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Protocol to grow phage in culture?

| posted 22 Sep, 2019 18:10
Does anyone have a general protocol for growing phage in culture? We need to increase titers for a few of our phage and would love a protocol to get us started.
Thanks
| posted 22 Sep, 2019 22:40
fogartym1
Does anyone have a general protocol for growing phage in culture? We need to increase titers for a few of our phage and would love a protocol to get us started.
Thanks

Hi Marie,

The Enriched Isolation protocol is essentially a modification for preparing liquid lysates. There's a commonly used protocol for amplifying phages of E. coli in liquid, which I provide below. Please also see the caveats, below.

1. To 1 ml of a saturated bacterial culture, add 10 ul of a phage lysate.
Note: Include a control that replaces phage with just phage buffer.
2. Allow to sit on bench, undisturbed, for 10 min.
3. Add the bacteria+phage mix to 10 - 15 ml of fresh media (e.g. PYCa, or 7H9 liquid media complete, depending on the bacterial host).
4. Incubate, with shaking, for 24 - 48 hrs.
5. Spin the culture, and filter the supernatant. Then determine the titer of your lysate.

Caveat:
1. Like preparing webbed plates, it is important that not all the host bacteria is lysed early. It is therefore important that you setup the experiment early in the day and check on the culture before you leave in the evening. If the culture is clear (and the control is cloudy), then you've run out of host bacteria for phage amplification.
2. Because it is hard to know how much phage to add, it is worth setting up at least one additional culture, using a 1/10 dilution of phage lysate. (The same logic for bracketing when preparing webbed plates).

Hope this is helpful.

Good luck.
| posted 25 Sep, 2019 12:39
Thanks Vic!
We will try it out and report back on how it goes.
Marie
 
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