Calling all Isochrysis Galbana Phytoplankton culture growers! Share your tips and tricks with us please! Isochrysis galbana growing guide

[Pepper Reefer]

ISOCHRYSIS GALBANA PHYTOPLANKTON

Hello,
The goal of this thread is to consolidate information, questions and answers for people looking to culture Isochrysis galbana phytoplankton
I appreciate those that have cultured isochrysis galbana sharing their secrets and knowledge with us
In this post I will write what I have learned so far - in the hopes that any wrong information can be corrected, and ask questions too

As I understand it there is
Isochrysis galbana T-ISO (Tahitian strain) (more nutritious and common)
Isochrysis galbana CCMP1323 (use in hatcheries)
Isochrysis galbana CCMP1850 (high growth rate / larviculture uses)
Isochrysis galbana strain S-ISO (colder waters)
Where each have different ideal growing conditions
However it appears (correct me if I’m wrong) that most hobbyists are growing Isochrysis galbana Tahitian strain, or it seems that this is the strain being sold by many vendors

I know that phyto can grow under a wide range of conditions, where some people can have a neglected, forgotten, half drank 2 liter of Mr. Pibb sitting in their basement that only gets hit by a patch of sunlight twice a year during the equinox, and they are successful at growing phyto in spite of the neglect; but if you had to ‘make it a science’, of growing Isochrysis galbana with the highest probability of success and lowest likelihood of crashing..

Ideal Isochrysis galbana (Tahitian strain) growing conditions:

Temperature:
68-78 degrees [however some threads mention that theirs may have been hotter in sunlight - others mention being much colder, but maybe they had S-ISO?) In general consensus seems to approve of room temperature for isochrysis galbana, that a heater is not necessary for success

Salinity:
35ppt 1.025/1.026S.G. seems to have the best success - however; the ‘official’ range is 20-35ppt - this is a very wide range, where I’ve seen some say it’s closer to 25-30ppt ~1.022 S.G. for isochrysis galbana (Tahitian), some say that slightly lower salinity is better; there doesn’t seem to be as much consensus on the ‘perfect’ salinity

Container:
Large glass jars / flasks are recommended, due to the ability to sterilize easier with boiling water, but any container can work - many use 2 liter bottles, others use large tubs or tanks. Protip was to use saran wrap instead of a hard lid to avoid lid contamination and cleaning, puncture one hole for gas exchange, the other for a hard acrylic tube to reach the bottom (or you can suction cup a soft airline tube in place at the bottom)
1-2 bubbles per second, not a roaring boil - just enough to keep the container moving [do not use airstone or will froth up]

Sterilization:
It appears that sterilization is extremely important for successful culture of isochrysis galbana compared to other commonly cultured phytoplankton - failure of sterilization seems to be the leading cause of iso culture crashes. You can either add boiling water to the container, pour it out, let it air dry; or you can add water + bleach and then use a bleach removing agent after. Any piece of equipment that touches the culture should be sterilized - bacteria/copepods/rotifers/other phyto species are all potential contaminants that can crash a culture of isochrysis galbana.
Consensus is to never use tank water or ocean water due to contaminants; to use freshly mixed salt water only for an isochrysis galbana culture. Many recommend to mix fresh saltwater, then boil the saltwater for 15 minutes to sterilize it, before adding it to the culture vessel (though this is ‘optional’) if boiling, to make the water slightly more diluted to account for the water that evaporates
It appears also that sometimes it may not be a failure of sterilization on your end, that it could be that the sample bought from various vendors may not be viable due to contamination on their end, and to try different vendors if one keeps crashing.

Nutrients:
What is needed 100% of the time is Guillard F/2 formula
Isochrysis galbana can benefit from supplements of the following, or can risk depleting these and crashing:

1. Silica
2. Iron
3. Manganese
4. Zinc
5. Cobalt for B12 synthesis
6. Amino acids
7. B12 + other vitamins
8. Detectable nitrates / phosphates (ranges I’ve seen are 5ppm nitrate 0.1ppm phosphate)
9. Seaweed extract for auxins/cytokinins etc

Ideal pH: 7.8-8.2

Lighting: 5000-6500K — cheap lights work fine; supposedly an adjustable LED light strip which you wrap around the container works well; any grow light, or light in this spectrum should work fine
Consensus seems to be that isochrysis galbana requires moderate to high lighting, ~200 PAR — in general requires more light than other phytoplankton species
However too much light can lead to photoinhibition / bleaching, and crashes

Ratio of start culture to seawater in vessel for a new culture:
It seems that isochrysis galbana is better to use 1-5% ratio, where if you had a 1 liter container of saltwater, to only place 10-50mL of isochrysis galbana, and allow it to grow and not start off very concentrated; this seems to differ from other phytoplankton species, where the starting ratio is much higher.

Misc:
On average, it should be harvested 50% taken out, 50% remaining, replace with sterile saltwater, every 7 days
It appears that isochrysis galbana can struggle to restart a culture if it has been placed in the freezer / refrigerator for any period - compared to other phytoplankton, where you can keep a reserve in the refrigerator to start up a culture if it crashes, isochrysis galbana does not work very well in this way to restart a culture if it has been chilled.


Questions:
Is there anything I’ve gotten wrong about culturing isochrysis galbana (Tahitian strain)?
What do you wish you had known at the start when you began culturing isochrysis galbana?
What has been the leading cause of crashes of isochrysis galbana cultures for you?
What change to your technique(s) led you to the greatest success?


Thank you very much for making it to the end of this long post!
I hope that you reefers out there successfully growing isochrysis galbana can share your tips and tricks here :^)
If you know anyone in the community who grows isochrysis galbana phytoplankton, please tag them here!

 

[Pepper Reefer]

Here are some helpful videos about culturing isochrysis galbana / phytoplankton

 

[Pepper Reefer]

@RSNJReef sorry to ping you brother, I saw your awesome post about isochrysis here:

Tips for Growing Isochrysis

Pretty isn't it? My wife found it and thought it would be a nice addition to the mix. It's Porphyridium curentum. Apparently one of the only true red pigments in a microalgae. You can read about it HERE if you scroll down a bit. Thanks. I have always had trouble with the other red one -...

www.reef2reef.com

I was hoping you could weigh in and correct me if I'm wrong about anything and share your great knowledge

 

[RSNJReef]

Hey Pepper, thanks for reaching out man. You actually caught me at a good time cause my schedule tends to be all over the place with the wife, kids, 3 reef systems, the phyto culture, amphipods culture, and 3 copepod cultures (yeah, I have no life……..).

So, I agree for the most part with everything you said, sterilization and a good/clean starter culture are the two keys to success. Also, the 50/50 harvest rule is a good one, though, always judge the harvest based on visual density of the culture. If it gets a nice dark tea color where you can’t see much light through it, it’s time to harvest. I myself do a 70/30 harvest and restart (harvesting 70%), and that gets me to a nice dark culture 7 days later.

Also, one of the things that most people don’t recognize until a few years in is any phyto, copepod, or any other type of culture is a living creature, which can adapt to any culture style within certain bounds over time. That’s why a neglected culture for some people can survive, because more than likely that neglect didn’t start up all of a sudden, there was a curve of normalcy to neglect, and the culture was lucky enough to adapt to the neglect. This rule should apply to any new culture, and people need to understand that any new culture needs to be handled a bit more delicately in the beginning, and you will have some die off / stress of the culture until it adapts to your style. Like with copepods, what typically happens is you won’t see rapid population growth for any home culture for the first generation or two of pods. It’s usually by the third generation or so that things take off, because by that time that generation has been born and adapted to your style of culturing, though, you still need to maintain it somewhat within bounds.

So generally if you have a starter culture on the lighter side (like in the case of a starter of isochrysis where you can see you hand through the jar of culture, you need to start on lower light, until you can’t see through the container, then you can turn up the light a bit. There are some that have been successful with higher light in the beginning, but it’s the luck of the draw if you go with higher light (what I do is put a sheet of printer paper between the light and the jar in the beginning to block some of the light, then when I can’t see through the culture I remove the paper).

Also, I agree that once you put it in the fridge, there is a lower likelihood of being able to restart the culture. What happens with most species of phyto is when you put it into the fridge, they loose their flagella, so they temporarily loose the ability to swim. Great for feeding to the tank, but bad for starting a new culture. If I was to re-start from the fridge, it would have to be a culture where the cells have not settled out. I would then take the refrigerated culture and put on a window sill for 24 hours so it gets some natural sunlight and comes to room temp (no aeration for the 24 hours). Then, after the 24 hours, I’d only collect some of the culture water where the cells haven’t settled out (do not collect the cells which settled to the bottom of the culture jar, those are good for feeding, but not for culturing.

Also, be careful with the silica. While some silica is helpful for iso, too much silica can cause issues, so generally I recommend an off the shelf guillards f2 without silica.

That’s all I can think of for right now. Everything else you got spot on man. Good job with this post.

 

[RSNJReef]

Actually, one other thing I’d mention is that I keep my iso culture with a somewhat vigorous boil (4-5 large bubbles a second), enough to elevate the water bubbles about a 1/4 to 1/2 inch above the water line, but again, your phyto may have to get adjusted to this over time.

 

[Pepper Reefer]

Thank you so much for your detailed response @RSNJReef - I truly appreciate you sharing your isochrysis galbana knowledge!

>always judge the harvest based on visual density of the culture. If it gets a nice dark tea color where you can’t see much light through it
For judging the color, your other post mentioned:
>If it turns white, then clear: your phyto bleached out and died, which means a chemical in the water stressed and killed it (like excess bleach or alcohol), or your light was too strong and it stressed and killed the cells. This can also happen with water that’s too hot.
>If it changes color (mostly to green): the culture was contaminated with another algae. If it turns yellow though and it’s not supposed to be, the cells died and they yellow is bacterial takeover.

>If you see brown or black buildup on the walls of your phyto container: you have a higher than normal bacterial load, which is causing the cells to clump and die. With a healthy starter, it can usually survive for a couple of days with the clumping building up until it crashes (so you want to move quickly when you see this to salvage the culture).
—— I think the hardest part visually (for me at least) would be distinguishing between normal, healthy yellow, and bacteria takeover
Do you have any more tips for assessing the culture by color
Eg. If you just split it less than a few days ago, and it’s too dark a yellow already, then it’s bacteria?

> Like with copepods, what typically happens is you won’t see rapid population growth for any home culture for the first generation or two of pods. It’s usually by the third generation or so that things take off, because by that time that generation has been born and adapted to your style of culturing, though, you still need to maintain it somewhat within bounds.
Very interesting!

>So generally if you have a starter culture on the lighter side (like in the case of a starter of isochrysis where you can see you hand through the jar of culture, you need to start on lower light, until you can’t see through the container, then you can turn up the light a bit.
Is it possible to have the light too low at the early stage, resulting in a crash? Or is it much more likely that you would run into the light would be too high, causing a crash?
If your light is too low, how could you best tell that it’s too low and you need to increase intensity (other than by PAR meter)?

Also, what do you think is the best photoperiod for isochrysis galbana?
There seems to be varying opinions of only 8 hours a day of light vs 12 or even 16 hours

>Actually, one other thing I’d mention is that I keep my iso culture with a somewhat vigorous boil (4-5 large bubbles a second), enough to elevate the water bubbles about a 1/4 to 1/2 inch above the water line
Good to know!

Thank you again for sharing your knowledge @RSNJReef - I’m sure it will help many people in the years to come who stumble across your posts!

 

[Pepper Reefer]

Just curious, what vendor did you end up getting the most viable culture from?
"For myself, I had to go through about 4-5 different vendors of iso before I found a vendor that provided a batch that stayed viable, and now I’ve had the same iso culture going for 3 years now."
I know that a lot may have changed in 3 years, where that same vendor may not have the same culture / methods, but it seems like a good first choice for people looking to get their first batch!

 

[Pepper Reefer]

FYI for phyto growers: I asked Mercer of Montana if it was recommended to boil the saltwater before or after adding Guillard F/2 (as there were varying opinions online about adding the Guillard F/2 and then boiling it and whether it would damage the vitamins)
He said quote "You should never boil the growth medium as it will become toxic!"
also said to use white vinegar for sanitizing

 

[taricha]

Here's a weird culture source:
Phycopure Copepod Blend from liveaquaria.
I used it to start cultures a half dozen times - one time it cultured up rhodomonas.
The other times, it cultured up T-iso (from an original mix of T-iso and chaetoceros diatom.)

 

[RSNJReef]

Hey Pepper, ok, took me a little bit to get some time to respond. Going to try and break down each response so this will be a bit long.

>always judge the harvest based on visual density of the culture. If it gets a nice dark tea color where you can’t see much light through it
For judging the color, your other post mentioned:
>If it turns white, then clear: your phyto bleached out and died, which means a chemical in the water stressed and killed it (like excess bleach or alcohol), or your light was too strong and it stressed and killed the cells. This can also happen with water that’s too hot.
>If it changes color (mostly to green): the culture was contaminated with another algae. If it turns yellow though and it’s not supposed to be, the cells died and they yellow is bacterial takeover.
>If you see brown or black buildup on the walls of your phyto container: you have a higher than normal bacterial load, which is causing the cells to clump and die. With a healthy starter, it can usually survive for a couple of days with the clumping building up until it crashes (so you want to move quickly when you see this to salvage the culture).
—— I think the hardest part visually (for me at least) would be distinguishing between normal, healthy yellow, and bacteria takeover
Do you have any more tips for assessing the culture by color
Eg. If you just split it less than a few days ago, and it’s too dark a yellow already, then it’s bacteria?

So, there’s a little bit of a variable curve here, and there are standards you can use on Lux based on cell density, but those curves change based on whether your culture is stressed or not, as well as the nutrition of the fertilizer you are using, and unfortunately some of this comes with experience. Generally speaking it’s always safer to go with slightly lower light than with light that’s too intense. Think of it in similar terms of coral for example. Years ago the thought was that to grow coral well was to blast them with light. What’s been found recently though is that the reason why blasintg them with light worked was because our filtration systems were not as good as they are now, and the nutrient/bio load of home tanks were much higher, so the corals would need the higher light to metabolize / cope with the higher nutrient load. In todays hobby, our filtration equipment works so well that too much of it can strip the water, and corals nowadays bleach under high settings of most leds nowadays where in the same tank / lights with older filtration systems, the coral may have done a little better (hence people dosing nutrients to their tanks now). It’s the same idea with most photosynthetic organisms. To Maximus growth, you want higher nutrient and higher light, but, your culture needs to get used to this first, on top of this, if you overdo it, your culture will crash into a wall.

Now, with all of this being said, it’s easy for someone to get overly stressed trying to calculate every single variable, get their curves exactly right, then have some unknown throw them a curve ball and then everything goes left field, which can make them want to quit entirely. To prevent yourself a lot of stress which isn’t worth it, remember that this is supposed to be interesting and fun, and keep things simple for yourself. This isn’t a job for most of us, we choose to do this, so don’t stress yourself out by going too hard too fast and crashing out. Start very simple, with a simple light and an easy phyto to grow. Once you have some simple failures then get that down, and you have the time, then take your knowledge and try something a little more advanced (not the top of the mountain, just a few feet further up). Just take it one step at a time and remember you will eventually get there.

Now, with all of that being said, what you can do is get a variable intensity led light (start with a warm white/yellow light strip), where you can change the light intensity via a flip/slide/rotation switch. I’d say get a 12in intensity adjustable bar light (that should cover 2 one gallon jars on each side if you put the bar light down and have it on the bottom facing up next to the foot of the jars. Then, think of where you’d have the light intensity if you had a dense culture ready to harvest. Now, take that setting on the light, and, if you can visibly see through your culture (see your hand clearly), turn the light down to 30-40%, and leave it there until you can no longer see through the jar, then turn it up to 70-80%, then, when it gets pretty dense (let’s say 2 days away from harvest day), put it to 100%. Once you go through this initial protocol for a new starter, your culture should be mostly adapted to your light and fertilizer (average reproduction cycle for isochrysis is around 24 hours, so you’re on your 5th to 7th generation of cells by that point), so don’t stress yourself with switching the light intensity when you spli. If you want to be on the safer side, you can do a 50/50 split so your starter density is higher and turn the lights down to 80% for a few days.

Remember, keep it simple, it’s not a sprint, but a marathon.


> Like with copepods, what typically happens is you won’t see rapid population growth for any home culture for the first generation or two of pods. It’s usually by the third generation or so that things take off, because by that time that generation has been born and adapted to your style of culturing, though, you still need to maintain it somewhat within bounds.
Very interesting!
Yeah it was a pretty cool thing to find. Over the years I’ve cultured tisbe, apocyclops, parvocalanus, Tigriopus (both Californicus and japonicus, though I like japonicus better, just harder to get), and amphipods, and while the culture techniques are different, it was the same reaction:

Stage 1, initial culture: culture is stressed, and the biome of your culture system isn’t established yet, so reproduction is slow. The good part is your water is cleaner so it eases the stress.

Stage 2, biome begins to build: harvesting is very limited, you’re a generation in, bacteria levels are rising, and the copepods are consuming some of it along with the phyto which is helping things. The new generation is also born into your water so from birth they will cope better than the adults.

Stage 3, biome is at peak, you’re now 3 or so generations in, your have your technique down, and your pods are used to you. Your water is also at its peak nutrient level now, and now your reproduction rate is at peak. Now you need to be diligent with gentle water changes (25-50% a week done over a couple days as long as you are not over feeding, but you need to not remove all of the detritus, leave at least 20% of the detritus behind each week because this is part of your biome, this is especially true for Tigriopus).

Stage 4 will be either a continuation of stage 3 or the neglect phase, where you’re getting tired of it, life gets in the way, and things slip. While your pods are used to your system, the nutrient level is getting too high, the pods begin to stress, and reproduction goes down. You can rectify this with multiple 20% water changes and 15-20% detritus removal each time (remember your culture is already stressed, so doing an aggressive water change might outright kill majority of the population). Recovery of the culture will now be setting you back to stage 2, so just be mindful of this.

>So generally if you have a starter culture on the lighter side (like in the case of a starter of isochrysis where you can see you hand through the jar of culture, you need to start on lower light, until you can’t see through the container, then you can turn up the light a bit.
Is it possible to have the light too low at the early stage, resulting in a crash? Or is it much more likely that you would run into the light would be too high, causing a crash?

There’s a higher likelihood of high light crashing the culture rather than light that’s too low. There is a lower boundary however, as your phyto needs both the nutrients and light (to convert the nutrients in their cells), with lower light there will be less nutrient consumption, and if they don’t convert the nutrients at a regular rate, they will starve and die. The rate of starving and dying is slower (longer duration) than bleaching (from light that’s too high) and dying though. You just need to look at your culture each day and assess, how did it change from yesterday. If no change, keep the course / if it changes to the better, keep the course till it’s ready for the next stage/ if it’s worse than yesterday , you need to adjust according to what you see.

If your light is too low, how could you best tell that it’s too low and you need to increase intensity (other than by PAR meter)?

What you can do is start at 40% of your lights total intensity (you can start off with a yellow led light which is 750 to 1000 lumen per foot length of light, with an adjustment feature to be able to turn the light down to at least 40%). Start at around 30-40% and work your way up slowly depending on what you see and density of the starter). Remember, move slowly at first, and don’t stress. If you fail a few times at first, remember that you needed to to learn what not to do.



Also, what do you think is the best photoperiod for isochrysis galbana?
There seems to be varying opinions of only 8 hours a day of light vs 12 or even 16 hours

I have seen all of these work for different people, but for an established healthy culture, if you want to maximize growth, you can do a 16 hour photoperiod. If starting a new culture, if you want to be safer, you can do 8 or 12.

What I do is my culture rack is by a window, and my lights are off in the morning until around 12, that way my phyto is getting just sunlight from the window. Then at 12 the leds come on at 100% until around 11pm; then the lights shut off. My belief is there are some wavelengths that an led cannot replicate when compared to sunlight, and, a little sunlight in a clean culture goes a long way:

>Actually, one other thing I’d mention is that I keep my iso culture with a somewhat vigorous boil (4-5 large bubbles a second), enough to elevate the water bubbles about a 1/4 to 1/2 inch above the water line
Good to know!

 

[RSNJReef]

FYI for phyto growers: I asked Mercer of Montana if it was recommended to boil the saltwater before or after adding Guillard F/2 (as there were varying opinions online about adding the Guillard F/2 and then boiling it and whether it would damage the vitamins)
He said quote "You should never boil the growth medium as it will become toxic!"
also said to use white vinegar for sanitizing

This is good to know, I need to pocket this information as well. Thanks pepper!!

 

[Pepper Reefer]

Hey Pepper, ok, took me a little bit to get some time to respond. Going to try and break down each response so this will be a bit long.

>always judge the harvest based on visual density of the culture. If it gets a nice dark tea color where you can’t see much light through it
For judging the color, your other post mentioned:
>If it turns white, then clear: your phyto bleached out and died, which means a chemical in the water stressed and killed it (like excess bleach or alcohol), or your light was too strong and it stressed and killed the cells. This can also happen with water that’s too hot.
>If it changes color (mostly to green): the culture was contaminated with another algae. If it turns yellow though and it’s not supposed to be, the cells died and they yellow is bacterial takeover.
>If you see brown or black buildup on the walls of your phyto container: you have a higher than normal bacterial load, which is causing the cells to clump and die. With a healthy starter, it can usually survive for a couple of days with the clumping building up until it crashes (so you want to move quickly when you see this to salvage the culture).
—— I think the hardest part visually (for me at least) would be distinguishing between normal, healthy yellow, and bacteria takeover
Do you have any more tips for assessing the culture by color
Eg. If you just split it less than a few days ago, and it’s too dark a yellow already, then it’s bacteria?

So, there’s a little bit of a variable curve here, and there are standards you can use on Lux based on cell density, but those curves change based on whether your culture is stressed or not, as well as the nutrition of the fertilizer you are using, and unfortunately some of this comes with experience. Generally speaking it’s always safer to go with slightly lower light than with light that’s too intense. Think of it in similar terms of coral for example. Years ago the thought was that to grow coral well was to blast them with light. What’s been found recently though is that the reason why blasintg them with light worked was because our filtration systems were not as good as they are now, and the nutrient/bio load of home tanks were much higher, so the corals would need the higher light to metabolize / cope with the higher nutrient load. In todays hobby, our filtration equipment works so well that too much of it can strip the water, and corals nowadays bleach under high settings of most leds nowadays where in the same tank / lights with older filtration systems, the coral may have done a little better (hence people dosing nutrients to their tanks now). It’s the same idea with most photosynthetic organisms. To Maximus growth, you want higher nutrient and higher light, but, your culture needs to get used to this first, on top of this, if you overdo it, your culture will crash into a wall.

Now, with all of this being said, it’s easy for someone to get overly stressed trying to calculate every single variable, get their curves exactly right, then have some unknown throw them a curve ball and then everything goes left field, which can make them want to quit entirely. To prevent yourself a lot of stress which isn’t worth it, remember that this is supposed to be interesting and fun, and keep things simple for yourself. This isn’t a job for most of us, we choose to do this, so don’t stress yourself out by going too hard too fast and crashing out. Start very simple, with a simple light and an easy phyto to grow. Once you have some simple failures then get that down, and you have the time, then take your knowledge and try something a little more advanced (not the top of the mountain, just a few feet further up). Just take it one step at a time and remember you will eventually get there.

Now, with all of that being said, what you can do is get a variable intensity led light (start with a warm white/yellow light strip), where you can change the light intensity via a flip/slide/rotation switch. I’d say get a 12in intensity adjustable bar light (that should cover 2 one gallon jars on each side if you put the bar light down and have it on the bottom facing up next to the foot of the jars. Then, think of where you’d have the light intensity if you had a dense culture ready to harvest. Now, take that setting on the light, and, if you can visibly see through your culture (see your hand clearly), turn the light down to 30-40%, and leave it there until you can no longer see through the jar, then turn it up to 70-80%, then, when it gets pretty dense (let’s say 2 days away from harvest day), put it to 100%. Once you go through this initial protocol for a new starter, your culture should be mostly adapted to your light and fertilizer (average reproduction cycle for isochrysis is around 24 hours, so you’re on your 5th to 7th generation of cells by that point), so don’t stress yourself with switching the light intensity when you spli. If you want to be on the safer side, you can do a 50/50 split so your starter density is higher and turn the lights down to 80% for a few days.

Remember, keep it simple, it’s not a sprint, but a marathon.


> Like with copepods, what typically happens is you won’t see rapid population growth for any home culture for the first generation or two of pods. It’s usually by the third generation or so that things take off, because by that time that generation has been born and adapted to your style of culturing, though, you still need to maintain it somewhat within bounds.
Very interesting!
Yeah it was a pretty cool thing to find. Over the years I’ve cultured tisbe, apocyclops, parvocalanus, Tigriopus (both Californicus and japonicus, though I like japonicus better, just harder to get), and amphipods, and while the culture techniques are different, it was the same reaction:

Stage 1, initial culture: culture is stressed, and the biome of your culture system isn’t established yet, so reproduction is slow. The good part is your water is cleaner so it eases the stress.

Stage 2, biome begins to build: harvesting is very limited, you’re a generation in, bacteria levels are rising, and the copepods are consuming some of it along with the phyto which is helping things. The new generation is also born into your water so from birth they will cope better than the adults.

Stage 3, biome is at peak, you’re now 3 or so generations in, your have your technique down, and your pods are used to you. Your water is also at its peak nutrient level now, and now your reproduction rate is at peak. Now you need to be diligent with gentle water changes (25-50% a week done over a couple days as long as you are not over feeding, but you need to not remove all of the detritus, leave at least 20% of the detritus behind each week because this is part of your biome, this is especially true for Tigriopus).

Stage 4 will be either a continuation of stage 3 or the neglect phase, where you’re getting tired of it, life gets in the way, and things slip. While your pods are used to your system, the nutrient level is getting too high, the pods begin to stress, and reproduction goes down. You can rectify this with multiple 20% water changes and 15-20% detritus removal each time (remember your culture is already stressed, so doing an aggressive water change might outright kill majority of the population). Recovery of the culture will now be setting you back to stage 2, so just be mindful of this.

>So generally if you have a starter culture on the lighter side (like in the case of a starter of isochrysis where you can see you hand through the jar of culture, you need to start on lower light, until you can’t see through the container, then you can turn up the light a bit.
Is it possible to have the light too low at the early stage, resulting in a crash? Or is it much more likely that you would run into the light would be too high, causing a crash?

There’s a higher likelihood of high light crashing the culture rather than light that’s too low. There is a lower boundary however, as your phyto needs both the nutrients and light (to convert the nutrients in their cells), with lower light there will be less nutrient consumption, and if they don’t convert the nutrients at a regular rate, they will starve and die. The rate of starving and dying is slower (longer duration) than bleaching (from light that’s too high) and dying though. You just need to look at your culture each day and assess, how did it change from yesterday. If no change, keep the course / if it changes to the better, keep the course till it’s ready for the next stage/ if it’s worse than yesterday , you need to adjust according to what you see.

If your light is too low, how could you best tell that it’s too low and you need to increase intensity (other than by PAR meter)?

What you can do is start at 40% of your lights total intensity (you can start off with a yellow led light which is 750 to 1000 lumen per foot length of light, with an adjustment feature to be able to turn the light down to at least 40%). Start at around 30-40% and work your way up slowly depending on what you see and density of the starter). Remember, move slowly at first, and don’t stress. If you fail a few times at first, remember that you needed to to learn what not to do.



Also, what do you think is the best photoperiod for isochrysis galbana?
There seems to be varying opinions of only 8 hours a day of light vs 12 or even 16 hours

I have seen all of these work for different people, but for an established healthy culture, if you want to maximize growth, you can do a 16 hour photoperiod. If starting a new culture, if you want to be safer, you can do 8 or 12.

What I do is my culture rack is by a window, and my lights are off in the morning until around 12, that way my phyto is getting just sunlight from the window. Then at 12 the leds come on at 100% until around 11pm; then the lights shut off. My belief is there are some wavelengths that an led cannot replicate when compared to sunlight, and, a little sunlight in a clean culture goes a long way:

>Actually, one other thing I’d mention is that I keep my iso culture with a somewhat vigorous boil (4-5 large bubbles a second), enough to elevate the water bubbles about a 1/4 to 1/2 inch above the water line
Good to know!

Thank you so much for your detailed response @RSNJReef!
I truly appreciate you going above and beyond to share your knowledge with the whole community and myself.
Thank you for the growing tips - you’re 100% right that I should enjoy the process as a hobby, that there will be trial and error, and a learning curve (as with everything in the hobby!)
Very good explanation on the starting lower and raising light intensity slowly and seeing how the culture reacts, just like coral.

I will use all of these tips that you have shared, and hopefully be able to post some nice, healthy dense cultures in the coming weeks / months :^)
My copepods, fish, corals, and tank thank you. I’m sure that many others will benefit from you sharing your knowledge here in the years to come
I will remember: Keep it simple, it’s not a sprint, but a marathon.

 

[Frank SM]

Great thread. Lots of good information.

I got my Iso culture from Algae Research and Supply. It's only been going 2 weeks but looks good. I've split it once when it got to a dark straw color and am just building up my cultures. I use plastic 1 gallon jugs and 2 liter bottles, Fritz Aquatic F/2, rolling air flow, 14 hour LED white grow lights. I'm also culturing Tetra and Nanno (as well as Tisbe, Apocyclops, and Tigriopus copepods and amphipods).

 

[djf91]

Great thread. Lots of good information.

I got my Iso culture from Algae Research and Supply. It's only been going 2 weeks but looks good. I've split it once when it got to a dark straw color and am just building up my cultures. I use plastic 1 gallon jugs and 2 liter bottles, Fritz Aquatic F/2, rolling air flow, 14 hour LED white grow lights. I'm also culturing Tetra and Nanno (as well as Tisbe, Apocyclops, and Tigriopus copepods and amphipods).

I’m about to start a few cultures of T-iso. What did you use to sterilize? I’m also thinking about growing mine in a window with natural sunlight as this has helped greatly for my Nanno cultures.

 

[RSNJReef]

Thank you so much for your detailed response @RSNJReef!
I truly appreciate you going above and beyond to share your knowledge with the whole community and myself.
Thank you for the growing tips - you’re 100% right that I should enjoy the process as a hobby, that there will be trial and error, and a learning curve (as with everything in the hobby!)
Very good explanation on the starting lower and raising light intensity slowly and seeing how the culture reacts, just like coral.

I will use all of these tips that you have shared, and hopefully be able to post some nice, healthy dense cultures in the coming weeks / months :^)
My copepods, fish, corals, and tank thank you. I’m sure that many others will benefit from you sharing your knowledge here in the years to come
I will remember: Keep it simple, it’s not a sprint, but a marathon.

Good luck with everything you do pepper, just remember to keep asking and never be afraid to fail, it’s the only way each of us and others can learn.

 

[Pepper Reefer]

I’m about to start a few cultures of T-iso. What did you use to sterilize? I’m also thinking about growing mine in a window with natural sunlight as this has helped greatly for my Nanno cultures.

With jars (culture vessels) that are new, I sterilize with citric acid scrubbing down with paper towels, then vinegar scrub, then thorough rinse with RODI (and dry)
If I've used them already for culturing phyto or copepods, then I boil water from the faucet, pour them in the glass jars and let them come to room temperature (takes ~2 hours to cool); then citric acid scrub down, vinegar scrub down, RODI rinse
I have not yet had to sterilize the rigid tubing I have been using to swap between cultures, as ones that I used for iso cultures I was able to swap them for copepod cultures and then buy more
However they are very expensive for what they are - I don't think reef2reef allows Amazon links, but search on Amazon

MECCANIXITY Clear Rigid Tubing 4mm(5/32'') ID x 5mm(3/16'') OD x 305mm/12inch Plastic Water Tube for DIY, Pack of 2 --- $9.99​

These are the only rigid tubing that fits inside of standard 3/16" airline tubing that I have been able to find (I've tried a bunch of stores in person, and practically every website online, and a lot of failed testing with too wide of tubes.. if someone else knows of a 5mm OD rigid tubing source that is better than $9.99 for two 1 foot pieces, please share!

I have heard that replacing the airline tubing used for the culture is one of the key parts of sterilization - to not use the tubing for one culture to another. I haven't had to face that yet, but the protocol I've been thinking about would be
>let tubes sit in container long enough to fit them + citric acid
>use pipe cleaner to scrub inside of tubing using citric acid
>rinse repeatedly
>let tubes sit in container + vinegar
>use pipe cleaner with vinegar
>rinse thoroughly
>air dry for a long time

 

[Pepper Reefer]

More information for people seeking to grow isochrysis:

I had been struggling to find a vendor for isochrysis that didn't refrigerate the culture before sending it to you. I emailed a lot of the big(ger) vendors, scientific research ones too, to ask if they refrigerated their cultures before shipping and they responded either outright "Yes they have been refrigerated" or deliberately avoided the question and said "We hibernate the culture!" (aka, we refrigerate it - lol)
As such, I would highly recommend that before buying an iso culture from any vendor, you should contact them and ask if they refrigerate the culture at all before sending it - don't buy from a vendor that refrigerates.
I don't want to bad mouth any vendors as it is their livelihood - instead I'll just say that out of all the vendors for iso, I had the best success with BasementReef on Etsy - every other vendor that I tried the bottle would come half dead, with soooo much detritus on the bottom. Their cultures never grew, would just go from dark yellow/brown tea to green or clear after a week - same growing process each time. I'm sure that their bottles are fine for feeding your tank / feeding your copepods.. but not for growing!

I also found out from BasementReef
>1.025 salinity is good
>1.5mL of Guillard F/2 per gallon is fine [I had been doing 4mL!]
>to start a culture, you can use as little as 50mL in 1 gallon (3785mL) of saltwater [I had been using 473mL!!]
>lights 6am to 10:30pm (16.5 hours)
>room temp
My first (successful) attempt was going well, got darker and darker everyday for ~1 week; I was planning on splitting it the following day, I woke up and saw that it had completely changed color (crashed) to a lighter yellow/green. It was upon this crash that I asked him for advice, and he said the above info
It's no wonder [no duh! moment] that it exhausted the nutrients, as I had used one 16oz bottle (473mL) in only half a gallon to start it, 20x the recommended starting dosage (50mL / 3785mL = 1.32%) vs (473mL / 1892.5 = 25%)
Dumb mistake for me to make - but at least others reading this post will not make the same mistake as I did of over-concentrating the initial addition of iso culture! I'm honestly amazed that it lasted / grew darker as long as it did, with such a high concentration.

I ordered more iso starting culture which should arrive in a day or two, will spread out the 16oz bottle in four 1 gallon vessels [which is still 118mL per gallon, double the recommended..!!]

 

[RSNJReef]

More information for people seeking to grow isochrysis:

I had been struggling to find a vendor for isochrysis that didn't refrigerate the culture before sending it to you. I emailed a lot of the big(ger) vendors, scientific research ones too, to ask if they refrigerated their cultures before shipping and they responded either outright "Yes they have been refrigerated" or deliberately avoided the question and said "We hibernate the culture!" (aka, we refrigerate it - lol)
As such, I would highly recommend that before buying an iso culture from any vendor, you should contact them and ask if they refrigerate the culture at all before sending it - don't buy from a vendor that refrigerates.
I don't want to bad mouth any vendors as it is their livelihood - instead I'll just say that out of all the vendors for iso, I had the best success with BasementReef on Etsy - every other vendor that I tried the bottle would come half dead, with soooo much detritus on the bottom. Their cultures never grew, would just go from dark yellow/brown tea to green or clear after a week - same growing process each time. I'm sure that their bottles are fine for feeding your tank / feeding your copepods.. but not for growing!

I also found out from BasementReef
>1.025 salinity is good
>1.5mL of Guillard F/2 per gallon is fine [I had been doing 4mL!]
>to start a culture, you can use as little as 50mL in 1 gallon (3785mL) of saltwater [I had been using 473mL!!]
>lights 6am to 10:30pm (16.5 hours)
>room temp
My first (successful) attempt was going well, got darker and darker everyday for ~1 week; I was planning on splitting it the following day, I woke up and saw that it had completely changed color (crashed) to a lighter yellow/green. It was upon this crash that I asked him for advice, and he said the above info
It's no wonder [no duh! moment] that it exhausted the nutrients, as I had used one 16oz bottle (473mL) in only half a gallon to start it, 20x the recommended starting dosage (50mL / 3785mL = 1.32%) vs (473mL / 1892.5 = 25%)
Dumb mistake for me to make - but at least others reading this post will not make the same mistake as I did of over-concentrating the initial addition of iso culture! I'm honestly amazed that it lasted / grew darker as long as it did, with such a high concentration.

I ordered more iso starting culture which should arrive in a day or two, will spread out the 16oz bottle in four 1 gallon vessels [which is still 118mL per gallon, double the recommended..!!]

Keep going pepper. Your post is proving that you’re learning from the failures, but, the rate you’re going, you’ll be there soon. Good luck!!

Also, as an alternative to a liquid culture, you can look for vendors that sell isochrisis on auger disks. These are semi dry disks covered in a layer of live micro algae, and the auger is a growth medium for the algae to grow semi dry. Making auger disks takes time and a bit of effort though, so you won’t find a lot of places that carry these, but the places that are willing to do this usually follow stricter protocols to prevent contamination.

 

[RSNJReef]

Also, one other thing to consider. Once you get a dense culture going, you may want to inspect some of the culture under a microscope to make sure that you have 100% isochrysis within your culture. Iso can turn green under specific conditions (i’d have to look it up), but it’s rare, and most of the time if you see green it’s because another phyto cell is within your culture, just at a much lower initial density than your iso concentration so you can’t see it at first. I had that repeatedly happen to me when ordering from different vendors. I couldn’t figure it out until i started looking at things under a microscope, and it was then I realized my supposed pure cultures weren’t so pure at all.

 

[RSNJReef]

Also, let us know how things go with this next round of culture you get. If this next batch fails, let me know and if you are in the US I can ship you a 32 oz container of my iso culture to get you going (of course no charge for the phyto itself, maybe we just split the shipping cost depending on how much overnight shipping is).