8/24/2018

enrichment

Objective: todays goal is to extract the phages from the soil that was collected on 8/20/2018, soil sample A.

Supplies: sample collected on Monday in 50 ml test tube, pipettes, tube top vacuum filter, shaking incubator, vacuum pump, 15 ml vials, 0.5 ml Arthrobacter host

Procedure:

1) set up an aseptic zone by cleaning your desk with Cidecon (wipe till dry) and ethanol ( 70%)( wipe on the desk and let it evaporate) after clearing the table.

2) lit the ethanol lamp to set up an air current to help keep other microbes from getting into tube when it is open.

3) divide the supernatant solution into two 15 ml vials and put it into the desk centrifuge for 17 minutes.

3) set up the tube top vacuum filter, put in the vacuum tube and put in the supernatant from the two vials using a pipette .

4) turn on the vacuum pump and let the supernatant filter.

5) After filtration, take out the lysate(solution filtered) and dispose of the waste appropriately .

6) Keep 10 ml of lysate in the tube and move the rest into an 15 ml vial in the aseptic zone.

7) put the lysate in the 15 ml vial in the fridge for direct isolation.

8) add the Arthrobacter host to the 10 ml of filtered lysate for enrichment.

9) put the enriched sample into the shaking incubator at 38 degrees Celsius for 76 hours

Results: the end product was the large particles deposited at the bottom of the tube. the solution was still very dark and dense.

Analysis: the lysate was filtered faster than initially predicted due to the second time use of the centrifuge. the aseptic zone was properly maintained and so far no apparent events that could have caused contamination. the filtered lysate that was acquired was 15.5 ml. the diagram for the process is below

enriched sample is labelled: ADP 08/24/2018 enriched ; and direct isolation sample is labelled: ADP 08/24/2018

Future notes: dividing the supernatant into the two vials and putting them into the centrifuge helps a lot with the washing process. next lab we will be to do the spot test and observe the plaque assay in the petri dish, which will be made from the enriched sample to test the presence of phages in the soil. we will know if there are phages in the soil if there are blank spots on the dish because that would represent dead bacteria, which will be the bacteria that were infected by bacteriophages.