Rationale:

The goal of today’s lab was to perform both a plaque assay and spot test as well as examine the soil metadata from the lab prior.

Materials:

• LB Broth
• 2X Top Agar
• 2x 0.5 mL of Arthobacter
• Calcium Chloride
• Serological pipettes
• Micropipettes
• Balance

Procedure:

1. This procedure began with establishing an aseptic zone using CiDecon to wipe the table down, 70% Ethanol to spray on the table and let evaporate, and a burner to create a convection current for air flow.
2. I began with a spot test first, grabbing my enriched isolation labeled “GJA Enriched 2 09/10/18” and sent it off to centrifuge for 5 minutes at 3,000 g.
3. While that was occurring, I found my soil sample that was left to evaporate and weighed it to find the mass of the dry soil and calculate the percent of water in the soil. This is the data below along with the equation used:
   

   Empty Petri Dish (mi)	6.90 g
   Petri Dish and Wet Soil (mf)	11.60 g
   Mass of Wet Soil (mwet soil)	4.70 g
   Petri Dish and Dry Soil (mf2)	10.62 g
   Mass of Dry Soil (mdry soil)	3.72 g
   Mass of Water (g)	0.98 g
   %Water	20.85%

    

4. After calculating my percent water, I retrieved my enriched sample from the centrifuge and began to make the top agar by first adding 2.00-mL of LB Broth to a 50-mL conical tube labeled “GJA Top Agar 2 09/12/18”.
5. Next I used a 20-200 µL micropipette to add 22.50-µL of Calcium Chloride to my LB Broth as well.
6. Following this, I filtered approximately 1.5-mL of my enrichment through a 2 micron syringe filter and into a micro centrifuge tube and left that to sit while I transferred 0.5-mL of our Arthrobacter into my top agar solution.
7. Once the bacteria was added, I used a serological pipette to add 2.5 mL of the 1x Top Agar solution. Then I swirled my 50-mL conical vial gently to allow everything to mix and I quickly poured the top Agar onto my plate and left it to solidify for 15 minutes.
8. While my solution was solidifying, I examined my soil separation and poured off the supernatant so I could examine the sand, silt, clay composition of my soil.  Out of the 8-mL of soil present, 3.8-mL was sand, 2.5-ml was silt, and the remaining 1.7-mL was clay. This makes a percentage of 47.5% sand, 31.25% silt, and 21.25% clay
9. After calculating the soil composition, my group decided to perform a plaque assay as well with the remaining time we had available.
10. The group decided to make enough top agar for 3 plates plus a control. This meant we had 8-mL of LB Broth, 10-mL 2X TA, and 90-µL of Calcium Chloride.
11. We began with adding the 8-mL of LB Broth with a serological pipette into one 50-mL conical vial.
12. After adding the LB broth, the 90-µL of calcium chloride was added using a 10-100 µL micropipette.
13. Once the calcium chloride was added, I combined 0.5-mL of Arthobacter with 10-µL of my lysate and let that infect for approximately 15 minutes.
14. At the end of the 15 minutes, we added 10-mL of 2X Top Agar to the 50-mL conical vial through serological pipette. Immediately following this, I extracted 5-mL of the top agar solution, added it to a 15-mL conical vial with my arthrobacter and lysate solution, mixed it very quickly, and poured it into my plate to let it solidify for 15 minutes.
15. With the remaining 5-mL of top agar left, we plated into our control plate
16. Once the 15 minutes were up, the plates were immediately put into the incubator for 48 hours.

Data/Results

• 
• Water percentage of the soil was approximately 20.85%. This was moderately surprising because the soil when it was extracted was very wet, almost clay like.
• The top agar came out looking very similar to previous top agars, despite my lysate being not as clear as everyone else’s.
• The soil contains an extremely large amount of sand in comparison to silt and clay. This was unsurprising as our soil once dry was very crumbly and exhibited sand-like qualities.

Conclusions:

• Soil was roughly 1/5 water. This could be a factor of its location to a nearby water source. There was also a man-made fountain nearby, which may contribute to the amount of water in the soil.
• In addition to the water amount, the soil was a majority of sand present in the soil. This is most likely due to the dry location that the soil and tree is in.

Next Steps:

• The next steps would be to analyze the plaque assay and spot test for the presence of phage in the soil. If there is phage present in the soil, then we will begin to analyze the amount of phage present. If there is no phage present, then I have to redo another plaque assay and spot test or gather a new sample.

9/12/18 Spot Test #2 and Completion of Metadata Collection

Objective:

The goal of this procedure is to use the previously created lysate to preform a spot test to test for phage presence in our new collected soil. During this lab period the results of the metadata tests previously run will be collected in order to help us better answer our questions.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

The question specific to my lab table is: Is the a difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine weather or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for Aseptic zone:

• CiDecon
• 70% Ethanol
• Ethanol Burner

Materials For Spot test

• .5 ml Arthrobacter
• refrigerator
• Pipette
• Test tube stand
• 50 ml tubes
• LB Broth
• 2X TA
• 1M Calcium Chloride
• Pipette cap
• Phage Buffer
• Agar plate
• Micropipette
• Syringe Filter

*In this lab period the results of soil metadata tests preformed last lab period were recorded. There were no procedural steps aside from recording information so the procedures for the metadata experiments will not be repeated here. However, the results of the metadata tests will be found both in this entry and the previous one to reflect both when they were collected and where they are applicable*

In order to complete the procedure an aseptic zone was created.

1. Clean off the work space (lab table) with CiDecon applied with a squeeze bottle and wiped away with a paper towel
2. Apply 70% Ethanol with a squeeze bottle, spread with a paper towel, and allow to evaporate
3. Light an ethanol burner in order to use the rising heat from the flame to form the aseptic zone

Then the spot test could be preformed.

1. Divide the bottom of an Agar plate into three sections and label enriched, direct, and PB for phage buffer *Note that the agar was made for four 5 ml plates*
2. Create a separate Agar plate for a top agar (TA) control, label and set aside
3. Gather previously created enriched lysate from incubator
4. Mass lysate and centrifuge at 5,000 g for 5 minutes to pellet Arthrobactor
5. Use a syringe to aseptically draw ~1.5 ml of the enriched lysate out of the 50 ml tube
6. Reseal tube
7. Attach a filter to end of syringe and gently push lysate through the filter and into a pipette tip
8. Cap the tip and set aside
9. Set aside a 50 ml tube
10. In the 50 ml tube add 8.0 ml of LB broth
11. Then add 90.0 μl Calcium Chloride
12. Add 10.0 ml of 2X Top Agar and pipette to mix
13. Add 4.5 ml of the mixture in the 50 ml tube to a culture tube and pipette to mix (repeat this twice more to create 3 culture tubes)
14. Pour the contents of culture tube into labeled agar plate, swirl contents, cap plate and set aside for 10 minutes
15. Pour the remaining liquid in the 50 ml tube into the agar plate labeled TA control
16. Swirl contents, cap plate and set aside for agar to solidify (10 minutes) *Note: one of the members in my group found a possible containment in his agar plate so as a group we made a new control and new agar plate for him detailed below*
    1. Divide the bottom of an Agar plate into three sections and label enriched, direct, and PB for phage buffer *Note that the agar was made for two 5 ml plates*
    2. Create a separate Agar plate for a top agar (TA) control, label and set aside
    3. Set aside a 50 ml tube
    4. In the 50 ml tube add 4.0 ml of LB broth
    5. Then add 45.0 μl Calcium Chloride
    6. Add 5.0 ml of 2X Top Agar and pipette to mix
    7. Add 4.5 ml of the mixture in the 50 ml tube to a culture tube and pipette to mix
    8. Pour the contents of culture tube into labeled agar plate, swirl contents, cap plate and set aside for 10 minutes
    9. Pour the remaining liquid in the 50 ml tube into the agar plate labeled TA control
    10. Swirl contents, cap plate and set aside for agar to solidify
17. Once agar has solidified use a Micropipette to pipette 10 μl of filtered enriched lysate onto the section of plate labeled enriched, then pipette 10 μl of direct isolation lysate onto the section of plate labeled direct, finally pipette 10 μl of phage buffer onto the section of plate labeled PB
    

    

18. Allow the plate to sit for about 15 minutes before being placed into the incubator
19. Leave to incubate until next class (approximately 120 hours)

Results:

The results of the spot test will not be discovered until Monday lab period and will be recorded in that lab entry. However, the results of the metadata experiments can be found below as they were collected during this lab period. The results show sand, acidic soil with a low water content.

Analysis:

The procedures conducted last lab and finished this lab are meant to provide more data that may inform future testing. Based on our results I can assert that my soil sample is sandy or potentially sandy loam if enough material was still in suspension. There is also a low percentage of water in the soil which could potentially affect if phage will be found. The information gathered  will help my group address our question further because having a collection of soil metadata will help us determine weather or not other factors besides tree species determine phage presence.

In addition, the results of the spot test will shed light on whether or not there is phage present in the soil we collected, which could help us address our question.

Future:

The next steps to be taken will depend on the results of the spot test. If the spot test is positive I will preform a plaque assay to confirm and the move on to phage isolation. If the test is negative, I will still preform a plaque assay to confirm, but I will likely also go collect new soil and begin the washing process again.

9.14.18 Spot Test Results and Plaque Assay

Rationale: Since the Spot Test returned negative results (indicating that the soil sample did not possess any bacteriophage), it was necessary to confirm the results by performing a Plaque Assay that would ensure that the results of the Spot Test were not due to an anomaly or a mistake.

Results from Wednesday (9.12.18):

• After observing the plate containing results from the Spot Test performed on Wednesday 9/12, it was clear that there was no presence of phage in the sample. There was perfect lawn growth with no interruption in the negative control or in any of the experimental sections. This is visualized in the image located below.

Procedure:

1. Lab surface was cleaned with Cidecon and 70% Ethanol by spraying on surface and then wiping dry. An ethanol burner was lit to establish an aseptic zone that was used for each step of the following procedure except for the steps where materials were obtained.
2. Obtained Filter Sterilized Enriched Lysate (labeled “HMB Filtered”) that was created on 9/12 from the fridge.
3. Obtained 0.5mL of Arthrobacter in tube.
4. Added 10µL of Filter Sterilized Enriched Lysate to tube containing 0.5mL Arthrobacter. Let sit for 15 minutes and allow Filter Sterilized Enriched Lysate to mix with Arthrobacter.
5. Obtained 50mL conical tube. Labeled “HMB Plaque Assay Top Agar 9.14.18”. Obtained separate 50mL conical tube for control labeled “NMN HMB 9.14.18 Control TA for Plaque Assay”
6. 2mL of LB Broth was added to both conical tubes.
7. 22.5µL of CaCl2 was added to both tubes.
8. 2 plates obtained. One labeled “HMB Plaque Assay 9.14.18” and the other was labelled “NMN HMB Control Top Agar for Plaque Assay Test 9.14.18”
9. 0.5mL Arthrobacter and 10µL Filter Sterilized Enriched Lysate added to tube labeled “HMB Plaque Assay Test 9.14.18”
10. Added 2.5mL of 2X Top Agar to both tubes, then swished to mix. Promptly poured on the respective plates, spread to all areas, then allowed to sit for 15 minutes.
11. After waiting 15 minutes, the plates were placed into the incubator to be examined on Monday (9/17).
12. Lab surface cleaned with Cidecon and 70% ethanol.

Observations

• Some particles appeared to be in Top Agar overlay that could have been caused by already-hardened agar. Their effect will be determined if the results are adverse on Monday (9/17).

No data was obtained from this experiment, as it will all be collected on Monday and updated in that entry.

Next Steps: If the results are negative yet again on Monday (9/17), it will be necessary to obtain new soil samples and possibly reevaluate the mini-question that our group examined. We will also converse with Group four about their results, as they also were examining the same mini-question as us. However, if the plaque assay reveals a positive result, then the plaque will need to be picked and held for later examination while further studying/sampling may need to occur to allow for trends in observations to be observed.

Conclusions: Based on the fact that Group four all had plaques and that Group 3 (Henry, Nathan, and Shepard) did not, there may be relevance or significance to the scientific question posed by table 2. This would need further data collection and repetition to be supported, but it would be a positive step in determining a question that the entire class could ponder and research.

SEA Phage Lab Journal

September 10th, 2018

Metadata and Enrichment

Objectives: Collect Metadata on soil sample 2, filter lysate and separate into direct and enriched portions.

Rationale: The metadata for each tree our unit collects from can be used to better understand the way the presence of Arthrobacter phage correlates with the health of different trees. Once we have discovered correlation, we could test causation. Filtering lysate allows me to test for phage effectively.

Results from last Lab: I collected soil from a large Live Oak tree right outside of the SLC, near a very popular walkway. I have the soil sample in a 15 mL vial and a plastic bag, as well as several leaves.

Procedure: Added 2 mL of soil sample 2 into a 15 mL tube, for enrichment. The other roughly 10 mL of soil was saved in the same plastic bag it was stored in, for metadata collection. Added 10 mL of LB Broth to the 15 mL enrichment tube. Then, I shook the tube for 15 minutes to mix the soil and LB Broth together. After shaking, I massed my tube, found it to be 18.72 grams, and ended up adding several drops of DI water to match a lab partner for centrifuging at 19.22 grams.

While the tubes were in the centrifuge, I began to collect metadata. Because I didn’t collect as much soil as I should have, I couldn’t complete the proportions as instructed. Instead of filling a tube to 10 mL of soil, I filled one to 4 mL, saving roughly half of the remaining soil for later. I then added DI water to 12 mL, and 3 drops of soil dispersion liquid. Shook for 30 seconds, and let the soil settle out. Afterwards, I massed a weigh boat at 2.45 grams, and my remaining wet soil in the same boat at 8.10 grams. This means the wet soil was 5.65 grams.

The soil and LB Broth finished the 10 min in the centrifuge. I set up my filter apparatus and used a bubble pipette to move the supernatant to the filter on the top. I was only able to collect around 9 mL of lysate, so I opted to stick with just an enriched sample rather than enriched and a direct. I added .5 mL of arthrobacter and set the enriched lysate in a shaking incubator at 26 degrees Celsius for the next 48 hours.

Finally, I took a few drops of the liquid from my dispersed soil sample and added DI water until it filled a small vial. I took pH paper and tested for 45 seconds. The pH was 6.0.

Analysis and Interpretation: The soil was light colored, especially when compared with other Live Oak tree soil in the area. The pH seemed to be acidic, but I don’t know what other trees are like.

Future Plans: Next lab I will complete a spot test with my enriched lysate, and determine the need for a plaque assay the class afterwards. I will also find the percent composition of my soil, and find the proper classification based on Lathan’s chart.

SEA Phage Lab Journal

September 12th, 2018

Spot Testing and Metadata

Objectives: Complete a Spot test with my second enriched lysate, and finish soil metadata.

Rationale: Testing for plaques and a basic understanding of the environment around my tree.

Procedure: Massed enriched lysate after incubation from the last lab. Found mass buddy (21.55g), and gave the 50 mL tube to Dr. Adair to centrifuge for 5 minutes at 3,000 g. I found the mass of my dry soil in the weigh boat, now 7.38g, and found the percent water loss, at 12.7% of the original mass.

I found the dispersed soil from the last lab to be 3.5 mL inside the tube. 1.75 mL was clay, (50%),, 1 mL was silt, (28.6%), and .75 mL was sand, (21.4%). When plotted, the readout was firmly in the Clay section of the sand/silt/clay chart.

I began to filter my lysate into a microcentrifuge tube with a syringe, and a 22 micrometer filter attached to it, but as my lab partners moved around me, I drifted out of the aseptic zone and needed to repeat. I got a new syringe and filter, as well as a microcentrifuge tube. I pulled slightly less than 2 mL of enriched lysate from my 15 mL tube, attached my new filter, and pushed the plunger slowly until I had filtered everything into the microcentrifuge tube. While I was doing this, my lab partners had made an Agar plate and separated it into quadrants. We used a pipette and transferred 10 microliters of each lysate into our respective quadrants, saving one quadrant as a control. It occurred to me after lab that I didn’t notice Henry switching pipette tips, but I likely just didn’t see it. Thought it was important to note, however.

Once that was done, we let the plate sit for around 10 minutes, then set it in the incubator.

Analysis and Interpretations: I suspect my soil has a different composition from others’, due to the high amount of clay and the distinct coloration as compared to others’. I will be interested to see where other people’s soil falls on the graph.

Future Plans: Depending on how my spot test goes, I will either complete a plaque assay next lab or reevaluate my soil and determine a new place to search for samples, based on our question.

Title: Metadata, Spot Test, and Plaque Assay

Date: September 12, 2018

Rationale: The sand-silt-clay results and the % water results are ready, and the lysate is done incubating, therefore metadata can be recorded, and tests be done to check for plaques.

Procedure: Aseptic zone created by the following procedure:

• Counter washed and wiped with CiDecon
• Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
• Ethanol lamp lit to create rising heat and a current that protests samples from falling contamination.

% Water: The dirt weighed 5.37 grams before evaporation and weighed 5.066 after evaporation. The following calculation yields the % water of the collected soil:

1-(5.066/5.37)= .057 = 5.7% water

Sand – Silt – Clay: 3.25 mL of soil was layered:

1 mL of sand      1.75 mL of silt   .5 mL of clay

= 30.77% sand     =53.85% silt    = 15.38% clay

1. The enriched lysate from Monday was pulled from the incubator, massed at 21.47g, and spun in a centrifuge to separate cells.
2. A spot test and a plaque assay were done to assure the best chances of a phage discovery.

Spot Test                                                                              Plaque Assay (All multiplied by 4 for 3 plates + negative control)

– 2.0 mL LB Broth                                                                            – 2.0 mL LB Broth

– 0.5 mL Arthrobacter                                                                    – 0.5 mL Arthrobacter

– 2.5 mL 2X Top Agar                                                                      – 2.5 mL 2X Top Agar

– 22.5 microliters CaCl₂                                                                  – 22.5 microliters CaCl₂

—————————————————————————————————————-

~ 10 microliters spotted onto plate –> put into incubator                       Set 15 min –> put into incubator