Rationale:

Finishing Wednesday’s part, more of the supernatant will filter through, so the steps of direct isolation of half of the lysate and enrichment of the other half can happen. By enriching the lysate, it allows for the determination of whether the bacteriophages in the sample target arthrobacter or not.

Procedure:

1. The 50 mL conical vial containing the remaining supernatant was centrifuged.
2. The remaining supernatant was filtered with a 0.22 μm top vacuum filter.
3. The lysate samples from Wednesday (8/22) and Friday (8/24) were combined into one 50 mL conical vial to an approximate total of 10 mL.
4. The rest of the lysate, from now on referred to as the direct isolation, was transferred into a 15 mL conical vial which was stored in the fridge for later use.
   • This vial was labeled “KEA 8/24/18 filter”

5. To the 50 mL conical vial with approximately 10 mL of lysate, 0.5 mL of the arthrobacter was added under a cleanhood.
   • This vial was labeled “KEA 8/24/18 enrichment isolation”

6. The 50 mL conical vial with the arthrobacter, from now on referred to as the enrichment isolation, was placed in the shaking incubator at room temperature.

Observations and Interpretations:

• The supernatant centrifuge faster than it did previously. This might be because the particles slowly started to combine back into the supernatant while waiting for others to complete the filtering on Wednesday.
• I forgot to use an EtOH (100%) lamp when transferring over some of the Friday’s sample into the Wednesday’s sample to have approximately 10 mL in the enrichment isolation conical vial. However, the lysate was transferred under the clean hood.

Next Steps:

On Monday, the enrichment isolation will be used in the spot test to determine whether there are any bacteriophages that target arthrobacter in the lysate.