Lab 5 & 6 Instructions and Procedures

Overview of Lab Schedule and Procedures

• Lab Timeline
  • Week five and six lab results are due tomorrow.
  • Last week’s results will be processed today.
  • Students are expected to complete their notebook entry for last week today.
  • Grading of week five notebooks will occur on Wednesday.
  • Future grading:
  • This week's notebook entries will be graded next Wednesday.
  • Notebooks due on Tuesdays will also have their entries graded subsequently.

Lab Materials

• 15 ml Conical Tubes
  • Tubes contain phage buffer with a small amount of glycerol.
  • Glycerol stabilizes phage particles during experimentation.

Procedure for Handling Phage Particles

• Setting Up Aseptic Field

  • Essential to prevent contamination when working with plates.
  • Begin by lifting the lid of the petri dish slightly to avoid exposure.

• Usage of Phage Buffer

  • Pour approximately half of the contents of one conical tube into one petri plate.
  • Pour the remaining half into a second petri plate.
  • Objective: Effectively divide the phage buffer between two plates without using a pipette.

Shaking Procedure

• Shaker Table Setup
  • Place both plates on the shaker table.
  • The glass plate on top allows for a slow-speed rotation to facilitate mixing.
  • The RPM (Revolutions per Minute) is set to a very slow rate for gentle mixing.
  • Duration: Plates will swirl for two hours.

Objective

• Phage Particle Recovery
  • After shaking, phage particles are released into the phage buffer on the plate’s surface.
  • Subsequent step: The solution will be collected for filtration.

Calculating Phage Concentration

• Spot Titer Determination
  • Following filtration, a spot titer will be performed to determine the concentration of phage particles present in the tube
  • Details regarding the process of calculating the concentration will be explained later in the session.