Detailed Procedure

1. Add 5.0 mL LB Broth to 50 mL vial in the aseptic zone. Labeled vial as control.
2. On a different 50 mL vial, labeled it as “E” and put 13.5 mL of LB Broth in aseptic zone.
3. Add 45 microliters of CaCl2 in control vial.
4. Add 135 microliters of CaCl2 in E vial.
5. Took my plate and labeled it into  sections (D,E,B)
6. Add 5.0 mL of 2X TA into control vial and used pipette to mix it.
7. Poured control vial onto control plate and let it sit for 15 mins.
8. While it dries, filter 2 mL of enriched lysate from Lab Day 1 using a syringe filter, and add it into a small cap in the aseptic zone.
9. Add 1.5 mL of Arthro in E vial
10. Add 5.0 mL of 2X TA (by mistake, therefore it did not work)
11. Took 4.5 mL of LB Broth into a separate 50 mL vial and add 4.5 microliters of CaCl2.
12. Went to the back and got 0.5 mL of Arthro into the vial with a pipette.
13. Add 5.0 mL of 2X TA and swirled vial to mix (did not use pipette to mix)
14. Steps 11-13 done in aseptic zone.
15. Poured separate vial onto plate that was labeled in step 5.
16. Wait 15 mins for it to dry.
17. Add 10 microliters of direct, enriched, and buffer onto correct labeled areas of the plate.
18. Try to prevent bubbles to appear on the plate.

Thoughts

• There were times where I wasn’t as careful such as spilling some TA on the side of my vial.
• Bubbles did appear onto the plate and tried to “pop” the bubbles with the pipette.
• Due to human errors above, plate may result in contamination or inaccurate measurement of spotting each solution.
  

  Plate for spot test

  

  control

  

  process of spot testing in aseptic zone

Detailed Procedure

1. Shake the bag of soil into vial Soil A. It was around 15 mL of lumpy soil.
2. Add the LB Broth to my vial at the 35 mL mark. (I had to use two bottles of LB Broth since the first one did not fill up to the 35 mL mark.)
3. Shake the vial by hand and vortex for 15 mins.
4. Weighed the tube= 52.21 grams
5. Centrifuge for 5 mins.
6. Tube is now separated  from soil pellet and supernatant. Looks very cloudy.
7. After two days, I filtered it out.
8. To filter, use a dropper to put the supernatant into the tube top filter.
9. After 23 mins, I poured 12.5 mL of filter supernatant into a 50 mL vial.
10. Went to aseptic zone to pour arthro into the 50 mL vial labeled as Enriched A
11. For the rest of supernatant that didn’t filter, put it in a centrifuge for 5 mins  in a 15 mL vial.
12. Used a syringe filter for 30 mins and got 4.5 mL of Direct Isolation A into a 15 mL vial.

Thoughts

• As I put the soil in the tube, it came out lumpy and slightly wet. Perhaps this might have affected the result of a cloudy appearance when put in the centrifuge
• Some large particles in the soil may have clogged up the filtering process.
• If the soil was dry, the supernatant filtering might have been easier.