SEA-PHAGES | Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???

Link to this post \| posted 15 Sep, 2015 13:52
GregFrederick@letu.edu	Well…. Well… Welll… I should have gone back into the lab prior to posting here and talking with Kevin on the phone this morning. On Friday afternoon, after 24 hours growth, I did not see any plaques in the positive control plates. Fred looked yesterday and said he hadn't seen anything. But this is what we see this morning! Sorry for the stress. Thanks for the trouble shooting. You guys are great! All systems go! Edited 15 Sep, 2015 16:09 1.17Mb

Link to this post | posted 15 Sep, 2015 13:52

Well…. Well… Welll…

I should have gone back into the lab prior to posting here and talking with Kevin on the phone this morning.

On Friday afternoon, after 24 hours growth, I did not see any plaques in the positive control plates.

Fred looked yesterday and said he hadn't seen anything. But this is what we see this morning!

Sorry for the stress. Thanks for the trouble shooting. You guys are great!

All systems go! smile

Edited 15 Sep, 2015 16:09

Link to this post \| posted 15 Sep, 2015 15:20
DanRussell	Yep, that looks like D29! Glad it's working.

Link to this post \| posted 15 Sep, 2015 16:15
GregFrederick@letu.edu	Dan Russell Yep, that looks like D29! Glad it's working. Me too. Still a little trouble shooting to be done as we did not see any likely plaques on the student isolate plates (11 post-enrichment plate sets analysed this morning. About 18 to be looked at more closely this afternoon.) Out of those 11 sets, only 1 had anything even remotely resembling plaques and those were 2 putatives from 50uL of an undiluted enrichment sample. If those are real, we calculated the PFUs of that stock to be ~40pfu/mL. Which seems very unlikely! But they were small plaques which might be predicted to have a smaller burst size. IDK. Edited 15 Sep, 2015 16:29

Link to this post | posted 15 Sep, 2015 16:15

GregFrederick@letu.edu

Dan Russell
Yep, that looks like D29! Glad it's working.

Me too. Still a little trouble shooting to be done as we did not see any likely plaques on the student isolate plates (11 post-enrichment plate sets analysed this morning. About 18 to be looked at more closely this afternoon.) Out of those 11 sets, only 1 had anything even remotely resembling plaques and those were 2 putatives from 50uL of an undiluted enrichment sample. If those are real, we calculated the PFUs of that stock to be ~40pfu/mL. Which seems very unlikely! But they were small plaques which might be predicted to have a smaller burst size. IDK.

Edited 15 Sep, 2015 16:29

Link to this post \| posted 15 Sep, 2015 17:41
debbie	so it sounds like you are testing your post-enrichment plates by putting 50 ul of sample into smeg and plating. Are you also doing spot testing? Sometimes 50 ul of enriched samples can so 'blow-out' the cells on a plate, you see no plaques. If the plaques are turbid, you will see lighter confluent growth of cells, so it can be quite confusing. Just thinking.

Link to this post \| posted 15 Sep, 2015 22:14
GregFrederick@letu.edu	Hmmmmm…. The plates with dilutions of putative phage stocks looked pretty much all the same and exactly like the plates with cells alone and with PB+cells. But I guess it is worth a try to do a quick spot test of each enrichment culture before we throw the entire lot out. We sent students back out with new collection kits. It seems that most did not actually follow (or possibly hear) our instructions on what the conditions of the most probably site would look like. Many collected soil from areas with little vegetation cover and areas lacking decaying matter such as wood mulch that sounded like they would not remain moist for long in the Texas heat and sunshine. But I think we should try a spot test before we discard the original enrichment stock isolates just in case. Thanks for the idea. Edited 15 Sep, 2015 22:15

Link to this post | posted 15 Sep, 2015 22:14

GregFrederick@letu.edu

Hmmmmm…. The plates with dilutions of putative phage stocks looked pretty much all the same and exactly like the plates with cells alone and with PB+cells. But I guess it is worth a try to do a quick spot test of each enrichment culture before we throw the entire lot out.

We sent students back out with new collection kits. It seems that most did not actually follow (or possibly hear) our instructions on what the conditions of the most probably site would look like. Many collected soil from areas with little vegetation cover and areas lacking decaying matter such as wood mulch that sounded like they would not remain moist for long in the Texas heat and sunshine.

But I think we should try a spot test before we discard the original enrichment stock isolates just in case. Thanks for the idea.

Edited 15 Sep, 2015 22:15

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Smeg grown in 7H9 Complete W/ or W/O Tween for enrichment???