22 August, 2018 ✷ Cleaning the Soil

After collection of soil from an oak tree in Zone 8 (near Gordon Teal Residential College), the soil was cleaned in order to isolate possible bacteriophage samples.

Procedure

• 20 mL of soil was removed from the collection vial and stored in a refrigerator for future tests. 15 mL of the original sample remained in the vial.
• The table was washed with Cidecon and 70% Ethanol solutions to promote an aseptic environment. An alcohol burner lamp was lit to clean the air surrounding the workspace.
• 20 mL of LB broth was added to the soil sample; the soil/LB broth mixture was then shaken by hand for 15 minutes to mix. At the end of 15 minutes, the vialwas massed and found to be 52.398 g.

• The vial was centrifuged at 3000 x g for several minutes. The mixture separated into 3 layers: heavier soil, a milky-yellow liquid, and some less dense wood chips at the top.

  click for centrifuged sample image
• Using a vacuum filter, the milky-yellow liquid from the center layer was filtered through a 20 micron filter. The resulting filtrate was paler yellow in color and more transparent.
• 5 mL of the lysate (filtrate) was poured into a 15 mL vial and refrigerated, and the remaining 10 mL were combined with 5 mL Arthrobacter (ATCC 21022). This sample was then stored in a shaking incubator for 5 days.

Observations/results

• As the lysate was cleaned and filtered, its color became lighter and more transparent.

Interpretations, Conclusion, Next Steps

• The purpose of this portion of the experiment was to purify the soil sample and isolate any bacteriophages in the soil sample. Thus, if any were present, they would be isolated in the enriched and direct isolation samples resulting from vacuum filtration.
• The next step is plaque assay and spot test–the potentially infected arthrobacter will be introduced to a nutrient-rich bacterial lawn and the presence of bacteriophages will be seen if plaques are present (places where the phage killed bacteria at the end of its lytic cycle).