Phage Titrering: Calculating Dilution Factor & Plate Volume

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SUMMARY

The discussion focuses on the correct calculation of dilution factors and volumes in phage titration experiments. The dilution factor is confirmed to be 5X, which only accounts for the diluted phage and does not include the additional volumes of XL1-Blue culture and melted top agar. The formula for calculating pfu/ml is clarified, emphasizing the use of the 1 µL of diluted phage for accurate results. Participants agree that the total volume of the mixture does not affect the dilution factor in this context.

PREREQUISITES
  • Understanding of phage titration techniques
  • Familiarity with the concept of plaque forming units (pfu)
  • Knowledge of dilution calculations
  • Experience with handling bacterial cultures, specifically XL1-Blue
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  • Research advanced phage titration methods and variations
  • Learn about calculating dilution factors in microbiological experiments
  • Explore the significance of plaque forming units in virology
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USEFUL FOR

This discussion is beneficial for microbiologists, virologists, and laboratory technicians involved in phage research and titration techniques.

mountain
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hi guys!

i am doing phage titrering but have difficulty in chosen the right values. please give me some ideas. thanks in advance!


here is what we did:
dilute phages 5X with dilution buffer. add 1ul of the diluted phage to 200ul of the XL1-Blue overnight culture. Add 2ml melted top agar. Quickly pour onto a plate. the day after count pfu/ml.

pfu/ml: (number of plaque*dilution factor*10^3ul/ml )/ul of diluted phage plated.

my problem is: what is the dilution factor? is that factor 5 or should i also take 200ul of the XL1-Blue cells and 2ml melted top agar in consideration? (practically it seemed like the phages is diluted more than 5X.)

what is the "ul of diluted phage plated"? is that the 1ul diluted phage i added to the other volumes or is that the total volume (1ul phage+200ul XL1-Blue cells+2ml top agar)?


thanks!
 
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5X is your dilution factor and does not include the 200 uL of culture and 2 mL of agar. Also, the 200 uL and 2 mL will not dilute your sample since your plating the whole volume and not just part of it. Therefore your are plating all the phage that are in your 1 uL sample.

pfu means plaque forming unit, so you need to use the volume of phage that you used (1 uL) not the total volume. You looking at counting the number of phage in your stock solution.
 
thanks! :approve:
 

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