Previous Results:

• The results of the metadata of soil composition were collected. The layers of the soil were measured by recording the markings on the tube. The total amount of soil was 16 mL with 59% silt, 37% clay, and 4% sand.
• Plaque assay from last lab was positive and had numerous clearings, too many to count so dilutions will be necessary to know concentrations.

Objective:

• Pick a plaque from the plaque assay made during last lab
• Conduct a series of serial dilutions and plate the diluted lysate

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. A plaque was picked by placing the tip of the pipet into a clearing on the plate. The tip was then put in 100 microL phage buffer and pipetted up and down 15 times to release phage in buffer, becoming 10^0 dilution
3. 10 microL of 10^0 was transferred to a microcentrifuge tube containing 90 microL of PB, creating 10^-1 dilution
4. Step 3 was repeated to create a 10^-2 dilution
5. The 3 dilutions were poured into separate tubes of 0.5 mL Arthrobacter to allow phage to interact with bacteria
6. A mixture of Overlay Agar for 4 plates was made using 8 mL of LB broth, 90 microL CaCl2, 10 mL 2xTA. Then the mixture was separated into 4 50 mL tubes, with 4.5 mL in each.
7. The mixture of Arthrobacter and dilutions were poured in their respective tubes, 10^0, 10^-1, and 10^-2
8. All experimental tubes and the control tube were plated and left to harden for 15 min
9. Plates were placed in incubator for 48 hours.

Results:

• Results will not be available until next lab (9/19) to see if the phage were plated correctly and there are positive results.

Conclusion:

• The results from the previous lab were positive and there are phage in soil sample B
• The serial dilution was correctly conducted using a picked plaque from the positive plate

Next Steps:

• During the next lab (9/19) the plates with the serial dilutions will be examined for phage. Another series of dilutions will be done to ensure only one strain of phage is being tested. The same steps will be conducted.