “NMN 9.17.18 Soil C Lysate Enriched” and filtered enough lysate with .22 micron syringe filter to fill a micro test tube, labeled the tube “Soil C FLE NMN 9.19.18”.
  6. The LB broth was found to be contaminated, the top agar control vial was kept to positively test for contamination of the LB broth and its lid was colored to denote its purpose, “NMN 9.19.18 Top Agar for Plaque Assay” was discarded, two new 50mL conical vials were then labeled the same as the discarded ones.
  7. Added 10 microliters of FLE to a vial with .5mL of arthrobacter. Left to infect for 15 minutes.
  8. Added 2mL of uncontaminated LB broth to both of the new 50mL conical vials.
  9. Added 22.5 microliters of 1M CaCl2 to both of the vials.
  10. Collected three plates with base agar, labeled them “NMN 9.19.18 Plaque Assay Soil C”, “Top Agar Contamination Control 9.19.18”, and “Top Agar Control 9.19.18”.
  11. Added .5mL of infected arthrobacter to “NMN 9.19.18 Top Agar for Plaque Assay”.
  12. Added 2.5mL of 2xTop Agar to all three vials.
  13. Shook vials to combine components for 10 seconds and poured them into their respective plates, let them sit for 15 minutes then they were inverted and incubated for 48 hours.
  14. Collected two 50mL conical vials, labeling one “TA Control for Spot Test 9.19.18 HMB NMN” and the other “TA for Spot Test 9.19.18 HMB NMN”.
  15. Added 2mL of LB broth to both vials.
  16. Added 22.5 microliters of 1M CaCl2 to both vials.
  17. Added .5mL of arthrobacter to “TA for Spot Test 9.19.18 HMB NMN”.
  18. Collected two plates labeling one “Top Agar Control Spot Test NMN HMB 9.19.18” and the other “9.19.18 HMB NMN Spot Test Soil C”, divide the latter plate into thirds labeling one NN, HB, and the other Control.
  19. Add 2.5mL of 2xTop Agar to both vials, swirl to combine and pour into their respective plates. Shake the plates slightly and let sit for 15 minutes.
  20. Retrieved the weigh dish labeled “NMN %H2O Soil C” and take the mass. The mass was 5.761, the mass of the water was thus found to be .599g, and the percent water of the soil was 14.975%.
  21. Retrieve the falcon tube from 9.17.18 and find the milliliters of sand, silt, and clay. Sand was 2mL. Silt was .5mL. Clay was 2.5mL.
  22. Added 10 microliters of each FLE and phage buffer to their respective sections of “9.19.18 HMB NMN Spot Test Soil C”, control and NN. Let sit for 12 minutes.
  23. Retrieved a pH vial and added 2mL of water from the falcon tube, top off the vial with DI water, shake 10 seconds, then place a 1-inch strip of pH paper into the tube for 30 seconds. The pH was found to be 6.
  24. Put the spot test plates in the incubator inverted, leave for 48 hours. Clean the workspace and leave.

Observations/Data)

We observed that our initial LB broth was cloudy, which indicates contamination, as our second container of LB broth was clear as LB broth is supposed to look. I also observed that there was 2mL of sand, .5mL of silt, and 2.5mL of clay in the falcon tube in which I dispersed the soil sample. Therefore Soil C was 40% sand, 10% silt, and 50% clay. The dried sample of Soil C was 5.761g with the tray and 3.401 without. Compared with the original 4 grams there was a total of .599 grams of water lost. Thus, the percent water of the Soil C was 14.975%. The soil pH was also observed to be 6.

Conclusions/Next-Steps)

From the data I gathered I can conclude that Soil C was a clay soil type, I can also determine the percent water was 14.975%, and the pH of the soil was slightly acidic at 6. Our next steps will include checking for the presence of plaques on both my plaque assay and spot test on Friday, which will determine if we can begin picking and purifying plaques or if we need to collect more soil. We will also check for the presence of bacterial growth in out top agar controls to determine if we need to reassess our process of conducting the procedures, as could occur with such things as contaminated LB broth which occurred when we conducted the procedures today.

Category: Nathan Newton, Uncategorized | LEAVE A COMMENT
September 20

9/17/18 Plaque Picking/Serial Dilution/Plaque Assay

Rationale: Pick Plaque from the plaque assay and to perform serial dilutions. Performed several plaque assays to isolate phage, and made a sample control to go along with our experiment.

Procedure: 

Before starting we had to create an aseptic zone to ensure that all bacteria were killed, and the working space would not contaminate our experiments.

  1. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
  2. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.
  3. We then got an ethanol burner, and our aseptic zone was created.
  • Picked Plaque from Plaque Assay, which was performed on 9/12/18.
  • Added Plaque to the 100 microliter PB, and pipetted/mixed well through the microcentrifuge. Labeled this solution as 10^0 solution on the microcentrifuge.
  • Got two more microcentrifuge caps, labeled one cap 10^-1 and the other solution 10^-2.
  • Added 90 microliters of PB to both caps.
  • Added 10 microliters of the 10^0 solution into the 10^-1 solution.
    • The 10^0 solution has the picked plaque. Transferred this to the 10^0 solution so that this could be added to the other two caps so that three Plaque Assays could be performed.
  • Added 10 microliters of the 10^-1 solution to the 10^-2 solution.
  • All microcentrifuge caps had plaques, added 10 microliters of Arthrophage to all three microcentrifuge caps (10^0,10^-1, and 10^-2).
  • Once this was done, went to get a 50mL vial to make the solution needed for the plaque assay.
    • This formula was used to make our solution for 10 plates (three for each of the three solutions and one for the control).
      • 20mL LB Booth (x10)
      • 22.5 microliters of Calcium Chloride (x10)
      • 25mL 2X TA (x10)
  • Added the TA last to each of the vials, shook the vial, and quickly poured the solution onto the plates.
  • Sat each plate for about 15mins to the solution solidify.
  • The remaining solution that was left in the 50mL vial was used for our control.
    • Added TA and poured that solution onto the last plate.
      • Side note: the control solidified <15 minutes.

Observations:

  • Group 4 had plaques on all plaque assays, but only Justin had a plaque from his spot tests.
  • On the plate of the plaque assay, it was very hard to identify a solid plaque but contained multiple little spots that could well be possible plaques.

 Plaque Assay with Plaque

  • Spot Tests had zero plaque but had air bubbles. Justin had spots form group four.

Additional Questions:

Question 1. Group four all had plaques from their plaque assays, but only Justin had a plaque from his spot test.  This could have resulted from the soil since all three group members got soil from three different trees. Justin’s soil could have different phage from his group members, resulting in Justin’s better-defined plaques. The soil samples from group 4 at least have some similarities, given everyone in the group had a successful plaque assay.

Question 2. 

Lathan needs 4.01 microliters to web his plate (work is shown above).

 

Next Steps/Conclusions:

On Wednesday, check all three plates to see if any plaques appear. If plaques do appear, perform the experiment again by picking the plaque of all three plates. If no plaque, simply redo the experiment with the plaques assay that does have plaque. Overall, the experiment was very easy since Lathan’s lecture video basically covered everything that was needed to be known for this week lab. The hard part was to determine where the plaques were, and whether if or not plaques were actually there, which they were.

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
September 19

9.19.18 Spot Test and Plaque Assay for Soil Sample C

9.19.18 Spot Test and Plaque Assay for Soil Sample C

Rationale: A Spot Test and Plaque Assay will be performed to analyze the composition of the lysates from Soil Sample C. The two tests will reveal if a phage is present through the presence or absence of a plaque on the bacterial lawn.

Procedure: (Metadata steps in italics)

  1. Cleaned lab bench and lit burner for aseptic zone.
  2. Obtained conical tubes and labeled “HMB Plaque Assay Soil C 9/19/18” and “Top Agar Plaque Assay Control 9/19/18”
  3. Added 2mL of LB broth to both tubes
  4. Added 25μL of CaCl2 to both tubes.
  5. Used syringe and filter to create at least 10μL of Filter Sterilized Enriched Lysate. Placed in microcentrifuge tube labeled “HMB FSEL 9/19/18”.
  6. It was observed that the LB Broth used in steps 3 and 4 was cloudy. This was thought to be due to contamination, so the control tube was kept and tested to confirm this thought while the other two tubes were remade (redid steps 3-4) with clear LB Broth.
  7. Added 10μL of Filter Sterilized Enriched Lysate to 0.5mL Arthrobacter. Let sit for 15 minutes.
  8. Obtained 3 new plates and labeled them “HMB Plaque Assay Soil C 9/19/18”, “Top Agar Contamination Control”, and “Top Agar Control 9/19/18”.
  9. Added 0.5mL Arthrobacter with Filter Sterilized Enriched Lysate to conical tube labeled “HMB Plaque Assay Soil C 9/19/18”
  10. 2.5mL of 2X Top Agar was added to all 3 tubes and was swished to mix.
  11. Poured Top Agar solutions on to respective plates. Let sit for 15 minutes before incubating.
  12. Obtained new 50mL conical tube labeled “TA Control for Spot Test HMB NMN 9/19/18” and “TA for Spot Test 9/19/18 HMB NMN”.
  13. 2mL of LB Broth and 22.5μL of CaCl2 were added to Conical Tubes
  14. 0.5mL of Arthrobacter added to experimental vial
  15. 2.5mL of Top Agar was added to both Conical Tubes.
  16. Conical Tubes were swished, then poured onto their respective plates.
  17. At this point, it was observed that after letting the “HMB Plaque Assay Soil C 9/19/18” plate sit for 15 minutes that the Top Agar had broken and slid around the plate. Therefore, a new plate was created using steps 3-4, 7, 9-11 in that order. It was also given a new 15 minutes, then placed in incubator.
  18. Weigh boat containing soil from Monday was reweighed and found to be 5.689g. 5.689g-2.43= 3.259g. 3.259g/4=0.815. 1-0.815= 0.185 –> 18.5% H2O.
  19. Reexamined Soil Separation: 6mL of soil was separated. Measurements were found to be 2mL of sand (33.3%), 1mL of silt (16.7%), and 3mL of clay (50%). Soil was properly disposed of and tubes were cleaned.
  20. pH tube and paper were obtained. Small amount of supernatant from soil separation sample was placed in bottom of pH tube and the rest of the tube was filled with Deionized Water. pH paper was placed in sample for 45seconds, then examined. Found to be a pH of 6.5 — more basic than previous sample.

Observations

  • pH of Soil Sample C is more basic than Soil Sample B. It will be interesting to see if this has anything to do with the presence of a plaque as a possible secondary study to the overarching question currently posed.
  • When Top Agar did not set, plate was shaken somewhat vigorously to attempt to prevent Top Agar from setting with bubbles present. This likely caused the plate to not set correctly, creating slipping of top agar. Important to note for next time.
  • There was much more clay in Soil Sample C than found in previous samples.
  • Cloudy LB Broth could be the result of contamination that has been previously observed throughout the lab.

Next Steps: The next component of the process is to reexamine the plates on Friday to determine whether or not there is a phage. If there is a phage, we will be able to move ahead in the process of picking a phage on Monday, but if not, I will obtain a new soil sample over the weekend.

Conclusions:

  • Over-swirling Top Agar likely results in slipping and dividing of Top Agar.
  • Cloudy LB Broth could have been the cause of contamination viewed on many of the plates.
Category: Henry Burns | LEAVE A COMMENT
September 19

SEA Bears Day 7

17 September 2018 ✷ Third Try’s a Charm?

Within Lab Group 6, two spot tests and one plaque assay were run, and all yielded negative results (see below). Thus, it was decided it was highly improbable that the sample was positive, so new soil was collected (again) in the same fashion as the previous time in order to try and locate a phage.

Lathan’s Questions

  1. The sample Justin used likely had a higher phage concentration in it (a stronger titer).

Procedure

  • As in the previous attempt at locating a phage, a soil sample was collected from a red oak outside of the Baylor Science Building (the difference from the last collection is the location of the tree).
  • Soil was collected from 2 ft away from the base of the tree, but no specific data was collected other than photos of the tree. Circumference, crown diameter, and tree damage data were collected.  These data and images of the tree can be seen below.
  • Upon returning to the lab, a small sample of the newly collected soil was set aside in a previously weighed weighboat to perform a test for percent water. It was left to dry under the fume hood until Wednesday’s lab period.
  • The remaining soil was refrigerated until Wednesday.

Observations/results/data

  • Circumference of tree: 102 cm; crown spread: 375 cm; some broken branches
  • component mass (g)
    empty weigh boat 2.41
    weigh boat + wet soil 6.66
    wet soil (6.66-weigh boat) 4.25
    dry soil + weigh boat 6.39
    dry soil (___-2.41) 3.98
    Water (wet soil – dry soil) 0.27
    % water (water/wet soil) 6.4% water
  • Unlike the last tree, the most recent tree had more grass covering the base of the tree/roots. The correlation between this and phage presence is unknown, but hopefully it leads to the discovery of a phage.

Interpretations/conclusions/next steps

  • The soil collected today will be enriched next time and then spot tested in order to hopefully isolate a phage. The percent mass test will be concluded and more metadata, including percent sand, silt, and clay will also be conducted.
  • The soil collected today is significantly less sticky (less clay) than the last soil sample. Again, the correlation is not known at this time, but this may possibly be a better environment for phages to infect arthrobacter and yield a positive spot test for presence of phage.
Category: Lily Goodman | LEAVE A COMMENT
September 19

SEA Bears Day 8: Enrichment 3+metadata

19 September 2018 ✷ Third Enrichment + Metadata

Soil will be washed and enriched in order to help isolate a phage (if there is one) and metadata will help compare positive/negative results with other groups’ data in order to look for trends.

Procedure

  • The workspace was cleaned with CiDecon and 70% ethanol and an alcohol burner was lit to promote an aseptic environment.
  • 2 mL of soil was collected in a vial and roughly 10 mL of LB Broth was added and the vial was vortexed for 15 minutes to mix. The sample was then centrifuged for 10 minutes at 10,000g.
  • In the meantime, the mass from the drying % Water test was massed and percent water was calculated to be 6.4% (see Day 7).
  • Another vial was filled with 4 mL of soil and roughly 8 mL of DI water. 3 drops of soil dispersion fluid was added, then the sample was shaken for 30 seconds, decanted into a falcon tube, and stored until Monday. This is the test for percent sand, silt, and clay.
  • pH was determined by placing a small amount of dirt in a pH tube and adding DI water, shaking to mix, and inserting a pH strip, which was compared to the test kit’s indicator chart.
  • After the enrichment sample was done spinning, it was filtered with a 20 micron syringe filter and 9 mL was set aside to be enriched and 1 mL was set aside to be the direct sample. The direct lysate was refrigerated.
  • 0.5 mL arthrobacter was added to the “enriched” sample and left in a shaking incubator until Monday.

Observatios/Results/Data

pH= 6.0

This sample is more acidic than the previous sample.

Interpretations, Conclusion, Next Steps

Next time, a spot test will be run in order to determine if phage is present or not.

Overall, the repetition of the same procedure three times because of two negative results has led to more confidence in the tests and hopefully the third attempt yields a positive result.

Both Group 5 and Group 6 had all negative results from the initial test location. This attempt compared the same types of trees in a different location, which limits the variables at play and only changes the research question from “near North Village” to “near the Science Building.”

Category: Lily Goodman | LEAVE A COMMENT
September 19

September 19, 2018 Soil Washing, Enrichment, and Metadata- Soil C

Rationale: After a negative plaque assay and a contaminated negative control, the purpose of this lab is to collect new soil, wash and enrich it, and collect metadata for the soil.

Description of Procedures

  1. Soil was collected from a red oak tree, as well as the tree’s dimensions.
  2. The work space was cleaned using aseptic technique. An ethanol burner was lit to create an aseptic zone.
  3. 2 ml of soil and 8 ml of LB broth were added to a tube. The tube was shaken for 10 minutes, and then massed. The mass was found to be 17.74 g.
  4. The soil was spun in the centrifuge for 10 minutes at 5000 x g.
  5. While spinning, the % water test was begun. The weigh boat was found to be 2.32 g, and the weigh boat with soil was found to be 5.85 g. This will sit in the vented hood until the next lab to allow the water to evaporate.
  6. Next the % concentration test was started. 10 ml of soil was added to a tube along with 20 ml of DI water. 3 drops of soil dispersion liquid was also added. The tube was then shaken for 30 seconds. It will sit under the vented hood until the next lab to allow the soil to separate into its layers.
  7. The pH was found next. A small amount of soil was added to the pH vial, and then it was filled with DI water. The tube was then shaken for 10 seconds and then allowed to sit for 10 minutes.
  8. While sitting, the tube containing soil was filtered with a 0.22 um filter and syringe. Approximately 7.75 ml of enriched lysate was obtained. No direct lysate was made. 0.5 ml of arthrobacter was added to this tube. It was labeled LIP 8-19-18 Enriched- Soil C. The tube will be shaken at room temperature until the next lab.
  9. Next the pH was found to be 6.0, using pH paper.
  10. The work station was cleaned using aseptic technique and the materials were properly stored and disposed of.

Observations/Results/Data:

  • Observations:
    • The soil collected was moist and dark in color.
    • The soil was collected from a red oak tree, along with a leaf.
    • The filtration process was easier than with Soil B.
  • Data:
    • Weigh boat- 2.32 g
    • Weigh boat and soil- 5.85 g
    • pH- 6.0
    • 7.75 ml of enriched lysate collected
    • Tree circumference- 60.5 cm
    • Tree canopy- 303 cm
    • Tree height- 800.5 cm

Red Oak Tree- Soil Source

Negative Plaque Assay

Contaminated Control Plate

Interpretations/Next Steps/Conclusions:

The soil collection, washing, and enrichment processes were complete. Of the metadata, the soil pH was found, and the procedures for % water and % soil concentration were started, and will be completed in the next lab. The next step will be to run a spot test or a plaque assay to look for plaques. Soil B yielded no plaques, which is why Soil C was collected. Also, the control plate for the plaque assay for Soil B was contaminated.

Category: Lucy Potts | LEAVE A COMMENT
September 19

Gram Staining Contamination 9/19/18

Rationale: The plaque assay resulted in a contamination, so I will gram stain the contaminant in my control plate and the arthrobacter lawn in my plaque assay plate to see if it was arthrobacter contaminating my control plate.

Procedure:

  1. Took loops and heated them to clear off contaminants. Pipetted 10μL of

    water onto two sections of a microscope slide.

  2. Dragged loop through large culture on contaminated control plate and placed

    into the water. Repeated with arthrobacter lawn from plaque assay after

    heating the loop.

  3. Let air dry then heat fixed the bacteria to the slide. Flooded slide with crystal

    violet dye and let sit for about 1 minute.

  4. Rinsed off dye then flooded slide with Lugol’s Iodine and let sit for 1 minute

    and rinsed off.

  5. Added a small amount of ethanol to slide and rocked around slide for about

    20-30 seconds. Then rinsed off.

  6. Finally flooded with safranin dye and let sit for another minute then rinsed

    off and dried.

  7. Observed under 1000x magnification using oil immersion.

Observations:
The contamination turned out to be a gram-positive bacilli bacterium. Whereas, the arthrobacter lawn looked more like a gram-negative cocci bacterium. This shows that the contaminant was, most likely, not from accidentally getting some of the arthrobacter onto the plate but instead a bacterium that may have fallen into the plate.

Image on the left is contaminant. Image on the right is arthrobacter.

Interpretations and Next Steps:
This means that we need to be extra carefully about not accidentally breathing on the plate or touching the inner parts of the lids of anything we use in class. Also, we should be more diligent about staying within the aseptic zone created by our flame. That way, the likelihood of something falling into our plate or top agar mixture is greatly reduced. Furthermore, we should try to be as quick as possible in order to minimize time that the plate is open to the air. For next steps, now that I have collected soil samples from saplings in Cameron park we can wash and run plaque assays on those. This time being more diligent about using aseptic technique so that this won’t happen again.

Category: Uncategorized | LEAVE A COMMENT
September 19

9.17.18 Soil Collecting and Washing

Rationale)

To collect another soil sample, and to collect metadata from the sample in addition to washing the soil sample in order to produce a direct and enriched lysate that can be used in plaque assays and spot tests in an effort to identify a plaque.

Results from 9.14.18)

My plaque assay tested negative for the presence of plaques when checked, our top agar control was contaminated as indicated by small bacterial colonies appearing throughout the top agar.

Questions)

  1. The reason had the most well-defined plaque was likely due to his lysate likely being a higher titer, or concentration of phage compared to the other lysates in his group, which likely had a lower titer, or concentration of phage in their lysate, which explains why they would have a less defined plaque than a plaque created with Justin’s higher phage concentration  lysate.
  2. 4.018 microliters of 10^0 lysate are needed to web the plate.

Procedures)

  1. Sat up an aseptic zone by wiping down the work area and lighting an ethanol flame.
  2. Collected a soil sample and a leaf from the tree it was near, I also recorded metadata for the soil, I stored the soil and leaf in a bag labeled “NMN 9.17.18  Soil C+Leaf Earle Hall”.
  3. Added 2mL of the soil from the bag to a 15 mL vial and labeled it “NMN 9.17.18 Soil C”
  4. Added enough LB broth to the vial to fill it to the 12.5mL mark.
  5. The vial was massed and shaken for 15 minutes. Mass was 20.128.
  6. Added enough DI water to the vial to reach 21.028g, in order for it to be centrifuged properly.
  7. Labeled a weigh tray and label “NMN Soil C %H2O”, added 4g of Soil C and placed under the vent hood for 48 hours. The weigh dish weighed 2.36g empty.
  8. Added 4mL of Soil C to a falcon tube and 10mL of water, added 3 drops of soil dispersion fluid and shook to mix. Let settle for 48 hours. The tube has “ss” on it.
  9. Centrifuged “NMN 9.17.18 Soil C”
  10. Filtered the supernatant of the centrifuged vial reserving 10mL for an enriched lysate in a 50mL conical vial labeled, “NMN 9.17.18 Soil C Lysate Enriched”, and .5mL for a direct isolation in a micro test tube labeled, “NMN 9.17.18 Direct Isolation”.
  11. Added .5mL of arthro to the vial containing the lysate designated for enrichment, placed the vial on the shaking table for 48 hours.
  12. Stored lysates and soil samples in the fridge and wiped down the table.

Data/Observations)

I observed that the supernatant was far clearer than previous supernatants I had filtered, however, the amount of time and the g’s that the vial was subjected to are unknown due to lack of information on the centrifuge. I also observed that the tree from which we gathered the soil was very healthy and relatively recently planted, additionally the soil was primarily brought in topsoil with only a little of what appeared to be native soil.

Conclusion/Next Steps)

Based on our negative results from our plaque assay conducted on Friday we proceeded to collect and wash another soil sample in order to produce a direct and enriched lysate. Our next step will be to conduct a plaque assay and spot test using the lysates produced in this procedure in an effort to find and isolate a phage via the formation of plaques in either or both tests.

Category: Nathan Newton | LEAVE A COMMENT
September 17

9.17.18 Obtaining and Washing Soil Sample C

9.17.18 Obtaining and Washing Soil Sample C

Questions posed by Lathan:

  1. The reason why Justin’s plate contained phage in the spot test and the plaque assay and the rest of group four only displayed plaque in the plaque assay was due to the titer of their solutions. Justin’s likely had the highest titer, or concentration, of phage in the plated sample, which would have allowed the spot to clear a plaque on the lawn growth. A lower titer would not be able to display a plaque in the same manner, which would have explained why the rest of group four had the results they did.
  2. 4.018μL

Rationale: Since previous soil samples (Soil Sample A and Soil Sample B) did not contain a plaque that contained phage, it became necessary to obtain a third sample and prepare it (by washing the soil and collecting metadata) for testing that will occur on Wednesday in the form of a Spot Test and Plaque Assay.

Data from Friday, September 14

The Plaque Assay performed on September 14 needed at least 48 hours to develop results, which were first available to be observed during this lab session. As predicted by the Spot Test, there were no plaques on the plate containing lysate from soil sample B. However, the negative control exhibited slight presence of small Arthrobacter colonies of unknown origin. These colonies could be due to an error in preparation, or they could be from general contamination in the incubator or otherwise. Furthermore, there are uneven areas present on the experimental plate that cause imperfections in the lawn growth. These breaks are likely caused by uneven top agar spreading rather than phage, so they have no bearing on result. Pictures below illustrate these occurrences.

   

Procedure: (Sections in italics are metadata related)

  1. Cleaned lab bench. Lit ethanol burner to create aseptic zone.
  2. Observed plates with results and observations listed above.
  3. Obtained materials for soil collection and obtained soil from an Earle Hall tree. Placed soil in bag labeled “HMB Soil Sample C Earle Hall”. Conical Tube labeled “HMB Earle Hall Soil Sample C 9/17/18”.
  4. 2mL of soil was added to Conical Tube.
  5. 12mL LB Broth added to Conical Tube.
  6. Mixture of LB Broth and soil was shaken for 15 minutes. During the 15 minute period, the mass was found to be 20.795g. To match Nathan’s sample for centrifuging, water was added to the tube to make it 21.028g.
  7. Sample was centrifuged for 10 minutes to pellet soil and waste.
  8. Metadata: Weigh boat was massed and found to be 2.43g. 4g of soil was added to weigh boat. After, the weigh boat labeled with a “2” and distinguished by a crack in the plastic was placed under the hood to be reexamined on Wednesday. 
  9. 4mL of soil was added to a 50mL Falcon Tube.
  10. 8mL of DI water was added to 50mL Falcon Tube.
  11. 3 drops of Soil Dispersion Solution were added to Falcon Tube. Tube was shaken for 30 seconds to mix soil. Soil let sit until Wednesday.
  12. Syringe and filter used to add 10mL of lysate to 50mL conical tube and about 1.5mL of lysate into microcentrifuge tube as a direct lysate (which was labeled “HMB D 9/17/18” and placed into the fridge for storage).
  13. 0.5mL of Arthrobacter was added to 50mL conical tube to create enriched lysate. Labeled “HMB Enriched 9/17/18”.
  14. Enriched lysate was stored in shaker to be recovered in 48 hours.
  15. Lab station cleaned and materials were returned to places they were found.

Observations

  • During the metadata section, the soil seemed to separate more evenly than the first analyzed sample.
  • As filter was used more during the filtration step, it became more and more difficult to push the lysate through the syringe. However, the tension never let up, which confirmed that the filter was at no point broken or dysfunctional.

Next Steps: On Wednesday, the enriched lysate will be used to perform both a Spot Test and a Plaque Assay. This will allow the sample to be read on Friday, which will reveal whether or not Soil Sample C had a viable phage that was causing plaques. Furthermore, if the testing process returns with a negative result, a new sample will be able to be collected more efficiently than if the washing process was put off until Wednesday.

Conclusions:
Since the second soil sample did not have a phage, it was necessary to repeat the process to obtain a third. Hopefully, this sample will contain phage and new procedures learned regarding picking the plaques and creating a webbed plate can be practiced.

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