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.