Date: Wednesday, September 12th, 2018

Title: Spot Test and Metadata Continued

Rationale: The purpose of today’s lab is to make a spot test, an agar control, and finish soil metadata.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
2. We lit a burner in the middle of the table in order to keep falling particles from contaminating the equipment or samples from above since the flame creates a circulating air current.
3. We mixed 8 mL LB broth and 90 microliters of CaCL2 in a 50 mL conical for our plates.
4. I used a syringe filter to transfer 1 mL of my enriched lysate to a culture tube containing .5 mL ATC 21022.
5. Next we added 10 mL of 2X top agar to our 50 mL conical. We pipetted 4.5 mL of this solution to our top agar control plate.
6. We quickly added 4.5 mL of our solution to our individual plates.
7. We waited for 10 minutes for our agar to harden.
8. We then took 10 microliters of our enriched lysate and put it on the plate. We also took 10 microliters of direct isolation and of phage buffer and added them to the plate.
9. We let our plates sit for 15 minutes then set them in an incubator at room temperature.
10. I took a pinch of my soil sample and added it to a small pH vial.
11. I filled the rest of the vial with water, dipped 1 inch of pH paper in the mixture, and waited for 45 seconds.
12. I compared the color of the paper and determined that the soil sample had a pH of 6.3.
13. I checked my Falcon tube and determined that my sample was 75% sand, 12.5% silt, and 12.5% clay. Some silt and clay particles were still in suspension.
14. I weighed my dry soil boat and found it to be 8.985 grams. This would make the soil by itself weigh 6.506 grams.
15. I subtracted today’s weight from Wednesday’s 7.259 grams of wet soil and calculated that my sample was 27.6% water.

Observations:

• My dispersion wasn’t completely settled, so soil composition percentages could be off.
• My soil pH was 6.3, so it was slightly acidic.

Results:

• This experiment yielded me a spot test as well as soil metadata.

Next Step:

• My next step is to observe the results from my spot test and check for possible phage plaques.