Rationale: Washing Soil Sample A will rid sample of waste products like dirt and bacteria and allow for possible bacteriophages found in sample to be isolated in a lysate.

Procedure:

1. Cleaned lab station using CiDecon (sprayed and fully dried) and 70% EtOH (sprayed, half-dried, and let to evaporate).
2. Obtained and lit an ethanol burner.
3. LB Broth (placed close to the burner to maintain aseptic technique) was poured into tube to 35 mL mark on Conical Tube.
4. Using a combination of hand-shaking and use of vortex machine, LB Broth and Soil Sample A (from oak tree by Baylor Science Building) were mixed for 15 minutes.
5. During mixing process, tube containing Soil Sample A and LB Broth was massed and found to be 53.454g. The purpose of this was to find another sample of similar mass (+/- 0.1g) to achieve symmetry in the centrifuge.
6. After mixing, tube containing Soil Sample A and LB Broth was centrifuged at 3000xg rotations/minute for 5 minutes.
7. Supernatant was added to top filter to remove bacteria and other particles from soil. Filtrate was obtained after it had passed through the white filter.
8. 26mL of filtrate that passed through filter was split (about 13mL left in top filter conical tube, about 13mL half placed in new 15 mL tube).
9. 0.5mL Arthrobacter was added to 50mL conical tube from top filter containing half of lysate to form an enriched isolation.  The other 15mL tube with half of lysate was left to act as the direct isolation.
10. Enriched and direct isolations were left to be shaken until Monday, 8/27.

Observations:

• After shaking process, overall volume appeared to have decreased from 35mL to 33mL – could have been due to soil absorbing LB Broth during shaking.
• Mixture was dark brown and had many bubbles immediately after shaking.
• After use of centrifuge, supernatant appeared to be a yellow-gold color that still had some soil particles and grass inside that was avoided when placing in the filter.
• After placing 0.5mL of Arthrobacter in the enriched solution, it appeared that there was a very slight amount of Arthrobacter left in the tube that did not get added to the solution. Adverse results could eventually be explained by this, however, it appeared that generally it was common for others to encounter the same situation.

Results:

• Procedure was accomplished without problems during creation of isolations. No issue with filter was had. Tangible results with more data will be possible to obtain on Monday (8/27) when a spot test could reveal any possible bacteriophages from sample.

Next Steps:

• During the next lab time, isolations will be examined to see if bacteriophages were present in the sample obtained from the soil. This will be accomplished by the spot test procedure.