SEA-PHAGES | All posts created by debbie

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

Posted in: Phage Discovery/Isolation → Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

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 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?

Posted in: Phage Discovery/Isolation → Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

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.

Posted in: Phage Discovery/Isolation → Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

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

Posted in: Phage Discovery/Isolation → Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

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