Link to this post | posted 03 Nov, 2016 18:35

viknesh

Hi Rodney, I can't say I've seen this, and have not yet heard from anyone with this problem. Perhaps someone who has will chime in. In the meanwhile, would you share a gel where you see the degradation only in the presence of restriction enzyme buffer? I assume the buffer isn't contaminated with DNAse, and that you are using brand new buffer when you used the new enzymes? Vic Edited 03 Nov, 2016 18:36

Link to this post | posted 02 Nov, 2016 15:51
viknesh	Emily, do you have a lysate of arthrobacter phage that you use as a control? Has this also stopped producing plaques? Vic

Link to this post | posted 02 Nov, 2016 15:07

viknesh

Hi Emily, We know that Arthrobacter lose viability when stored on plates or in liquid culture over time. In the A. sp Phage Discovery Guide, I think the guidelines are to use colonies or cultures that are no older than 2 weeks in order to obtain consistent results. The phage we have isolated on Arthrobacter, however, have been pretty stable in lysates. It is surprising that your students are not able to recover any phage from 2-3 week old plates. Is it worth flooding the plate 3 mls media, collecting 1 - 2 mls of the lysate, and the trying to infect with that? Are you able to streak purify directly from a plaque? Vic Edited 02 Nov, 2016 15:07

Link to this post | posted 27 Sep, 2016 18:42
viknesh	Christine, While you are doing your tests, it may be worth asking for smeg from your buddy school. That way, you'll at the very least have a strain to compare to. Vic

Link to this post | posted 22 Sep, 2016 21:09

viknesh

Hi Christine, It's going to be hard to tell what you have without photos of your plates with colonies. I've attached a figure with smeg on streak plates over time when incubated at 37C. You'll see from the figure that small specks become visible at day 2, and they gradually grow into larger well-defined colonies with a dry and irregular morphology by day 4 - 5. They are white to tan, depending on the age of the colonies and the properties of the plate. An important test for you to perform is a plaque assay (or a spot test) with D29. If you see plaques or a cleared spot on your bacterial lawn, then you can be confident that you are working with smeg. Vic 686Kb

Link to this post | posted 07 Jul, 2016 21:26

viknesh

Hi Emily, I've seen this happen before - lawns and cultures of Arthrobacter take an extra day or two to become dense or saturate, and lawns tend to be thin and spotty/clumpy. We found that this happened unexpectedly from time to time when we grew cells at 30C. Once we switched to doing everything at 26C, this problem went away. I suggest starting from your glycerol stock and try to do everything (streak, culture in liquid, plate and incubate) at 26C (or at room temperature). I'll be curious to know if this solves the problem for you too. Vic