Soil Metadata and Enrichment (9/10/18) 

Rationale: Find the metadata of our soil, such as the PH, the % water, silt, sand ,and clay, and to create a enriched and direct lysate for our spot or plaque assay testing.  

Procedure: 

1. We first cleaned our table with Cidecon 70% ethanol and then we used a ethanol flame to create an aseptic zone for us to work in.
2. Got a 15mL test tube and with the soil we had collected from the prior week we added 2mL of soil into it.  
3. Added 9mL of LB broth using a glass pipette into the 15 mL test tube, for a total closer to 11mL of solution.  
4. Shook the test tube for the next 15 minutes, since the solutions were going to be centrifuged 
5. In the meantime, we started to obtain the metadata results.  
6. Started with percent water and measured the dish without the soil to find a value of 2.33 grams.  
7. Added about 5 grams of soil onto the petri dish and found the total of the petri dish and the soil was 7.91 grams.  
8. In order to find the percent water, let the soil and the petri dish sit for 48 hours in the fume hood.  
9. After 10 minutes of shaking we took the masses of our solution and found that to be 18.231 grams.  
10. We then found a partner to centrifuge with, since there must be a balance of weight in the machine while the solutions are being centrifuged  
11. After the test tubes came back after being centrifuged, we used a syringe to get our enriched and direct lysate.  
12. After getting 5 mL of filtered solution, we realized that the liquid was too low and the syringe would not go in more.  
13. We then transferred the liquid into a 50 mL test tube and used a syringe to filter out the solution into another 50mL test tube, which made it easier to reach the liquid.  
14. Only obtained 8mL of solution for lysate, so decided to not have a direct sample.  
15. We then obtained Arthro and added it to the 50mL conical tube, and then shook up the tube to make sure that it was well mixed.  
16. Started with soil PH testing by obtaining a small PH vial and adding a little bit of soil to it, and then filled up the rest of the tube with DI water. 
17. We shook the small vial for 10 seconds and let it settle for the next 2 minutes.  
18. We waited 45 seconds and then put a 1 inch PH testing strip inside the small vial, and took it out as the color started changing, and quickly recorded our results by comparing the colors.  

Observations/  Results : 

We found that the petri dish weight was 2.33 grams, and with the soil the total of grams was 7.91. 

We found out that the Ph of the soil was 6.0

Next Step:  

Since there were limited test tubes for performing the salt, silt, clay percentage tests for our metadata, we will have to set up and perform the data next time. Also next time we will set up our testing, either a spot test or a plaque assay, to test for the presence of plaques and phages.  

Metadata and Spot Testing (9/12/18) 

Rationale: Procedure: Continuing to find soil metadata and setting up the spot test, using the enriched lysate we created from last week, to find phages in our soil sample.  

Procedure: 

1. Clean tables with Cidecon and 70% ethanol and create an aseptic zone with an ethanol burner.  
2. Mass the enriched sample and found its weight to be 21.53g 
3. Found a partner with a similar mass and let the enrichment sample centrifuge for five minutes at 3,000 g to pellet the Arthro.  
4. While waiting for centrifuged sample, take the mass of the soil left in the hood to find the % water in the soil.  
5. Recorded the results and found the total mass to be 7.34 grams.  
6. Obtain the enriched sample after being centrifuged and filtered 2.5 mL of the enriched sample into a 15mL conical tube.  
7. Next start on the process for creating the top agar solution for a spot test.  
8. First pipette 2.0 mL of Lb Broth into a 50 mL conical tube for the top agar.  
9. Add 22.5 mL of 1 M CaCl2 into 50 mL conical tube using a micropipette  
10. Next add 2.5 mL of 2x Top Agar using a pipette.  
11. Mixed all the materials for creating a top Agar solution and poured it into a plate and let it sit for 10 minutes  
12. Created a 5mL solution for our entire plate for spot test.  
13. Divided and labeled our plate into 4 sections: control, SS, EAG, and LIP.  
14. While waiting for the top agar to set, we started on our soil metadata for percent sand silt and clay.  
15. Obtain a 50 mL vial and added about 9.9 mL of soil to the conical tube, and added DI water until the 30 mL mark on the tube.  
16. Add 3 drops of soil dispersion liquid into my 50 mL vial and shook the solution for 30 seconds, and let it sit in the hood for 48 hours.  
17. 10 minutes passed, so we returned back to our spot testing.  
18. Added 5 uL of enriched lysate, using a pipette, to each of the three sections of the plate labeled SS, EAG, and LIP.  
19. Add 5 uL of PH buffer to the control part of our plate, and let the plate sit for 10 minutes. 
20. Placed the top spot test plate in the incubator upside down.  

Observations/  Results : 

Found the percent water in my soil was 10.22% 

Before centrifuging today, realized that my enriched lysate still had some pellet in it.  

While setting the top agar, realized that there were 2 bubbles. Also noted that the bubbles were around the control section of our plate.  

Next Step:  

Next time we will view the results of our spot test and check the plates for plaques. We will also observe and take data on our soil metadata for percent silt, sand, and clay.