Cooper Johnson

Title:  Serial Dilution

Date: 17 September 2018

Rationale/Past Results: The spot test was negative, however, at least one plaque was found on the plaque assay. A lab partner grew many plaques on his plaque assay, perhaps the aforementioned phage underwent the lysogenic cycle, while the assays with less plaques experienced the lytic cycle, producing less phage. A serial dilution will be done to begin isolation of a phage.

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 and then transferred and mixed with 100 microliters of Phage Buffer solution. This solution is labelled “10⁰” to represent a 10⁰ serial dilution. Then, 10 microliters of the 10⁰ solution was transferred to another vial of 90 microliters Phage Buffer and mixed to create a 10^-1 solution. This process was repeated one more time to create a 10^-2 solution. The three solutions are then plated on a plaque assay.

The following recipe was used to make 10 plaque assays:

• 20 mL LB Broth
• 225 microliters CaCl₂
• 25 mL 2X Top Agar

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

One plaque assay done each of 10⁰, 10^-1, and 10^-2 lysate. Plus, one TA Control.

Plates left to set for 10-15 minutes and then put into incubator for 48 hours.

Conclusions/Observations: The 10⁰ lysate should yield the most plaque-forming-units, however all 3 are tested to eventually web and amplify.

1. Group 4 got plaques on the plaque assays, but not on the spot test, also, another lab partner had much more defined plaques. This could result from a different phage discovery and a difference between lytic and lysogenic cycles.

2. 14 pfu/10 microliters * 1000 microliters/1 mL = 1400 pfu/mL || 1400 pfu * 10³ = 1.4 x 10⁶ pfu/mL

(37.5²)π/(0.5²)π= 5625

5625/1.4 x 10⁶ = .004 mL = 4.01 microliters. Lathan needs 4.01 microliters to web his plate.