SEA-PHAGES | Strep problems

Link to this post \| posted 16 Sep, 2015 14:05
scaruso	Hello all, I have started working with Strep. griseus and would like to solicit some advice. Lee generously provided me with a lot of information, but, honestly, these are critters that I have seen for the first time. I am growing them in nutrient broth (NB) + PEG to prevent clumping then using NB + supplements described by Lee for enrichments, and nutrient agar with the same supplements for plating. I have a culture being maintained at 30-deg w/ shaking in NB+PEG which I am sub-culturing for students, and I am starting it fresh on Fridays from a spore stock I created. So, my issues. Lawns with Strep. are not dense since TA is not used, so plaques are really tough to see. So far, those that the students have tested have proven not to be such. So, no phage so far, which is discouraging, of course. The cultures are pretty dense, but I am wondering how/if I can make thicker lawns or increase the likelihood of picking up a phage. Any thoughts are appreciated. Steve

Link to this post | posted 16 Sep, 2015 14:05

Hello all,

I have started working with Strep. griseus and would like to solicit some advice.

Lee generously provided me with a lot of information, but, honestly, these are critters that I have seen for the first time. I am growing them in nutrient broth (NB) + PEG to prevent clumping then using NB + supplements described by Lee for enrichments, and nutrient agar with the same supplements for plating.

I have a culture being maintained at 30-deg w/ shaking in NB+PEG which I am sub-culturing for students, and I am starting it fresh on Fridays from a spore stock I created.

So, my issues. Lawns with Strep. are not dense since TA is not used, so plaques are really tough to see. So far, those that the students have tested have proven not to be such. So, no phage so far, which is discouraging, of course. The cultures are pretty dense, but I am wondering how/if I can make thicker lawns or increase the likelihood of picking up a phage.

Any thoughts are appreciated.

Steve

Link to this post \| posted 16 Sep, 2015 14:36
dedrick	Hi Steve, Why aren't you using top agar? I have used it when isolating S. lividans phage. Bekah Bekah Dedrick

Link to this post \| posted 16 Sep, 2015 16:40
cdshaffer	We are using top agar with lividans and getting good plaques. Students are simply spreading phage solution on the plate, then adding top agar onto the plate with the spores already added to the top agar. Very Convenient, since it avoids the 15 min incubation step we did with phage + mycobacteria. Always nice in a class setting with limited time. So far so good for us, looks like we have at least a dozen phage isolated by direct plating. The hard part is growing and collecting the spores it adds time to prep work but they are expected to be stable throughout the phage isolation and purification stage so we only have to collect spores once. Edited 16 Sep, 2015 16:43

Link to this post | posted 16 Sep, 2015 16:40

cdshaffer

We are using top agar with lividans and getting good plaques. Students are simply spreading phage solution on the plate, then adding top agar onto the plate with the spores already added to the top agar. Very Convenient, since it avoids the 15 min incubation step we did with phage + mycobacteria. Always nice in a class setting with limited time.

So far so good for us, looks like we have at least a dozen phage isolated by direct plating. The hard part is growing and collecting the spores it adds time to prep work but they are expected to be stable throughout the phage isolation and purification stage so we only have to collect spores once.

Edited 16 Sep, 2015 16:43

Link to this post \| posted 16 Sep, 2015 16:52
lhughes	Hi All: Bekah - we've been using spread plates with liquid cultures of Streptomyces instead of the spore/top agar method at UNT. Steve got his protocols from me, so I'm to blame I sent him the following information by e-mail this morning about how we get good lawns with this method: We usually grow up 50 mL culture until turbid (for a subculture usually 2 days, for an initial culture from spores usually 4 days). Then we centrifuge in a 50 mL conical at 3000rpm (Sorvall tabletop with buckets), pour off all but about 10 mL of the supernatant, and resuspend. 100 uL of this usually gives a nice confluent lawn. Lee

Link to this post | posted 16 Sep, 2015 16:52

lhughes

Hi All:

Bekah - we've been using spread plates with liquid cultures of Streptomyces instead of the spore/top agar method at UNT. Steve got his protocols from me, so I'm to blame smile

I sent him the following information by e-mail this morning about how we get good lawns with this method:

We usually grow up 50 mL culture until turbid (for a subculture usually 2 days, for an initial culture from spores usually 4 days). Then we centrifuge in a 50 mL conical at 3000rpm (Sorvall tabletop with buckets), pour off all but about 10 mL of the supernatant, and resuspend. 100 uL of this usually gives a nice confluent lawn.

Lee

Link to this post \| posted 17 Sep, 2015 00:24
dedrick	Hi Lee, Do you use this method for all Streptomyces species? Are you using liquid media specific for sporulation? I know some do not sporulate in liquid (coelicolor)…not sure if it matters. I have always used spore stocks and top agar. Of course your method sounds easier!!!! Best, Bekah Bekah Dedrick

Link to this post \| posted 17 Sep, 2015 00:50
lhughes	It does seem that this method primarily works with those that sporulate in liquid media. So far we've had great success with S. griseus and S. venezuelae using the spread plates. Others are not so easy to do. I think we've tried S. albus this way and have had trouble getting phage. I am pretty sure my student has switched back to spores on that one (he had a few positives, but lost them early in the process so he's trying again). It is definitely much easier to do these plates and avoid the whole top agar issue. Chris - We haven't tried that particular method, but does seem like it would be faster than waiting for the incubation step as long as you get good infection. Lee

Link to this post | posted 17 Sep, 2015 00:50

lhughes

It does seem that this method primarily works with those that sporulate in liquid media. So far we've had great success with S. griseus and S. venezuelae using the spread plates. Others are not so easy to do. I think we've tried S. albus this way and have had trouble getting phage. I am pretty sure my student has switched back to spores on that one (he had a few positives, but lost them early in the process so he's trying again). It is definitely much easier to do these plates and avoid the whole top agar issue.

Chris - We haven't tried that particular method, but does seem like it would be faster than waiting for the incubation step as long as you get good infection.

Lee

Link to this post \| posted 17 Sep, 2015 02:10
cdshaffer	Well, I cannot say if we are getting "good" infections. We haven't tried to titer a stock with the different plating techniques. What I can say is every pair of students has a phage from their soil samples. So it seems to be working well enough.

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