My method is 1. inoculate host bacterium in broth (250 ml) to get a OD=0.2-0.3; 2. add 2ml phage lysate (108pfu), then culture for 18-24h (sometimes the culture is quite clear); 3. Centrifuge and filtre to remove bacterial cells and debris. However, this way worked for some of my phages (I can get 109pfu/ml), but not the other ones (only 104-5pfu/ml can be got). Does anybody have experience with this and could tell me how to improve my methods?
A few things to consider:
- The same medium will probably not be equally appropriate for all types of phages. For instance, it's pretty straightforward to get high titers of T4 and T7 and many other coliphages using regular LB or TSB, but to get high lambda titers, it's helpful to supplement the medium with calcium. There are lots of papers out there that grow phages differently, so you might select a few that look promising and try supplementing your media accordingly.
- A high-titer lysate should definitely have visible signs of lysis.
- If your low-titer phages are not sensitive to chloroform, you can add some chloroform (a few hundred microliters to 250 ml media) to the flask 30-60 minutes before the end of your death curve to lyse bacterial cells and liberate the phages within.
- Before you centrifuge and filter the lysate, add stock NaCl for a final concentration of 1M. I've found that this step helps remove phages from membranes and "debris" and will contribute to a higher end titer.
- You might want to consider changing your starting MOI -- a lower starting MOI typically leads to a higher end titer. I often use a 1:20 inoculum, let the culture grow for 20 minutes, and infect at a low MOI.
Good luck!
Another idea: growing the phage for ~24 hours may actually be counterproductive because phage adsorption to debris (and, to a lesser extent, the growth of phage-resistant mutants in the culture) will lower the final titer. More time isn't necessarily better. With that in mind, it might be worth trying some smaller scale experiments (e.g. 20 ml media in 250 ml flasks) with smaller inocula, lower MOIs, and different supplementation based on educated guesses from the literature. If you get strong lysis in any of the flasks within a shorter period of time (6-8 hours), add chloroform (after separately confirming the phage is tolerant to chloroform), add NaCl, and spin down and titer as you normally would.
I've been assuming that your phages are virulent, but if they're temperate, your task may be complicated by the fact that it's harder (though not impossible) to get complete lysis of such a culture because lysogeny is favored at high MOIs. If that's the case, you may need to explore other strategies (e.g. prophage induction from lysogens) in order to get a really good titer.
Hi Eric, thank you very much for your ideas. I have read some of the ideas before, but since I'm not working with coliphages, and didn't find any explaination about why people did that, so I didn't apply them in my trial. Now, after your explaination, it's quite clear. I will try your suggestions.
I'm quite new with phage study. If you won't mind I want to ask some more questions according to your answers.
1. what is the 'death curve', is it the curve that shows the number changing of bacteria after phage inoculation, how it looks like, and 'the end' of it means the time point of the lowest bacterial number?
2. you said the low starting MOI normally would give a high end titer. so I am thinking, if I want to use phage to inhibit the bacterial host, is it also better to use a low starting MOI?
3. I have two types of phages: one forms clear plaques, and the other one forms turbid ones. What do you think the latter, is it virulent or temperate?
4. Do the phages with similar morphology have the similar characters? e.g. if my phage is like T4, then I'd better follow the protocols that work with T4?
Many thanks.
Yes, by death curve, I meant the changing concentration of bacteria (with more difficulty, you could also measure the phage concentration via serial dilutions/plaque assays) after you add phage. Obviously, different levels of bacteria/phage, different media, different incubation conditions, etc. will all affect the shape of the death curve.
If your goal is to totally inhibit the bacterial growth, you might as well use a high MOI. You can easily see this on plates: if you have a very low dilution (e.g., an undiluted or 10^-1 lysate) of phage and do a plaque assay, the bacteria will be killed before they can form a lawn -- the only growth you'll see will be individual resistant colonies.
In my experience, a turbid plaque does not necessarily mean that the phage is temperate. If you want to find out if your turbid phage is temperate, you can observe its growth kinetics: you're more likely to have incomplete lysis of the culture if the phage is temperate (again, because a high MOI will favor the lysogenic pathway over the lytic one). You can also take cells from that culture, dilute them down, irradiate them with UV, and add the sample to fresh media -- a viable temperate phage should be induced by UV radiation/DNA damage, causing cell lysis. This lysis may be visible or may need to be confirmed by serial dilution and plating in subsequent plaque assays.
I'm not sure about your last question -- it's an interesting one.
I have the same problem. Most common propagation techniques doesnt work for sea phages. Instead I use high polluted water samples.
Hi ,
I have stocks of MS2 in SM buffer frozen at -80 . The stocks are aprox 6 month old but i'm having issues recovering the MS2 ( using E. coli ATCC® 15597-B1) .- Basically, no growth at all on the plate or in some cases the plaque size is very small. I managed to recover MS2 from those frozen aliquotes a few times with no problem. Any one has experience the same problem ?
I have a similar problem recovering the ATCC 15597-B1 stock from frozen aliquots. I am currently using E. coli C3000 as a host but I have heard E. coli C600 may be better for generating high titers of MS2. I will try and use this host as a next step in my attempt to generate high titers of MS2.
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