09/10/2018

Washing And Enrichment of Soil B

Research Question:

Does the presence of arthrobacter appear more dominant in the soil of one oak species than the others? Is there a correlation between the presence of Arthrobacter Phage and the presence of oak wilt fungus?

Objective:

• Prepare direct and enriched lysate for soil sample B
• Collect metadata for soil sample B ( %water, pH, and sand silt clay percentages)

Materials Required:

50 ml conical vials, 15 ml conical vials, pH vials, pH paper, serological pipettes, Cidecon, 70% ethanol, LB broth, Soil Sample B, Falcon tubes, DI water, soil dispersion liquid, scooper, syringe, syringe tip filter (22 microns), shaking incubator, weighing boat

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.

Washing and Enrichment:

1. retrieved Soil Sample B from the refrigerator.
2. retrieved the 15 ml conical vial with 2 ml of soil in it.
3. retrieved the LB broth
4. using a serological pipette, transferred LB broth to the 15 ml conical vial until it reached the 11 ml mark.
5. closed the 15  ml conical vial and shook it by hand for 12 minutes
6. after  12 minutes, find the mass of the vial ( 19.31g), and find another lab partner with a tube of mass within 0.05g of your tube mass.
7. take the vials to the centrifuge and put them in for 10 minutes at an acceleration  10000 times g.
8. take the vial back to the aseptic zone.
9. using a sterile syringe and a syringe tip filter (22 microns), transfer 10 ml of filtered lysate to a 50 ml conical vial. ( take care to use the aseptic zone to avoid contamination of lysate.
10. after you acquire 10 ml of filtered lysate, add 0.5 ml of arthrobacter to the vial. this is the enriched sample.
11. place this tube, loosening the cap a little bit , into the shaking incubator for 48 hours
12. filter the rest of the lysate into another 15 ml conical vial ( 1/2 ml for my sample). this will be your direct isolation sample
13. store direct isolate in the fridge

% Water

1.  weigh the weighing boat on the scale ( g)
2. pour some soil onto the boat and weigh the weight of the boat plus the wet soil
3. take the difference of the final weight ( boat and soil ) and initial weight (boat) to find the weight of the soil.
4. put the soil outside for 48 hours to allow the water to evaporate.
5. weigh the soil again.
6. take the difference of the weight of the wet soil and dry soil to find weight of the water in the soil ( the water that evaporated)
7.  calculate the percent of water in the soil by using     percent water= (weight of water/ weight of wet soil) x 100

Sand, Silt, Clay

1. take 50 ml conical vial and fill it with soil sample to the 10 ml mark
2. add DI water to the vial until it reaches the 30 ml mark.
3. add soil dispersion liquid to the vial.
4. shake vial vigorously for 30 seconds
5. let the vial rest for 48 hours.

pH

1. add a small amount of sand to the pH vial
2. add water so that the level reaches the top of the vial
3. shake vial for 10 seconds and then let is rest for 120 seconds
4.  after 120 seconds, dip 1 inch of the pH paper in the vial for 45 seconds
5. immediately compare the color of the strip to the scale to acquire pH

Analysis and Data:

there is no data that can yet be calculated. that will be done in pre lab calculation for the lab on 09/12/2018.

the following was collected for today

Mass of wet soil = 6.370 g

pH of soil= 6

the aseptic zone was properly maintained and there were no apparent events that may have caused contamination.

Future:

calculate % water, %sand, %silt, and % clay.