I have performed the quantification of MS2 phage with ATCC media (medium 271). Briefly, this was the protocol:
- I prepared plates with 10 mL of ATCC agar medium, ATCC soft medium (7 g/L agar) and broth. All with the ATCC 271 medium procedure.
- First, I put a culture of E. coli ATCC 15597 in 50 mL of broth ATCC 271 at 37 ºC, 100 rpm, overnight (day 1).
- The next day, I took 300 uL from that culture to infect 25 mL of broth and left it at 37 ºC 100 rpm for an hour and a half. The culture from the previous day was stored in a refrigerator at 4 ºC, which was the one I used for the quantifications.
- Then I added the phage to a final concentration of approximately 1E10 PFU/mL. The culture was incubated at 37 ºC, 100 rpm for 3 hours. Finally, this culture was centrifuged for 10 min at 4000 g, the supernatant was filtered with sterile 0.22 um filters and stored in a refrigerator at 4 ºC.
- The next day, I take the samples and the E. coli culture from the first day out of the refrigerator, prepare the soft medium and keep it in the bath at 42 ºC, temper the plates, etc. I make the phage dilutions in 200 uL. The first 3 dilutions were 1/100, 2 uL in 198 uL of ATCC broth (10^-2, 10^-4 and 10^-6). And the last four were 1/10, 20 uL in 180 uL, up to 10^-10. These last ones were the ones that were quantified. Once a dilution was prepared, it was vortexed and the next one continued.
- The mixture was: 4.5 mL of soft ATCC medium (approx. 42 ºC), 100 uL of the MS2 dilution and 250 uL of E. coli. This mixture was quickly pipetted onto one of the ATCC agar medium plates and incubated at 37 ºC until the next day. The same with the other plates.
I quantified 5 different samples with this procedure and the results were as follows:
- In two samples the results were satisfactory, 18E10 and 20E11 PFU/mL.
- In two other samples, dilution 8 had much less PFU than 9 and 10, which seemed similar to each other, uncountable.
- In the last sample, dilution 9 was uncountable but dilution 10 had only 8 PFU.
What could have happened?
- Perhaps in the more concentrated sample the phage particles interact with each other, rendering many of them inactive, thereby decreasing the effective phage concentration.
- It could be a similar phenomenon as when transforming bacteria with plasmids - transformation efficiency increases only up to a certain ammount of the plasmid DNA used and then its starts to decline - see e. g. An efficient transformation method for Bacillus subtilis DB104, Applied Microbiology and Biotechnology 94(2):487-93.
When you say "uncountable" do you mean no plaques or way too many plaques to count?
But I would say that you should normally have 2-3 plates which are somewhat countable for each dilution series (one of them being optimal to count) and if you don't then there is some other issue. For example things are not being mixed well, or the pipets are contaminated so you get PFU at highest dilutions that shouldn't be there or some other source of cross contamination.
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