Cooper Johnson

Title: Serial Dilution pt. 2

Date: 19 September 2018

Rationale/Past Results: The 10⁰ and 10^-1 plaque assays from before yielded no plaques, however the 10^-2 yielded one plaque-forming-unit. This could have been caused by a lytic/lysogenic cycle differential causing one phage to grow rapidly and another to not flourish as obviously. The TA control was also positive, signaling potential contamination among one or more of the plaque assay ingredients. The individual ingredients will be labelled to further isolate the cause.

Procedure:

Aseptic zone created by the following procedure:

• Counter washed and wiped with CiDecon
• Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
• Ethanol lamp lit to create rising heat and a current that protests samples from falling contamination.

A pipet tip was inserted into the selected plaque spot of the 10^-2 plaque assay then transferred and mixed with 100 microliters of Phage Buffer solution. This solution is labelled “10⁰” to represent a 10⁰ serial dilution. Then, 2 plaque assays of the 10⁰ lysate were made to further analyze the present phage(s).

The following recipe was used to make 7 plaque assays:

• 14mL LB Broth
• 5 microliters CaCl₂
• 5 mL 2X Top Agar

~4.5 mL pipetted into Arthrobacter culture tube containing 0.5 mL Arthrobacter and 10 microliters of 10⁰ dilution lysate.

The 2 plaque assays were left to set for 10-15 min before placing into incubator for ~48 hours.

Conclusions: The plaque that was grown from the previous 10^-2 lysate has been plated for 2 plaque assays in hope of growing a plaque and eventually being able to identify, isolate, and amplify a singular phage for further analysis and answering the research question