Results:

No contamination, however, the “KEA 11/5 PA 10^-2” plate looked strange since it folded up when solidifying.

Rationale:

The titer will be calculated from the “KEA 11/5 PA 10^-1” plate. From this, the amount of lysate needed to webbed the plate will be calculated. Three plaque assays will be run with slightly varying amounts to increase the likelihood of receiving a webbed plate.

Procedure:

1. Counted the total number of plaques on “KEA 11/5 PA 10^-1” plate.
2. Calculated the average diameter of plaques, the titer, and the amount of lysate needed to web the plate.
3. Once an aseptic zone was established, 97 µL, 125 µL, and 150 µL of “KEA 11/5 FS lysate 10⁰” were added to correlated test tubes which already contained 0.5 mL of Arthrobacter.
4. 8 mL of LB Broth, 90 µL of CaCl₂, and 10 mL of 2X TA were combined into a conical vial.
5. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
6. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the TA mixture was poured onto a plate labeled “KEA 11/7 Control.”
7. These plates were placed in the incubator at room temperature.

Observations:

• 132 plaques were counted from the “KEA 11/5 PA 10^-1” plate. The following photos show the plates from previously.

• The following measurements were recorded from 10 random plaques to approximate the average diameter: 0.3, 0.3, 0.5, 0.6, 0.5, 0.6, 0.2, 0.2, 0.3, and 0.3 mm. The average diameter calculated was 0.38 mm.
• The approximate plaque diameter from the “KEA 11/5 PA 10^-1” plate were slightly less than half the size of the approximate plaque diameter from the “KEA 10/29 10⁰PA” plate.

Calculations:

• After the plaque assays had been performed, it was noticed that the webbed calculations were done using the plaque diameter instead of the radius. The following shows the correct calculations.

• The following calculations were performed to determine enough LB Broth, 2X TA, and CaCl₂needed for 4 plates.
  

  Original Recipe	X4
  2 mL LB Broth	8 mL LB Broth
  2.5 mL 2X TA	10 mL 2X TA
  22.5 μL CaCl2	90 μL CaCl2

  Next Steps:

  In open lab, plaque assays will be performed with the correct amount of lysate.

Results:

The webbed plate was not completely webbed as shown below.

Rationale:

The “KEA 11/2 Web” plate will be flooded to collect lysate with a higher titer. Serial dilutions and plaque assays will be performed in order to make a plate from which the new titer can be calculated.

Procedure:

1. Once an aseptic zone was established, 4 mL of phage buffer was poured onto “KEA 11/2 Web” plate.
2. The plate was shaken on an incubator for one hour.
3. Filtered lysate from flooded plate through a 0.22 µm syringe filter into a conical vial labeled “KEA 11/5 FS lysate 10⁰.”
4. 10 µL of “KEA 11/5 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1
5. 10 µL of “KEA 11/5 10^-1” was added with 90 µL of phage buffer to create 10^-2
6. 10 µL of 10⁰, 10^-1, and 10^-2dilutions were added to correlated test tubes which already contained 0.5 mL of Arthrobacter.
7. 8 mL of LB Broth, 90 µL of CaCl₂, and 10 mL of 2X TA were combined into a conical vial.
8. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
9. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the TA mixture was poured onto a plate labeled “KEA 11/5 Control.”
10. These plates were placed in the incubator at room temperature.

Observations:

• The following calculations were performed to determine enough LB Broth, 2X TA, and CaCl₂needed for 4 plates.

• When pouring the TA mixture, it started to solidify causing bubbles to form on the plates.
• The “KEA 11/5 PA 10^-2” plate did not solidify, and the TA mixture folded over on itself as shown below.

Next Steps:

If there is contamination, a plaque assay will be run again with the same dilutions. If not, the titer will be calculated. If the titer is not high, the plates will be flooded and a web plate will be made. If the titer is high, the experiment will go on to characterization procedures.

Rationale:

After the flooded plate lysate goes through a filter, a plaque assay will be performed with 524 µL of lysate to make a web plate.

Procedure:

1. Filtered lysate from flooded plate “KEA 10⁰10/29” through a 0.22 µm syringe filter into a conical vial labeled “KEA 11/2/18 FS lysate 10⁰.”
2. 524 µL of “KEA 11/2/18 FS lysate 10⁰” was added to a test tube which already had 0.5 mL of Arthrobacter.
3. 4 mL of LB Broth, 45 μL of 1 M CaCl₂, and 5 mL of 2X TA were combined into a different conical vial.
4. Transferred and mixed 4.5 mL of the Top Agar mixture from the conical vial into the test tube with the Arthrobacter and enriched lysate.
5. Poured the test tube mixture onto a plate labeled “KEA 11/2 Web.”
6. The rest of the conical vial mixture was poured onto the TA control plate.
7. The “KEA 11/2 Web” plate was placed in the incubator at room temperature.

Observations:

• The following calculations were performed to determine necessary amount of LB Broth, 2X TA, and CaCl₂needed for 2 plates.

Next Steps:

If contaminated, a plaque assay will be performed again with 524 µL of the filtered 10⁰ lysate. If not contaminated, the titer will be calculated and the next steps will be determined.

Results:

As expected, the control plate was contaminated. However, it was apparent that a plaque was picked. Plaques formed on both the “KEA 10/29 10⁰‘10/22’” and the “KEA 10/29 10⁰A” plates. No plaques formed on the “KEA 10/29 10⁰B” plate.

Rationale:

Although the phage has only gone through two passages, the plaques formed are distinct enough to move past the purification stage and go onto the amplification. Titer calculations along with flooding a plate will take place to form enough lysate to form a high titer.

Procedure:

1. Drew quadrants and counted total number of plaques on “KEA 10/29 10⁰A” plate.
2. Calculated the average diameter of plaques, the titer, and the amount of lysate needed to web the plate.
3. Once an aseptic zone was established, 8 mL of phage buffer was poured onto “KEA 10/29 10⁰A” plate.
4. Placed parafilm and stored “KEA 10/29 10⁰A” plate at 8ºC.

Observations:

• The control plate had a strange, contaminated yellow dot. The following pictures show the results.

• 514 plaques were counted from the “KEA 10/29 10⁰A” plate as shown in the picture below.

• The following measurements were recorded from 10 random plaques to approximate the average diameter: 0.1, 0.1, 0.05, 0.12, 0.04, 0.07, 0.08, 0.1, 0.1, and 0.1 mm. The average diameter calculated was 0.82 mm.

Calculations:

Next Steps:

In open lab, the flooded plate’s lysate, phage buffer mixture will go through a 0.22 µm filter to isolate the phage. A plaque assay will also be run with the mixture to receive a high titer.

Results:

No contamination or plaques were observed from the plaque assay performed on 10/26 as shown below.

Rationale:

Since it does not appear a phage was picked previously, two plaques will be picked from the “KEA 10/22 10⁰-1 PA” plate.  To continue the purification process, plaque assays will be performed with a plaque, phage buffer mixture solution made from both the newly picked plaque along with the “KEA 10/22 10⁰-1” mixture.

Procedure:

1. Once an aseptic zone was established, 100 µL of phage buffer was placed into both microcentrifuge tubes “KEA 10/29 10⁰ A” and “KEA 10/29 10⁰ ”
2. Used a micropipette tip to touch a plaque from the “KEA 10/22 10⁰-1 PA” plate and then swirled the tip in the “KEA 10/29 10⁰ A” microcentrifuge tube.
3. Used a micropipette tip to touch a different plaque from the “KEA 10/22 10⁰-1 PA” plate and then swirled the tip in the “KEA 10/29 10⁰ B” microcentrifuge tube.
4. Both “KEA 10/29 10⁰ A” and “KEA 10/29 10⁰ B” microcentrifuge tubes were vortexed.
5. 25 µL of “KEA 10/22 10⁰-1”, “KEA 10/29 10⁰ A” , and “KEA 10/29 10⁰ B” mixtures were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
6. 8 mL of LB Broth, 90 µL of CaCl₂, and 10 mL of 2X TA were combined into a conical vial.
7. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
8. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the TA mixture was poured onto a plate labeled “ML KEA 10/29 Control.”
9. These plates were placed in the incubator at room temperature.

Observations:

• The following calculations were performed to determine enough LB Broth, 2X TA, and CaCl₂needed for 4 plates.

• The plaques circled belowed were picked to make “KEA 10/29 10⁰ A” and “KEA 10/29 10⁰ B” plaque, phage buffer mixtures. To increase the chances a phage was picked, the plaques were picked under a light microscope.

• When pouring the TA mixture, the TA mixture started to solidify causing bubbles and lumps to form on the plates.

Next Steps:

If there is contamination, a plaque assay with be run again with the same mixtures. If there are plaques, a third passage will be performed. If there are no plaques, a different plaque will be picked.

Results:

Since the plates had no signs of contamination or plaques, it is safe to assume that no phage was present in the plaque picked on Wednesday (10/24). The pictures below show these plates.

Rationale:

Another plaque will be chosen and a plaque assay will be run on it to continue the purification process.

Procedure:

1. Once an aseptic zone was established, 100 µL of phage buffer was placed into a microcentrifuge tube labeled “KEA 10/26 10⁰.”
2. Used a micropipette tip to touch a plaque from the “KEA 10/22 10⁰-1 PA” plate and then swirled the tip in the “KEA 10/26 10⁰” microcentrifuge tube.
3. The “KEA 10/24 10⁰” microcentrifuge tube was vortexed.
4. 25 µL of 10⁰were added to a test tube which already had 0.5 mL of Arthrobacter.
5. 4 mL of LB Broth, 45 µL of CaCl₂, and 5 mL of 2X TA were combined into a conical vial.
6. Transferred and mixed 4.5 mL of the TA mixture from the conical vial into the test tube.
7. The test tube was poured onto a plate and the rest of the TA mixture was poured onto a different plate.
8. These plates were placed in the incubator at room temperature.

Observations:

• The following calculations were performed to determine the amount of LB Broth, 2X TA, and CaCl₂needed for 2 plates.

Next Steps:

If there is contamination, a plaque assay with be run again with the same dilutions. If there are plaques, a third passage will be performed. If there are no plaques, a different plaque will be chosen.

Results:

Many plaques formed on the “KEA 10/22 10⁰-1 PA” plate. The “KEA 10/22 10⁰-0 PA” plate was contaminated. One small plaque appeared on “KEA 10/22 10⁰-2” plate. The shared control plate was contaminated. The pictures below show these plates.

Rationale:

To continue the purification process, a plaque will be chosen from the “KEA 10/22 10⁰-1 PA” plate. Two plaque assays will be performed with this chosen plaque, with one using 10⁰and the other 10^-1.

Procedure:

1. Once an aseptic zone was established, 100 µL of phage buffer was placed into microcentrifuge tube “KEA 10/24 10⁰.”
2. Used a micropipette tip to touch a plaque from the “KEA 10/22 10⁰-1 PA” plate and then swirled the tip in the “KEA 10/24 10⁰” microcentrifuge tube.
3. The “KEA 10/24 10⁰” microcentrifuge tube was vortexed.
4. 10 µL of “KEA 10/24 10⁰” was added with 90 µL of phage buffer to create 10^-1dilution which was then vortexed.
5. 10 µL of 10⁰and 10^-1were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
6. 6 mL of LB Broth, 67.5 µL of CaCl₂, and 7.5 mL of 2X TA were combined into a conical vial.
7. Transferred and mixed 4.5 mL of the TA mixture from the conical vial into each test tube.
8. Each test tube was poured onto their correlating plate.
9. These plates were placed in the incubator at room temperature.

Observations:

• The following calculations were performed to determine the amount of LB Broth, 2X TA, and CaCl₂needed for 3 plates.

• The plaque circled bellowed was picked to make “KEA 10/24 10⁰” plaque, phage buffer mixture. The plaques on this plate were not as distinct as the plaques found on the “KEA 10/12/18 Soil E PA” plate.

• Air bubbles formed when pouring the “KEA 10/24 10^-1PA” plate.
• Strange contamination spots formed on the shared control plate as shown below.

Next Steps:

If there is contamination, a plaque assay will be run again with the same dilutions. If there are plaques, a third passage will be performed. If there are no plaques, a different plaque will be chosen.

Results:

Since the TA mixture started to solidified when placed on plate, it is hard to tell if the plates were contaminated or not as shown in the picture below.

Rationale:

It is not certain if a phage was picked previously, so plaque assays will be run with the 10⁰dilution from last time, a 10⁰dilution made from a newly-picked plaque from the “KEA 10/12/18 Soil E PA” plate, and a 10⁰dilution made from a newly-picked plaque from the “KEA 10/17 10⁰ PA” plate. From this, maybe one of the plates will have many plaques, and then a second passage can be performed.

Procedure:

1. Once an aseptic zone was established, 100 µL of phage buffer was placed into both microcentrifuge tubes “KEA 10/22 10⁰-1” and “KEA 10/22 10⁰-2.”
2. Used a micropipette tip to touch a different plaque from previous plaque assay from the “KEA 10/12/18 Soil E PA” plate and then swirled the tip in the “KEA 10/15 10⁰-1” microcentrifuge tube.
3. Used a micropipette tip to touch a plaque from the “KEA 10/17 10⁰ PA” plate and then swirled the tip in the “KEA 10/15 10⁰-2” microcentrifuge tube.
4. Both microcentrifuge tubes were vortexed.
5. 10 µL of 10⁰, 10⁰-1, and 10⁰-2 dilutions were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
6. 6 mL of LB Broth, 67.5 µL of CaCl₂, and 7.5 mL of 2X TA were combined into a conical vial.
7. Transferred and mixed 4.5 mL of the Top Agar mixture from the conical vial into each test tube.
8. Each test tube was poured onto their correlating plate.
9. These plates were placed in the incubator at room temperature.

Observations:

• The plates with the dilutions did have strange marks which most likely were air bubbles.
• The plaque circled bellowed was picked for the “KEA 10/15 10⁰-2” dilution.

• The following calculations were performed to determine the amount of LB Broth, 2X TA, and CaCl₂needed for 3 plates.

• Made a shared control plate with a different group.
• Since the “KEA 10/12/18 Soil E PA” plate had been left in the incubator, not the fridge, it appears that another plaque had formed.

Next Steps:

If there is contamination, a plaque assay with be run again with the same dilutions. If there are plaques, a second passage will be performed. If there are no plaques, a different plaque will be picked.

Rationale:

Soil metadata will be completed for future correlations. Also, plaque assays will be run with 10⁰, 10^-1, and 10^-2 dilutions. From the results of these plates further calculations will be made to determine what steps need to find a high titer.

Procedure:

1. To complete the soil metadata, the “KEA 10/15 soil E” weigh boat’s weight and the “KEA 10/15 soil E” falcon tube different layers were recorded.
2. Once an aseptic zone was established, 10 µL of 10⁰, 10^-1, and 10^-2dilutions were added to correlation labeled test tubes which already had 0.5 mL of Arthrobacter in them.
3. 8 mL of LB Broth, 90 µL of CaCl₂, and 10 mL of 2X TA were combined into a conical vial.
4. Transferred and mixed 4.5 mL of the Top Agar mixture from the conical vial into each test tube.
5. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the Top Agar mixture was poured onto a plate labeled “KEA 10/17 Control TA.”
6. These plates were placed in the incubator at room temperature.

Observations:

• The following calculations were performed to determine the amount of LB Broth, 2X TA, and CaCl₂needed for 4 plates.

• The falcon tube, as shown below, had a layer of dark brown sand with a layer of cinnamon-brown sand on top of that. The silt layer on top of these two layers appeared a whitish-gray. The remaining layer on top of this was a thin tannish-brown color which was clay.

• When pouring the TA mixture, bubbles and lumps formed on the plates.

Metadata:

Percent Water

Data Table

Equation for Percent Water

Percent Water = 18.61%

Percent Sand, Percent Silt, and Percent Clay

Data and Calculation Table

Percent Clay = 9.09%

Percent Silt = 9.09%

Percent Sand = 81.82%

Classifying Soil Texture

Based off the percent sand, percent silt, and percent clay, calculations using a soil texture triangle classified the soil sample’s texture to be loamy fine sand. The point is plotted on the soil texture triangle below.

This image was retrieved from the Natural Resources Conservation Service website at https://www.nrcs.usda.gov/wps/portal/nrcs/detail/soils/survey/?cid=nrcs142p2_054167.

Next Steps:

If the plates are contaminated, the plaque assays will be run again with the 10⁰, 10^-1, and 10^-2 dilutions. If the plates are positive, another plaque will be picked, serial dilutions, and more plaque assays will be run.

Results:

From the plaque assay ran on Friday (10/12), 4 plaques were found as shown below.

Rationale:

Since plaques did appear, soil metadata will be collected for future correlations. Also, a plaque will be picked and serial dilutions will be made to be ready to plate to start the quest to find a high titer.

Procedure:

1. Once an aseptic zone was established, about 3 grams of soil E was poured out into a weigh boat labeled “KEA 10/15 soil E” and left under the flume hood to dry out.
2. A falcon tube labeled “KEA 10/15 soil E” was filled with 10 mL of soil E, 20 mL of de-ionized (DI) water, and three drops of texture dispersion liquid.
3. The “KEA 10/15 soil E” falcon tube was shaken for 30 seconds and then left under the flume hood for the next 48 hours.
4. A small amount of soil E was placed in a pH vial and the rest was filled with DI water.
5. The pH vial was shaken for 10 seconds and then settled for 2 minutes.
6. Used pH paper to determine the pH of the soil.
7. 100 µL of phage buffer was placed into a microcentrifuge tube labeled “KEA 10/15 10⁰.”
8. Used a micropipette tip to touch the plaque and then swirled the tip in the “KEA 10/15 10⁰” microcentrifuge tube.
9. The “KEA 10/15 10⁰” microcentrifuge tube was vortexed.
10. 10 µL of “KEA 10/15 10⁰” was added with 90 µL of phage buffer to create 10^-1
11. 10 µL of “KEA 10/15 10^-1” was added with 90 µL of phage buffer to create 10^-2

Observations:

• The 4 plaques found on the “KEA 10/12/18 PA Soil E” plate were small and distinct.
• The weigh boat weighed 2.39 grams. The weigh boat with wet soil weighed 6.15 grams.
• The pH of the soil was determined to be 6.0 as shown in the picture below.

• Dilutions were created all the way out to 10^-7.

Next Steps:

On Wednesday, the dilutions will be plated.