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Determination of adsorption time

Requirements

TSA (Some other media can be used read here)
SM Buffer (for preparation click here)
MgCl2
Host bacteria
Phage isolate
PRE/FeSO4 solution
Tween-80

Procedure

  1. Place 200μl of an appropriate SM buffer dilution of the bacterial host cell at 105 CFU/ml in a sterile Eppendorf micro-centrifuge tube.
  2. Add 200μl of the selected phage from the highest phage titer in SM buffer. Mix gently to avoid causing any bubbles.
  3.  Hold the phage-bacteria mixture at room temperature for time intervals 1, 2, 3, 4, 5, and 10 min.
  4.  Transfer 20μl from phage-bacteria mixture into a sterile Eppendorf micro-centrifuge tube.
  5. Add 150μl of the PRE/FeSO4 solution and mix gently to avoid causing any bubbles then incubate at room temperature for 5 min.
  6. The activity of the virucidal agent is neutralized by adding an equal volume 150μl of 2% (v/v) Tween-80 in SM buffer.
  7. After 30 s transfer the mixture into a 15 ml sterile tube and add 680μl of109 CFU/ml of 18 h bacterial host TSB culture and mix.
  8. Immediately add 2.5 ml of top layer agar cooled to 45°C and pour over TSA plates then incubate at 37°C for 18 h.
  9. Calculate the phage titers for each contact time. Contact time is given by the lowest time interval required to give the highest phage progeny for a given host at a given concentration.

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Last modified: March 14, 2021
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