Rationale: Centrifuge and filter contaminated samples and enrich soil to get fresh direct and enriched isolations using filters.

results from the heating and killing bacteria method

The results showed high levels of contamination, but we were advised to centrifuge and re-filter the samples and then plate them

Process: 

Soil Metadata

• mass of weigh boat and dry soil = 8.651 g
• mass of weigh boat and wet sample = 14.225 g
• mass of weigh boat = 2.315 g
• mass of H2O
  • 14.225 g – 8.651 g = 5.574 g
• mass of wet soil
  • 14.225 g – 2.315 g = 11.910 g
• percent water
  • 5.574 / 11.910 * 100% = 46.8% H2O

Re-purification of Lysate

1. transferred ~1 mL of samples from plate wells into micro-centrifuge tubes
   1. wells with arthro were put into one tube and the wells without arthro were put into another
2. the tubes were centrifuged for 2 minutes at 13,300 G
3. supernatant was emptied into a clean dish and then filtered with a syringe filter into two fresh micro-centrifuge tubes
4. filtered lysates were stored in the fridge

Soil Enrichment

1. a 15 mL conical tube was filled with soil to the 3 mL mark
2. LB broth was added to the 12 mL mark
3. mixture was shaken and vortex-ed for ~10 minutes
4. tube was centrifuged for 5 minutes at 3,000 G
5. the supernatant was filtered using a syringe filter into two 15 mL conical tubes
   1. One tube was labeled direct and stored in the fridge
   2. 0.5 mL of arthro was added to the other tube (enriched) and it was left in the shaking incubator at 27 °C and 150 RPM

Observations:

The enriched sample had very little lysate in it – only 1.5 mL.

Next Steps:

Perform a spot test with the new direct and enriched isolations, as well as the re-filtered lysates (arthro and no arthro) from the previous week.