SEA-PHAGES | Orange Cells

Link to this post \| posted 12 Sep, 2022 16:19
cellokiwi	Has anyone ever had experience with Arthrobacter turning orange rather than yellowish? We are pretty sure there is no contamination, but the culture is orange suddenly.

Link to this post \| posted 12 Sep, 2022 17:25
debbie	Hi Alison, Could it be that you have a mutant of "WT" Arthrobacter sp.? So you still have your original stock in the freezer? You will want to use that until you confirm what the orange culture is. (Maybe a 16S to confirm). I believe G. terrae CAG3 is a mutant of G. terrae 3612, and is also a mutant in the gene that produces the color. In order to use it for phagehunting, you will want to confirm what you have. With propagation, does it stay orange? or does it go back to yellow. (Is this a mutable change or an environmental change?) Did you make any changes in the prep materials (a different brand of agar)? You will want to test your previous phages on the the new host to see if this change is results in a different infection pattern. If it is now a different bacteria than you started with, meaning it is an inherited trait, it is a new strain, subspecies and should be labeled as a new thing. Keep us at Pitt in the loop so that you enter your phage information correctly. We can add a new subspecies to the list if this proves to be true. Show us a picture of the pretty orange! debbie

Link to this post | posted 12 Sep, 2022 17:25

debbie

Hi Alison,
Could it be that you have a mutant of "WT" Arthrobacter sp.?
So you still have your original stock in the freezer?
You will want to use that until you confirm what the orange culture is. (Maybe a 16S to confirm).
I believe G. terrae CAG3 is a mutant of G. terrae 3612, and is also a mutant in the gene that produces the color.

In order to use it for phagehunting, you will want to confirm what you have. With propagation, does it stay orange? or does it go back to yellow. (Is this a mutable change or an environmental change?) Did you make any changes in the prep materials (a different brand of agar)?
You will want to test your previous phages on the the new host to see if this change is results in a different infection pattern.

If it is now a different bacteria than you started with, meaning it is an inherited trait, it is a new strain, subspecies and should be labeled as a new thing.

Keep us at Pitt in the loop so that you enter your phage information correctly. We can add a new subspecies to the list if this proves to be true.

Show us a picture of the pretty orange!
debbie

Link to this post \| posted 13 Sep, 2022 12:52
cellokiwi	So when you pass it, it goes back to yellow for a bit then turns orange again. We sadly don't have the resources to check 16S. Is there a resource for this through the program? The first time we saw it was in a lysogen so I just attributed it to wherever the genome had integrated itself messing with the color. Now, there has been zero change whatsoever. Same media, same CaCl2, same buffer, same incubator, same everything. One colleague suggested it is a carotenoid response to oxidative stress? Does that sound possible? Since we're using the same everything what would suddenly be stressing them out that wasn't before? In the picture the one on the left is a normal Arthrobacter culture and the one on the right is just starting to turn orange. They get darker than that. 107Kb

Link to this post | posted 13 Sep, 2022 12:52

cellokiwi

So when you pass it, it goes back to yellow for a bit then turns orange again. We sadly don't have the resources to check 16S. Is there a resource for this through the program? The first time we saw it was in a lysogen so I just attributed it to wherever the genome had integrated itself messing with the color. Now, there has been zero change whatsoever. Same media, same CaCl2, same buffer, same incubator, same everything. One colleague suggested it is a carotenoid response to oxidative stress? Does that sound possible? Since we're using the same everything what would suddenly be stressing them out that wasn't before?

In the picture the one on the left is a normal Arthrobacter culture and the one on the right is just starting to turn orange. They get darker than that.

Link to this post \| posted 13 Sep, 2022 23:38
viknesh	cellokiwi So when you pass it, it goes back to yellow for a bit then turns orange again. We sadly don't have the resources to check 16S. Is there a resource for this through the program? The first time we saw it was in a lysogen so I just attributed it to wherever the genome had integrated itself messing with the color. Now, there has been zero change whatsoever. Same media, same CaCl2, same buffer, same incubator, same everything. One colleague suggested it is a carotenoid response to oxidative stress? Does that sound possible? Since we're using the same everything what would suddenly be stressing them out that wasn't before? In the picture the one on the left is a normal Arthrobacter culture and the one on the right is just starting to turn orange. They get darker than that. Hi Alison, Since we began using M . foliorum for phage-hunting in the SEA, I've yet to hear from anyone about it turning orange. Can you confirm the following? 1. If you streak a plate from your freezer/glycerol stock and incubate the plate at 30C, colonies start out yellow but then turn orange? How long before they turn yellow, and how long before they turn orange? If you have photos, please share. 2. If you setup a culture, they saturated culture is yellow but then eventually turns orange? If so, as before, please share the timing of these color changes. 3. If you streak a plate from the culture (from #2), colony formation and timin gof color changes proceed similar to streaking from the glycerol? 4. You have several phages that can still form plaques when plated with bacteria from the orange culture? Thanks. Vic Edited 13 Sep, 2022 23:39

Link to this post | posted 13 Sep, 2022 23:38

viknesh

cellokiwi
So when you pass it, it goes back to yellow for a bit then turns orange again. We sadly don't have the resources to check 16S. Is there a resource for this through the program? The first time we saw it was in a lysogen so I just attributed it to wherever the genome had integrated itself messing with the color. Now, there has been zero change whatsoever. Same media, same CaCl2, same buffer, same incubator, same everything. One colleague suggested it is a carotenoid response to oxidative stress? Does that sound possible? Since we're using the same everything what would suddenly be stressing them out that wasn't before?

In the picture the one on the left is a normal Arthrobacter culture and the one on the right is just starting to turn orange. They get darker than that.

Hi Alison,

Since we began using M . foliorum for phage-hunting in the SEA, I've yet to hear from anyone about it turning orange. Can you confirm the following?

1. If you streak a plate from your freezer/glycerol stock and incubate the plate at 30C, colonies start out yellow but then turn orange? How long before they turn yellow, and how long before they turn orange? If you have photos, please share.

2. If you setup a culture, they saturated culture is yellow but then eventually turns orange? If so, as before, please share the timing of these color changes.

3. If you streak a plate from the culture (from #2), colony formation and timin gof color changes proceed similar to streaking from the glycerol?

4. You have several phages that can still form plaques when plated with bacteria from the orange culture?

Thanks.
Vic

Edited 13 Sep, 2022 23:39

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