phytoplankton culture settling

[TWYOUNG]

Do phytoplankton cultures need to be shaken? I started my first culture six days ago with 50% Nanno and 50% sw. I'm bubbling air in at several bubbles per second but settling is occurring. Is this normal, especially since Nanno is nonmotile? Also when should I divide and dilute it? Thanks

 

[fryman]

It is not normal to have alot of settling. Live phyto (even non-motile ones like nanno) are able to maintain bouyancy even with no water movement. Dead phyto will settle out at the bottom but this should be minor amounts.

I've seen alot of settling in badly contaminated cultures, especially with bacteria. But in a new culture this is not uncommon so don't lose hope. You can clean it up with a good sanitization protocol. Sanitize your media and equipment with every split and it should eventually clean up. Also don't carry over the particate/settled out stuff when splitting.

I split every 6-7 days.

 

[TWYOUNG]

It is not normal to have alot of settling. Live phyto (even non-motile ones like nanno) are able to maintain bouyancy even with no water movement. Dead phyto will settle out at the bottom but this should be minor amounts.

I've seen alot of settling in badly contaminated cultures, especially with bacteria. But in a new culture this is not uncommon so don't lose hope. You can clean it up with a good sanitization protocol. Sanitize your media and equipment with every split and it should eventually clean up. Also don't carry over the particate/settled out stuff when splitting.

I split every 6-7 days.

Thanks! I don't think it's contaminated as I've been very cautious maintaining sterility. So when I split the cultures I should discard the plankton which has settled on the bottom, and divide my cultures into two NEW bottles? Or can I continue to use the current container for half of the product?

 

[fryman]

It's up to you how much risk you want to take but I guess I would carefully pour out the top of the culture into a new/clean container, stopping before any particulate. Then I'd split this into two bioreactors and add 50% sanitized media (whatever that is). You don't need to fill bioreactors to the top.

I always sanitize equipment between splits. There is a significant amount of precipitate on the airline due to high pH in the culture which is a breeding ground for bacteria.

 

[TWYOUNG]

It's up to you how much risk you want to take but I guess I would carefully pour out the top of the culture into a new/clean container, stopping before any particulate. Then I'd split this into two bioreactors and add 50% sanitized media (whatever that is). You don't need to fill bioreactors to the top.

I always sanitize equipment between splits. There is a significant amount of precipitate on the airline due to high pH in the culture which is a breeding ground for bacteria.

Thanks for all the helpful advice. Are you experienced at copepod culture bc I started one of those as well?

 

[TWYOUNG]

It's up to you how much risk you want to take but I guess I would carefully pour out the top of the culture into a new/clean container, stopping before any particulate. Then I'd split this into two bioreactors and add 50% sanitized media (whatever that is). You don't need to fill bioreactors to the top.

I always sanitize equipment between splits. There is a significant amount of precipitate on the airline due to high pH in the culture which is a breeding ground for bacteria.

Here are pics of cultures taken today!

 

[fryman]

Here are pics of cultures taken today!

2nd pic looks good to me. Do you have a microscope? I'm curious of the sediment in that first pic, not sure what's going on there.

I do have some experience with rotifers, artemia, tisbe, tigriopus, and apocyclops. But I'm just a hobbiest like yourself so still learning.

 

[TWYOUNG]

2nd pic looks good to me. Do you have a microscope? I'm curious of the sediment in that first pic, not sure what's going on there.

I do have some experience with rotifers, artemia, tisbe, tigriopus, and apocyclops. But I'm just a hobbiest like yourself so still learning.

Yea, there was sediment in the initial culture. What initiated this thread was my concern as to whether I should agitate to sample. I followed the stated advice and disposed of that. I would imagine the phyto which was shipped to me may have had some die off before I added it, or some of my culture may have died. I've seen instructions to use a microscope to see if phyto is alive but since Nannochloropsis is non-motile I'm not sure what I'm to look for.

 

[TWYOUNG]

Here are the two cultures I split last weekend. The one on the left lightened significantly the day after split and although darkening has lagged behind the one on the right. What to do now with the one on the left, a) clean, add F2 and allow to continue to grow b) split as usual c) dump d) use for tank possibly adding a larger amount daily?

 

[fryman]

Here are the two cultures I split last weekend. The one on the left lightened significantly the day after split and although darkening has lagged behind the one on the right. What to do now with the one on the left, a) clean, add F2 and allow to continue to grow b) split as usual c) dump d) use for tank possibly adding a larger amount daily?

Hard to say. Depending on visual appearance alone is not reliable in my experience. Do you have a microscope?

Did you maybe forget to add f/2 to the lighter one? It could also have had bleach residue or something like ciliates or bacteria contamination. But I also get suspicious now when a culture gets darker quickly, since this can be a sign of cyano contamination.

When odd things happen to me now I usually just pull fridge backups. Only problem with this is not all phyto types handle the fridge well (like tisochrysis). But for nanno and tetraselmis, having backups in the fridge has been a game changer for me.

Assuming you don't have a backup I'd split as usual, and then wait & observe.

 

[TWYOUNG]

Hard to say. Depending on visual appearance alone is not reliable in my experience. Do you have a microscope?

Did you maybe forget to add f/2 to the lighter one? It could also have had bleach residue or something like ciliates or bacteria contamination. But I also get suspicious now when a culture gets darker quickly, since this can be a sign of cyano contamination.

When odd things happen to me now I usually just pull fridge backups. Only problem with this is not all phyto types handle the fridge well (like tisochrysis). But for nanno and tetraselmis, having backups in the fridge has been a game changer for me.

Assuming you don't have a backup I'd split as usual, and then wait & observe.

Here are some microscopic images. I definitely added the F2. It would be nice if I could come up with a method of determining the correct density for harvesting/splitting. For example, when I could no longer see the rigid tubing in the center, or perhaps something behind the jar. I'm using Nanno bc I read it's the best/easiest to culture however I wish I could culture a MOBILE species so I could easily see it was alive. I guess as long as it's green it's alive? I appreciate you sharing your knowledge and experiance.

 

[fryman]

That looks like a reasonably clean nanno culture, good job! Personally I stick to weekly splitting with a good sanitization protocol and over time the cultures seem to get much more consistent.

Tetraselmis was actually the easiest for me, although nanno is pretty close. Tertaselmis is larger and motile and so it is more satisfying to look at it under the microscope.

 

[pmcmahon]

Hoping I can jump into this thread since I'm having the same problem. I started a new culture with a bottle of @AlgaeBarn ocean magic two days ago. I put the whole bottle in 2.5g of fresh saltwater reading 1.025. I checked on it this afternoon and the water is looking significantly lighter and there is a lot of the phyto that has settled on the bottom of the container. The airline is around 2 bubbles a second I would guess. There are 3 more containers with pods next to this and I added some phyto in each of those. It looks like most of the phyto has settled in those as well

Could this just be die off from transit or is there something crashing my culture?

Second question, do you guys have any good resources on culturing? I've just seen a few youtube videos that breeze over the process. I don't know how often to dose f/2, best practice for sanitizing and splitting, etc.

Third question, the back of the bottle of F/2 says there is a part A and part B and both should be dosed. I thought there was just one kind (I have A apparently). Is it critical to have both A and B?

 

[TWYOUNG]

I'm FAR from any expert but have been culturing phyto and pods for a few months. First Ocean Magic is a mixture of several different types of phyto so I'm not sure what species will dominate, or if it my be more complicated to work with. Nannochloropsis is the species most easily and most ofter cultured. Nanno is nonmotile so you can look at it under a scope but will just see green circles. The only way to tell it's viable is the cultures darken as they become more dense. Tetraselmus has flagella and can be seen moving under the scope. Some settling is normal and seems much more pronounced in my tetra cultures. Right after they're split they go almost clear and colorless for a day before bouncing back. I split every 7 days or so and do a 50/50 dilution with 1 ml of F2 added to 32 total ounces in each drawer. I don't know anything about two part fertilizer. The settling could be limited by more vigorous aeration. Whereas copepods are cultured with very little air flow, phyto can be aerated much more aggressively.
Bleach can be used for sanitizing but rinse thoroughly. Many people also use 70% rubbing alcohol.
This information is based on my limited experience and others may have more/better advice.

 

[fryman]

Hoping I can jump into this thread since I'm having the same problem. I started a new culture with a bottle of @AlgaeBarn ocean magic two days ago. I put the whole bottle in 2.5g of fresh saltwater reading 1.025. I checked on it this afternoon and the water is looking significantly lighter and there is a lot of the phyto that has settled on the bottom of the container. The airline is around 2 bubbles a second I would guess. There are 3 more containers with pods next to this and I added some phyto in each of those. It looks like most of the phyto has settled in those as well

Could this just be die off from transit or is there something crashing my culture?

Second question, do you guys have any good resources on culturing? I've just seen a few youtube videos that breeze over the process. I don't know how often to dose f/2, best practice for sanitizing and splitting, etc.

Third question, the back of the bottle of F/2 says there is a part A and part B and both should be dosed. I thought there was just one kind (I have A apparently). Is it critical to have both A and B?

Probably die off from transit but don't lose hope. There could still be live cells in there and it only takes a few to get started. Give it some time and maybe a couple splits.

I recommend fao.org for sanitization, it's an aquaculture industry resource but they have guidelines and techniques described in simple terms and they can be adopted to home culture.

2.3. Algal production

There are multiple types of f/2 available, some are 2-part (e.g. fritz or algagen), while others are 1-part (florida aqua farms, mercer of montana). If you get a 2-part fertilizer you must use both parts.

*edit* Sounds like you only used part A of a 2-part fertilizer, which won't work. Probably that's the main issue.

 

[TWYOUNG]

Probably die off from transit but don't lose hope. There could still be live cells in there and it only takes a few to get started. Give it some time and maybe a couple splits.

I recommend fao.org for sanitization, it's an aquaculture industry resource but they have guidelines and techniques described in simple terms and they can be adopted to home culture.

2.3. Algal production

There are multiple types of f/2 available, some are 2-part (e.g. fritz or algagen), while others are 1-part (florida aqua farms, mercer of montana). If you get a 2-part fertilizer you must use both parts.

*edit* Sounds like you only used part A of a 2-part fertilizer, which won't work. Probably that's the main issue.

I have the 1 part Guillard’s F2 and it has begun to show some dark pieces of debris in it. Is this some kind of normal precipitation or should I pitch it. I don’t think I contaminated it in any way.

 

[Koh23]

No matter how strong i bubble culture, i have habbit that everyday i swirl container a bit, it helps a lot with settling.

 

[TWYOUNG]

Probably die off from transit but don't lose hope. There could still be live cells in there and it only takes a few to get started. Give it some time and maybe a couple splits.

I recommend fao.org for sanitization, it's an aquaculture industry resource but they have guidelines and techniques described in simple terms and they can be adopted to home culture.

2.3. Algal production

There are multiple types of f/2 available, some are 2-part (e.g. fritz or algagen), while others are 1-part (florida aqua farms, mercer of montana). If you get a 2-part fertilizer you must use both parts.

*edit* Sounds like you only used part A of a 2-part fertilizer, which won't work. Probably that's the main issue.

Does it matter how GREEN a pod culture is. Is just a tint of green enough or should it look more like the Tetraselmus in the attached photo?

 

[pmcmahon]

Probably die off from transit but don't lose hope. There could still be live cells in there and it only takes a few to get started. Give it some time and maybe a couple splits.

I recommend fao.org for sanitization, it's an aquaculture industry resource but they have guidelines and techniques described in simple terms and they can be adopted to home culture.

2.3. Algal production

There are multiple types of f/2 available, some are 2-part (e.g. fritz or algagen), while others are 1-part (florida aqua farms, mercer of montana). If you get a 2-part fertilizer you must use both parts.

*edit* Sounds like you only used part A of a 2-part fertilizer, which won't work. Probably that's the main issue.

Yeah I've never seen two part before so I just assume when I bought it. I have part B coming but it will be a couple of days before I have it. Hopefully the culture can hang in there until it gets here. I have a second bottle of phyto in the refrigerator that I can use if this culture fails. I figured there would be a few mistakes so I came prepared with a backup plan.

Sounds like since I have a multistrain culture eventually one will become dominant. Do you culture multiple strains? Is the work worth the diversity between strains?

Do you only dose f/2 right after the split or is it something you do on a more regular schedule?

 

[pmcmahon]

No matter how strong i bubble culture, i have habbit that everyday i swirl container a bit, it helps a lot with settling.

My tanks are 2.5g so I might have a little trouble swirling them without sending water everywhere. Maybe I can keep some sort of spoon or something in a little sanitizer to mix it up ever few days.