Rationale:

Conduct a spot test to see whether or not there are phages in either Direct Isolation or Enriched.

Procedure:

• To prevent contamination, wipe table with CiDecon and ethanol and set up an aseptic zone using an ethanol lamp.
• Take enriched lysate out of the refrigerator and bring it to the table.
• Prepare a microcentrifuge tube and 0.22 um filter as they will be in use later on in the procedure.
• In the aseptic zone, use a syringe and remove 0.2 mL from the tube and place it in the microcentrifuge tube. Label it “Enrich 2.”
• Take an agar plate and label it into three sections: Enrich, Direct Isolation, and Negative Control.
• To make the top agar, calculate how much Calcium Chloride is needed from one mole using the C1V1=C2V2 formula to get a concentration of 4.5 mM.
• Combine LB Broth, 42.75 uL of Calcium Chloride, and LB 2X Top Agar (last) to have a total volume of 9.5 mL.
• Shake the tube and pour over the negative control agar plate.
• Calculate the amount of Calcium Chloride needed to conduct the spot test.
• Combine LB Broth, 45 uL of CaCl2, 0.5 Arthrobacter, and at last, add LB 2X Top Agar.
• Pour the top agar into the agar plate for the spot test.
• Allow the top agar to solidify.
• Add 0.1 mL of phage buffer to the negative control using a pipette.
• Prepare a 0.22 um filter, a microcentrifuge tube, and a syringe to filter the direct isolation.
• Add 5 mL of Enrich and Direct Isolation into their designated areas in the agar plate.
• Allow the plate to sit to avoid mixing of the spots.

Spot Test

• After 10 minutes, place both plates in the incubator.
• Clean the table with CiDecon and ethanol and place all equipment in their proper places.

Results and Analysis

• For the spot test, we used C1V1=C2V2 to find the concentration needed for a 10 mL solution out of 1M of CaCl2.

C1V1=C2V2

(1 M)(V1)=(4.5 mM)(10 mL)

(1000 mM)(V1)=(4.5 mM)(10000 uL)

V1= ((4.5 mM)(10000 uL)) / (1000 mM)

V1= (45000 mM x uL) / 1000 mM

V1= 45 uL

• For the top agar control, we used the same formula to find the concentration needed for a 9.5 mL solution.

C1V1=C2V2

(1 M)(V1)=(4.5 mM)(9.5 mL)

(1000 mM)(V1)=(4.5 mM)(9500 uL)

V1= ((4.5 mM)(9500 uL)) / (1000 mM)

V1= (42750 mM x uL) / 1000 mM

V1= 42.75 uL

• Between the time of cleaning the table and starting the experiment, my elbows were on the table and instruments were also placed on the table.
• The narrow end of the filter was touching the table and a little more than 0.2 mL from the tube was put into the microcentrifuge tube.
• After I added the enriched to the plate, two bubbles appeared in the middle but was moved to the side.

Conclusion and Future Plans

• Using both lysates (Direct and Enriched), a spot test was conducted to see if phages are present within the sample. Two separate sets of Top Agar were made: one was without the arthrobacter to serve as the Top Agar control which show if the top agar was contaminated and the other was for the spot test. The experiment was completed with few mistakes; none that cause drastic changes in the results.
• In the future (8/29/18), I plan on conducting the plague assay experiment to further prove the existence of phages in my sample.