Soil Metadata and Plaque Assay (9/17/18) 

Rationale:  

Performed a plaque assay, since the results from our spot test from last class were negative, and therefore resulted in no plaques.  

Procedure: 

1. Observed spot test plates, and then cleaned the table with Cidecon and ethanol.  
2. Set up a ascetic zone with an ethanol burner.  
3. Obtained our enriched lysate from the fridge and added 10 uL to the red capped vial with 0.5mL of Arthro. 
4. Let the solution sit and waited for 10 minutes . 
5. Made a combined Top Agar plate for our 3 individual plates and a control plate.  
6. Obtained a tube and transferred the lysate and Arthro mixture to a bigger tube 
7. Added 8mL of LB broth and 90uL of CaCl2 to the tube.  
8. After letting it sit for 10 minutes, added 10 mL of Top Agar.  
9. Mixed the solution using the pipetting up and down method.  
10. Quickly, pipetted 4.5mL of our Top Agar solution into our 3 individual tubes.  
11. After pipetting into our individual tubes, directly poured the solution into our individual plates.  
12. Poured the remaining solution into our individual tubes and let the 5mL solution set.  
13. Then poured the rest of the Top Agar (4.5 mL) into our control plate and let the plates sit for 10 minutes to solidify.  
14. While waiting for our plates to solidify, measured our soil metadata for percent sand, silt, and clay.  
15. Incubated the plates and let it stay for 48 hours.

Observations/ Results:  

Spot Testing:

• Spot testing plate was negative.  

• 

  Negative Spot Test Plate

• Did notice that there was one spot of contamination in my part of the plate
• While Plating Plaque Assay noticed that there were some bubbles in our agar while pouring it in.  

Soil Metadata:

• Total : 7 mL 
• Sand  : 5mL = 66.67%  
• Silt : 1.75 mL = 23.33% 
• Clay : 0.75 mL = 10% 

• 

  Soil Metadata

Class Observation:

• Everyone in group 4 had phage, but Justin had the most defined phage. Group 4 did both a spot and plaque test, and Justin’s spot and plaque tests had plaque. One reason for this could be the tree they chose from. The group mentioned that they chose neighboring trees, so if the tree that Justin had picked had more phage or more defined phage it could proceed to more positive results.  

Lathan’s Problem:

• Answer : 4.01 uL.  
• (14pfu/ 10) * (1,000mL/1mL) = 1400 x10^3  
• (3.75^2)/(0.5^2) = 5,625 
• 5,625/1,400,000 = 0.00401 = 4.01 uL  

Next Step: 

We will observe our plaque assay plates. If our plaque assay plates have plaques, then will continue with more plaque assay plates to amplify the plaque. If there are no plaques then will obtain another soil sample, find the soil metadata and make another plaque assay.  

Soil Sampling and Washing + Soil 2 Metadata (9/19/18) 

Rationale: Collected and washed soil because our plaque assay plates were contaminated, and since these results came out negative we got new soil sample and found some of the soil metadata.  

Procedure: 

1. Collected soil from a read oak tree across from the BSB, collected 2mL of soil in a tube, and filled ¼ of a Ziploc bag with the same soil.  
2. Found the tree measurements and returned back to the class.  
3. Using the tube with 2mL soil, added about 8.5 mL of Arthro into the tube, and shook for the next 10 minutes. Found the total solution was 10.5 mL.  
4. Massed the solution and found it to be 17.73 grams 
5. Centrifuged the soil for 10 minutes at 5,000 g.  
6. Meanwhile started on collecting soil metadata for percent water.  
7. Measured the weighing boat which was 2.478g and then the soil and the weighing boat which was 6.549 grams, and then placed it in the hood to let it evaporate until we check it again.  
8. Set up the test for percent sand, silt and clay.  
9. Started off by adding 10mL of soil into tube, 20 mL of DI water, until it was a total of 30 mL.  
10. Lastly added 3 drops of dispersion liquid and shook the solution for 45 seconds. Let it sit until we are back in lab. Placed it in hood as well.  
11. Next tested pH of the soil. First added a little soil into the tube about 1/5 of it and added DI water till the top. Let it sit for a few minutes and then shook for 10 seconds. Wait 10 minutes and measure pH.  
12. Once the enriched sample is ready, syringe out just the liquid part or the supernatant.  
13. Used a filter, to make sure that the bark and other bigger components of the soil was filtered out.  
14. Resulted in about 8.5mL of enriched lysate, and therefore don’t have an direct lysate.  
15. Measured pH using a small pH paper and let it sit in the solution for 45 seconds, and found the pH of the soil.  
16. Obtained 0.5mL of Arthro and poured it into the enriched lysate tube.  
17. Transferred the enriched lysate into a 50mL tube and placed the tube into a shaking device.  

Observations/ Conclusions  

Plaque Assay Results

• The control plate for the spot test was contaminated  
• Seemed to be a bubble in my individual plate but otherwise clear.  
• 

Soil Metadata:

• PH of soil = 6  

Next Step: 

Next time, will use our enriched sample to create plaque assay or spot test plates in hopes of finding plaque on the plate. We will also measure our soil metadata for percent sand silt clay.