Hello everyone in the Bacteriophage Research Society,
I hope you're all doing well! I'm thrilled to be part of this incredible community as I embark on my first research journey in the exciting field of bacteriophages. Recently, I've encountered some intriguing and puzzling challenges in our lab, and I would greatly appreciate your professional insights and guidance.
As the first, have you ever achieved a 10^15 PFU/mL high titer of phage-enriched stock? In my recent experiments, I saw 30 plaques in 100 µL of the -14 dilution, which translates to 3 × 10^16 PFU/mL from a simple 50 mL bacteria and phage liquid enrichment with an MOI of 0.01 (also I had got 10^9 CFU/mL host bacteria at start time). This seems unusually high to me. Has anyone else observed such results, and what factors could contribute to this?
I'm eager to learn from your experiences and expertise. I would greatly appreciate any suggestions, advice, or shared knowledge.
Looking forward to your valuable responses!
Best regards, Mina
Having bacteria at 10^9 CFU/mL sounds pretty typical to me for a growing culture with visible turbidity. E. coli will usually hit around 10^10 CFU/mL with good aeration.
However your phage titer of 10^15 PFU/mL is probably not possible. I think it is most likely that you have some phage contamination. It could be that you have some phage either in your diluent so that you get some plaques at any concentration, or there was some phages on your pipets. Especially if you are using a pipette with tips, you should be sure to use barrier tips to prevent phage from contaminating the pipette itself.
It is really easy to get phage contamination so this is a common problem.
Simple calculation shows that 10^15 PFU/mL = about 0,1 g of phage per mL.
if 1 phage is 6,2 X 10^3 kDa and 1 kDa= 1.6605402^-21 g.
Michael J. Benedik
Many thanks, Prof. Benedik for your insightful guidance.
You are absolutely right and phage contamination is a common problem. However, another surprising challenge in my work is that after 3 days of storing filtered enriched bacteriophages at 4°C, the phage titer dropped to 10^9 PFU/mL, in which I had just around 30 plaques in 100 µL of -7 dilution and NO other plaques on higher dilutions.
Did you have about 300 plaques on -6 dilution also?
So phage titers often decrease a bit with time (depending upon the phage and storage). Or maybe this is the actual titer and the previous titers were incorrect due to contamination. But the titer decrease would normally be a few fold (rarely even one log).
Michael J. Benedik
Unfortunately, I didn't check the -6 dilution by the double agar layer plaque assay, but based on the results of the spot titration assay, I think I had more than 100 plaques on 100 µL of -6 dilution (averaging 6 plaques per 5 µL spot).
Also, my experimental conditions, including the use of SM buffer as a diluent and pipettes, remained consistent on both the first and third days of titration.
The unusually high titer of bacteriophage at first and the significant reduction after 3 days are mysterious and puzzling to me especially since these results were consistent across three independent replications.
I would appreciate any insights or suggestions you might have on this matter professor.
I've occasionally seen 10e13 with filamentous phages like M13 but usually 10e11 is about the normal max I've seen. So I really believe the 10e15 is not correct. I've never heard of a phage titer that high.
Thank you so much, Professor Benedik, for your valuable and helpful insights.
How can I prevent phage contamination during work?
What are the essential practices in bacteriophage labs to avoid phage contamination and also cross-contamination with other phages?
Just use good aseptic technique. But if you are using pipets with disposable tips be sure to use barrier tips and not regular tips or you are likely to get phage in the pipet barrel. This is very common.
Mina Bahador I'm working on the same theme and I'm having the same problem of the title decreasing after a few days. Have you found a solution?
Imane el Belghiti
Dear Imane
Unfortunately, we have not found the reason behind it yet. However, our current solution involves further and regular enrichment to maintain a high titer of bacteriophage.
Hi Mina,
One of possible cause of a relatively fast titer decreasing is the interaction with OMVs (gram-negative bacteria) or MVs (gram-positive). These vesicles are produced by bacteria and can be a part of their defense, acting as decoys for phages. If you check your phage stock, which was stored for a few weeks and significantly lost concentration, using TEM with negative staining, likely you'll find there lots of virions which are "discharged" into the vesicles. See the attached example.
In terms of the unusually high concentration, you can simply estimate the volume of the virions, e.g. just approximating the heads with icosahedrons and find how many virions would fill the whole 1 ml volume. For example, let's approximate the virion with a a = 60 nm icosahedron. To avoid common conversion errors, let's calculate it in cubic meters:
Vsingle_phage = 2.182 * (6 * 10^-8)^3 = (approximately) = 4.7 * 10^-22 [m^3]
1 ml = 10^-6 [m^3]
Thus, (10^-6)/(4.7 * 10^-22) = (approximately) = 2 * 10^15 virions will fill the whole 1 ml volume forming something crystal-like. Obviously, it's nonsense, that's why we should be concerned when measured such titers.
Good luck!
Denis
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