8/24/18 Enrichment One Part Two

Objective:

The goal of this procedure was to finish preforming soil washing and enrichment on soil samples collected from oak trees around Baylor University’s campus in order to isolate bacteria-phages. The particular sample used in this procedure was found at 31*32’40” N 97*7’9″ W near Waco Hall. This is a continuation of 8/22/18 lab journal.

Procedures and Protocols:

Materials:

• CiDecon
• 70% Ethanol
• Ethanol Burner
• Top filter with 50 ml tube attached
• Fume hood with a vacuum tube
• .5 ml Arthrobacter
• 15 ml conical vial
• refrigerator
• Pipette
• Test tube stand
• 50 ml tube

In order to complete the procedure an aseptic zone was created.

1. Clean off the work space (lab table) with CiDecon applied with a squeeze bottle and wiped away with a paper towel
2. Apply 70% Ethanol with a squeeze bottle, spread with a paper towel, and allow to evaporate
3. Light an ethanol burner in order to use the rising heat from the flame to form the aseptic zone

Then the soil washing and enrichment procedure was complete (see photo below for full procedure and see last entry for previous steps).

1. Remove the refrigerated sample (in a 50 ml tube) from the fridege
2. Open the sealed top filter under the fume hood and attach to the vacuum tube
3. Turn the vacuum on
4. Use a small pipette to transfer the supernatant from the 50 ml tube to the top filter
5. Wait for the supernatant to filter to a minimum of 10-15 ml (completely is preferred and 18 ml were collected in this instance)
6. Once the desired about of lysate has been obtained discard the filter in the bio-hazard container and seal the 50 ml tube with the filtered lysate
7. Under aseptic conditions pipette 5 ml of lysate into a 15 ml conical vile, label, and set aside
8. Add .5 ml of Arthrobacter to the remaining lysate in the 50 ml tube *note that this tube should have been labeled but I forgot to do so* and seal the tube
9. Place tube into shaking device and let it set until Monday

Important to note:

During this lab I was responsible for completing two separate soil washings, my own and my lab partners. I have only detailed my soil washing in the procedure above.

Results:

This procedure yielded a direct isolation (5 ml) and an enriched isolation (13 ml) of the supernatant. Both appeared of these isolations were yellow in color and could contain phages.

Analysis:

The procedure was more difficult than I initially assumed, and filter times were longer than expected. One possible way to improve upon the design of this lab could potentially be to centrifuge the soil and LB broth for a little bit longer in order to have better separation. It is possible, although unlikely that the results could have been contaminated during the course of this procedure and that may affect results. Assuming that this is not the case then future procedures will reveal weather or not phages are present.

Future:

My plans for the future are to use these two isolations to preform a spot test during Monday’s lab period. The results of this spot test will allow me to discover if there are phages present in the soil sample I collected.