Link to this post | posted 07 Jul, 2016 15:37

estoweva

After a very successful year of Arthrobacter phage hunting this summer we are having issues with our cells. We grow our cells in LB (tried PYCa, they seemed to grow more slowly), shaking in a baffled flask at 30 C. Originally we'd get a good culture within a day or two, this summer we seem to need at least three days to get a dense enough culture. We've had cells from one flask work well one day and then not the next (we store the cells in 4C after three days at 30 shaking, this worked before). Lawns are not as dense or as smooth. We bought a new vial from ATCC. Those cells worked, I froze them down, next time I started a culture from frozen, no plaques. The culture I started for students to work with on Tuesday the phage formed decent plaques, though the lawn was not as dense as before, I used those cells on Wednesday and this morning no plaques. We've tried increasing incubation time, increasing shaking time, doubling the amount of cells per plate (1 ml instead of 0.5ml), different media, different batches of LB, different batches of the components for PYCa, cleaning the flasks differently, restreaking, I keep track of eactly what plate I use to start the cultures. I'm starting to feel like someone's put a curse on us this summer–any ideas?

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

Link to this post | posted 02 Nov, 2016 13:56

estoweva

New issues! Ok We figured out some issues after the summer, lower temp, specifically using a sigma quality LB (Never got PYCa to work for us). As plates age, cells will grow but phage won't infect–now only using plates less than 2 weeks old. Every student had a phage, two weeks ago all but two phage stopped forming plaques. We pulled Arthro out of the freezer to start from new cultures instead of passaged ones but we can't recover those other 13 phage (of course this is the year I made everyone enter their phage by mid term instead of later). Calcium is at 5 mM. Plate with phage on them are now 2-3 weeks old, we've tried picking multiple plaques, higher volumes mixed with cells, struggling to find an explanation and any ideas would be welcome. Emily

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 02 Nov, 2016 15:37

estoweva

Hi Vic, Our plates for starting the Arthrobacter cultures are never more than 2 weeks old to start a culture from. The cells we used yesterday (that failed) were from a plate I started last Thursday directly from our frozen stock. They grew well overnight. The lawns are good now compared to the summer. We'll try soaking plates, though I did that with one student's plate earlier this semester and got nothing. E

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 18:23
estoweva	I tired from a plate last week and earlier in the semester we did use control lysate–will try that again tomorrow.