Link to this post | posted 04 Sep, 2015 17:49

GregFrederick@letu.edu

It may be in the Resource guide. But I just saw this line on the Complete with Tween recipe. "Do not add Tween 80 to media if the bacteria being grown will be used for phage isolation or propagation." So my questions are these: Can the 5ml of O.N. smeg culture that is used in the enrichment process be grown with Tween added? The 7H9 medium used during the enrichment will not have Tween. I just want to make certain that the trace Tween in the host smeg volume should not have too much negative impact. What about the smeg mixed with the top agar for plating phage? Please let me know ASAP. We are inoculating today. Thanks.

Link to this post | posted 05 Sep, 2015 19:42
isern	Hi Greg, Tween inhibits phage adsorption and infectivity. So you may see some plaques but fewer than if you had used smeg without Tween. Sharon

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

Link to this post | posted 07 Sep, 2015 22:58
GregFrederick@letu.edu	Thanks Ladies! We should know shortly. Hopefully we will still see phage after the enrichment step. I'll let you know. The plaque plating media will not have Tween in it. So that should help with the absorption step. Hopefully. Greg

Link to this post | posted 14 Sep, 2015 17:55

GregFrederick@letu.edu

Just FYI. We got zero visible plaques using a Smeg culture grown with Tween 80 in the shaker culture. only the liquid culture had Tween 80. Plates and top agar did not. I am not certain if this was the problem or if there may have been problems with the phage buffer or CaCl2 solution concentrations. We did not even see plaques on the D129 plates. I am remaking everything today to try and troubleshoot before we have the entire class proceed and burn up a lot more plates and reagents. Thoughts? QUESTIONS: Am I right that even the overnight Smeg culture should be grown WITHOUT Tween 80 for phage plaquing?

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 15 Sep, 2015 12:13

GregFrederick@letu.edu

Thanks Debbie. I am in the process of remaking all the buffers and solutions. I allowed an undergrad to make them and so I am not certain I can trust the CaCl2 and PB solutions. I have played with a lot of E. coli phages and some from other Gm+'s in the past. The shaker cultures were started from single colonies. Since we did not have the 7H9 powder when we first cultured those plates I substituted Brain Heart Infusion agar. After 24hrs, there was a haze on the plates but no discernible colonies. After 2-3 days there were visible rough textured colonies, all of one apparent type. The other caveat is that Fisher was back ordered on Carbenicillin so we were trying to culture without it. It came in on Friday. So we will have it in the culture media from now on. There was some obvious contamination on the student plaque plates. So this should at least help with some of that issue. CLARIFICATION QUESTION: I want to be certain I am doing this correctly. I know the Top Agar and Luria agar should not have Tween 80. Am I correct that the overnight shaker culture used for plaque plating should NOT have tween 80 in it? The Smeg will not clump without the Tween 80 in the 7H9 broth???

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 12:24
debbie	Also - what flasks are you using for growing smeg?

Link to this post | posted 15 Sep, 2015 12:32
GregFrederick@letu.edu	We have been growing the liquid cultures in 250ml and 1L baffled Erlenmeyer shaker flasks. Those have either Al foil or steel slider caps. We have the disposable vented flasks in stock now. So for time sake I was going to use those today. Suggestions? (P.S. Thanks for the quick replies!) Edited 15 Sep, 2015 12:32