Rationale:

To pick a new plaque, dilute it using phage buffer, and create plaque assays

Procedure:

• First, wipe the table with CiDecon and Ethanol and set up an aseptic zone to prevent the spread of contamination.
• Arrange three small tubes in order, from 10^0 to 10^-2. Fill each tube with 90 µL of phage buffer.
• From a plate with a high concentration of plaques, locate a plaque and pick it.
• Place the picked plaque in the 10^0 tube of phage buffer.
• Transfer 10 µL of lysate from the 10^0 dilution tube to the 10^-1 dilution tube.
• After, transfer 10 µL of 10^-1 diluted lysate to the 10^-2 dilution tube.
• After performing the serial dilution, 2 mL of LB Broth and 22.5 µL of CaCl2 were combined together for as many plaque assays being made.
• Each dilution was combined with 0.5 mL of Arthrobacter and left alone for 10 minutes to infect.
• After infection, combine the Arthrobacter and dilution to the LB Broth and CaCl2 mixture.
• Add 2.5 mL of 2X Top Agar to the solution then pour over the assigned plate.
• Allow for the plate to solidify for 15 minutes before placing it invertedly in the incubator.

Results and Analysis:

There are no plaques in both dilutions. This may be due to the many mistakes that were made when conducting the dilutions and creating the plaque assays. Due to the errors, the entire procedure was conducted again with a new picked plaque.

Conclusion:

After setting up the correct measures to prevent contamination, three small tubes (10^0, 10^-1, and 10^-2) were filled with 90 µL each. From the plaque assay, pick a plaque and place in the 10^0 tube. From that tube, transfer 10 µL to another tube, creating the 10^-1 dilution. From the 10^-1 dilution, transfer 10 µL to the 10^-2 tube to create the dilution. Next, add each dilution to their respective 0.5 mL of Arthrobacter and allow it to infect for 10 minutes. While infection, combine LB broth and CaCl2. After 10 minutes, combine the lysate with the culture and add 2X Top Agar. Pour onto their respective plates and allow to sit for 15 minutes to solidify. Then place plate inverted in the incubator.

Future Plans:

In the future, dilutions will continue if there are plaques present on the plate. If not, another plaque will be picked.

Rationale:

Due to the possible presence of plaques on the plate, two will be picked and put into phage buffer and a plaque assay will be made with the original lysate.

Procedure:

• First, the previous plaque assays were examined to find some contamination in the negative control and some presence of phage in the plates.
• The lab table was wiped down with CiDecon and Ethanol, after which an aseptic zone was set up to prevent contamination.
• The phages were located and picked using a pipette tip, then inserted into 90 µL of phage buffer and stored away.
• Then, 0.5 mL of Arthrobacter and 1 µL were mixed together and left alone for 10 minutes.
• Next, LB broth (2 mL) and CaCl2 (22.5 µL) were combined.
• After 10 minutes, the 2x Top Agar (2.5 mL) was added to the top agar solution along with the original lysate and Arthrobacter.
• The solution was then poured onto the plate and given enough time to solidify.
• After solidification, the plates were placed into the incubator and left there for 48 hours.

Results and Analysis:

The amount of contamination in the negative control has significantly decreased compared to other plaque assays conducted in the past.

The phages in the plaque assay are located in the center and are very small.

Conclusion:

First, the tables were cleaned with CiDecon and ethanol along with an aseptic zone. Plaques were spotted, picked (2), then put into 90 µL of phage buffer. However, due to the contamination in the negative control, another plaque assay was made. The lysate and Arthrobacter were mixed together to infect. The LB broth and CaCl2 were added together. After 1o minutes, the 2X top agar was added along with the lysate and Arthrobacter onto the plate. It was left alone to solidify then put into the incubator.

Future Plans:

In the future (10/10/18), the plaques that were picked and put into phage buffer will be used to make a plaque assay. Before doing that, the lysates will be diluted.

Rationale:

To make plaque assays with the plaques picked last class and to also pick new plaques from the latest plaque assay made.

Procedure:

• First, the lab table was wiped down with ethanol and CiDecon and an aseptic zone was set up to prevent contamination.
• Using the most recent plaque assay, phages were spotted, picked, then put into 90 µL of phage buffer, making the 10ˆ0 dilution.
• Then, 10 µL of lysate and phage buffer from the 10ˆ0 dilution was added to 90 µL of phage buffer to make 10ˆ-1 dilution.
• Then, 0.5 mL of Arthrobacter and 100 µL of 10ˆ0 dilution were mixed together and left alone for 10 minutes.
• The same process occurred for the 10ˆ-1 dilution.
• Next, two sets of combined LB broth (2 mL) and CaCl2 (22.5 µL) were made.
• After 10 minutes, the 2x Top Agar (2.5 mL) was added to the top agar solutions along with the lysate and Arthrobacter.
• The solutions were then poured onto their respective plate and given enough time to solidify.
• After solidification, the plates were placed into the incubator and left there for 48 hours.

Results and Analysis:

The possible plaques are the small, clear areas in the center of the plate.

Conclusion:

Due to the possibility of plaques on the recent plaque assay, those plaques were picked and placed in phage buffer, becoming 10ˆ0 dilution. Then, 10 µl of lysate was transferred from the 10ˆ0 dilution to another vile of phage buffer which made 10ˆ-1 dilution. Arthrobacter was added to both dilutions and left alone for 10 minutes to infect. While infection, two sets of LB broth and calcium chloride were mixed. After 10 minutes, the dilutions along with the Arthrobacter were added to their culture. 2X Top Agar was added then poured onto their respective plates.

Future Plans:

In the future (10/15/18), the plates will be checked for the presence of plaques. If there are plaques, then dilutions of the plaque will be continued. If not, another picked plaques will be tested to determine whether or not it is a plaque.

Rationale:

For this lab, a plaque assay will be created, the % soil composition will be determined, the enriched lysate will be filtered, and % water experiment will be set up.

Procedure:

First, the lab table was wiped down with CiDecon and Ethanol, after which an aseptic zone was set up to prevent contamination. The soil composition was determined by removing the water from the top. After, the procedure to determine % water was set up by weighing a weigh boat along with 3-5 g of soil. 1.2 mL of enriched lysate was added to a tube then put into the centrifuge for 10 minutes to be filtered. After 10 minutes, the lysate was filtered using a syringe and filter. Then, 0.5 mL of Arthrobacter and 1 µL of lysate were mixed together and left alone for 10 minutes. Next, LB broth (2 mL) and CaCl2 (22.5 µL) were combined. After 10 minutes, the 2x Top Agar (2.5 mL) was added to the top agar solution along with the lysate and Arthrobacter. The solution was then poured onto the plate and given enough time to solidify. After solidification, the plates were placed into the incubator and left there for 48 hours.

Results and Analysis:

% Soil Composition 

Out of 4 mL

% Soil (.75 mL)= (.75/4) x 100= 18.75%

% Sand (2.75)= (2.75/4) x 100= 68.75%

% Clay (.5)= (.5/4) x 100= 12.5%

Rather than having a smooth layer, the control plate had a bumpy texture.

Conclusions:

First, the tables were cleaned with CiDecon and ethanol along with an aseptic zone. The soil composition was determined then a % water procedure was set up. 1.2 mL of lysate was centrifuged for 10 minutes then taken out and filtered using a syringe and filter. Then, the lysate and Arthrobacter were mixed together to infect. The LB broth and CaCl2 were added together into a vial of which would become the top agar solution. After 1o minutes, the 2X top agar was added along with the lysate and Arthrobacter onto the plate. It was left alone to solidify then put into the incubator.

Future Plans:

The plaque assays will be checked for the presence for plaques. If there are no plaques with contamination, another plaque assay will be created. If there are no plaques and no contamination, then a new soil sample will be collected, If there are plaques with no contamination, a plaque will be picked and will be diluted using phage buffer. After diluting it, a plaque assay will be created to begin the process to get a high concentration of plaque.

Rationale:

To wash soil sample 4 and enrich it using Arthrobacter, to determine the pH of the sample, and to also set up the experiment to determine the soil components

Procedure:

First, 2 mL of the soil sample was taken out and transferred to a new vial. 10 mL of LB broth was added to the vial and vortexed for 15 minutes. Then, the vial was weighed and placed in the centrifuge for 10 minutes. While the sample is in the centrifuge, prepare the soil composition experiment by adding 4 mL into a tube along with 8 mL of water. Vortex for 30 seconds then add 3 drops of soil dispersion liquid and set aside. After 10 minutes, the vial was taken out and the supernatant was separated from the rest of the contents. Using a syringe and 0.22 µm filter, the supernatant was filtered to become the lysate. 0.5 mL of Arthrobacter was then poured into the lysate. Finally, the pH of the soil was taken using some of the soil sample, water, and pH paper. Water was filled to the top and shaken for 45 seconds. After it was left alone for 2 minutes, the pH was taken using the pH paper.

Results and Analysis:

On the right, the control with little amounts of contamination. On the left, the plaque assay with a negative result.

When the LB broth is used, it is clearly uncontaminated; however, the next class there was contamination.

The contamination may have been due to the contaminated LB broth.

The soil was a mostly a mixture of clay and soil.

pH of soil sample: 5.7

Mass of Soil Sample and LB Broth: 18.54 g

Mass after adding water drops: 19.43 g

Total amount of lysate: below 5 mL

Conclusion:

In order to wash the soil sample, 2 mL of soil and 10 mL of LB broth was combined together then vortexed for 15 minutes. After the 15 minutes, the vial was weighed then placed in the centrifuge. While the vial was in the centrifuge for 10 minutes, a method used to determine the soil composition was made. The method called for 4 mL of soil with 8 mL of water and an addition of 3 drops of soil dispersion liquid. After centrifuged, the supernatant was separated from the rest and then filtered using a syringe and filter. Then, the pH of the soil was taken.

Future Plans:

Next time, the lysate will be filtered and will be used to prepare a plaque assay. Also, a plaque assay will be made using the previous lysate (soil sample 3).

Rationale: To collect a new soil sample and to also perform another plaque assay due to the contamination in the control

Procedure:

First, a new soil sample was collected from a white oak. Measurements (diameter, canopy height, and tree height) and leaves were taken and soil (2 feet from base). Next, a plaque assay was made due to the contamination in the control. The lab table was wiped down with CiDecon and Ethanol, after which an aseptic zone was set up to prevent contamination. Then, 0.5 mL of Arthrobacter and 1 µL were mixed together and left alone for 10 minutes. Next, LB broth (2 mL) and CaCl2 (22.5 µL) were combined. After 10 minutes, the 2x Top Agar (2.5 mL) was added to the top agar solution along with the lysate and Arthrobacter. The solution was then poured onto the plate and given enough time to solidify. After solidification, the plates were placed into the incubator and left there for 48 hours.

Results and Analysis:

The latest control (right) was negative and the plaque assay (left) was negative.

A reason for the contamination may be due to the carelessness in performing the experiment (not remaining close to the aseptic zone.)

The Top Agar became contaminated.

Tree Data:

Diameter: 277 cm

Height: 2118.36 cm

Shortest Side of Canopy: 721.36 cm

Longest Side of Canopy: 1249.68 cm

Average: 985.52 cm

The soil was obtained from the largest tree on the large area of grass by iHop.

A large clump of grass had to be removed to access the soil. The soil became accessible 1.5 inches from the surface. Rather than being small particles, the soil was composed of large chunks of soil mixed with small pieces of grass. It had a dark coloration and was dry.

The leaves at eye level were dead and were decaying in random areas on the leaves. However, as the branches get higher and higher, the leaves looked healthier. Not only were there leaves on the tree, but many big acorns also the tree and would fall once in a while.

Due to the age and location of the tree, it is clear that there was no garden soil added.

Conclusion:

Due to the contamination of the negative control and negative result on the plaque assay, a soil sample was collected from a Burr Oak near iHop. Data such as the diameter, height, average canopy length, soil composition, and leaves were taken. After collecting a sample, a plaque assay was created using the current lysate to make sure there are no plaques. First, lysate and Arthrobacter were combined and left alone for 10 minutes to infect. Then, LB broth (checked for contamination) was added along with the calcium chloride. After the allotted time, the lysate and Arthrobacter were added to the LB broth and calcium chloride. 2X Top Agar was then added to the solution and poured onto the agar plate. For 15 minutes, the plates remained near an aseptic zone to solidify. After, the plates were placed in an incubator.

Future Plans:

If there are plaques in the current lysate, a plaque will be picked, diluted using phage buffer, then used to create a plaque assay. If there are no plaques along with no contamination, the new soil sample will be washed and enriched. If there are no plaques and contamination on the control, a new plaque assay will be made with methods being carefully used to avoid contamination.

Rationale:

To filter the enriched lysate using a syringe, collect the data to understand the soil’s % water and soil composition, and complete a plaque assay using the filtered enriched lysate.

Procedure:

The data for the soil composition and % water was first collected. To determine the % water, a scale was used to determine the initial and final masses which were used for calculations. For the soil composition, the different layers were observed and the volume of each layer was taken down for later use in calculations. After collecting all the data, the next step was to filter the enriched lysate. It was filtered using a syringe and 0.22 µm filter into a tube. In another tube, 1 mL of filtered enriched lysate and 0.5 Arthrobacter were combined and left alone for 10 minutes to infect. Next, 22.5 µL of CaCl2 and 2mL of LB broth were combined to form what was to become the top agar. After 10 minutes, 2.5 mL of 2X Top Agar was added to the Top Agar solution. The Arthrobacter and lysate were added to the top agar solution and poured onto the agar plate. The plate was left alone for 10 minutes for solidification. Then, the plates were placed in the incubator.

Results and Analysis:

% Water

(3.336/3.79)x100=88.0%

Soil Composition

Total: 4 mL

2 mL of sand

.7 mL of silt

1.3 mL of clay

(2/4)x100= 50% of sand

(.7/4)x100= 17.5% of silt

(1.3/4)x100= 32.5% of clay

Plaque Assay Plate

Conclusions and Future Plans:

First, data was collected from procedures completed previously (soil composition and % water) and used to perform calculations to determine the percentages. Then, the lysate was filtered using a syringe. Lysate and Arthrobacter were combined and left alone for 10 minutes. During that time, CaCl2 and LB broth were combined in a conical vial. After the 10 minutes, the Arthobacter and lysate were added to the CaCl2 and LB broth along with 2X Top Agar. The top agar was poured on a plate and left to solidify for 10 minutes then placed in the incubator.

Future Plans:

On Monday (9/23/18), the plates will be checked for plaques. If there is contamination of the control with no plaques on the plate, another plaque assay will be made. If there is no contamination along a negative plate, another soil sample will be collected in another area. If there is no contamination with a positive plate, the plaque will be picked and will be diluted using a phage buffer.

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.