Link to this post | posted 14 Apr, 2021 20:54
kmaclea	viknesh Thrilled to hear it, Kyle. Hope your students had fun figuring this out. It actually did turn out to be a fun, unscripted, sleuthing exercise! Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 29 Sep, 2023 03:47

uOttawaPHAGE

Hi Kyle. We think we are having an issue similar to what you describe above. We have just tried new agar and it seems to have helped but not completely. Given the contaminant survives the autoclave, it could be in some of the bottles that are filled with top agar, so I suppose it could persist. I’m hopeful this will fix the problem. Two questions: 1. In your thread you mention you were also having issues with phage propagation, which it sounded like you linked to the top agar issue. Did the propagation issue resolve when the top agar issue resolved? I’m assuming yes, but you didn't say it above. We are seeing propagation issues, but only for a subset of phages. The phages were found on new hosts for us, A. glob 2979 and A. sulf, and we aren't sure if this issue could be more common for these hosts, or if it is linked to the top agar. 2. We aren't seeing contamination in our plates, which is confusing to me. We wonder if this could be because of the cycloheximide. Did you ever consider this? This would suggest a fungal contaminant. Adam

Link to this post | posted 03 Oct, 2023 13:51

bcutsor

Hi Kyle and Adam, We have been having issues with top agar contamination also. I've even tried adding CHX to the top agar and ended up with contamination in my last batch. What's frustrating is that I'm using the same agar for PYCA plates + CHX with no issues. I am using the Fischer Brand agar BP1423. What brand are you using? I am going to make another batch and put the media in 15 mL sterile tubes that were purchased sterile. I'm not sure if it's the agar, the tubes that we autoclave inhouse, the black screw caps or a water batch issue. We are also trying to solve the issue. Any advice or info would be appreciated. Thanks, Bernadine 67Kb

Link to this post | posted 03 Oct, 2023 15:59

uOttawaPHAGE

Hi Bernadine. Based on an email exchange with Kyle (posted below), he thinks his issue was contamination with Geobacillus stearothermophilus, a thermophile that grows at 55-60 degrees and forms spores, so something that could be hard to get rid of. I also learned that these spores are used to test autoclaves and it is ubiquitous in the environment. My current thinking is our autoclave may not be killing every last spore, but we only see this in our top agar. We've increased the cycle time to 55' until we can have the autoclave checked. We wouldn't see problems in our plates/liquid (unless we put them at 55 - we are trying this) because the bacteria won't grow at 30-37. Not sure if you run the spore tests for your autoclave, but you might want to. Kyle is trying to avoid an extended time at 55, which may work as well, but we can't really make that work for our course. Good luck! Adam Kyle's email to me: Happy to talk this out sometime if it’s helpful. I went to reply on the Forum but I need to reset my password and just decided to do this instead. Re-reading the forum entries and both remembering things from our first bout and also probably forgetting some things, I can say that I feel pretty sure of what was the original cause of our problem. We isolated bacteria from the top agar. We sequenced it. It was Geobacillus stearothermophilus. Naturally, as a thermophile this makes sense. But where did it come from? I believe for us the source was our still. We still have an old distilled water still in our prep lab. We have always used it to make media with. For making regular old media to use at 37C, the presence of Geobacillus would never be noticed. But our phage work is the ONLY work with a hold step at 55C, which is just getting a Geobacillus going…. Given that we have sequencing data to show it is definitely this species, I had our lab manager replace the outflow hose from the still and the problem seemed to go away. Now I give this all to you as preface because, lo and behold!, it is BACK, RIGHT NOW. My TAs this semester were IN the course in SP21 when we had all the problems. And they are very very careful. We’ve done the usual stuff, replacing everything, using water from a different system we have upstairs, autoclaving the hell out of everything, before using containers and then again with media in them. We’ve put the TA in disposable sterile 50 ml conicals. We get contamination everywhere. It seems for us like ANY hold step at 55C is just asking for trouble. Vic suggested at first it could just be calcium precipitation and not contamination, but this looks really biological based on how it grows in the bottles. I am at the moment convinced the only thing we can do is basically never hold the TA at 55C except for the shortest period of time possible. So my plan for Monday is this: We made a couple large glass bottles of top agar. All the usual safeguards, autoclaved the hell out of it, and now we have let the TA bottles cool, and have placed them in the fridge to be solid, happy, and definitely not growing thermophiles, over the weekend. On Monday, we will boil the top agar to liquefy it, then let it cool, aliquot into sterile conicals, and place in the 55C bead baths. When the students are done with their Monday TA we will have them dispose of the TA. We will then rinse and repeat for Weds. This seems like vast overkill, but unless we absolutely minimize our TA time in 55C it seems like a continuous problem right now. Sounds crazy, doesn’t it? We also don’t see contamination in our plates OR in broth. Only the TA.

Link to this post | posted 03 Oct, 2023 17:49

bcutsor

Hi Adam, Thanks for forwarding me the information from Kyle. It's very helpful. We have a very old, steam autoclave so it could very well be contaminated with a thermophile which is what's giving us grief. For the time being I will prepare the TA the morning of the lab, alliquot into 15 mL sterile conical tubes and treat them as "one time use" instead of making the TA ahead of time and keeping in the 55 water bath for several hours or days as we have been doing. Hopefully this will work out. Thanks again, Bernadine

Link to this post | posted 03 Oct, 2023 18:22

kmaclea

Bernadine and Adam I had to reset my password, so thanks to Adam for sharing what I had sent him on email. Yes, it's clear that least in our old mill building we have some serious thermophile (or thermophile spore) contamination. Since this is the only class we see it, but also the only class where we had commonly held anything at 55C for any length of time, it tracks. There could be other issues above that were separate issues, but our main continuing issue seems to be the thermophile, and our immediate solution to it as has been the single-use top agar, and only either making it fresh or melting it only right before use out of the fridge. We have had a separate issue with making sure that A. glob. 2979 or 2880 are healthy and uncontaminated, so we have become very paranoid and rigorous in our handling, making single-use host cultures as well. Thus far, our single-use approach has been working, and quickly got us back to numerous plaques on direct isolation with A. glob. 2979. If you have any further questions, please reach out! Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129