Titer Determination

[Mathguy104]

Why is there a greater number of PFUs per miliLiter for a lesser number of bacteriophage?

[deleted-116311]

Hi Mathguy104,

I just want to make sure that I understand your question. Are you are finding more plaques on a lawn of bacteria when you add a lower phage titer than when you add a higher phage titer? How did you set up your experiment?

CHeers,
Emily

[Mathguy104]

So I had various dilutions of phages. 10^-1, 10^-2, 10^-3, etc. I tested the dilutions from 10^-6 to 10^-10. So in my mind, 10^-6 should have more bacteriophage than 10^-10 dilutions, right? So then I took my phage dilutions, added them to e. coli agar test tubes bathed in a water bath (so that the agar would remain liquid and the e. coli would stay alive), and then I poured the contents of each agar test tube (while it was still hot) onto 5 different petri dishes containing an agar medium. I allowed the dishes to cool and turned them over for the night. Next morning i checked the plaques in each petri dish, and just as I expected, the 10^-6 phage/e. coli mixture had the most plaques, because it had the most phage, which killed the most e. coli growths, which couldn't go any further because the hardening of the agar made them stay in place. Thus forming plaques.

So anyways, the 10^-6 had the most phage, which produced the most plaques, however when i tried calculating titer, i took the plaque number, divided it by the dilution put into the petri dish, and obtained my answer in PFUs per milileter (plaque forming units per milileter). For example, there were 223 plaques on the 10^-6 dilution petri dish, so I said 223/(10^-6)=PFU/mL=2.23*10^8 PFUs /mL. Then I calculated the titer for the 10^-10 tube (which had less phage and less plaques). It had 13 plaques, so 13/10^-10=1.3*10^11 PFU/mL. Comparing the two titers, isn't the 10^-10 petri dish a larger number? I'm confused as to why I'm getting 1.3*10^(ELEVEN) plaque forming units in a petri dish with less phage versus 2.23*10^(EIGHT) plaque forming units in a petri dish with more phage. Am I calculating titer correctly? Please help, and thank you so much for your time.

[deleted-116311]

Hey Mathguy104,

So I think that I see the problem. The phage in the 10E-9 may be too dilute for an accurate reading. I found a protocol published by the America Society for Microbiologists (http://www.microbelibrary.org/component ... -protocols) that recommends you only count plates with 30-300 plaques. If you have enough phage to generate more than 300 plaques you could be saturating your E. coli with phage and the results could get weird. If you have plates with plaques in that range (30-300), there should be an interesting relationship between the ratios of PFU and dilution across samples.

I hope that this helps.

Cheers,
EMily