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 06 Nov, 2015 20:04

debbie

Joe, Minor question: The precipitate is the beads. Some folks don't realize that the resin needs to be well(evenly) suspended. Precipitate is probably not the right word. Major question: I would try anything but PEG precipitating. The literature says that you lose about 1/2 of the phage concentration when you do it. So if your lysate is close to 5 x 10^9, then I would just try an extraction. If it is not, concentrate by centrifugation and resuspend in a smaller volume. (and that is not cheating!)

Link to this post | posted 06 Nov, 2015 18:49
debbie	Hi Joe, I heard that you spoke with Kevin yesterday about this. I am just adding a link to the protocol that is already on seaphages.org. http://seaphages.org/media/docs/NewDNAProtocol.pdf

Link to this post | posted 15 Sep, 2015 17:41
debbie	so it sounds like you are testing your post-enrichment plates by putting 50 ul of sample into smeg and plating. Are you also doing spot testing? Sometimes 50 ul of enriched samples can so 'blow-out' the cells on a plate, you see no plaques. If the plaques are turbid, you will see lighter confluent growth of cells, so it can be quite confusing. Just thinking.

Link to this post | posted 15 Sep, 2015 12:24
debbie	Also - what flasks are you using for growing smeg?

Link to this post | posted 15 Sep, 2015 12:23

debbie

Greg, Hi. In your final working cultures and media, no Tween is recommended. (nor needed, unless you are having issues. I have never added Tween to anything but my liquid cultures.) So no Tween in the Top agar, Luria agar or broths used to grow the cultures. Tween is used when you are making the 'starter' cultures for your big plating batches of smeg. By that I mean, I inoculate a series of 3-5 ml cultures tubes that contain media (L or 7H9, antibiotics, ADC, and Tween -Calcium if you want)from single colonies on my streak plate of smeg. That gets the smeg into liquid with no clumps. I let those grow up until they are a thick culture. I discard any of these that are clumpy. Wwith the best heavy growth liquid smeg, I sub a small aliquot (usually a drop) into the larger volume of media WITHOUT TWEEN that I will then use for plating. Make sense?

Link to this post | posted 15 Sep, 2015 11:36
debbie	Hi Greg, So without seeing, I can only guess. If you have no plaques with D29, it could be that you are not growing smeg. Streak out the liquid culture. If there are ANY colonies in 24 hrs., it is not smeg - or the smeg is contaminated. So to be thorough, culture everything to be sure nothing is contaminated. Start your smeg cultures from a single colony and follow regular protocols.

Link to this post | posted 06 Sep, 2015 01:06
debbie	Hi Greg. Tween can also be used by the bacteria as a carbon source, so it will be broken down by the time you plate for phage. I would not fret too much about using a Tween culture for enrichment. Sharon is correct that Tween can inhibit phage adsorption, but once it is hydrolyzed, there should be no worries. debbie Hatfull Lab