SEA-PHAGES | Protocol to grow phage in culture?

Link to this post \| posted 22 Sep, 2019 18:10
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

Link to this post \| posted 22 Sep, 2019 22:40
viknesh	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.

Link to this post | posted 22 Sep, 2019 22:40

viknesh

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.

Link to this post \| posted 25 Sep, 2019 12:39
fogartym1	Thanks Vic! We will try it out and report back on how it goes. Marie

Link to this post \| posted 15 Nov, 2021 17:17
stpage	fogartym1 Thanks Vic! We will try it out and report back on how it goes. Marie Hi, did you ever have any luck with this? I have a student who has apparently lost their phage that they isolated a couple of weeks ago. We'd like to take his old plaques and PB and amplify them back up. Thanks.

Link to this post \| posted 30 Nov, 2022 19:47
stpage	Vic wrote: "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." Hi, what is the concern with "early lysis"? Is it just that it limits phage amplification or that too many bacterial degradative products are released early or…..? (just wondering, we've had trouble getting students to be consistent with timing) Edited 30 Nov, 2022 19:48

Link to this post | posted 30 Nov, 2022 19:47

stpage

Vic wrote: "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."

Hi, what is the concern with "early lysis"? Is it just that it limits phage amplification or that too many bacterial degradative products are released early or…..? (just wondering, we've had trouble getting students to be consistent with timing)

Edited 30 Nov, 2022 19:48

Link to this post \| posted 05 Dec, 2022 05:17
viknesh	stpage Vic wrote: "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." Hi, what is the concern with "early lysis"? Is it just that it limits phage amplification or that too many bacterial degradative products are released early or…..? (just wondering, we've had trouble getting students to be consistent with timing) Yup - if all host cells are lysed after only a few rounds of infection, the amplification will be low. You really need to get to later rounds of infection… 7 ish and above in order to have a high titer.

Link to this post | posted 05 Dec, 2022 05:17

viknesh

stpage
Vic wrote: "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."

Hi, what is the concern with "early lysis"? Is it just that it limits phage amplification or that too many bacterial degradative products are released early or…..? (just wondering, we've had trouble getting students to be consistent with timing)

Yup - if all host cells are lysed after only a few rounds of infection, the amplification will be low. You really need to get to later rounds of infection… 7 ish and above in order to have a high titer.

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Protocol to grow phage in culture?