Rationale:

If there is a phage, create a plaque assay to dilute lysate. If the plate is negative with no contamination, collect more soil. If the plate is negative with contamination, perform another plaque assay using the enriched lysate.

Procedure:

The plaque assay and spot test conducted previously were checked to see whether or not there were plaques and to also see if there was contamination. Both tests came out negative with contamination; therefore, a plaque assay was performed again. The tables were thoroughly wiped down with CiDecon and ethanol and an aseptic zone was also set up to prevent contamination. 10 µL of enriched lysate and 0.5 mL Arthrobacter were placed in a new tube and left alone for 10 minutes to allow for infection. Next, 90 µL CaCl2 was combined with 2.0 mL of LB broth. However, a problem occurred which led to another method being used. Four vials were being used to create the top agar to ensure correct concentrations. 2.0 mL of LB broth,  22.5 µL of CaCl2, and 2.5 mL 2X Top Agar was added to each vial which was then combined with the lysate and Arthrobacter. The mixture was poured over the plate and then left alone to solidify before being placed in the incubator. After, more soil was collected in case there are no phages present after conducting a second plaque assay.

Results and Analysis:

A problem occurred with the calculations of the concentrations of the CaCl2 and the 2X Top Agar which led to another method to create the top agar (top agar made individually).

Contaminated Negative Control

Negative Spot Test

Negative Plaque Assay

After pouring the solution onto the agar plate, there was some lysate and Arthobacter solution left in the microcentrifuge tube. Although it was a small amount, it was a considerable amount.

Titer Problem

4.02 µL

Conclusion and Future Plans:

Because there was contamination in the negative control and no plaques present in the plate, performing another plaque assay was done to ensure that there were no plaques in the sample. While performing this plaque assay, proper methods were used to prevent contamination in our tests. From chances that there are no plaques in the sample, more soil was collected more future use.

Future Plans:

If there is no sign of plaques on Wednesday (9/19/18), the new collected soil will be washed and enriched in order to conduct another spot test and plaque assay test. If there are signs of phages with no contamination, a plaque will be picked and diluted.