September 21

SEPTEMBER 17TH AND 19TH- Lab

  • SEPTEMBER 17TH 2018
    • RESULTS: (following results and conclusion are from 9/12/18 and were not viewed until 9/17/18)
      • Spot test, seen in figure 7, was cloudy, had a yellow liquid in the bottom of dish, and was contaminated due to it not being inverted 
      • The control, seen in figure 8, was cloudy from contamination, and also had yellow liquid  gathered in the bottom of the dish, was most likely contaminated from not being inverted 
    • CONCLUSION: 
      • As a result of the contamination the plates were cloudy and had no signs of plaques being present. A plaque assay will be conducted next to determine if there are plaques or not. One group member did have plaques present, and her sample was obtained from Baylor’s campus meaning her soil had been exposed to pesticides. 
    • SPOT TEST ANALYSIS + PLAQUE ASSAY 
    • OBJECTIVE: Conduct a plaque assay with no contamination 
    • PROCEDURE:
      • Tables were cleaned and lamp was lit
      • .10𝝁L of lysate was pipetted into a test tube containing .5mL of arthrobactor and was left to sit for 15 minutes
      • A large test tube (50mL)  was then filled with: 
        • 8mL LB broth
        • 90𝝁L CaCl
        • 10mL 2X TA
      • The control plate accidentally had 9.5 mL of the top agar solution added to the plate, therefore there was only enough for 2 group members to have the solution added to their lysate
      • Next the 4.5mL of the original solution was added to the test tube containing the lysate and arthrobactor 
      • The test-tube was then poured onto the plate and left to solidify for 15 minutes 
      • During the 15 minutes a different group member (SA) made a double batch of the TA solution and conducted a separate control
    • RESULTS: 
      • The control plate, seen in figure 9 had many spots on it from contamination, also had yellow 
      • The spot test is cloudy from contamination as seen in figure 10
      • As seen in figures 9 and 10 there is contamination on both plates. Contamination could possibly be from Micropipettes or LB.   
    • CONCLUSION: 
      • Due to the plates showing no plaques after a spot test and plaque assay, the group had decided to collect new soil samples, and keep continuing investingng the question: Do trees sprayed with pesticides have a higher concentration of phage than trees that have not been treated? (species of tree being test has yet to be determined). Also due to contamination from now on, LB bottles used will be labeled by groups, and all micro pipettes have been cleaned with ethanol to help diminish the chanced of future contamination. 
    • NEXT STEPS:
      • Collect new soil samples from Cameron Park and the Baylor campus 
    • EXTRA- Critical Thinking Problems:
      • 1)Group 4 all had plaques on their plaque assays. Justin had the most and most well defined  plaques (but all 3 had plaques). They each did a spot test in. Addition to their play assays but only Justin had plaque on his spot…what do you think is going on? I think that since Justin had the most, and most well defined plaques that his phages were probably very stable when put into the spot test so his pages were probably the most likely to survive since he had a higher concentration of pace and had “stronger” or more well preserved plaques. The other group members phages probably weren’t in high enough concentrations, or were in a “weaker” state and were unable to be put into a spot test with out falling apart.
      • 2) Lathan Checked a purified lysate by doing a play assay of a 10^-3 lysate. He counted 14 plaques. How many 𝝁L of Lathan’s 10^0 lysate should he add to web a plate (75mm in diameter) if his average plaque diameter is 1mm? .004017 mL
  • SEPTEMBER 19TH 2018
    • GRAM STAINING OF CONTAMINANTS 
    • OBJECTIVE:
      • Determine the source of contamination 
    • PROCEDURE: 
      • .10 𝝁L of water was put on 2 spots onto a slide
      • Inoculating loop was dragged across plate and mixed into one water spot
        • Was done for control and play assay 
      • Water was then left to air dry
      • Then the slides were dragged over a flame multiple times to heat fix the bacteria 
      • Then crystal violet was applied to both samples for 1 minute and were then rinsed 
      • Next, potassium iodine was applied to both samples for one minute and was the washed away with water
      • Then ethanol was applied to the samples for 30 seconds, and was then washed away with water 
      • Lastly, Zafrin was applied to both sampled for 1 minute and was then washed away by water 
      • Samples were dried, then viewed under a compound microscope
    • RESULTS: 
      • As seen in figures 11 and 12 the control and plaque assay were both contaminated by a cocci gram-negative bacteria 
    • CONCLUSION:
      • It appears that arthrobactor was the possible bacteria present on both plates. It was a gram negative cocci, which correlates with arthrobactor. 
    • NEXT STEP: 
      • Collect new soil sample from Cameron Park.

FIGURES 7-12:

Category: Lauren Foley | LEAVE A COMMENT
September 21

Plaque Assay 2 on Soil Sample 2 (9/17/18)

Rationale:

If there is a phage, create a plaque assay to dilute lysate. If the plate is negative with no contamination, collect more soil. If the plate is negative with contamination, perform another plaque assay using the enriched lysate.

 

Procedure:

The plaque assay and spot test conducted previously were checked to see whether or not there were plaques and to also see if there was contamination. Both tests came out negative with contamination; therefore, a plaque assay was performed again. The tables were thoroughly wiped down with CiDecon and ethanol and an aseptic zone was also set up to prevent contamination. 10 µL of enriched lysate and 0.5 mL Arthrobacter were placed in a new tube and left alone for 10 minutes to allow for infection. Next, 90 µL CaCl2 was combined with 2.0 mL of LB broth. However, a problem occurred which led to another method being used. Four vials were being used to create the top agar to ensure correct concentrations. 2.0 mL of LB broth,  22.5 µL of CaCl2, and 2.5 mL 2X Top Agar was added to each vial which was then combined with the lysate and Arthrobacter. The mixture was poured over the plate and then left alone to solidify before being placed in the incubator. After, more soil was collected in case there are no phages present after conducting a second plaque assay.

 

Results and Analysis:

A problem occurred with the calculations of the concentrations of the CaCl2 and the 2X Top Agar which led to another method to create the top agar (top agar made individually).

Contaminated Negative Control

 

Negative Spot Test

 

Negative Plaque Assay

 

After pouring the solution onto the agar plate, there was some lysate and Arthobacter solution left in the microcentrifuge tube. Although it was a small amount, it was a considerable amount.

 

Titer Problem

4.02 µL

 

Conclusion and Future Plans:

Because there was contamination in the negative control and no plaques present in the plate, performing another plaque assay was done to ensure that there were no plaques in the sample. While performing this plaque assay, proper methods were used to prevent contamination in our tests. From chances that there are no plaques in the sample, more soil was collected more future use.

Future Plans:

If there is no sign of plaques on Wednesday (9/19/18), the new collected soil will be washed and enriched in order to conduct another spot test and plaque assay test. If there are signs of phages with no contamination, a plaque will be picked and diluted.

Category: Sabin Patel | LEAVE A COMMENT
September 21

Washing of Soil Sample 3 and Collecting Metadata (9/19/18)

Rationale:

To wash Soil Sample 3 using a syringe and 0.22 µm filter and to also perform experiments to collect Metadata on the soil sample.

 

Procedure:

Due to the negative results from the second plaque assay, the newest soil collection was washed. To determine the next step to be taken, the spot test, plaque assay, and negative control were checked from 2 mL of the sample and 10mL of LB broth were combined in a 50 mL conical vial, then vortexed for 15 minutes. The vial was then weighed to be placed in a centrifuge for 10 minutes at 10,000 g. While it was spinning, procedures were done to determine the soil composition, pH, and % water of the soil. For the pH, a small amount of soil was added to a pH vial in which was filled to the brim with DI water. After shaking for 45 seconds, the vial was left alone on the table for 2 minutes. A strip of pH paper was inserted into the vial for 45 seconds which was then used to determine the pH. The soil composition consisted of mixing 4 mL of soil, 8 mL of DI water, and 3 drops of soil dispersion liquid. Then, the tube was shaken for 30 seconds and left alone for 30 seconds. To measure the % water, the weight of a weigh boat was taken (initial) along with the soil (3 g-5 g)in the weigh boat (final). After the allotted time in the centrifuge, the samples were taken out and filtered using the serological pipette, syringes, and some filters. Out of the lysate, 2 mL became the Direct Isolation and the rest became the Enriched. 0.5 mL of Arthrobacter was added to the Enriched vial and shaken for 5 seconds.

 

Results and Analysis:

Plaque Assay 3 and Negative Control 

It was later found out that the reason for the contamination was because the LB broth was the contaminated.

The LB broth on the left was contaminated and the one that was used.

% Water

Weigh boat- 2.32 g

Weight boat and soil- 6.11 g

Soil- 3.79 g

pH

pH of 6.3

Soil Composition

A little more than 4mL of soil was added due to the large spaces at the bottom despite the many tries to break the clumps down.

Soil Sample 3

Weight: 19.74 g

Direct Isolation and Enriched (without Arthrobacter)

5 mL Enriched

2.5 mL Direct Isolation

 

Conclusion and Future Plans:

The 3rd plaque assay that was conducted on 9/17/18 came back negative and the control was contaminated. It was soon found out that the reason for the contamination was due to the contamination of the LB broth. Today (9/19/18), Soil Sample 3 was washed using a syringe and filter and metadata for % water, soil composition, and pH was collected. There was a total of 7.5 mL of lysate after centrifuging the sample. The procedures to collect the soil metadata was successfully completed.

In the future (9/21/18), the enriched lysate will be filtered again using the same method and the metadata will be collected. If there is enough time, a plaque assay will be performed.

Category: Sabin Patel | LEAVE A COMMENT
September 21

Results of Soil B PA, Collecting, and Enrichment of Soil C (09/19/18)

Results:

The plaque assay from Monday (9/17) was contaminated. Just like from the Spot Test ran on 9/12, there was a strange yellow ochre liquid in both the control and enriched plate. This amount was smaller than in the Spot Test. The pictures below show the plates.

Rationale:

Since there were no more positive plates, the experiment will start all over and try with new soil samples. By selecting white oak trees in a different area of campus, perhaps the sample will have a bacteriophage. Through filtration and enrichment of this sample, the sample will be ready to run a plaque assay to determine whether or not it contains a bacteriophage that specifically targets Arthrobacter.

Procedure:

  1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
  2. Through aseptic technique (over an EtOH (100%) flame),10 mL of LB Broth were added with a serological pipette to the conical vial with 2 mL of soil C in it.
  3. This conical vial was shaken for 15 minutes.
  4. This conical vial was weighed and approximately 0.5 mL of water was added so the conical vial would be within 0.05 grams of another conical vial tube.
  5. The conical vial was centrifuged at 5,000 g for 5 minutes.
  6. With the use of a 0.22μL top vacuum filter, the supernatant of the centrifuged conical vial was filtered into a 50 mL conical vial which was labeled “KEA 9/19/18 Soil C enriched filtered lysate.”
  7. Poured into “KEA 9/19/18 Soil C enriched filtered lysate” conical vial was 0.5 mL of Arthrobacter.
  8. The“KEA 9/19/18 Soil C enriched filtered lysate” conical vial was placed in the incubator at room temperature for the next 48 hours.
  9. The counter area was cleaned with CiDecan and EtOH (70%).

Observations, Metadata, and Interpretations:

  • As far as the strange liquid on the plaque assay plates, it has been determined, from a gram-strain, that it is most likely from contamination in the LB Broth used.
  • The tree selected for soil C was located outside Teal by East Village. There was mulch surrounding the tree. From the leaves, the tree was identified as a post-oak. The tree’s leaves contained a few brown patches. No broken branches or any other unusual signs. The pictures below show the leaves.

  • The tree trunk had a circumference of 48.1 cm. The tree had a canopy of approximately 177.4 cm. The tree’s height was approximately 6.858 meters. The picture below was used to approximate the trees height.

  • Before the conical vial was centrifuged, it weighed 18.42 g.

Next Steps:

In open lab on Friday, soil metadata will be collected from the soil C sample. On Monday, the remaining soil metadata will be collected and a plaque assay will be run with soil C.

Category: Kathryn Adkins | LEAVE A COMMENT
September 21

09/19/18- Serial dilutions and Plaque Assays.

Objectives:

  • Extract bacteriophages from plaques on plaque assay from 09/17/18
  • make serial dilutions of extracted plaque to 10^0, 10^-1, 10^-2
  • make plaque assays for each dilution

Pre Lab observations:

  • The plaque assays prepared on 09/17/18 had plaques on them.
  • Therefore, soil sample will not be washed and enriched at the time
  • Bacteriophages must now be extracted from a plaque and serial dilutions must begin to acquire a more purified sample.
  • The control plate was yet again contaminated, possibly by arthrobacter in a similar pattern as the control plate that was contaminated from the previous spot test.

Procedure:

  1. Cidecon was poured on the desk and wiped till the desk was dry. Then, 70% ethanol was poured and wiped until it was all over the table and then it was allowed to evaporate. After the ethanol had evaporated, an ethanol lamp was lit, setting up the aseptic zone.
  2. Phage buffer was acquired from the lab instructor and microcentrifuge tips.
  3. Using the micropipette, 100μl of phage buffer was transferred to a microcentrifuge tube.
  4. In the aseptic zone, at a 90° angle, a plaque on the plaque assay was stabbed using a micropipette tip ( attached to the micropipette) and the tip was then put into the microcentrifuge tube with 100μl of phage buffer and stirred to properly remove bacteriophages on the tip.
  5. This microcentrifuge tube was then vortexed for 30 seconds and was labelled 10^0.
  6. Using the micropipette, 90μl of phage buffer was transferred to a microcentrifuge tube.
  7. 10μl of the 10^0 bacteriophage and phage buffer mixture was transferred to the microcentrifuge tube with 90μl of phage buffer and the tube was labelled 10^-1.
  8. Using the micropipette, 90μl of phage buffer was transferred to a microcentrifuge tube.
  9. 10μl of the 10^-1 bacteriophage and phage buffer mixture was transferred to the microcentrifuge tube with 90μl of phage buffer and the tube was labelled 10^-2.
  10. One Top Agar mixture was made for the group.
  11. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
  12. While in the aseptic zone, 20 ml of LB broth was transferred to the 50 ml conical vial.
  13. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
  14.  Using the micropipette, 225 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
  15. The vial was then set on the rack.
  16. 3 of the 0.5 ml of arthrobacter test tubes were retrieved from the lab instructor
  17. Using the micropipette, 10 microliters of the 10^0 bacteriophage mixture was transferred to a arthrobacter vial.
  18. the same was done using the other 2 test tubes with arthrobacter and the 10^-1 and 10^-2 bacteriophage mixtures
  19. The vials were then allowed to rest on the test tube rack for 10 minutes
  20. After the 10 minutes had ended, 25 ml of the 2X TA was added to the LB broth and Cacl2.
  21. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to each of the test tubes with the arthrobacter and the lysate.
  22. the contents of each of the test tube were then poured into the 3 separate agar plates.
  23.  Part of the top agar mixture was poured into the top agar control plate for the group after each member had followed step 22.
  24. To let the top agar solidify, the plates were allowed to rest for 10 minutes.
  25. The plates were placed upside down in the incubator, where they will remain for 48 hours.

Analysis and Conclusion

Proper methods and measurements were used. All procedures were performed properly in the aseptic zone. The contaminated plate from the plaque assay control from 09/17/18 was similar to the contamination on the spot test control 09/12/18. Many of the other lab groups have also had contaminated control plates. It seems that there is a greater chance that the LB broth and 2X TA were contaminated. It is also possible that everyone is making the same mistakes in protocol, but the previous is more likely.

Future Notes:

to keep track, the LB broths and 2X TA used will now labelled so as to locate possible contaminated LB broths and TA

Category: Dr. Adair | LEAVE A COMMENT
September 21

09/17/2018 – Plaque Assay and Soil Collection

09/17/2018

Plaque Assay and Soil Collection

Pre- lab Observations:

The spot test made for sample soil B on 09/12/18 was retrieved from the incubator and analyzed. The plate had no plaques. The Top Agar control plate did have peculiar features that seemed to indicate contamination, which was not reflected in the spot test plates. According to the Teaching Assistant, they seemed to be arthrobacter, which may have entered the plate due to contact of the bacteria with an instrument used during the procedure.

Objectives:

  • For verification, make a plaque assay from the filtered enriched lysate to test the presence of arthrobacter
  • Collect soil sample C

Procedure:

  1. Cidecon was poured on the desk and wiped till the desk was dry. Then, 70% ethanol was poured and wiped until it was all over the table and then it was allowed to evaporate. After the ethanol had evaporated, an ethanol lamp was lit, setting up the aseptic zone.
  2. The group decided to make 1 top agar for the group.
  3. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
  4. While in the aseptic zone, 8 ml of LB broth was transferred to the 50 ml conical vial.
  5. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
  6.  Using the micropipette, 90 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
  7. The vial was then set on the rack.
  8. 0.5 ml of arthrobacter was retrieved from the lab instructor ( 0.5 ml for each group member)
  9. Using the micropipette, 10 microliters of filtered enriched lysate was transferred to the arthrobacter vial.
  10. The vial was then allowed to rest on the test tube rack for 10 minutes
  11. After the 10 minutes had ended, 10 ml of 2X TA was added to the conical vial.
  12. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to the test tube with the arthrobacter and the lysate.
  13. The contents of the tube were then poured onto the agar.plate
  14. a part of the top agar mixture was into the top agar control plate.
  15. To let the top agar solidify, the plate was allowed to rest for 10 minutes.
  16. The plates were placed up side down in the incubator, where they will remain for 48 hours.

Soil Collection

  1. Due to the low probability of finding plaques, a new soil was acquired in case a new lysate was to be extracted for testing for phages again
  2. The lab group went into the field ( went outside ) to find a live oak tree.
  3. The group picked a live oak and metadata was collected as the survey required.
  4. First, numerous pictures of the tree were taken.
  5. The diameter of the tree trunk was measured 137 cm from the ground.
  6. Average diameter of the canopy was measured by taking the average of the longest and shortest diameter of the tree canopy.
  7. Using the known height of an individual, the length of the individuals shadow and trigonometry, the angle of elevation of the sun was calculated, which was later used with the length of the shadow of the tree and trigonometry to find the height of the tree.
  8. A soil sample was collected a certain distance from the tree trunk, one sample per group member, and the sample was put into a Ziploc bag. A leaf sample was also collected.
  9. Store the sample in the fridge.

 

Conclusion and Interpretation:

The analysis of the control plates indicates that there was contamination. It indicates that more caution is required to maintain the aseptic zone and the methods used at the time of this procedure require more care and attention. There is also a chance that the LB broth or Top Agar. Currently it is not clear which is more probable.

Other Notes:

  1. Group 4 had plaques on their Plaque Assays. They group also did  spot tests in addition to plaque assays, but only one group member (Justin) had plaques on the spot test whereas the entire group had plaques on their plaque assays. The same group member also had the most well defined plaque on the plaque assay. One possible reason could be that the soil sample collected by Justin was more concentrated with bacteriophages than the samples of the rest of the group, yielding him the best results.
  2. Calculations for Lathan’s (lab instructor) webbing plate

no. of plaques= 14

dilution of sample= 10^-3

titer of sample = (14/10 μl)x(1000ul/1μl)(10^3)= 1.4×10^6 pfu/ml

Diameter of plate= 8 cm

Diameter of the plaque= 1mm

Area plate=πr^2=6400π

Area plaque=πr^2=π

area plate/ area plaque=6400π/π=6400

volume of lysate required= 6400/1.4×10^6=4.57×10^-3

 

 

 

Category: Dr. Adair, Uncategorized | LEAVE A COMMENT
September 21

Plaque Assay results for Soil Sample (9.5.18) Soil Washing, Enrichment and metadata for Soil Sample (9.17.18) 9/19/18

Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

A Plaque Assay helps us determine if there is a presence of bacteriophages by adding Arthrobacter directly to the lysate. We can tell the existence of bacteriophages by checking the presence of plaques on the agar plate.  The new sample would provide a larger data size for the research, so besides the washing and Enrichment, the sample’s metadata is also very important for more insight into our research question.

Soil Washing and Enrichment for Sample (9.17.18):

Material:

  • Two 15 ml Conical Tube
  • One 50 ml Conical Tube
  • LB Broth
  • Syringe Filter
  • Vortex machine
  • Centrifuge
  • Micropipette
  • Serological pipette

Procedure:

  1. Set up an Aseptic zone
  2. Placed sample Soil (9.17.18) to the 2 ml mark in the 15ml Conical Tube.
  3. Poured LB Broth to the 12 ml mark in the 15 ml Conical Tube (15 ml Conical Tube Soil (9.17.18) weight: 19.23 g)
  4. Vortexed for 15 min to mobilize the soil.
  5. Centrifuged at 3000 G for 10 min.
  6. Filtered the supernatant from the 15 ml Conical Tube Soil 9.5.18 with syringe filter to a new 50 ml conical tube to 10ml mark and placed the rest in a new 15 ml conical tube. ( 50 ml conical tube labeled (9.17.18) Enrich, 15 ml conical tube labeled (9.17.18) Direct)
  7. Added 0.5 ml Arthrobacter into the 50 ml conical tube with the filtered supernatant and place on the shaker.
  8. Direct Isolation tube was stored in the fridge.

Soil Metadata for (9.17.18):

Soil Composition:

  1. Added soil to the 4 ml mark on the falcon tube.
  2. Added water to the 12 ml mark.
  3. Added 3 drops of Soil Dispersion Liquid
  4. Wait for 48 hrs to see the results

% Water:

  1. Weigh the empty weigh boat: 2.40 g
  2. Weigh the weigh boat with wet soil: 7.40 g
  3. Wait for 48 Hrs for Soil to dry and check results

Soil pH:

  1. Added a small amount of soil to the pH vial
  2. Added water to 80% full
  3. Shook the vial for 45 sec and waited for 2 min
  4. put in a pH paper(should not exceed 1 inch)
  5. Waited for 45 sec and compared the color with the pH scale to determine the pH of the soil
  6. The pH level for Soil Sample 9.17.18 is  pH 6.0

Observations, Results & Data:

Plaque Assay for Sample (9.5.18):

  • The Control again showed Contamination like the last run, white dots of colonies spreading on the plate.
  • The Plaque plate showed negative results.

Interpretations & Conclusions:

Plaque Assay for Sample (9.5.18):

  • The LB broth used for both Plaque Assays for Sample (9.5.18) is contaminated, which explains the reason why the control plate was still contaminated after being more cautious during the preparation of top agar solution.
  • The LB broth and 2x Top Agar both must be double checked before use in case of a contamination of the bottle causing the inaccuracy of the experiments.
  • The tests for Sample (9.5.18) are temporarily suspended to test the new sample (9.17.18) due to the fact that no traces of plaques were identified on both runs.

Next Step:

In the next lab, I will be running Plaque Assay for Sample (9.17,.18)

Category: Yang-En Yu | LEAVE A COMMENT
September 21

Plaque Assay, Spot test results, Plaque Assay for Soil Sample (9.5.18) and Soil Sampling for Sample (9.17.18) 9/17/18

Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

A Plaque Assay helps us determine if there is a presence of bacteriophages by adding Arthrobacter directly to the lysate. We can tell the existence of bacteriophages by checking the presence of plaques on the agar plate.  Since the last plaque assay yielded contamination on the control plate, plaque assay rerun would provide a more accurate result. Also, more samples were collected to expand the data size and provide more insight into whether the presence of Arthrobacter phages in the soil around red or white oaks has a correlation with the health condition of oak trees.

Plaque Assay for Soil (9.5.18):

Materials:

  • Micropipette
  • Serological pipette
  • Centrifuge tube(1.5ml)
  • 50 ml conical tube
  • LB Broth
  • CaCl2(aq)
  • Sample (9.5.18) Lysate
  • 2x Top Agar
  • Agar plate
  • Arthrobacter

Procedure:

  1. Set up an Aseptic zone.
  2. Add 0.5 ml Arthrobacter and 10 ul Filtered Enriched Lysate to a Centrifuge tube 20 min for infection.
  3. Add 2 ml of LB Broth, 22.5 ul Calcium Chloride (aq) to a 50 ml conical tube
  4. Add the infected lysate to the 50 ml conical tube
  5. Add 2.5 ml of 2x Top Agar to the 50 ml conical tube, pipette up and down then decant the solution to the agar plate
  6. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Soil Sample (9.17.18):

  1. Location: 31.5450 N, -97.1187 W
  2.  Species: Burr Oak
  3. More information would be taken later this week.

Observations, Results & Data:

Plaque Assay (9.5.18):

  • The control plate was contaminated with white dots of colonies.
  • The Plaque Assay plate showed negative results.

Spot Test (9.5.18):

  • The Stop Test showed some uneven solidification of top agar.
  • The plate showed negative results.

Interpretations & Conclusions:

Considering this:

  1. Group 4 all had plaques on their plaque assays. Justin had the most and well-defined plaque (but all 3 got plaque). They each did a spot test in addition to their plaque assays, but only Justin had a plaque on his spot test. What do you think is going on?
  • The possibles reasons for having the most and well-defined plaque on the plaque assay are:
  1. His experimental techniques were more in the bacteriophage’s favor.
  2. The phages and Arthrobacter were more active in his sample and his enrichment lysate.
  • The possible reasons for being the only one having a plaque on the spot test:
  1. The amount of sample he put down on the plate.
  2. The phage activity on the plate.
  1. Lathan checked a purified lysate by a plaque assay using 10 µL of a 10^-3 lysate. He got 14 plaques. How many µL of Lathan’s lysate should he add to web a plate (8 cm in diameter) if his average plaque diameter is 1 mm.
  • 10^0 Lysate = 14(pfu) / 10(ul) * 10^3
  • Plate(Area) / Plaque(Area) = [40(mm)]^2 / [0.5(mm)]^2
  • Final quantity needed:   {[40(mm)]^2 / [0.5(mm)]^2 } / (10^0 Lysate) = 4.6 (ul)

Spot Test:

  • The unequal solidification of the top agar might be caused by the mixing of the solution since the top agar was prepared in a 50 ml conical tube for the whole group so it could be possible that a part of the solution had started solidifying when it was decanted to the plate.

Plaque Assay:

  • The reason causing the control plate to be contaminated might be the last time when the agar solution was being prepared it was too far away from the aseptic zone, lettiing microbes in the environment to contaminate the plate.

Next Step:

If the results of the Plaque Assay on (9.5.18) is negative, the main focus would be to wash and enrich sample (9.17.18) for Plaque Assay. If it yields positive results then further purification process would be done.

Category: Yang-En Yu | LEAVE A COMMENT
September 21

9/19/18 ~ 2nd Plaque Assay

Rational:

Performed another round of plaque assays to further isolate the bacteriophage in the sample

 

Procedure:

  • Created an aseptic zone to prevent bacteria contamination
  • Picked a plaque from the 9/17 plaque assay (10^0), and added it to 100μL of PB
    • This became the new 10^0 dilution for which the plaque assay would be performed with
  • Added 10μL of 10^0 to 0.5 arthrobacter
  • Obtained a 50mL conical vial and added 14mL LB Broth, 22.5 μL CaCl2, and 17.5 2X TA
    • Made enough TA for seven plates; two plates per member of the group, and one control plate
  • Immediately added 4.5 mL TA to the 10^0 + arthrobacter tube and allowed to mix, and then plated
  • Let the plates sit for 15 minutes and then incubated

 

Observations:

  • The 10^0 had many plaques, similar to the 9/12 plate
  • The 10^-1 only had one, singular plaque
  • The 10^-2 only had one, singular plaque

    Contaminated TA control for 9/17

    The 9/17 plaque assay (10^-2) with one plaque (Circled)

    The 9/17 plaque assay (10^0) with noticeable plaques

    The 9/19 plaque assay plate

    The 9/17 plaque assay (10^-1) with one plaque (Circled)

    9/19 control plate for TA

     

  • Plates turned out as expected, with 10^0 with the most plaques, and 10^-1 and 10^-2 having less plaques
  • Contamination of the 9/17 Control TA is concerning, and may entail a contaminated LB Broth

 

Conclusion/Next Steps:

The lab group was able to complete the procedure quickly since it was a repeat of the procedure of Monday’s lab. The next steps would be to see the results of the current plaque assay, and repeat another plaque assay to continue to isolate the bacteriophage

Category: Justin Yu | LEAVE A COMMENT
September 21

Soil Metadata and Plaque Assay (9/17/18) 

Rationale:  

Performed a plaque assay, since the results from our spot test from last class were negative, and therefore resulted in no plaques.  

Procedure: 

  1. Observed spot test plates, and then cleaned the table with Cidecon and ethanol.  
  2. Set up a ascetic zone with an ethanol burner.  
  3. Obtained our enriched lysate from the fridge and added 10 uL to the red capped vial with 0.5mL of Arthro. 
  4. Let the solution sit and waited for 10 minutes . 
  5. Made a combined Top Agar plate for our 3 individual plates and a control plate.  
  6. Obtained a tube and transferred the lysate and Arthro mixture to a bigger tube 
  7. Added 8mL of LB broth and 90uL of CaCl2 to the tube.  
  8. After letting it sit for 10 minutes, added 10 mL of Top Agar.  
  9. Mixed the solution using the pipetting up and down method.  
  10. Quickly, pipetted 4.5mL of our Top Agar solution into our 3 individual tubes.  
  11. After pipetting into our individual tubes, directly poured the solution into our individual plates.  
  12. Poured the remaining solution into our individual tubes and let the 5mL solution set.  
  13. Then poured the rest of the Top Agar (4.5 mL) into our control plate and let the plates sit for 10 minutes to solidify.  
  14. While waiting for our plates to solidify, measured our soil metadata for percent sand, silt, and clay.  
  15. Incubated the plates and let it stay for 48 hours.

Observations/ Results:  

Spot Testing:

  • Spot testing plate was negative.  

  • Negative Spot Test Plate

  • Did notice that there was one spot of contamination in my part of the plate
  • While Plating Plaque Assay noticed that there were some bubbles in our agar while pouring it in.  

Soil Metadata:

  • Total : 7 mL 
  • Sand  : 5mL = 66.67%  
  • Silt : 1.75 mL = 23.33% 
  • Clay : 0.75 mL = 10% 

  • Soil Metadata

Class Observation:

  • Everyone in group 4 had phage, but Justin had the most defined phage. Group 4 did both a spot and plaque test, and Justin’s spot and plaque tests had plaque. One reason for this could be the tree they chose from. The group mentioned that they chose neighboring trees, so if the tree that Justin had picked had more phage or more defined phage it could proceed to more positive results.  

Lathan’s Problem:

  • Answer : 4.01 uL.  
  • (14pfu/ 10) * (1,000mL/1mL) = 1400 x10^3  
  • (3.75^2)/(0.5^2) = 5,625 
  • 5,625/1,400,000 = 0.00401 = 4.01 uL  

Next Step: 

We will observe our plaque assay plates. If our plaque assay plates have plaques, then will continue with more plaque assay plates to amplify the plaque. If there are no plaques then will obtain another soil sample, find the soil metadata and make another plaque assay.  

 

Soil Sampling and Washing + Soil 2 Metadata (9/19/18) 

Rationale: Collected and washed soil because our plaque assay plates were contaminated, and since these results came out negative we got new soil sample and found some of the soil metadata.  

Procedure: 

  1. Collected soil from a read oak tree across from the BSB, collected 2mL of soil in a tube, and filled ¼ of a Ziploc bag with the same soil.  
  2. Found the tree measurements and returned back to the class.  
  3. Using the tube with 2mL soil, added about 8.5 mL of Arthro into the tube, and shook for the next 10 minutes. Found the total solution was 10.5 mL.  
  4. Massed the solution and found it to be 17.73 grams 
  5. Centrifuged the soil for 10 minutes at 5,000 g.  
  6. Meanwhile started on collecting soil metadata for percent water.  
  7. Measured the weighing boat which was 2.478g and then the soil and the weighing boat which was 6.549 grams, and then placed it in the hood to let it evaporate until we check it again.  
  8. Set up the test for percent sand, silt and clay.  
  9. Started off by adding 10mL of soil into tube, 20 mL of DI water, until it was a total of 30 mL.  
  10. Lastly added 3 drops of dispersion liquid and shook the solution for 45 seconds. Let it sit until we are back in lab. Placed it in hood as well.  
  11. Next tested pH of the soil. First added a little soil into the tube about 1/5 of it and added DI water till the top. Let it sit for a few minutes and then shook for 10 seconds. Wait 10 minutes and measure pH.  
  12. Once the enriched sample is ready, syringe out just the liquid part or the supernatant.  
  13. Used a filter, to make sure that the bark and other bigger components of the soil was filtered out.  
  14. Resulted in about 8.5mL of enriched lysate, and therefore don’t have an direct lysate.  
  15. Measured pH using a small pH paper and let it sit in the solution for 45 seconds, and found the pH of the soil.  
  16. Obtained 0.5mL of Arthro and poured it into the enriched lysate tube.  
  17. Transferred the enriched lysate into a 50mL tube and placed the tube into a shaking device.  

Observations/ Conclusions  

Plaque Assay Results

  • The control plate for the spot test was contaminated  
  • Seemed to be a bubble in my individual plate but otherwise clear.  

Soil Metadata:

  • PH of soil = 6  

Next Step: 

Next time, will use our enriched sample to create plaque assay or spot test plates in hopes of finding plaque on the plate. We will also measure our soil metadata for percent sand silt clay.  

 

Category: Sona Subramanian | LEAVE A COMMENT