Date: Monday, August 27, 2018

Title: Spot Test for Lysate

Rationale: By conducting a spot test, the phage (if present) will be given an opportunity to grow on a bacterial lawn and establish a plaque for further research.

Materials: Arthrobacter culture, LB Broth, CaCl₂, 2X Top Agar, 50 mL test tubes, micro centrifuge tubes, pipettes, micropipettes, 22-micron filter, plates, phage buffer

Preparation: To create an aseptic zone, the following was done:

• 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 contamina

Procedure:

• Enriched lysate filtered through 22-micron filter into micro centrifuge tube to prepare for spotting.
• With a new 50 mL vial, the following was added:
  • 5 mL ArthrobacterI (pipetted by TA)
  • 5 mL LB Broth
  • 0 mL 2X Top Agar
  • 45 microliters CaCl₂ (via micropipette)

~ 10 mL added to plate to solidify for 15 minutes

• After the plate solidified, 10 micro liters of filtered lysate was spotted onto the section labelled “E” for enriched. 10 microliters of direct lysate were spotted onto the section labelled “D” for direct. There was a third section labelled “C” that is designated as a negative control area. The plate was incubated for 48 hours to discover if a phage was present.

Results/Observations: After 48 hours, the spot test showed a potential phage growth from the direct isolation spot, and was put into a walk-in fridge to further incubate in case of a negative plaque assay. (See pictures)

Potential Plaque from Spot Test

Potential Plaque from Spot Test

Potential Plaque from Spot Test (View from Bottom)