SEA-PHAGES | Clumpy smeg

Link to this post \| posted 17 Dec, 2015 21:02
debbie	Recently I was asked via email to give my best "tricks' to avoid clumpy smeg. After my suggestions worked, I was asked if I could post them, so here they are. Here are my recommendations: Streak out the smeg that you have – frozen if possible. Once you have colonies (in 3 -4 days) that you can recognize as smeg - flat, wrinkled, dry, and tan – more yellow if you are growing on LB – sub into small aliquots (2-4 mls) of growth media with Tween into test tubes. Final Tween concentration is recommended at 0.05%, but I would increase that 5x. Inoculate with a small inoculum from a single smeg colony – the smaller the better. Place on a shaker and shake vigorously for 2 days. Vortex and then check this culture. Is it homogenous and creamy or do you have clumps? I set up 3- 5 tubes this way. Invariably, one doesn’t grow, one is clumpy and one is perfect – smooth and creamy. This is my started culture for the entire semester. To set up my big cultures – and I mean big – because I set up 1-2L of smeg at a time. I add all of my media ingredients to a flask and then add 1 drop of the starter culture. It will take at least 3 days to grow this up to a saturated culture, but if I need smeg in the meantime, I remove what I need. (always putting the flask back on the shaker). I also avoid allowing others to enter my flasks of smeg to avoid contamination. I also aliquot by day, meaning I only permit one entry into that flask/day. Eventually, contamination will occur, so drop the number of occurrences. If your students have phage, but just can’t purify and amplify – you can try adding Tween to all of your liquid cultures – you will know if the Tween is problem or not. It usually is not. You can’t do that if you have not found any phages. Short list of things to do: Start your ‘starter’ cultures from single colonies that you recognize as smeg. Use Tween (lots of it) in your starter cultures Grow big volumes so you don’t have to keep growing it. You could grow ALL that you need for the remainder of the term right now (if you have flasks that are big enough). Keep the flasks on the shaker until you are sure the culture is not growing any more (has reached saturation). Edited 17 Dec, 2015 21:03

Link to this post | posted 17 Dec, 2015 21:02

Recently I was asked via email to give my best "tricks' to avoid clumpy smeg. After my suggestions worked, I was asked if I could post them, so here they are.
Here are my recommendations:
Streak out the smeg that you have – frozen if possible. Once you have colonies (in 3 -4 days) that you can recognize as smeg - flat, wrinkled, dry, and tan – more yellow if you are growing on LB – sub into small aliquots (2-4 mls) of growth media with Tween into test tubes. Final Tween concentration is recommended at 0.05%, but I would increase that 5x. Inoculate with a small inoculum from a single smeg colony – the smaller the better.
Place on a shaker and shake vigorously for 2 days. Vortex and then check this culture. Is it homogenous and creamy or do you have clumps? I set up 3- 5 tubes this way. Invariably, one doesn’t grow, one is clumpy and one is perfect – smooth and creamy. This is my started culture for the entire semester.

To set up my big cultures – and I mean big – because I set up 1-2L of smeg at a time. I add all of my media ingredients to a flask and then add 1 drop of the starter culture. It will take at least 3 days to grow this up to a saturated culture, but if I need smeg in the meantime, I remove what I need. (always putting the flask back on the shaker). I also avoid allowing others to enter my flasks of smeg to avoid contamination. I also aliquot by day, meaning I only permit one entry into that flask/day. Eventually, contamination will occur, so drop the number of occurrences.

If your students have phage, but just can’t purify and amplify – you can try adding Tween to all of your liquid cultures – you will know if the Tween is problem or not. It usually is not. You can’t do that if you have not found any phages.

Short list of things to do:
Start your ‘starter’ cultures from single colonies that you recognize as smeg.
Use Tween (lots of it) in your starter cultures
Grow big volumes so you don’t have to keep growing it. You could grow ALL that you need for the remainder of the term right now (if you have flasks that are big enough).
Keep the flasks on the shaker until you are sure the culture is not growing any more (has reached saturation).

Edited 17 Dec, 2015 21:03

Link to this post \| posted 05 Sep, 2018 02:27
jeremy	So M. Smeg grown early in the semester will be fine to use even 3 months later? Two consecutive semesters (Fall2017/Spring201 we were not able to get plaques (first time we got 10/12). We requested fresh M. Smeg but were referred back to our original stocks. It was suggested that perhaps the bacteria had to be in a particular phase of growth and/or aerobic state to work but we still had issues with plaque formation after growing/using fresh cultures every 5 days. We are able to get some plaques now, and hopefully it works this Fall semester, but are not sure what the problem was/is. Anyway, I know the guide says once a culture is grown to saturation it should be good for "weeks to months", and we were able to successfully use a few weeks-old culture the first time. I've not worked with Mycobacteria before this and was curious if "old" bacteria could be a cause of any of our problems..? Thanks!

Link to this post | posted 05 Sep, 2018 02:27

jeremy

So M. Smeg grown early in the semester will be fine to use even 3 months later?

Two consecutive semesters (Fall2017/Spring201 smile

we were not able to get plaques (first time we got 10/12). We requested fresh M. Smeg but were referred back to our original stocks. It was suggested that perhaps the bacteria had to be in a particular phase of growth and/or aerobic state to work but we still had issues with plaque formation after growing/using fresh cultures every 5 days. We are able to get some plaques now, and hopefully it works this Fall semester, but are not sure what the problem was/is.
Anyway, I know the guide says once a culture is grown to saturation it should be good for "weeks to months", and we were able to successfully use a few weeks-old culture the first time. I've not worked with Mycobacteria before this and was curious if "old" bacteria could be a cause of any of our problems..? Thanks!

Link to this post \| posted 05 Sep, 2018 13:12
debbie	Jeremy, I think the simplest way to think about that is that "old" bacteria would be a selection bias. Because of the large number of phages that plaque on "old" bacteria, I don't think that is what would stop you from finding phages. But, it will stop you from finding phages we haven't ever found before (because we may have been using "old" bacteria). We have an anecdotal report from Northwestern College that they found a Mycobacterium phage singleton because they selected for tiny plaques and they were using "fresh" smeg. It is an N of 1, but still interesting. Good microbiology says to not use old cultures. So it will never hurt to grow them weekly. The most likely causes of not finding phages from enriched samples are: The smeg is clumpy. Use a Tween starter culture, so you start with individual cells. Keep shaking the culture for 3 days (you can use it as soon as you have a dense enough culture, but then put it back on the shaker. Do not let it sit still on your bench until you are sure cells have reached stationary phase. Otherwise a biofilm will form and clump your culture. Enrichment time is not long enough (or too long). How many days are you enriching? The number of phages found increases with the right timing. Are you enriching at 37? Plate at the same temperature as your enrichments. When (and how often) do you check your plates for plaques? Sometimes, the plaques are so turbid, they can overgrow to look like an uninfected bacterial lawn. Other times, a plaque - especially from the original sample - takes a few days longer to 'appear'. Good luck, debbie

Link to this post | posted 05 Sep, 2018 13:12

debbie

Jeremy,
I think the simplest way to think about that is that "old" bacteria would be a selection bias. Because of the large number of phages that plaque on "old" bacteria, I don't think that is what would stop you from finding phages. But, it will stop you from finding phages we haven't ever found before (because we may have been using "old" bacteria). We have an anecdotal report from Northwestern College that they found a Mycobacterium phage singleton because they selected for tiny plaques and they were using "fresh" smeg. It is an N of 1, but still interesting.

Good microbiology says to not use old cultures. So it will never hurt to grow them weekly.

The most likely causes of not finding phages from enriched samples are:
The smeg is clumpy. Use a Tween starter culture, so you start with individual cells. Keep shaking the culture for 3 days (you can use it as soon as you have a dense enough culture, but then put it back on the shaker. Do not let it sit still on your bench until you are sure cells have reached stationary phase. Otherwise a biofilm will form and clump your culture.

Enrichment time is not long enough (or too long). How many days are you enriching? The number of phages found increases with the right timing. Are you enriching at 37? Plate at the same temperature as your enrichments.

When (and how often) do you check your plates for plaques?
Sometimes, the plaques are so turbid, they can overgrow to look like an uninfected bacterial lawn.
Other times, a plaque - especially from the original sample - takes a few days longer to 'appear'.

Good luck,
debbie

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