Plaque Assay and Soil Metadata for Soil C (9/24/18) 

Rationale: Performed a plaque assay for the new soil sample, found the soil metadata specifically percent sand, silt, clay and percent water.  

Procedure: 

1. Cleaned tables with Cidecon and 70% ethanol.  
2. Set up an aseptic zone using the ethanol flame  
3. Obtained lysate and soil sample.  
4. Micro pipetted 10 uL of our enriched lysate into the tube with 0.5 mL of Arthro, and let it sit for 10 minutes. 
5. Obtained 50 mL tube and added 8 mL of LB broth for our 4 plates.  
6. While agar plates were being set, found the soil metadata – % water, % sand, silt, and clay.  
7. Obtained the mass of the soil sample that was sitting in the hood for percent water; found the weight to be 5.879 g.  
8. Obtained the tube containing our sand, silt, and clay metadata from the hood and calculated the results.  
9. Once the plates were set, added 90 uL of CaCl2 into our 50 mL tube.  
10. Added 10 mL of Top Agar into our 50mL tube, and then added 4.5 mL into each of the individual tubes containing the Arthro and lysate.   
11. Poured the individual tubes into agar plates.  
12. Took remaining top agar solution and poured into control plate.  
13. Let it set for 10 minutes and store it upside down.

Observations/ Results : 

Soil metadata: 

• Water = (4.071 – 3.401) / (4.071) = 16.45% 
• Sand = (6.5mL / 8.5mL)= 76.47% 
• Silt =  (1.25mL / 8.5mL) = 14.70% 
• Clay = (0.75mL / 8.5 mL) = 8.82%
• Total = 8.5 mL

Plaque Assay – realized that there was an air bubble in the middle of my individual dish.  

Next Steps: 

We will next observe all the results of the plaque assay plate. If the plate is negative then we will might have to collect new soil sample and wash the soil to create a enriched and direct sample. If the plate is positive we will amplify the plaque by creating more plaque assay plates and eventually a webbed plate.  

Plaque Assay #2 for Soil C (9/26/18) 

Rationale: Perform and repeat plaque assay for soil sample, since Arthro was contaminated and produced a negative plaque assay plate.  

Procedure: 

1. Obtained plaque assay plates from Monday’s lab and observed the results. 
2. Cleaned the table ,and created an aseptic zone with the ethanol burner.  
3. Picked up enriched lysate from fridge and started the second plaque assay.  
4. Observed that the enriched lysate was different in color and there were pellets at the bottom of the tubes.  
5. Decided to filter the enriched sample one more time.  
6. Divided the enriched into two equal samples into two 15mL tubes. 
7. Massed each of them and found the values to be 10.80 and 10.82 grams.  
8. Centrifuged them for about 5 minutes.  
9. Obtained a 50 mL tube and started to make our Top Agar mixture for the plate. 
10. Added 8.4 mL of LB broth into the tube and 90 uL of 1M CaCl2.  
11. Obtained our two tubes with enriched sample, and filtered them using a syringe.  
12. Found that the remaining amount of enriched lysate to be approximately 8 mL. 
13. Added 10 uL of filtered enriched lysate into 0.4 mL of Arthro, and let it sit for 10 minutes.  
14. Pipetted 10 mL of Top Agar into 50mL tube containing LB Broth and CaCl2.  
15. Added 4.5 mL of solution into each of our individual tubes containing enriched lysate and Arthro.  
16. Poured this solution directly into Top Agar plates creating a 5.0 mL top Agar Plate 
17. Remaining solution of 4.5 mL was poured into control plate.  
18. Waited for 10 minutes to let the Top Agar to set.  
19. Incubated the plates upside down until next class period.  

Observations/ Results: 

Plaque Assay from Monday’s Lab: 

• The control plate was contaminated.  
• My individual seemed to have some contamination as well. 
• Resulted in a negative plate 
• Plaque Assay created today had some air bubbles on the sides, and one almost in the center.  
• Next Steps: 

  Observe results of the plaque assay performed. If there is plaque and our plates are positive then we can continue with a amplifying the plaque. If the plate is negative, we will obtain another soil sample, and wash the soil.