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!

 

[DaJMasta]

My pipettes are 2.5mL or so each to the top of their graduation marks, so 5mL total between the two parts.

 

[bozo]

Could I buy some isochrysis from you?

thanks

 

[Pepper Reefer]

Could I buy some isochrysis from you?

thanks

sorry for my late reply!
In my experience, the absolute best vendor for live isochrysis (never refrigerated) is BasementReef on Etsy

https://www.etsy.com/listing/1459371935/live-phytoplankton-16-oz-isochrysis

Very good, kind guy too
I tried 3 other vendors, including a fancy shmancy supplier for colleges, and all of them arrived dead
BasementReef was the only one that came live - he also charges a very reasonable price
For the Guillard I would buy from Mercer of Montana on Etsy as well
(not sure if I'm allowed to post that URL link!)

 

[Mikeltee]

I have 48oz of Isochrisis arriving Thursday from Pod Your Reef. Has anyone started their culture from them? I hope its legit because I spent $80 for 3 bottles and a bottle of Apocyclops pods. I do have a decent triocular Swift microscope, and I will be checking for contamination. I am ditching the nanno in favor of iso as it seems that it is rather useless if other strains of phyto can be successfully cultured.

I have been culturing nanno and tetra for a couple of months now, and I have been lucky to not have any crashes or contamination. I use seperate, dedicated tooling and wipe down the workspace in between harvests. I use the Posideon Reef system with fresh bags and airlines after each harvest and wipe everything down with 100% rubbing alcohol.

I see that most people struggle with sterilization. I have a spare, 40 watt UV that I plan to run for several hours in my mixing station. The pump will push the flow to roughly 200gph but I can lower it with a valve if recommended. It was $40 with a new bulb, so the purchase was a no-brainer. What is your guy's opinion on this method of sterilization? I guess if it hasn't been done, we will find out because I am too lazy to be boiling and bleaching water in order to produce brown gold.

The phyto will be used to feed my pod cultures (tigger, tisbe and apo) and dose my one month cycled sterile rock 130g display. My fish room is in the basement which is 65F year round. I use the Posideon Reef system with the Eheim air pump which has a dedicated line and Hepa filter, and I run the flow pedal to the metal. I run a 16 hour light cycle and i'd estimate those lights bring the cultures up to about 70F with the lights on. If temperature matters that much I will record my samples on Friday when I harvest and adjust if necessary. I plan to run a 7-day cycle and dose 3ml of Mercer's f2 to the 3.5l culture which is a 50/50 split of the harvest. I mix my phyto's medium at 1.025 verified with the Milwaukee MA977 digital refractmometer. I also have two-part fertilizer from Posideon if that is recommended.

FWIW... I have experimented with doubling the f2 and running the culture 12 days, but to be honest, I did not see a difference in density when comparing samples side by side with the previous harvest. Tetra seperates every day and the level of algae on the bottom of the jar was not much different between samples either. I did not empirically test this as I don't have the tools to conduct such a test.

One last question... I keep a backup 16oz in case of a crash in the refrigerator. Should I not refrigerate my Iso backup if I am lucky enough to harvest? The one video I watched showed the dude have his sample on the kitchen counter so he could give it a shake each time he walks by. Will it be okay to mix with tetra and refrigerate for my dosing allotment? It's only a weeks worth.

Are there any suggestions?

 

[Mikeltee]

After reading several posts, I see room temp is good for this as well as Tetra. So what is stopping us from putting an air pump in a gallon jug and dosing our tanks with a peristaltic pump throughout the week? The skimmer? We can automate that. Is there any harm done in sending the phyto via the return pump? Considering that I have a newly cycled tank with no animals in it but pods, I don't mind giving this a shot.

 

[Mikeltee]

It's not looking too good. I could see my hand through the bottle when I received it. I put a sample under the microscope and had to search for a spec of algae. The good news is that I didn't see any contamination. As a matter of fact, I didn't see much at all. Unfortunately I can say the same thing for my bottle of Apex pods as well. Oh well... you live and you learn. I wish I would have saw this thread before dropping $80.

 

[Mikeltee]

Let's try this again. I got 16oz from Nate at Coral Hub. It's the thickest I've seen. It was harvested 2 days ago. This is a 16oz innoculation for a total of 3.5l using his homemade 2 part and 50% light for a few days. 12 hours of UV and I strained the Saltwater. If this don't work out I'm going back to nanno.

 

[El Toro]

I got 32 oz Iso from my trusted guy for Tetra and it was a rich dark brown. I split it for 3x 30% concentration and held back 30% in the shipping bottle. My culture after 3 days is no longer dark brown but dark green. It was sitting on the same shelf as my Nano and Im wondering if/how it got cross contaminated. All sterilization was followed and the only thing shared is the Air Manifold.

I did note the Iso smelled stinky when I opened the shipping bottle. My first time with Iso so dunno if thats normal?

 

[Mikeltee]

I got 32 oz Iso from my trusted guy for Tetra and it was a rich dark brown. I split it for 3x 30% concentration and held back 30% in the shipping bottle. My culture after 3 days is no longer dark brown but dark green. It was sitting on the same shelf as my Nano and Im wondering if/how it got cross contaminated. All sterilization was followed and the only thing shared is the Air Manifold.

I did note the Iso smelled stinky when I opened the shipping bottle. My first time with Iso so dunno if thats normal?

It wouldn't have contaminated that quickly from anything that you may have done. It's probably on it's way to turning clear. If it smells it means that it's bad. My podyourreef batch smelled and went clear within 36 hours. Try coralhubus and you may as well grab his 2part. 1ml part A 1ml partB / 1l of water. I'm going to harvest 3l on Saturday. I'll post pics. It's super thick already. He runs his 10-14 days. I used 500ml innoculation for this batch. I'll probably use 1l next time. I'm going to start upping my f2 dose by 1ml on my tetra to match the 10 day harvest. It might be in your best interest to get rid of the nanno and run tetra. I'll run nanno again if I get a third culture. It's good for the tank. Not so much the pods.

 

[Mikeltee]

What elements do we test to verify that all of our fertilizer has been used up which I would assume correlates to the correct time to harvest? I've read Phosphates and Nitrates. How do we have Nitrates without a biological filter? Is it supposed to be Nitrites? I use salifert test kits for nitrites and it always shows a high reading so I don't trust it.

I'd perfer to harvest weekly so that I can stay in a rhythm and harvest every Saturday. As long as I add the correct volume of nutrients, I should be okay to add a higher volume to innoculate, correct? So let's say 50/50 and 2ml f2 for a week vs 25/75 and 4ml of f2 for 2 weeks.

 

[El Toro]

It wouldn't have contaminated that quickly from anything that you may have done. It's probably on it's way to turning clear. If it smells it means that it's bad. My podyourreef batch smelled and went clear within 36 hours. Try coralhubus and you may as well grab his 2part. 1ml part A 1ml partB / 1l of water. I'm going to harvest 3l on Saturday. I'll post pics. It's super thick already. He runs his 10-14 days. I used 500ml innoculation for this batch. I'll probably use 1l next time. I'm going to start upping my f2 dose by 1ml on my tetra to match the 10 day harvest. It might be in your best interest to get rid of the nanno and run tetra. I'll run nanno again if I get a third culture. It's good for the tank. Not so much the pods.

What bubble rate do you run on Iso? I didnt think of it being motile and ran a bit of a roil which I wondered if it would kill them off. I normally roil Tetra and Chloro.

 

[Mikeltee]

What bubble rate do you run on Iso? I didnt think of it being motile and ran a bit of a roil which I wondered if it would kill them off. I normally roil Tetra and Chloro.

Full blast. The guy I got it from runs 2 pumps in his. I have the eHeim with 2 outputs. Both tetra and iso are cranked. There is some settling in my iso. After evaluating this harvest I may consider adding another. I can take a video if you would like. I think the purpose of air is to keep it stirred. I don't think less air would do anyone any good.

 

[Mikeltee]

Today is day 9 with 500ml -> 3l innoculation. It started to lighten up on me today so I must have ran out of nutrients. I'm sticking to a 7 day harvest. I strained it. We will see what happens. I used 1l-3l on this one. The culture is a shade lighter than my last post. 2.5ml part A and 2.5ml part B.

 

[El Toro]

Full blast. The guy I got it from runs 2 pumps in his. I have the eHeim with 2 outputs. Both tetra and iso are cranked. There is some settling in my iso. After evaluating this harvest I may consider adding another. I can take a video if you would like. I think the purpose of air is to keep it stirred. I don't think less air would do anyone any good.

Good, I didnt kill it. I may let my supplier know his Iso turned on me. Im 99.9% certain I didnt x-contaminate it or kill it with too much light/air.

This stuff grows in the ocean am I right?

 

[Mikeltee]

Good, I didnt kill it. I may let my supplier know his Iso turned on me. Im 99.9% certain I didnt x-contaminate it or kill it with too much light/air.

This stuff grows in the ocean am I right?

That's a great question! I wonder if the different stains come from different parts of the world. Can any experts chime in?

What are you doing to sterilize your medium? I'm using a spare 40 watt uv for a few hours in a 20g mixing station. You will want to take this out of the equation for your next attempt. Some boil their water. Some add bleach. I wouldn't go out and buy a UV just for this. I stumbled on this beast for $40 from a dude I dude I bought a couple of canister filter from.

 

[Mikeltee]

I pulled another harvest of iso today. On day 5 it was so thick that I could barely see the light (see pic below). I was scared that it wouldn't make it to day 7. I used 1l innoculation. Today, I only did .75l. It's safe to say coralhubus has a great product! It's also safe to say that circulating your saltwater mix in UV will sterilize your medium.

 

[El Toro]

Wow that's nice and thick!

So to be clear, you used to do a 1ml initial inoculation when you split. Now your dropping it to .75ml? Why if you got such good results?

I inoculate my culture 1ml initial and .5ml after 5 days if it's still not thickening.

 

[Mikeltee]

Wow that's nice and thick!

So to be clear, you used to do a 1ml initial inoculation when you split. Now your dropping it to .75ml? Why if you got such good results?

I inoculate my culture 1ml initial and .5ml after 5 days if it's still not thickening.

The reason I used less was because at day 5 it was ready. The light wasn't penetrating. I want to get on a 7 day cycle.

One liter for 3.5 liter total. I did .75 liter this time (1:4 ratio roughly). Nate recommended his 2 part so I use it for iso and Mercers f2 for the tetra. I inoculate tetra at a 1:1 ratio. He runs 5g cultures with 500ml innoculation for 10-14 days. He also has a ton of air circulation as well. It appears to be a rapid boil bubbling several inches off the surface. What he shipped was much thicker than mine. It was soupy. https://coralhubus.com/product/ch-phyto-food/

 

[Stephers]

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.

I know this thread is old, but hoping you have an answer. I've been culturing T-iso for months. After the first couple weeks, it is now a yellow-green color. It's absolutely still pure T-iso. I've confirmed with a microscope several times. It grows great. But I'm wondering why it's turned more green and if I can get it back to the nice yellow/brown color. Please let me know if you know the reasons as you implied here. I've searched on Google a bunch and couldn't find much.

 

[RSNJReef]

I know this thread is old, but hoping you have an answer. I've been culturing T-iso for months. After the first couple weeks, it is now a yellow-green color. It's absolutely still pure T-iso. I've confirmed with a microscope several times. It grows great. But I'm wondering why it's turned more green and if I can get it back to the nice yellow/brown color. Please let me know if you know the reasons as you implied here. I've searched on Google a bunch and couldn't find much.

Hey Stephen’s,

I wasn’t able to find a ton of information on it as to why the t-iso turns green, but from what I found the researchers stated that it was either salinity or an imbalance of trace elements within the source water used to make the F2 fertilizer or the source culture water (my guess is an overload of iron or some form of trace metal because those are usually the suspects for “greening” in a reef tank).


Before I go further though, are you certain it’s still a pure strain? T-iso and nannochloropsis cells look very similar to one another. Typically even a drop of nanno being introduced into a t-iso culture is usually the beginning of the end for the culture because eventually the nanno will catch up population wise to the iso, and sometimes it will take months for people to notice the color change.

If you’re certain it’s a pure strain, then, a couple of questions:

What brand of f2 fertilizer are you using?

Are you using rodi I water as your source for making the culture water, and, are you sterilizing the culture water before use?

Whatever salt you are using to make the culture water, try and find out if it is high in iron or any other trace metals (I personally use reef crystals / Red Sea pro in a 75/25 ratio).

Also, during your time culturing, did your t-iso culture ever get to a nice dark brown color before you split the culture, or did you get it to a strong tea color then split (I’d always recommend getting your culture to a nice dark brown color at least a couple of times a year because from what I’ve read the lipid content of the iso increases once it has reached a high population density and turns a dark brown color (it slows down in growth then and begins fattening up apparently).