Rationale: Washing of the soil will get rid of the unwanted particles such as dirt and other debris, and will leave us with the bacteriophage to become our lysate which we can use.

Procedure:

• Firstly, we had to create an aseptic zone using CiDecon and Ethanol (70%). This zone is necessary when experimenting to ensure that no other microorganisms may contaminate the experiment.
• We sprayed down our workspace with CiDecon and wiped dry. We then sprayed the Ethanol and wiped, letting the ethanol evaporate.
• We then used a burner in the middle of our work space to ensure that no microparticles or microorganisms would land in our open vials (The open flame creates an area of low pressure and creates a circling air current)
• LB broth was added to my vial of soil sample until it measured 35mL. The addition and pouring of the LB broth was done within the aseptic zone
  • When pouring LB broth into my vial of soil, we over-poured and ended up with 37mL of solution.
• The solution was then mixed for 15 minutes. This was done with hand shaking as well as through the use of a vortex machine.
• During the mixing process, I weighed my sample and found that it was 53.89 g. The reasoning of finding the mass of the sample was for centrifugation, where another sample of similar weight (±0.1g) was needed to balance the machine.
  • When finding another sample that was similar to my weight, I needed to add 1.29g of water to ensure my sample was within ±.1g of my partners.
•  After mixing was complete, the sample was centrifuged at 3,000 revolutions per minute for five minutes to separate the more dense particles in the solution from the smaller micromolecules and microorganisms.
• After centrifugation, I was left with a conical vial separated between the supernatant (Smaller particles in the solution, hopefully with bacteriophage) and the solid, more dense materials at the bottom of the vial.
• The supernatant was then run through a .22 micrometer filter to get rid of any remaining particles, resulting in 10 mL of lysate
• I only had enough lysate to create an enriched sample, which was done through the addition of .5 mL arthrobacter to the lysate, and was stored; the addition of arthrobacter to the lysate was done in an aseptic zone.

Observations:

• When mixing my sample, my vial resembled the consistency and coloration of chocolate milk.
• After centrifugation, the contents were separated into visually noticeable “sections” where the more dense particles were at the bottom of the vial, and was darker in color, while the top “sections” were more clear.
• The supernatant was yellowish-clear in coloration.

Results:

• The experiment resulted a usable vial of lysate which will be used in the next step of my experiment.

Next Steps:

• The next step would be to examine the lysate to see if there are any bacteriophage present via the spot test technique/procedure.