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.

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.

Rationale: To perform a washing to separate the phages and bacteria in which the lysate will be divided into what will become enriched and direct isolation. To also determine the pH, % sand, % silt, and % clay

Materials:

• 15 mL conical vial
• 10 mL of LB broth
• 20 mL of DI water
• Soil Dispersion Liquid
• Petri Dish
• Syringe and Filters
• Microcentrifuge tubes

Procedure:

• Add 2 mL of Soil Sample 2 to a 15 mL conical vial.
• Add LB broth up to the 12 mL mark in the vial.
• Vortex the vial for five minutes, shake the vial for another five, and vortex the vial for the last five minutes.
• While vial is on the vortex, add 10 mL of soil into a Falcon tube.
• Add DI water up to the 30 mL mark in the Falcon tube.
• Add three drops of soil dispersion liquid.
• Shake the tube for 30 seconds.
• Let the tub sit for 30 seconds.
• The 15 mL vial now goes into the centrifuge at 10,000 g for 5 minutes.
• While the 15 mL vial is in the centrifuge, weigh a petri dish then add soil and weigh it.
• Set aside the petri dish with soil under a vacuum hood for 48 hours
• FIlter the Soil Sample 2 supernatant using a syringe.
• Use as many filters as needed to filter the supernatant.
• Pour the lysate into a microcentrifuge tube labeled “Filtered Enriched 2.”
• Take some soil from Soil Sample 2 and put it in a small glass vial to measure the pH.
• Add some DI water and shake.
• After shaking, take an inch of pH paper and put it in the mixture for about 45 seconds.
• Take out the paper and determine the pH based on the color of the water at the top of the vial.
• Dispose of the contents of the vial and clean it thoroughly.

Results and Analysis:

• The weight of the Petri Dish: 7.19 g
• The weight of the Petri Dish and soil: 11.80 g
• pH of soil: 7.6
• I had a lot of difficulty in filtering my supernatant using the syringe resulting in a shortage of lysate.

Soil Sample 2 immediately after shaking the Falcon Tube

Supernatant before filtering

Determination of pH by the color of the water

Conclusion and Future Plans:

• To determine pH, remove a small sample of the soil sample and place it into a vial. Fill the vial up with water all the way to the rim. Shake the vial until the two components are evenly mixed. Take a piece of pH paper and determine the pH of the soil by using the color chart that is provided. Another procedure was to take some soil from Soil Sample 2 bag and to place it on a petri dish under a vacuum hood. The purpose is to determine the % water after 48 hours. The last procedure that was conducted is the filtering of the supernatant. Using a syringe and many filters, filtering the supernatant was a bit difficult and long due to the debris that was restricting the flow of the phages through the filter. Remove all the supernatant from the conical vial and filter for the phages which would go into a microcentrifuge tube labeled “Filtered Enriched 2.”
• In the future, I plan to conduct a spot test to check for phages.

Rationale:

We (the class) narrowed down the scientific question to make it possible for testing. We also collected soil samples that was related to the question to see whether or not there is a relation between the three factors: area, species and presence of phage.

Procedure:

• We discussed, as a class, of what the scientific question should be, which would result in clear results. We also determined the question based on which question would require the least amount of variables which would allow us to receive more clear results. We soon decided that the question should involve whether or not the presence of phages is determined by which area the tree is in and the species of the tree.
• After consulting with the others at the table, my group decided to collect soil samples from a white oak in the area around Texana.
• I collected my measuring tape, plastic scoopers, 15 mL vial, and a plastic bag labeled “Soil Sample 2.”
• We walked over the area and spotted a white oak (bur oak) in a garden near Texana.
• I collected soil that filled a little over 1/4 of the plastic bag.
• I collected leaves which would help to determine the health of the tree.
• My group and I measured the diameter of the tree; measured about 4.5 ft from the base of the tree.
• We measured the longest and shortest side of the canopy and found the average.
• We then found the height of the tree.

Results and Observations:

The diameter of the tree is 61 cm.

The longest side of the the canopy is 304 cm.

The shortest side is 141 cm.

The average being 222.5 cm

Using proportions, we found the height of the tree to be 740.50 cm.

The tree (white oak – bur oak) was in a shaded area with many other trees surrounding it.

The soil was wet and dark, looked like clay.

The leaves looked like a combination of green and yellow, with yellow being mostly in the middle of the leaf and little spots on the edges. 

Conclusions and Future Plans:

Overall, the bur oak in our area was in a shaded area in a garden near Texana with wet soil. The leaves looked healthy which is an indication that the tree is also healthy.

In the future, I plan to “wash” the soil which means to perform a filtration which would filter the phages and the bacteria to create my enriched and direct isolation samples.

Rationale:

Due to the limited amount of agar plates, a plaque assay for the enriched will be conducted rather than both enriched and direct.

Scientific Question:

Does the presence of Arthrobacterphage appear more dominant in one oak tree species than the others? If so, in this species is there a correlation between the presence of Arthrobacterphage and the presence of oak wilt fungus growth?

Procedure:

• To prevent contamination, wipe the table with CiDecon and ethanol and also set up an aseptic zone.
• Remove the spot test plate from the incubator to check for plaques.

Plaque

• Use a pipette to add the enriched lysate to 0.5 mL of Arthrobacterphage, label it Culture 1, and allow it to sit for 15 minutes.
• Add 8mL of LB Broth to a 50 mL conical vial.
• Add 90 uL of CaCl2 to the 50 mL conical vial using 10uL – 100 uL pipette.
• When the 15 minutes for “Culture 1” is almost over, add 10 mL 2X Top Agar to the solution of LB Broth and CaCl2 in the 50 mL vial.
• Shake the vial several times to get an even mixture of the solution.
• Using a pipette, add 4.5 mL of the top agar into a new vial.
• 1 mL of the top agar was added to the top agar control plate (shared by four tables.)
• Pour “Culture 1” onto the agar plate and label it “PA 1 (enriched).”

Plaque Assay 1

Results and Analysis:

• The materials need to create the top agar was multiplied by four.
• It was a little difficult to get an exact amount of LB Broth and Top Agar due to the difficulty of seeing the marks.

Conclusion and Future Plans:

• Due to the presence of plaque in my spot test which indicated the presence of phage, the plaque assay provided a way to further prove that there are phages within soil sample 1.
• In the future (9/5/18), I will complete the plaque assay for the direct isolation lysate if there are enough agar plates.

Rationale:

Conduct a spot test to see whether or not there are phages in either Direct Isolation or Enriched.

Procedure:

• To prevent contamination, wipe table with CiDecon and ethanol and set up an aseptic zone using an ethanol lamp.
• Take enriched lysate out of the refrigerator and bring it to the table.
• Prepare a microcentrifuge tube and 0.22 um filter as they will be in use later on in the procedure.
• In the aseptic zone, use a syringe and remove 0.2 mL from the tube and place it in the microcentrifuge tube. Label it “Enrich 2.”
• Take an agar plate and label it into three sections: Enrich, Direct Isolation, and Negative Control.
• To make the top agar, calculate how much Calcium Chloride is needed from one mole using the C1V1=C2V2 formula to get a concentration of 4.5 mM.
• Combine LB Broth, 42.75 uL of Calcium Chloride, and LB 2X Top Agar (last) to have a total volume of 9.5 mL.
• Shake the tube and pour over the negative control agar plate.
• Calculate the amount of Calcium Chloride needed to conduct the spot test.
• Combine LB Broth, 45 uL of CaCl2, 0.5 Arthrobacter, and at last, add LB 2X Top Agar.
• Pour the top agar into the agar plate for the spot test.
• Allow the top agar to solidify.
• Add 0.1 mL of phage buffer to the negative control using a pipette.
• Prepare a 0.22 um filter, a microcentrifuge tube, and a syringe to filter the direct isolation.
• Add 5 mL of Enrich and Direct Isolation into their designated areas in the agar plate.
• Allow the plate to sit to avoid mixing of the spots.

Spot Test

• After 10 minutes, place both plates in the incubator.
• Clean the table with CiDecon and ethanol and place all equipment in their proper places.

Results and Analysis

• For the spot test, we used C1V1=C2V2 to find the concentration needed for a 10 mL solution out of 1M of CaCl2.

C1V1=C2V2

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

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

V1= ((4.5 mM)(10000 uL)) / (1000 mM)

V1= (45000 mM x uL) / 1000 mM

V1= 45 uL

• For the top agar control, we used the same formula to find the concentration needed for a 9.5 mL solution.

C1V1=C2V2

(1 M)(V1)=(4.5 mM)(9.5 mL)

(1000 mM)(V1)=(4.5 mM)(9500 uL)

V1= ((4.5 mM)(9500 uL)) / (1000 mM)

V1= (42750 mM x uL) / 1000 mM

V1= 42.75 uL

• Between the time of cleaning the table and starting the experiment, my elbows were on the table and instruments were also placed on the table.
• The narrow end of the filter was touching the table and a little more than 0.2 mL from the tube was put into the microcentrifuge tube.
• After I added the enriched to the plate, two bubbles appeared in the middle but was moved to the side.

Conclusion and Future Plans

• Using both lysates (Direct and Enriched), a spot test was conducted to see if phages are present within the sample. Two separate sets of Top Agar were made: one was without the arthrobacter to serve as the Top Agar control which show if the top agar was contaminated and the other was for the spot test. The experiment was completed with few mistakes; none that cause drastic changes in the results.
• In the future (8/29/18), I plan on conducting the plague assay experiment to further prove the existence of phages in my sample.

Rationale:

Filter the Direct Isolation lysate

Procedure:

• Take the Direct Isolation out of the refrigerator and get a new vial.
• Fill the new vial with water with the same mass as the Direct Isolation.
• Place the tubes directly across from each other in the centrifuge and allow it to spin from 5-10 minutes.
• Take the Direct Isolation out of the centrifuge and place it in the test tube rack in the refrigerator.

Results and Analysis:

• Upon receiving my direct isolation, I noticed that the sample was already separated. Some minutes later, without me shaking the tube, the sample became evenly mixed again.

Conclusion

• I did not fulfill the purpose of this lab which was to filter the direct isolation. However, I did complete half of the procedure which was to put it in the centrifuge to separate the phage from the dirt and bacteria.

Future Plans

• In the future (8/28/28), I plan to finish filtering my Direct Isolation lysate. I also plan to conduct a spot test in which the filtered Direct Isolate will come in play.

Rationale:

Separate the phages and bacteria from an environmental sample with the purpose of obtaining phages for experimentation

Procedure:

• To prevent contamination, wipe the tables with CiDecon and ethanol (70%) and set up an aseptic zone by using an ethanol lamp
• Near the lamp, pour the LB Broth up to the 35 mL mark in the 50 mL conical vial containing the soil sample and then close the sample as quickly as possible
• Shake the sample vigorously for 15 minutes.
• After shaking for 15 minutes or more, place the sample in a centrifuge for five minutes
• Using the tube top vacuum filter under the vacuum hood, filter the supernatant. Use a bulb pipette to transfer the supernatant to the filter.
• Filter supernatant until reaching 20 mL of lysate.
• After reaching 20 mL, carefully unscrew the filter from the vial filled with lysate and quickly screw on the lid to prevent contamination
• Separate the lysate evenly; half of the sample will stay in the vial while the other half will go into a new one (15 mL conical vial).
• The lysate in the new vial, labeled “Direct Isolation”, will go into the fridge to be filtered later.
• Combine the other half of the lysate with 0.5 mL of Arthrobacter (the host) near the lamp and label it “Enriched 1.”
• Close the lid and place the lysate in a test tube rack to be placed in the refrigerator.
• Place all equipment back to their proper place and wipe the table with CiDecon and ethanol (70%).

Results and Analysis:

• Rather than placing the sample in the bag, I placed it in the vial and sealed it shut. Over the course of two days, the vial remained in my room with cold temperatures (68 F). During this time, soil particles and perspiration lined the walls.
• Due to the large spaces between the clusters of soil, I tapped the vial against the table to fill those spaces. In doing so, my soil sample went from 17 mL to 10 mL; 2 mL less than the minimum requirement. Due to this problem, I did not produce as much lysate as I should have to create my Direct Isolation and Enrichment.
• The rate at which the supernatant flowed through the filter was incredibly slow and I lightly tapped the vacuum filter against the table.
• After pouring the Arthrobacter in the lysate, I noticed that there was still some left in the vial.
• The soil sample combined with the LB Broth reached a mass of 51.4 g.
• The Direct Isolation had a mass of 13.8 g.

Conclusion and Future Plans:

• The procedure was carried with little mistakes but none that can cause much contamination. Despite having less soil than what was required, I have enough to carry on for further experimentation.
• In the future (Friday- 8/24/18), I will filter the Direct Isolation using the tube top vacuum filter.