Date: Wednesday, September 12th, 2018

Title: Spot Test and Metadata Continued

Rationale: The purpose of today’s lab is to make a spot test, an agar control, and finish soil metadata.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
2. We lit a burner in the middle of the table in order to keep falling particles from contaminating the equipment or samples from above since the flame creates a circulating air current.
3. We mixed 8 mL LB broth and 90 microliters of CaCL2 in a 50 mL conical for our plates.
4. I used a syringe filter to transfer 1 mL of my enriched lysate to a culture tube containing .5 mL ATC 21022.
5. Next we added 10 mL of 2X top agar to our 50 mL conical. We pipetted 4.5 mL of this solution to our top agar control plate.
6. We quickly added 4.5 mL of our solution to our individual plates.
7. We waited for 10 minutes for our agar to harden.
8. We then took 10 microliters of our enriched lysate and put it on the plate. We also took 10 microliters of direct isolation and of phage buffer and added them to the plate.
9. We let our plates sit for 15 minutes then set them in an incubator at room temperature.
10. I took a pinch of my soil sample and added it to a small pH vial.
11. I filled the rest of the vial with water, dipped 1 inch of pH paper in the mixture, and waited for 45 seconds.
12. I compared the color of the paper and determined that the soil sample had a pH of 6.3.
13. I checked my Falcon tube and determined that my sample was 75% sand, 12.5% silt, and 12.5% clay. Some silt and clay particles were still in suspension.
14. I weighed my dry soil boat and found it to be 8.985 grams. This would make the soil by itself weigh 6.506 grams.
15. I subtracted today’s weight from Wednesday’s 7.259 grams of wet soil and calculated that my sample was 27.6% water.

Observations:

• My dispersion wasn’t completely settled, so soil composition percentages could be off.
• My soil pH was 6.3, so it was slightly acidic.

Results:

• This experiment yielded me a spot test as well as soil metadata.

Next Step:

• My next step is to observe the results from my spot test and check for possible phage plaques.

Date: Monday, September 10th, 2018

Title: Soil Filtration and Metadata

Rationale: The purpose of today’s lab was to filter out our soil, get an enriched and direct sample, and record soil metadata.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
2. We lit a burner in the middle of the table in order to keep falling particles from contaminating the equipment or samples from above since the flame creates a circulating air current.
3. Next I filled a 50 mL conical with my soil up to the 2 mL mark.
4. I filled the same conical up to the 12 mL mark with LB broth and shook for 14 minutes.
5. I massed my conical vial and recorded it to weigh 19.79 g. I spun this for 5 minutes at 10,000 g.
6. I filtered the supernatant with a 22 micrometer syringe filter to a new 50 mL conical.
7. I added .5 mL ATC 21022 to my lysate, labelled it BJR and E for “enriched,” and set it in the incubator at 3:50 pm.
8. I then filtered 1 mL of my supernatant to a 15 mL vial to use as my direct isolation, storing it in the fridge.
9. While waiting for my soil to soin, I added soil to a Falcon tube to the 10 mL mark.
10. I added DI water to the 30 mL mark, added 2 drops of dispersion fluid, shook it for 30 seconds, and left it to disperse.
11. I set some soil on a plastic boat to let it dehydrate to find the percent water. The boat weighed 2.479 grams, weighing 9.738 grams after adding soils. This means I had 7.259 grams of wet soil in the boat.

Observations:

• The soil in the Falcon tube already began to separate by the time I left, showing different layers of different sized particles.

Results:

• This project yielded me a direct isolation, an enriched isolation, soil metadata, and a dispersed Falcon tube allowing me to record soil composition.

Next Step

• My next step is to use my enriched lysate, direct isolation, and a phage buffer to make a spot test. I will also finish recording metadata.

Date: Wednesday, September 5th, 2018

Title: Plaque Assay Evaluation and Soil B Collection

Rationale: The purpose of today’s lab was to check on our plaque assays from the previous lab and collect new samples of soil to test in order to work towards an answer to our class question.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. We began by removing our plaque assays from the incubator.
2. Upon viewing the plaque assay, my plate had negative results. The plate was covered with a vein-like layer of agar. It’s possible I didn’t let the agar harden long enough before moving the plate to the incubator or that my measurements for making the agar were incorrect.
3. Next I went outside with my group to find a live oak to take samples from.
4. We chose a large live oak at coordinates 31°32’57” N 97°6’54” W.
5. Using a tape measure, we concluded that the circumference of the the tree at 137 cm from the ground was 299 cm.
6. The canopy was 1,672 cm at its largest and 912 cm at its smallest, averaging out to 1,292 cm in diameter.
7. We used shadows and trigonometry to determine the tree height. Since I stand at 6’1″ tall, converted this to 182.5 cm. My shadow was measured to be 173 cm. The tree’s shadow was 1013 cm, so using similar triangles we determined that the tree was 1068.64 cm tall.
8. We placed a soil sample and a tree leaf into a plastic bag labelled BJR 9-5-18 Soil B and transferred dirt from the bag into a 15 mL conical to the 2 mL mark.

Observations:

• My plaque assay was webbed.

Results:

• My plaque assay was negative and the control plate was clean therefore I didn’t have a phage in Soil A.
• This lab day yielded me a new soil sample to test for phages.

Next Step:

• My next step is to wash, filter, and enrich the sample for phages.

Date: Wednesday, August 29th, 2018

Title: Spot Test Results and Plaque Assay Preparations

Rationale: The purpose of today’s lab is to examine my spot test for positive or negative results and test my remaining lysate with a plaque assay to check for possible missed phages.

Preliminary Class Question: Is there a correlation between certain concentrations/species of bacteriophages and different species of oak trees?

Procedure:

1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
2. We lit a burner in the middle of the table in order to keep falling particles from contaminating the equipment or samples from above since the flame creates a circulating air current.
3. We checked our plates for results. My plate was negative, with a few spots being attributed to bubbles. My plate sat in the incubator for ~46 hours.
4. Group 5 received 3 plates to set up a plaque assay on. In addition, my side of the room (four groups, 1, 2, 5, and 6) received one more plate to share as a control.
5. We used a P200 micropipette to transfer 22.5 microliters 1M CaCl2 to a 50 mL conical vial to be used in the top agar control.
6. We added 2 mL LB broth to our control vial before realizing that we had miscalculated how much material we needed, forgetting to multiply the values by four to have enough solution for each plate.
7. We added 67.5 microliters 1M CaCl2 to the group conical using a P200 micropipette.
8. We then added 6 mL LB broth to our group conical vial.
9. I used a P10 micropipette to transfer 10 microliters of my enriched lysate to to a vial containing .5 mL arthrobacter ATC 21022 and let this solution sit for 21 minutes in order to let any phages infect the arthrobacter.
10. We added 10 mL x2 top agar to the group vial then quickly transferred 1 mL of the group vial solution to the group control plate so the agar didn’t harden early.
11. I transferred 5 mL  of x2 top agar to my personal vial with arthrobacter and enriched  lysate before adding the mixture to my agar plate in the aseptic zone.
12. I moved the agar plate side-to-side in order to cover the plate completely. I let this sit for 10 minutes to solidify.
13. I set my plate in the incubator at room temperature.

Observations:

• The spot test and plaque assay seem very similar on the surface, but have important differences. In the spot test, the plate is divided into separate sections to see if certain isolations or samples yield results. In a plaque assay, the whole plate has the same solution spread all over it and is less organized.

Results:

• Our group spot test control was contaminated, and my personal spot test yielded negative results.
• This experiment yielded me a plaque assay set up which will hopefully be uncontaminated and yield something positive.

Next Step:

• My next step is to evaluate my plaque assay for positive results next week.

Date: Monday, August 27th, 2018

Title: Spot Test

Rationale: The purpose of today’s lab is to set up a spot test on an agar plate with an ATC 21022 lawn in order to find a bacteriophage.

Procedure:

1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
2. We lit a burner in the middle of the table in order to keep falling particles from contaminating the equipment or samples from above since the flame creates a circulating air current.
3. I filtered 2 mL of my enriched isolation using a 22 micrometer filter into a 3 mL microcentrifuge tube.
4. My group (Group 5) then received four agar plates. We each used one individually, then used the fourth plate as a top agar control to make sure our top agar wasn’t contaminated, thus invalidating any potential results.
5. I labelled my plate with my initials (BJR) and divided the plate into three equal sections, labelled E, D, and B for Enriched isolation, Direct isolation, and Phage buffer respectively.
6. We calculated how much of each material we would need for our plates: for my personal plate, I would use .5 mL arthrobacter ATC 21022, 4.5 mL LB Broth, 5 mL x2 top agar, and 45 microliters 1M CaCL2. For Group 5’s top agar control, we would use 4.5 mL LB broth, 5 mL x2 top agar, and 42.5 microliters 1M CaCl2.
7. We used a P200 pipette to transfer 42.5 microliters 1M CaCl2 to a new 50 mL conical vial. This was to be used for Group 5’s top agar control.
8. I used the same P200 pipette to transfer 45 microliters  1M CaCl2 to my own 50 mL conical vial.
9. We used another pipette to transfer 4.5 mL LB broth into the group conical and 4.5 mL LB broth into my personal conical.
10. I then added 5 mL of x2 top agar to my conical.
11. I received .5 mL arthrobacter ATC 21022 to add my conical from the TA Lathan.
12. I transferred the contents of my 50 mL conical to my agar plate then let it sit for  15 minutes. We also added the top agar to the control plate and let it sit for 10 minutes.
13. I used a P10 pipette to drop 10 microliters of my enriched sample to the division of my plate marked “E,” 10 microliters of my direct sample to the division of my plate marked “D,” and 10 microliters of a phage buffer to the division of my plate marked “B.”
14. We placed our plates into a room temperature incubator.

Observations:

• Top agar solidifies quickly, so it’s better to add top agar last. I had added the top agar to my conical before adding arthrobacter, leading rushing and possible sloppy work.

Results:

• This experiment yielded me a spot test set up to evaluate at a later date.

Next Steps:

• My next step is to examine the plate in ~48 hours to see if there is a positive result.

Date: Friday, August 24th, 2018

Title: Supernatant Filtration and Lysate Enrichment

Rationale: The purpose of today’s lab was to filter my supernatant from my 50 mL conical vial to two new vials in order to examine them and use them for a Spot Test next week.

Procedure:

1. I began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
2. I lit a burner in the middle of the table in order to keep falling particles from contaminating my equipment or sample from above since the flame creates a circulating air current.
3. I used a pipette in the aseptic zone to transfer my supernatant from the 50 mL conical to two 15 mL vials.
4. The two smaller vials filled with supernatant were then spun for five minutes in a small centrifuge in order to separate any heavier particles that may have drifted up since 8-22-18 when I originally centrifuged my 50 mL conical.
5. Under the fume hood, I used a pipette to transfer the supernatant from my two smaller vials to a 22 micrometer filter in order to separate potential bacteriophages from bacteria too large to fit through the filter.
6. I transferred 10.5 mL of the filtered lysate to a 15 mL vial and kept 10 mL of filtered lysate in another 50 mL conical.
7. I then added .5 mL arthrobacter (ATC 21022) to the Enriched sample (50 mL conical vial) in the aseptic zone.
8. The direct sample was then refrigerated, while the enriched sample was incubated.

Observations:

• Soil and other particles were present in the supernatant after the day spent out of the lab, meaning it’s important to filter out supernatant soon after being centrifuged in order to keep an uncontaminated sample as well as one that won’t clog the filter.

Results:

• This experiment yielded me both an enriched and a direct isolation.

Next Step:

• My next step is to set up a spot test next week to test for plaques and therefore bacteriophages.

Date: Wednesday, August 22, 2018

Title: Soil Washing

Rationale: The purpose of today’s lab was to isolate potential bacteriophages from dirt, bacterium, and other particles in the soil sample I collected (Soil A*) by filtering the sample into a direct and enriched lysate.

Procedure:

1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
2. We lit a burner in the middle of the table in order to keep falling particles from contaminating our equipment or samples from above since the flame creates a circulating air current.
3. I transferred the sample of Soil A from a plastic storage bag to a 50 mL conical vial in the aseptic zone until the soil reached the 15 mL mark.
4. I transferred LB broth to the 50 mL conical vial in the aseptic zone until Soil A and the LB broth together reached the 35 mL mark.
5. I shook the 50 mL conical vial with LB broth and Soil A for 15 minutes (15:11-15:26). This was done in intervals switching between my hand and a vortex machine.
6. During the mixing process, I weighed my 50 mL conical vial, finding it to be 52.01 g. The weight of the vials are important so that the centrifuge can be balanced without a risk to the rotor being posed by uneven weights.
7. My 50 mL conical vial was placed in the centrifuge along with other students’ samples at 3,000 revolutions per minute for five minutes. This was done to separate possible bacteriophages from heavier particles such as dirt.
8. Some students at this point filtered their supernatant to isolate their lysate; however, due to time constraints, my centrifuged 50 mL conical was placed in the fridge to be worked on later in the week.

Observations:

• The mixed solution of LB broth and Soil A was dark and larger particles were unevenly distributed throughout.
• After being centrifuged, the dirt was all at the bottom of the 50 mL conical. It was tightly packed and smoothed over.
• The resulting supernatant was mid-to-dark yellow in coloration after being centrifuged, similar to a slightly dehydrated urine sample.

Results:

• This experiment yielded me a 50 mL conical with a separated supernatant to be filtered at a later date.

Next Steps:

• My next step is to filter my supernatant before examining my lysate for bacteriophages.

*Soil A is my first sample of soil, found near a red oak with a few dying branches outside of the Dawson residence hall at Baylor University.