Phyto culture - colors?

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kilnakorr

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Sorry to hear it, that sucks. The cultures look about to crash honestly. I will try to offer suggestions but keep in mind I'm mostly guessing based on my experience. I'm just a fellow hobbyist like yourself, not an expert.

Where did you get your f/2, and how much did you add? If you don't use enough fertilizer the culture can run out and turn yellow. But usually in my experience not so quickly. If you used the recommended amount of f/2 then running out in 48 hours doesn't make alot of sense to me.

Contamination is another possibility but again, it's odd to happen so fast. Usually when setting up a new system with new equipment contamination isn't an issue. Contamination is my biggest problem long-term, but not at first and you have a brand new setup so I wouldn't expect that particular issue to pop up. Did you use rodi water for your salt mix? Hopefully you didn't use old tank water or anything like that?

I will say that your 24 hour growth was very surprising to me - phyto can grow fast but that's really, really fast growth for the first 24 hours in a brand new setup. Usually phyto takes a couple days to get used to a new bioreactor, at least in my experience - and then growth explodes after it's more acclimated.

Is this nanno? I recommend looking under a microscope to see if there is contamination, or if the cells look funny in any way.

I've attached a pic of some of my nanno for reference. I recommend looking for contamination - basically anything that doesn't look like the nanno. Big cells moving fast like ciliates or very small cells like bacteria. Anything that doesn't look like this, basically.
It is nano, and I use RODI.
I did notice a few things.
I was recommended 1ml f/2 for 2L, however the bottle says '20 drops' per liter, which I just measured to be just a bit more than 1 ml.
So per direction I've only added about half.

I also seem to have a lot more light duration than I thought, running just 2 hours of darkness which I also suspect to increase the temperature a bit to high.

I've now added more f2/2, set the light duration for 16 hours a day, added a carbon filter on the air tube, and a fan to remove some of the hot air.
20210924_193017.jpg


Not sure if I can save this batch, but we'll see.
If not I guess I need to try again.
 
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kilnakorr

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Update.
Cultures did get a bit greener after a day, but turned back to yellowish, and seems to stay that way.
Before changes bottom, day after top.

20210926_104716.jpg


Today, they look like the bottom picture again.
Since the issue could still be from previous overheating I decided to add one more culture:

20210926_104215.jpg


Will update tomorrow on progress.
 
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kilnakorr

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It seems the problem is the f/2 fertilizer.

Bottom picture: yesterday 'morning'.
Middle: A few hours after adding 1 ml f/2 to left container.
Top: This morning after adding x ml of Tropica standard plant fertilizer to right container 1 hour before lights out.
It looks quite obvious, that the phyto is starving and adding nutrients brings it back within hours.
20210927_083822.jpg
 

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So perhaps you aren't using enough f/2? I get my fert from florida aqua farns, and I think recommended dose is 1.5 ml fert per gallon saltwater. But this could be different for fert from other sources, I'm not sure.

I do not halve the recommended dose, which is listed to achieve f/2 media concentration. Actually I add a little excess. I've tested nitrate & phos a few times on tetraselmis cultures and found that all phos was consumed in about a week. Going over a week results in yellow/crashed culture.

I've not had good results "resurrecting" cultures after they turn yellow. They will green back up but often not the same organism. Competing microorganisms such as cyano can take advantage of the situation and take over.
 
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kilnakorr

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So perhaps you aren't using enough f/2? I get my fert from florida aqua farns, and I think recommended dose is 1.5 ml fert per gallon saltwater. But this could be different for fert from other sources, I'm not sure.

I do not halve the recommended dose, which is listed to achieve f/2 media concentration. Actually I add a little excess. I've tested nitrate & phos a few times on tetraselmis cultures and found that all phos was consumed in about a week. Going over a week results in yellow/crashed culture.

I've not had good results "resurrecting" cultures after they turn yellow. They will green back up but often not the same organism. Competing microorganisms such as cyano can take advantage of the situation and take over.
The recommended dose is 1ml per liter, which seems to grow the phyto for about a day.
I'm not sure I'll keep these resurrected cultures, but I might after a little microscope peak.
I'm quite sure the issue is the f/2 that seems very diluted / poor quality.
 
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kilnakorr

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Sorry to hear it, that sucks. The cultures look about to crash honestly. I will try to offer suggestions but keep in mind I'm mostly guessing based on my experience. I'm just a fellow hobbyist like yourself, not an expert.

Where did you get your f/2, and how much did you add? If you don't use enough fertilizer the culture can run out and turn yellow. But usually in my experience not so quickly. If you used the recommended amount of f/2 then running out in 48 hours doesn't make alot of sense to me.

Contamination is another possibility but again, it's odd to happen so fast. Usually when setting up a new system with new equipment contamination isn't an issue. Contamination is my biggest problem long-term, but not at first and you have a brand new setup so I wouldn't expect that particular issue to pop up. Did you use rodi water for your salt mix? Hopefully you didn't use old tank water or anything like that?

I will say that your 24 hour growth was very surprising to me - phyto can grow fast but that's really, really fast growth for the first 24 hours in a brand new setup. Usually phyto takes a couple days to get used to a new bioreactor, at least in my experience - and then growth explodes after it's more acclimated.

Is this nanno? I recommend looking under a microscope to see if there is contamination, or if the cells look funny in any way.

I've attached a pic of some of my nanno for reference. I recommend looking for contamination - basically anything that doesn't look like the nanno. Big cells moving fast like ciliates or very small cells like bacteria. Anything that doesn't look like this, basically.

nannochloropsis.jpg
I know I'm asking a lot from you @fry-man , but can you tell from my kiddy-scope and smartphone if this looks like nanno phyto:
20210928_112326.jpg


They do look round, but it's dang hard to tell.
 

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Hmm, sorry but I can't tell from that pic. What magnification is it? I know it's hard to get good pics on a microscope.

Nanno is round, synnechococcus is more oblong, and smaller. You could have a mix too.

Taking a step back, what's your goal with the phyto? Maybe synnechococcus gets in there (it's happened to me, and I expect it happens alot) but perhaps this is not such a big deal if you are just learning about setting up bioreactors and trying your hand at culturing phyto. Alot of people dose synnechococcus to their reef tanks; some on purpose, others who maybe don't realise it. Most report positive outcomes either way. It would be a different story if you were planning to feed rotifers / raise clownfish fry or something similar but I don't get the impression that's the case.

Don't get me wrong, I think it's best practice to use a microscope and know what you're culturing. This will be especially important if you're going to improve the cultures and progress to the next level. But I don't want you to keep spending money on new starter cultures, unless there's a good reason.

Maybe just split the culture, let it grow out for awhile and see what happens?
 

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Sorry to hear it, that sucks. The cultures look about to crash honestly. I will try to offer suggestions but keep in mind I'm mostly guessing based on my experience. I'm just a fellow hobbyist like yourself, not an expert.

Where did you get your f/2, and how much did you add? If you don't use enough fertilizer the culture can run out and turn yellow. But usually in my experience not so quickly. If you used the recommended amount of f/2 then running out in 48 hours doesn't make alot of sense to me.

Contamination is another possibility but again, it's odd to happen so fast. Usually when setting up a new system with new equipment contamination isn't an issue. Contamination is my biggest problem long-term, but not at first and you have a brand new setup so I wouldn't expect that particular issue to pop up. Did you use rodi water for your salt mix? Hopefully you didn't use old tank water or anything like that?

I will say that your 24 hour growth was very surprising to me - phyto can grow fast but that's really, really fast growth for the first 24 hours in a brand new setup. Usually phyto takes a couple days to get used to a new bioreactor, at least in my experience - and then growth explodes after it's more acclimated.

Is this nanno? I recommend looking under a microscope to see if there is contamination, or if the cells look funny in any way.

I've attached a pic of some of my nanno for reference. I recommend looking for contamination - basically anything that doesn't look like the nanno. Big cells moving fast like ciliates or very small cells like bacteria. Anything that doesn't look like this, basically.

nannochloropsis.jpg
when you look at these are they moving or rather static? How can you tell live algae from dead?
 
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kilnakorr

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Hmm, sorry but I can't tell from that pic. What magnification is it? I know it's hard to get good pics on a microscope.

Nanno is round, synnechococcus is more oblong, and smaller. You could have a mix too.

Taking a step back, what's your goal with the phyto? Maybe synnechococcus gets in there (it's happened to me, and I expect it happens alot) but perhaps this is not such a big deal if you are just learning about setting up bioreactors and trying your hand at culturing phyto. Alot of people dose synnechococcus to their reef tanks; some on purpose, others who maybe don't realise it. Most report positive outcomes either way. It would be a different story if you were planning to feed rotifers / raise clownfish fry or something similar but I don't get the impression that's the case.

Don't get me wrong, I think it's best practice to use a microscope and know what you're culturing. This will be especially important if you're going to improve the cultures and progress to the next level. But I don't want you to keep spending money on new starter cultures, unless there's a good reason.

Maybe just split the culture, let it grow out for awhile and see what happens?
Thanks for the insight.
I wouldn't think there is any synne-whats-it-called in it. I sanitized and restarted everything, with new nanno cultures.
My concern is more if something else could have found its way in there, given the ups and downs of the cultures.
For now, I'm just using it for copepods cultures, as I'm unsure I want to add it to my tank.
I have one last culture going from the original culture, so hoping it will come out positive.
 

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when you look at these are they moving or rather static? How can you tell live algae from dead?
Good question. Nanno does not move so...if there are lots of cell fragments or it's a funny color then I'd guess it's dead. It may be hard to tell, at least for me. Maybe a biologist would know other ways? I just go by color and if it's getting denser.

There are motile phyto like tetraselmis and isochrysis then it's much easier to tell if these are alive/healthy.
 
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Thanks for the insight.
I wouldn't think there is any synne-whats-it-called in it. I sanitized and restarted everything, with new nanno cultures.
My concern is more if something else could have found its way in there, given the ups and downs of the cultures.
For now, I'm just using it for copepods cultures, as I'm unsure I want to add it to my tank.
I have one last culture going from the original culture, so hoping it will come out positive.
There's definately other stuff in the culture. All phyto cultures have bacteria but having a good sanitization protocol keeps it (hopefully) under control.

I grow copepods too right now I do nanno, tetraselmis, and isochrysis phyto and feed this to tigtiopus, tisbe, and apocyclops copepods. For copepods, in my experience nanno, tet, and isochrysis blend is good but nanno alone is prob ok. They may even grow on cyano (synnechoccus) I think but expect not so well. Tigriopus copepods grow pretty well on about anything and were easy for me to start although they do not reproduce as quickly as my tisbe do now. It took awhile for the tisbe to get going but once they did I got alot of them. I'm still working on apocyclops - the cultures I have are a mix of different copepods and I'm not sure what I can do to change that. It's not as productive for me but I'm not buying any more starters so just gonna let it grow out and see what happens.

Good luck!
 

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The recommended dose is 1ml per liter, which seems to grow the phyto for about a day.
I'm not sure I'll keep these resurrected cultures, but I might after a little microscope peak.
I'm quite sure the issue is the f/2 that seems very diluted / poor quality.
Hey! Great to see some phytoplankton cultures!

They could just be hitting stationary phase (becoming nutrient limited, which I think you observed) and starting to die. Cultures that green are likely super dense. Cells get stressed out when they sit in stationary phase and eventually die. I think this is what you're observing with the change in color of your cultures.

Maybe you should transfer the cells to new growth medium before they start to turn yellow? I don't think your f/2 is bad, I just think the cultures are getting so dense they are running out of nutrients.
 
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kilnakorr

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Hey! Great to see some phytoplankton cultures!

They could just be hitting stationary phase (becoming nutrient limited, which I think you observed) and starting to die. Cultures that green are likely super dense. Cells get stressed out when they sit in stationary phase and eventually die. I think this is what you're observing with the change in color of your cultures.

Maybe you should transfer the cells to new growth medium before they start to turn yellow? I don't think your f/2 is bad, I just think the cultures are getting so dense they are running out of nutrients.
Thanks for your input.
I can't imagine I can get too dense a culture within 48 hrs.
The below pictures are taking afternoon, and the next two days in the morning:
20210924_160139.jpg

Getting nice dark green after 16 hrs or so, and the next morning dying.
 

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It's hard to say without knowing what cell density you're starting your cultures at and without knowing what cell density they can reach before hitting stationary phase. That being said, even the top cultures in your picture are probably pretty dense too.

I've never cultured nannochloropsis before, but with some of the cultures that I grow (some marine haptophytes) when I start to see "color" the cells have a density between 10^5-10^6 cells per ml. If I don't bubble those cultures, with the f/2 that I make, the cells usually hit a max cell density of ~3x10^6 cells per ml.

One thing you could try is by doing a larger dilution of your cell culture in fresh f/2 when you transfer them. Perhaps that's what you've done with the middle culture in this picture. That way it will take longer for the cells to reach the dark green color on the top left.
1632857525355.png

You could also check to make sure they aren't getting too much light and that they are growing at the recommended temperature. Just to rule those out too. Looking at them under the microscope (as others have suggested) would also be a good confirmation as well. Sounds like you're doing all the right things though!
 
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kilnakorr

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It's hard to say without knowing what cell density you're starting your cultures at and without knowing what cell density they can reach before hitting stationary phase. That being said, even the top cultures in your picture are probably pretty dense too.

I've never cultured nannochloropsis before, but with some of the cultures that I grow (some marine haptophytes) when I start to see "color" the cells have a density between 10^5-10^6 cells per ml. If I don't bubble those cultures, with the f/2 that I make, the cells usually hit a max cell density of ~3x10^6 cells per ml.

One thing you could try is by doing a larger dilution of your cell culture in fresh f/2 when you transfer them. Perhaps that's what you've done with the middle culture in this picture. That way it will take longer for the cells to reach the dark green color on the top left.
1632857525355.png

You could also check to make sure they aren't getting too much light and that they are growing at the recommended temperature. Just to rule those out too. Looking at them under the microscope (as others have suggested) would also be a good confirmation as well. Sounds like you're doing all the right things though!
I think my setup is decent now, with some temperature control and all.
My concern at the moment, is mostly weather the culture I brought from yellow back to green is still good.
The color is a more dusty olive green, than a jade green color.

The lack of light does make the right look a bit darker, but the color is still a lot different than the middle one.
20210928_222637.jpg
 
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I think this will be the last update, unless someone has anything to add.
I harvested half of the culture. As it was my last clean portion of nanno, I wanted to make sure I could restart a culture just in case, so it might have been a couple of days early.

Left is the new harvest, right is from the culture that was brought back from yellow with more fertilizer.
I find the left one looking a lot greener and healthier.
20210930_081400.jpg
 

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Good question. Nanno does not move so...if there are lots of cell fragments or it's a funny color then I'd guess it's dead. It may be hard to tell, at least for me. Maybe a biologist would know other ways? I just go by color and if it's getting denser.

There are motile phyto like tetraselmis and isochrysis then it's much easier to tell if these are alive/healthy.
Can you still feed phyto that has died like the ones shown in this thread (assuming the yellow ones are dead)? The reason I ask is that I feed Seachem's Reef Phyto which isn't alive but I don't know if there is a difference between their "non-living" phyto and dead phyto in home cultures.
 

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Can you still feed phyto that has died like the ones shown in this thread (assuming the yellow ones are dead)? The reason I ask is that I feed Seachem's Reef Phyto which isn't alive but I don't know if there is a difference between their "non-living" phyto and dead phyto in home cultures.
Commercial phyto for feed from a reputable source like Seachem or reef nutrition (I.e. phytofeast) works well ime. It's also way easier. Some of them sell live versions as well as preserved and it doesn't make much difference as far as I can tell.

But I expect they harvest the phyto when it's most nutritious and then preserve it for use as feed. A home phyto culture that's crashed is not the same.
 
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