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Shallee Page posted in Plaques appear on spot test, don't appear in titer
Viknesh Sivanathan posted in Plaques appear on spot test, don't appear in titer
Debbie Jacobs-Sera posted in Plaques appear on spot test, don't appear in titer
Shallee Page posted in Plaques appear on spot test, don't appear in titer
Plaques appear on spot test, don't appear in titer
| Link to this post | posted 16 Feb, 2024 14:01 | |
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Hi, I pulled my high titer lysates from the -80 and did a spot test and had excellent results. The spot was completely clear of the host (M. foliorum) and I took this to mean that the phage had survived the -80 storage. However, when I tried to do a titer I had no plaques on any of my plates. I tried this twice, first by picking the spot around the margins, and second by using the lysate directly. Neither produced plaques, and I used the same plates, top agar, phage buffer, and M. foliorum culture that I used in the successful spot test. I'm not sure what I'm doing wrong in the titer. Any help would be appreciated. Thanks, Ryan |
| Link to this post | posted 19 Aug, 2024 15:31 | |
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Hey, Ryan. Did you ever figure this out? We're seeing "spots" forming when we do spot titers and host-preference assays, but then no real plaques forming. This actually seems to be a random issue on our plates, where we have a clear spot in our host lawns that don't yield any plaques when picked. But we're also seeing it very clearly on spot titers, where the spots clear, but there definitely are not plaques. It seems to be less bad when we use PYCa for dilutions, but still obvious. Attached is a spot titer plate showing the spots we get. Thanks! Justin |
2.05Mb| Link to this post | posted 19 Aug, 2024 16:45 | |
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Hi, That's the exact problem I had. It came from a contaminant in the M. foliorum stocks. I streaked a new plate and selected a single colony, which I used to streak a new plate and select another single colony and grew up that colony for fresh bacteria stocks. This solved the problem for me. -Ryan |
| Link to this post | posted 20 Aug, 2024 01:04 | |
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JustinA Hi Justin and Ryan, One explanation for what you are observing is called "lysis from without". This happens when phages that are not able to successfully infect and replicate in particular bacteria are nevertheless still able to lyse those bacterial cells when they are applied to cells at high concentrations. Such lysis is not the result of replication and lysis from within the cells but instead is happeneing from the outside of cells, hence the term "lysis from without". Such lysis is likely due to the mass action of lots of hydrolase enzymes that phages typically have as part of their tails and that are used to locally depolymerase the cell wall in order to deliver the phage genome into the cell. At some point, there is too much of these "local" depolymerization activity that the cells are overwhelmed and lyse as a result. Because lysis from without only happens at high concentrations, you will not see lysis when the phage sample is diluted. Because lysis from without does not involve phage replication, you will not see individuals plaques form. When performing host-range assays, it is typical to observe clearing of spots at high phage concentrations but no individual plaques at the lower concentrations. Such results are regularly interpreted as phage not being able to infect that particular host. I hope this helps explain some of your observations. |
| Link to this post | posted 20 Aug, 2024 01:26 | |
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viknesh Vic, that might be an explanation, except we are seeing the clearing on all dilutions down to -8. Lysis from without should rapidly dilute away. Notice that we have rows of "clearing", except they are not really clear. Sort of hazy, which naturally could be due to some lysogeny. But I wouldn't really expect a phage to spot like this further than the host on which it was isolated. |
| Link to this post | posted 20 Aug, 2024 01:37 | |
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Ah, I didnt see the image you attached. Very odd looking. Is each row a dilution series for a different phage? And is this on the isolation host or a different bacterium you are testing for host range? Do you have a photo of the second plate? |
| Link to this post | posted 20 Aug, 2024 01:42 | |
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viknesh This is not the isolation host. I'll try to get a picture of that when I'm back in the lab tomorrow. I should not that this image is from a project looking at Citrobacter phages, but we have seen similar "spots" on Arthrobacter. |
| Link to this post | posted today, 19:10 | |
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So, we have had a lot of trouble amplifying, with great spot tests and poor plaque assays (a lot of killing at high concentration but no nice plaques at any concentration). I suspect lysis from without. So, after checking to make sure that we are plating from a single colony of the correct host, what would be the next step? Some spot test plate pictures attached. |
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2.44Mb| Link to this post | posted today, 19:22 | |
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Hi all, I think there is bacterial contamination at play. But hard to tell, no phage or host is named. anyone want to help a girl out? debbie |
| Link to this post | posted today, 19:32 | |
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stpage Hi Shallee, It looks to me that you are indeed seeing individual plaques form for the one dilution after the last spot that resulted in a full clearing. However, it looks like your lawn is not particularly even. As a result, it is hard to see those plaques. Because those plaques are forming within a defined spot on the plate, it is not too hard to see those plaque when we compare that defined area to the rest of the plate. However, I imagine that we wouldnt be able to see those plaques if they were distributed across the entire plate, as they would be in a plaque assay. So the first thing we need is a nice lawn. I suspect the lawn is uneven due to the top agar. I would suggest melting new/fresh top agar and preparing a top agar lawn with freshly prepared bacteria too. I think you'll then be able to see the plaques. Hope this is helpful. Vic |