September 17

September 17, 2018 Plaque Assay and Soil Metadata – Soil B

Rationale: To attempt to isolate a phage by performing a plaque assay, after a negative spot test. This procedure will also finish calculating the soil concentrations of sand, silt, and clay.

Description of Procedures:

  1. The work space was cleaned using aseptic technique and a 100% ethanol burner was lit to create an aseptic zone.
  2. 10 ul of enriched lysate was added to 0.5 ml of arthrobacter. This was allowed to sit for 10 minutes.
  3. Next, the top agar solution was made. One tube was used to make four 5 ml plates. 8 ml of LB broth and 90 ul of CaCl2 were added to the tube.
  4. 2x Top Agar was then added to the tube and pipetted up and down to mix the top agar solution.
  5. 4.5 ml of the solution was added to each of the groups arthrobacter and phage mixture, and immediately poured onto petri dishes. The remaining 4.5 ml was poured onto a control top agar plate. The experimental plate was labeled LIP 9-17-18 Plaque Assay. The control was labeled EAG, SS, LIP 9-17-18 Control Plaque Assay. The plates were then allowed to sit for 10 minutes.
  6. While the plates were sitting, the soil metadata collection was finished. There were 7 ml of soil total, and it was found that 5.5 ml were sand, 0.5 ml were silt, and 1.0 ml was clay. The percent sand found was 78.57%. The percent silt was 7.14%. The percent clay was 14.29%.
  7. After 10 minutes, the plates were inverted and placed in the incubator for 48 hours.
  8. The work station was cleaned using aseptic technique and the materials were properly disposed of.

Observations/Results/Data:

  • Observations:
    • There were small bubbles on the control plate, and one larger one on the edge.
    • There were 1-2 small bubbles on the control plate.
    • The soil had many layers of silt, all small, but different colors.
  • Data:
    • % Sand: 78.57%
    • %Silt: 7.14%
    • %Clay: 14.29%

Interpretations/Next Steps/Conclusions:

The procedures were complete. The soil composition was found and the plaque assay, both the control and experimental, were completed. The next step will be to wait 48 hours to allow the bacteria to grow and plaques to form. If there are plaques, they will be picked. If not, then new soil will need to be found and the procedures will be started again.

Additional Questions:
1. Group 4’s Plaques: The group members in Justin’s group did not all collect soil from the same tree. The soil was from three different trees in the same area. This could account for the differing results. The soil Justin collected could have different phages from his group members, allowing him to have more and better defined plaques. However, the soil was most likely at least similar, as all three members of group 4 had plaques.

2. Lathan will need 4.01 ul to web his plate. (Work shown below).

Category: Lucy Potts | LEAVE A COMMENT
September 17

Plaque Assay 9/17/18

Rationale: After the spot test turned up negative, so I will run a plaque assay to double check my spot test result.

Procedure:

  1. Pipetted 10μL of lysate into 0.5mL of arthrobacter. Then began making top

    agar.

  2. Using aseptic technique and fresh pipettes each time I put 8mL of LB broth

    in a 50mL tube, then 90μL of CaCl2, then 10mL of 2X TA.

  3. Accidentally spilled some of a lab mates TA with their arthrobacter and

    lysate onto plate so started over.

  4. This time only needed enough for 2 plates so mixed 4mL of LB broth, 45μL

    of CaCl2, and 5mL of 2x TA and poured 4.5mL into a control plate and poured the other 4.5mL into my arthrobacter and lysate mixture. I pipetted up and down to mix them then poured onto a plate.

  5. Inverted and incubated for 48 hours.

Observations:
My control turned out contaminated, and the plaque assay had no plaques. This contamination could be from lack of aseptic technique, so we cleaned our pipette tips and made sure to mark our own LB broth bottle. Group 4 all had plaques in their plaque assay and Justin’s the most well-defined plaque.
Claire had a lot of plaques in her plaque assay, and her tree was from Morrison. Image on the left is plaque assay and Image on the right is control.

Interpretations and Next Steps:
Lathan checked a purified lysate by doing a plaque assay (10μL of lysate) of a 10-3 lysate. He counted 14 plaques. With an average plaque diameter of 1mm he would need 4μL of 100 lysate to web a plate with a diameter of 75mm. Arthrobacter has been known to have an affinity for a compound in pesticides so the findings thus far would support the positive correlation between pesticides and the presence of arthrobacter phage, as where there are bacteria there are phages. The next steps would be to collect more samples from different trees in Cameron park. This time I will be gathering samples from living Oak saplings. I will also be gram staining my control contaminant and the arthrobacter lawn from my plaque assay to see if the contaminant was arthrobacter or some other bacteria that may have come from the surroundings.

Category: Sriram Avirneni | LEAVE A COMMENT
September 17

9.14.18 Spot Test Results and Plaque Assay

Rationale)

Since the spot test from 9.12.18 was negative it is necessary to conduct a plaque assay in order to confirm that the lysate derived from Soil B was negative or deny it by having a positive result from the plaque assay.

Procedures)

  1. Setup an aseptic zone by wiping down the work area with CiDecon and 70% ethanol, and lighting an ethanol flame.
  2. Retrieve “Filtered Lysate Enriched 9.12.18″(FLE) and a vial of .5mL of Arthrobacter, under aseptic conditions add 10 microliters of FLE to the vial of arthro and let sit for 15 minutes.
  3. Collect two 50mL conical vials, labeling one “Top Agar for Plaque Assay NMN 9.14.18” and the other “Control Top Agar for Plaque Assay HMB, NMN, 9.14.18”.
  4. Retrieve LB broth and using a 10mL serological pipette add 2mL of LB broth to both the 50mL conical vial labeled “Control Top Agar for Plaque Assay HMB, NMN, 9.14.18” and the 50mL conical vial labeled “Top Agar for Plaque Assay NMN 9.14.18”, under aseptic conditions.
  5. Retrieve the micro test tube of 1M CaCl2 from 9.12.18 and add 22.5 microliters of CaCl2 to both of the 50mL conical vials under aseptic conditions using a 10-100 microliter micropipette.
  6. Collect two plates with base agar, labeling one “NMN, HMB Control Top Agar for Plaque Assay, 9.14.18” and the other plate “NMN Plaque Assay 9.14.18”.
  7. Add the red-capped test tube containing both the Arthro and FLE to the 50mL conical vial labeled “NMN Plaque Assay 9.14.18” under aseptic conditions and swirl to combine.
  8. Add 2.5mL of 2xTop Agar to both 50mL conical vials using a 10mL serological pipette under aseptic conditions.
  9. Swirl both 50mL conical vials for 3 seconds to combine all ingredients, then proceed to pour their contents into their respectively labeled plates, shaking the plates slightly to distribute the top agar evenly. Let the plates solidify for 15 minutes.
  10. After they have solidified invert the plates and place them in the incubator, letting them incubate over the weekend.
  11. Clean the work area with CiDecon and 70% ethanol, and dispose of all used items into their appropriate receptacle.

Results From Spot Test)

The spot test I conducted came back negative, however, we did not create a top agar control thus this could have affected the results of the spot test.

Observations)

I observed that there were strands and particles that appeared when the top agar was mixed and when the top agar was poured into the plate.

Conclusions/Next Steps)

Due to the negative results of the spot test we had to conduct a plaque assay in order to positively confirm that the lysate from Soil B was negative in the presence of phages if it comes back negative or confirms the presence of phage in Soil B if the plaque assay comes back positive. The next step will be to check the plaque assay on Monday for plaques, wherein if present we will pick and begin isolating the plaques; if plaques are not present we will have to collect another soil sample.

Category: Nathan Newton | LEAVE A COMMENT
September 16

Results of Spot Test and Starting Plaque Assay for Soil B (09/14/18)

Results:

Both the “LEF KEA 9/12/18 Spot Test Control” and the “KEA 9/12/18 Spot Test” plates were contaminated. There was a yellow ochre liquid present in both plates. This might be because the plates were accidentally placed right side-up and were not inverted. The pictures below show these plates and the liquid.

  

Rationale:

Since the spot test was contaminated, I decided to run a plaque assay with the enriched soil B lysate. This will reveal whether or not there are bacteriophages in the soil sample that target Arthrobacter.

Procedure:

  1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
  2. Performed the following calculations to determine how much of the materials were required.

Calculations

Original Recipe

 X2

2 mL LB Broth

4 mL LB Broth

2.5 mL 2X TA

5 mL 2X TA
22.5 μL CaCl2

45 μLCaCl2

 

  1. Through aseptic technique (over an EtOH (100%) flame), used a cartwheel serological pipette with 10 mL tip to transfer 4 mL of LB Broth into 50 mL conical vial.
    • Labeled this 50 mL conical vial “KEA TA 9/14/18.”
  1. Added 45 μL CaCl2 with P200 micropipette into “KEA TA 9/14/18” conical vial.
  2. Transferred 5 mL of 2X TA using a cartwheel serological pipette with 10 mL tip into “KEA TA 9/14/18” conical vial.
  3. Used a P10 micropipette to add 10 μL of the enriched lysate from the “KEA 9/12/18 enriched filtered lysate” microcentrifuge tube into the test tube with 0.5 Arthrobacter.
  4. Allowed 10 minutes for the mixture to set.
  5. Used a cartwheel serological pipette with 5 mL tip to separate 4.5 mL of the Top Agar mixture into another 50 mL conical vial.
    • Labeled this 50 mL conical vial “KEA TA 9/14/18 control.”
  1. Obtained two plates, one to run the enrich plaque assay on, which was labeled “KEA PA 9/14/18 enrich lysate” and the other plate served as a control, which was labeled “KEA PA 9/14/18 control.”
  2. Used a cartwheel serological pipette with 5 mL tip to add and mix the remaining 0.5 mL of LB Broth into “KEA TA 9/14/18 control” conical vial.
  3. Used the same cartwheel serological pipette with 5 mL tip to transfer the mixture onto “KEA TA 9/14/18 control” plate and swirled to even out the mixture.
  4. Once the 10 minutes for the Arthrobacter and enrich lysate were up, poured this mixture directly into the “KEA TA 9/14/18” conical vial.
  5. Used a cartwheel serological pipette with 5 mL tip to mix and transfer the mixture in “KEA TA 9/14/18” conical vial onto “KEA PA 9/14/18 enrich lysate” plate.
  6. Allowed both plates to solidify.
  7. Placed both plates inverted in incubator at 48ºC for the weekend.
  8. Cleaned lab counter with CiDecan and EtOH (70%).

Observations:

  • The mixtures in both “KEA TA 9/14/18” and “KEA TA 9/14/18 control” conical vial had started to solidify. The “KEA TA 9/14/18” conical vial mixture more than the “KEA TA 9/14/18 control” mixture since it had to wait 10 minutes before putting in the Arthrobacter with enriched lysate.
  • The solidifying caused many air bubbles to form when mixing as shown below in the pictures.

Next Steps:

On Monday, I will examine both plates. If the plates are contaminated, I will run another plaque assay. If the plates are negative, I will find new soil. If the plates are positive, I will move on to do purification.

 

Category: Kathryn Adkins | LEAVE A COMMENT
September 15

SEA Bears day 6

12 September 2018 ✷ Metadata Conclusion & Spot Test

Metadata (% silt, % sand, % clay, % water, and pH) was collected in order to further examine the links between tree type and phage presence. Today it will be finalized by recording final results. A spot test will be performed to indicate the presence of phage from the soil collection.

Procedure

  • The enriched lysate was spun at 3000g for 5 minutes.
  • The dried soil from Monday’s lab was massed and the percent water was calculated (see SEA Bears Day 5).
  • I worked with one of my lab partners to mix the agar solutions for our plates and a control plate. We cleaned the workspace with CiDecon and 70% ethanol to promote an aseptic environment and then lit an alcohol burner. The control plate was made separately, but our two plates were made together, thus the volumes of the components were doubled. LB Broth was added first, then Calcium Chloride, followed by arthro to the spot test plates (not added to the control plate) and finally top agar. The volumes and concentrations of the components can be seen below.
  • component volume (control) concentration volume (spot test) concentration
    2X Top Agar 2.5 mL 1X 5 mL 1X
    LB Broth 2 mL – 4 mL –
    1M Calcium Chloride 23 µL 4.5 M 45 µL 4.5 M
    Arthro 0 mL – 0.5 mL –
  • The test plate was poured immedietally to prevent it from hardening in the conical vial; it was allowed to set for 10 minutes.
  • The spot test plates were poured next from a serological pipette, which was also used to mix the mixture. Each plate was allowed to set for 10 minutes.
  • The enriched sample was filtered through a 22 micron syringe filter and stored in a microcentrifuge tube. 5 µL of the filtered enriched sample was added to the designated space on the plate. 5 µL of phage buffer was added to the plate as a control, and 5 µL of the direct sample was added to the plate.
  • The excess samples were stored in the refrigerator and the plates were placed in an incubator to sit until Monday.
  • Because the % silt/sand/clay falcon tube was difficult to read, it was reshaken and repoured to attempt to get more accurate results for this metadata.

Observations/results/data

The “recipe” for a spot test plate requires 0.5 mL arthro for EACH plate, and we forgot to double this quantity when we mixed our plates. Thus, there may not be enough arthrobacter on the plate for the phage to infect and kill to make a noticeable plaque on the plate.

Results from the metadata collection are recorded on Day 5’s post.

Interpretations/Conclusion/Next Steps

The presence of arthrobacter in the plate is important for the plaques to form, and the limited amount in my plate may pose an issue. If results are inconclusive, I will run a plaque assay with my enriched sample in order to confirm the presence of a phage.

Category: Lily Goodman | LEAVE A COMMENT
September 15

SEA Bears Day 5

10 September 2018 ✷ Metadata & Enrichment

Metadata (% silt, % sand, % clay, % water, and pH) was collected in order to further examine the links between tree type and phage presence. While it isn’t imperative to the research process, the metadata may provide some interesting connections between phage presence in different groups/locations. Additionally, the soil was cleaned and enriched to help isolate any phage that may have been present in the original sample.

 

Procedure

  • First, the workspace was cleaned with CiDecon and 70% ethanol and then an alcohol burner was lit to promote an aseptic work environment.
  • 2 mL of soil was added to a 15 mL conical vial and then 10 mL of LB Broth was added to the same vial (reaching 12 mL line on tube). The vial was then shaken for 5 minutes, vortexed for 5 minutes, and shaken by hand again for a final 5 minutes. The vial was massed at the end of the 15 minutes and found to be 19.329 g.
  • The vial was taken to be centrifuged at 10,000g for 10 minutes. In the time the Dr. Adair had my sample, I worked on the metadata.

% Water

  1. I found the mass of a dry, empty weigh boat to be 2.31 g. I added some of my soil sample to the boat and found the mass of the soil + weigh boat to be 8.62 g. The soil/weigh boat was placed uncovered under the fume hood in the lab to dry until Wednesday, when it will hopefully be dried out and massed again.

% sand, % silt, % clay

  1. 4 mL of dirt was added to a falcon vial and the vial was then filled to the 12 mL line with deionized (DI) water. 3 drops of soil dispersion liquid was added, the vial was covered, and the mixture was shaken by hand for 30 seconds. The supernatant was removed and stored in a separate 50 mL conical vial. Both were stored at room temperature under the fume hood.

pH

  1. A small amount of soil was added to a pH tube and then the vial was filled with DI water, closed, and shaken for 10 seconds. A pH indicator strip was inserted for 30 seconds and read immediately after. The strip was a light green consistent with a pH of 6.5 (slightly acidic).

 

 

  •  Once the sample was separated via centrifuge, 3 mL was removed with a syringe and filtered through a 22 micron filter attached to the end of the syringe. This was 3 times; each time the filter was removed to collect more lysate, the ends of it were cleaned with 70% ethanol. The intent was to have 10 mL lysate in a 50 mL conical vial and 1 mL in a 15 mL conical vial, but it ended up being 8 mL in the larger vial and 0.75 mL in the smaller. The smaller vial became the direct lysate and it was stored in the refrigerator.
  •  0.5 mL of arthrobacter was added to the larger vial and stored in a shaking incubator at 28 degrees Celsius until Wednesday.

Observations, Results, Data

Pictured above is a leaf collected from the tree we collected soil from and an analysis of the traits of the leaf. This is more metadata that can be utilized in future steps of answering our scientific question.

day 5 data
component mass (g)
soil/LB broth mix 19.329
empty weigh boat 2.31
weigh boat + wet soil 8.62
wet soil (8.62-weigh boat) 6.31
dry soil + weigh boat 7.69
dry soil (7.69-2.31) 5.38
Water (wet soil – dry soil) 0.93
% water (water/wet soil)*100 14.7%
soil pH 6.5

 

interpretations/conclusion/next steps

The % sand, silt, clay separation didn’t work as well as hoped and the test will be redone next time to get better, more usable results.

Enriching the soil sample will hopefully lead to the discovery of a phage because the increased amounts of nutrients from LB broth will allow the arthrobacter to flourish, and because the phage feeds on bacteria, increased amounts of phage should be present.

The next lab period will consist of a spot test and conclusion of the metadata collection in order to isolate a phage and analyze the conditions it was found in (i.e. wetness from water percentage).

 

 

Category: Lily Goodman | LEAVE A COMMENT
September 14

9/12/18 Plaque Assay of Soil B and Metadata

Objective:

  • Plaque assay will be conducted to test for the presence of Arthorbacter phage in soil sample “Soil B” and help to answer the experimental question.

Procedure:

  1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
  2. Enriched lysate that was collected in the previous lab (9/10) was gathered and centrifuged @ 3000 g for 5 minutes. This was done to pellet the Arthrobacter that had multiplied from being in contact with the LB broth for 48 hours.
  3. Then a syringe and filter were used to filter 1 mL of the enriched lysate into a microcentrifuge tube.
  4. 10 microliters of the filtered enriched lysate was added to 0.5 mL of Arthrobacter in a test tube. The mixture was left for 15 minutes while the Overlay Agar was made. This was done to ensure the possible phage were able to interact with the Arthrobacter before being added to the Overlay tube.
  5. To make the Overlay Agar, a 50 mL tube was obtained. The agar was created for 3 soil samples in addition to a control sample. This was done to ensure the same base of Overlay Agar was used and no contamination occurred. 8 mL of LB broth, 90 microliters of CaCl2, and 10 mL 2x Top Agar were added to the tube.
  6. After swirling the Overlay Agar in the 50 mL tube, 5 mL of the mixture was piped into a new 50 mL tube that would then receive the Arthrobacter and lysate mixture that was created earlier. The mixture was then poured onto a petri dish label “Soil B Plaque Assay” and allowed to solidify for 15 minutes.
  7. Next, 5 mL of the Overlay Agar stock was plated and labeled “Control Group” to ensure their was not any contamination for Group 1. The plate was left to solidify for 15 minutes.
  8. Both plates were incubated and left to be checked during next lab day (9/17)

Metadata=

  1. The “wet soil” plate that was made during the previous lab (9/10) was collected and massed. It was found to be 10.15 g and labeled as “dry soil”. The difference in mass between the “wet soil” and “dry soil” was found to be 1.07 g. After calculations, it was found that soil sample Soil B was 26.6% water.
    1. Calculations:
      1. wet soil= 4.03 g      dry soil = 2.96 g
      2. 4.03 g – 2.96 g = 1.07 g
      3. (1.07 g / 4.03 g) x 100 = 26.6% water
  2. In order to know the ratio between sand, silt, and clay in the Soil B sample, 10 mL of the soil was put in a 50 mL falcon tube. The tube was filled to the top with water and 3 drops of Soil Dispersion Liquid. The tube was hand-shaken for 2 minutes then left to sit for 48 hours to separate.

Results:

  • It reviewing the metadata experiments that were conducted on 9/10, it was found that Soil B was 26.6% water.
  • The plaque assay was not completed, so there are no results. Results will be recorded during next lab (9/17)

Next Steps:

  • During the next lab, the plaque assays will be examined for clearings in the Arthrobacter lawn. If there are clearings, then there is the presence of Arthrobacter phage in Soil B and the phage will be picked for further testing. If no clearings are found, then a spot test will be conducted to confirm negative results. Results of the plaque assay/spot test will help to answer the experimental question.
Category: Claire Wentzlaff | LEAVE A COMMENT
September 14

SEPTEMBER 10TH AND 12TH- Lab

  • SEPTEMBER 10TH, 2018
    • SOIL WASHING AND METADATA
  • OBJECTIVE: To wash new soil samples collected and to perform metadata on the soil as well
  • PROCEDURE:
    • Tables were cleaned 
    • Next soil samples were used to fill a test tube to the 2mL mark, filled to the 12mL mark with LB, and were then shaken and centrifuged for 15 minutes
    • The tubes were then taken to spin @10,000G for 5 min 
    • During that time 10mL of soil was added to a conical vile 
    • DI water was added to the vile until it hit the 30mL mark
    • Then 3 drops of soil dispersion fluid was added to the vile, where it was then shaken for 30 seconds 
    • The test tube of soil and broth was taken to a fume hood where the supernatant was poured into a top filter, where 10mL was filtered out 
    • Then the 10mL filtered supernatant  then has .5 mL of arthrobactor added to it and was left to incubate
    • Wet soil sample was weighed out and then left to dry 

– NEXT STEPS: Conduct spot test

    • SEPTEMBER 12TH, 2018
      • SPOT TEST
      • OBJECTIVE: Conduct a spot test without any contamination 
      • PROCEDURE: 
        • Tables were cleaned 
        • Dry soil sample was weighed out 
          • Plate without soil: 7.20g
          • Wet soil: 10.02g
          • Dry Soil: 9.45g
          • Water: 0.57g 
          • %Water: 
      • Then meta data was analyzed: 
          • %sand: 54.02%
          • %clay: 26.44%
          • %silt: 19.54%
      • My sample was then masses and a “spin buddy” was found
      • The sample was spun at 3000G for 5 min
      • Then a syringe was used to push the spun supernatant through a .22𝝁m filter out 
      • Then a test was made with the following:
          • 6mL LB
          • 1.5mL Arthro
          • 7.5mL 2X TA
          • 67.5𝝁L of 1M CaCl 
  • 5mL of the contents of test tube were pipetted onto 3 plates and left to sit for 15 min
  • After 15 min .5𝝁L of lysate of was pipetted onto plate
  • Was left to incubate
  • NEXT STEPS: wait to see results from spot test
Category: Lauren Foley | LEAVE A COMMENT
September 14

Spot Test and Plaque Assay of Soil Sample 2 (9/12/18)

Rationale: To conduct a spot test and plaque assay on the enriched sample on Soil Sample 2 and to gather the data from the procedures conducted last class

Materials:

Spot Test

  • Lysate (direct and enriched)
  • 2.0 mL of LB broth
  • 2.5 mL of 2X Top Agar
  • 22.5 µL of CaCl
  • 0.5 mL Arthrobacter
  • two agar plates
  • pipettes
  • microcentrifuge tubes
  • syringe and filters

Plaque Assay

  • 8 mL of LB broth
  • 50 mL conical vial
  • 1 mL enriched lysate
  • 90 µL of CaCl
  • 10 mL of top agar
  • agar plate
  • pipettes

Spot Test Procedure:

  • Weigh the enriched sample in order to be put back in the centrifuge then set aside.
  • Place the sample in a centrifuge to be spun at 3000 g for 5 minutes.
  • Weigh the petri dish filled with soil and calculate the percentage of water.
  • Pipette the supernatant out of the Falcon tube using a bulb pipette and determine the volume each component of the soil takes up.
  • Serological pipette 2.0 mL of LB broth into a new vial labeled “TA 2.”
  • Pipette 22.5 µL of CaCl into TA 2.
  • Filter the now centrifuged enriched sample and label a microcentrifuge tube “Filtered Enriched 2” using a syringe and 0.22 µm filter.
  • After filtering, add 0.5 mL of Arthrobacter to TA 2.
  • Add 2.5 mL of 2X Top Agar to TA 2, stir immediately then pour the mixture in the agar plate labeled “Spot Test 2.”
  • Allow the top agar to solidify for 10 minutes.
  • After 10 minutes, add the direct, enriched, and phage buffer to spot test in the designated area in drops of 3-5 mL.
  • Allow it to remain in an incubator for 48 hours.

Plague Assay Procedure: 

  • Add 8 mL of LB to a 50 mL conical vial using a serological pipette
  • Add 90 µL of CaCl to the conical vial
  • Transfer 1 mL of enriched lysate from the tube labeled “FIltered Enriched 2” to a new microcentrifuge tube.
  • Add 0.5 mL Arthrobacter to the 1 mL enriched and allow it to sit for 10 minutes
  • Pipette 10 mL of top agar to the CaCl and broth
  • Add Arthrobacter to a new vial and pipette 5 mL of TA with the enriched and Arthrobacter mixture
  • Stir and pour onto the agar plate labeled “PA 2” and allow it to sit for 10 minutes.
  • After 10 minutes, place the plate in an incubator for 48 hours.

Results and Analysis:

  • The weight of Enriched Sample: 21.22 g
  • The weight of Soil Sample 2 soil including the petri dish: 10.84
  • Percentage of water in Soil Sample 2
    • (3.65/4.61)×100= 79.18%
  • Components of Soil Sample 2
    • 8.5 mL / 10.5 mL is soil
      • (8.5/10.5)×100= 81%
    • 1.5 mL /10.5 mL is silt
      • (1.5/10.5)×100= 14%
    • 0.5 mL / 10.5 mL is clay
      • (0.5/10.5)×100= 5%

Soil Sample 2 Components without Supernatant

 

Soil Sample 2 without Water

 

Spot Test

(note: there are bubbles in the top agar when trying to find phage, be careful when trying to identify phages)

 

Plaque Assay with bubbles in Top Agar

While pipetting the filtered enriched into the microcentrifuge tube, a considerable amount of lysate did not come of the pipette.

Conclusion and Future Plans:

  • We successfully completed the spot test and plaque assay procedures to see whether or not there are phages present in our soil samples. For the spot test, group 5 made one collective sample of top agar and evenly distributed it to three separate vials. We then poured the solution on the plate and then added our own samples of enriched and direct and the same phage buffer which was the third area on the plate. For the plaque assay, we also made one collective sample of top agar and separate it evenly. We then poured our Arthrobacter and filtered enriched sample onto the plate.
  • In the future plan, I will check for phages in both of my tests. If there are phages present, I will go on to reconduct the test to check again and to also do a plaque assay on my direct sample.
Category: Sabin Patel | LEAVE A COMMENT