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]

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).

That's very kind of you to offer @RSNJReef! I would pay for everything, of course! I really appreciate it. I'll keep you posted, I inoculated the cultures yesterday - hopefully they will thrive and grow.

This attempt:
In 1 gallon glass jar, 1.025
>4 drops brightwell koralle vitamin/amino acid
>4 drops seachem vitamin/amino acid
>2 drops brightwell silica
>12 drops brightwell neonitro
>4 drops brightwell neophos
>3mL Guillard F/2
>~118mL Isochrysis Galbana culture
>12 hours a day to start, low intensity - will turn up / lengthen as culture grows more dense

 

[skylernelson]

I have found that most isochrysis cultures come dead, I ordered one that came today that was green with tons of settlement on the bottom.

I have one that I’m not sure is monoculture strain. I need to grab a microscope. Isochrysis seems to be as tricky as Rhodomonas salina.

I am having success at a lower salinity 1.020-1.022. I keep my lights for 16 hours on and air at 4-5 bubbles per second.


I’d be interested in getting another culture going and I can pay for the phytoplankton and I can send a label.

 

[Haim]

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

אור: 5,000-6500K אורות זולים עובדים בסדר; לכאורה רצועת אור LED מתכווננת אשר אתה עוטף סביב המכולה עובד היטב; כל גידול אור, או אור בספקטרום זה צריך לעבוד בסדר
קונקונקונס נראה כי איזוצ'יריזה גלבאנה דורשת תאורה גבוהה, 200 PAR באופן כללי דורש יותר אור מאשר מינים אחרים של פיטופלנקטון.
עם זאת יותר מדי אור יכול להוביל להעלאת תמונות / הלבנה, והתרסקויות

Ratio of start culture to half in vessel for a new culture:
נראה כי איזוצ'יריזה גלבאנה עדיף להשתמש יחס של 1-5%, שבו אם היה לך מיכל של 1 ליטר של מי מלח, כדי למקם רק 10-50mL של איזוצ'יריזיס גלבאנה, ולאפשר לו לגדול ולא להתחיל מרוכז מאוד; נראה שזה שונה ממינים אחרים של פיטופלנקטון, שבו יחס ההתחלה הוא הרבה יותר גבוה.

Misc:
בממוצע, יש לקצור אותו 50% הוצאו, 50% נותרו, להחליף במי מלח סטריליים, כל 7 ימים.
נראה כי איזוצ'יריס גלבאנה יכולה להיאבק כדי להתחיל מחדש תרבות אם היא הוצבה במקפיא / מקרר במשך כל תקופה - בהשוואה לפיטופלנקטון אחר, שם אתה יכול לשמור על שמורה במקרר כדי להתחיל תרבות אם היא מתרסק, איזוצ'יריזיס גלבנה לא עובד טוב מאוד בדרך זו כדי להפעיל מחדש את התרבות אם היא הייתה קרירה.


שאלות:
האם יש משהו שהשתבתי על תרבויות איזוצ'יריזה גלבאנה (זן טהיטי)?
מה היית רוצה לדעת בהתחלה כשהתחלתית לפתח את איזוצ'יריזיס גלבנה?
מה היה הגורם המוביל להתרסקויות של תרבויות גלובנה איזוצ'יריזה בשבילך?
איזה שינוי בטכניקה שלך (s) הוביל אותך להצלחה הגדולה ביותר?


תודה רבה לך על כך שהגעתי לסוף הפוסט הארוך הזה!
אני מקווה שאתה מחליק שם בחוץ בהצלחה גדל איזוכיזה גלבנה יכול לחלוק את הטיפים והטקים שלך כאן ^)
אם אתה מכיר מישהו בקהילה שגדל איזוצ'יריזה גלבנה פיטופלנקטון, בבקשה לתייג אותם כאן!

Not sow any academic experience that phytoplankton of any kind contribut for any aspect to grow SPS or LPS.

 

[DaJMasta]

It sounds like my basic methods are a bit different than yours, but here's a rundown of mine:
1 gallon cultures, take about 1/6 to 1/8 of the previous culture at harvest to start the next one
Add ~5mL of f/2 (I'm using Fritz two part, so one full pipette squeeze of each)
Fill with 30ppt fresh seawater
Aerate heavily (I try to keep it below what can be heard rumbling nearby, but more air means an ultimately darker culture)
Currently a 17h photoperiod a couple inches from a Stingray 2 light (I've run 24h photoperiods no problem, but don't need the growth rate and appreciate some darkness in the room).

Takes about 6 days to mature, but as I haven't needed it as much, I tend to let it go longer - not a great habit as it makes it easier for other things to take hold.

For cleaning/sterilization, I add an inch or two of fresh water and then a jigger of bleach to the used container, put its top on, and shake it around. I typically leave it for a day, shake it again, rinse throroughly, and let it dry. I use a dry container and lid for each new culture. I use pipettes to dose the fertilizer, so those get dried, then filled with bleach water, drained, filled with fresh water, drained, dried, and then put back in the cup of them. If a pipette touches the side of a culture container when adding fertilizer, it's immediately replaced, and I keep cultures separate when refilling with seawater to reduce chance of splashing.

Been going this way for more than a year now, letting it bubble away in my kitchen. The current batch has a benthic bright green algae as a contaminant that makes it more effort to clean up after, so I've just started a replacement batch's grow in period. The green algae definitely grows slower than the isochrysis, so I imagine if I harvested at 6-7 days instead of the 8-10 I'm averaging, it would lessen with each batch and be better managed.

I arrived at the fertilizer amount by slowly increasing it and seeing if it had any impact on culture opacity, then backing off a step at the end. With high aeration and that much fertilizer, my cultures are basically opaque in the jug, and are probably close to americano coffee opacity.

 

[Pepper Reefer]

It sounds like my basic methods are a bit different than yours, but here's a rundown of mine:
1 gallon cultures, take about 1/6 to 1/8 of the previous culture at harvest to start the next one
Add ~5mL of f/2 (I'm using Fritz two part, so one full pipette squeeze of each)
Fill with 30ppt fresh seawater
Aerate heavily (I try to keep it below what can be heard rumbling nearby, but more air means an ultimately darker culture)
Currently a 17h photoperiod a couple inches from a Stingray 2 light (I've run 24h photoperiods no problem, but don't need the growth rate and appreciate some darkness in the room).

Takes about 6 days to mature, but as I haven't needed it as much, I tend to let it go longer - not a great habit as it makes it easier for other things to take hold.

For cleaning/sterilization, I add an inch or two of fresh water and then a jigger of bleach to the used container, put its top on, and shake it around. I typically leave it for a day, shake it again, rinse throroughly, and let it dry. I use a dry container and lid for each new culture. I use pipettes to dose the fertilizer, so those get dried, then filled with bleach water, drained, filled with fresh water, drained, dried, and then put back in the cup of them. If a pipette touches the side of a culture container when adding fertilizer, it's immediately replaced, and I keep cultures separate when refilling with seawater to reduce chance of splashing.

Been going this way for more than a year now, letting it bubble away in my kitchen. The current batch has a benthic bright green algae as a contaminant that makes it more effort to clean up after, so I've just started a replacement batch's grow in period. The green algae definitely grows slower than the isochrysis, so I imagine if I harvested at 6-7 days instead of the 8-10 I'm averaging, it would lessen with each batch and be better managed.

I arrived at the fertilizer amount by slowly increasing it and seeing if it had any impact on culture opacity, then backing off a step at the end. With high aeration and that much fertilizer, my cultures are basically opaque in the jug, and are probably close to americano coffee opacity.

That's really interesting in regards to your aeration - I was under the impression that if you do that much air it would have a negative impact on the culture; where guides / consensus seemed to be 1-3 bubbles a second, as little air as possible. I've had times where I've noticed that the bubble rate had increased (without me touching it!) and I feel panic to rush over and turn down the aeration. I won't feel as panicked now after reading your post, haha.

That's also very interesting re: contamination, that you can avoid it / tone it down by harvesting on shorter intervals, to not allow the longer growing contaminants time to take over (and each progressive split 'dilutes' the contaminant)

I didn't know that Fritz made their own F/2, good to know!
Thank you for sharing your experience and knowledge @DaJMasta!!

 

[MikeTheNewbie]

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.

This thread is excellent, thanks for sharing all your knowledge.
@RSNJReef do you have any pictures that show how the culture should look like under the microscope or a good resource where one can compare the most popular cultures to identify the ones we ended up with?

 

[RSNJReef]

This thread is excellent, thanks for sharing all your knowledge.
@RSNJReef do you have any pictures that show how the culture should look like under the microscope or a good resource where one can compare the most popular cultures to identify the ones we ended up with?

Hey MTN:

Here’s a video of what the micro algae cells should look like under a microscope. The one thing I caution on is that some of these videos are not 100% pure algae strains, so you need to pay attention to the 80% majority in each of the sections (whatever you see the majority of within each section is the actual algae). For example, in the isochrysis section you will see larger cells moving around. Isochrysis is non-motile, which means they don’t move around, they just drift in the water and follow the current.

Hopefully this helps.

 

[DaJMasta]

Isochrysis is non-motile, which means they don’t move around, they just drift in the water and follow the current.

Every time I've seen isochrysis described, it's always described as motile. When examining under my microscope, my samples have been able to move under their own power, and they have flagella.

 

[ncfalco]

Should nitrates and phosphates read zero when the culture is being harvested, meaning the phyto used up all the available nutrients in the water?

 

[DaJMasta]

I don't test mine, if you ramp up how much phyto you feed and judge by density (opacity of the water) you should be at least pretty close to full utilization. If you test and read some, you could let it cook for another day or two or turn up the light and wait a day or two and see if it drops all the way, but I would presume as long as the levels are readable on a normal test kit, the tiny amount added to the tank wouldn't be a problem for it (a few hundred mL of 80ppm nitrate culture wouldn't end up being much except for the smallest nanos.)

 

[ncfalco]

@DaJMasta Thank you, I went and havested 16oz (480ml), roughly a third of my 1.5 liter culture and then topped off the culture container with more salt water. I’m not going to add any
more nutrients (f2) to this next batch since I overdosed my initial culture and will measure periodically throughout the week to see if the rest get used up.

I’m getting a green mandarin tomorrow, which is finally possible since live phyto seems to make my pod population explode. So far dosing live phyto has been best thing I’ve done for my tank. I don’t know why culturing phyto isn’t considered an essential part of the hobby yet, as it is such a vital part of the marine ecosystem. If I had to choose between my refugium and live phyto, I would choose the phyto all day long.

 

[Levinson]

How do you guys store isochrysis?
I culture them and store them in the fridge but they seem to die when kept in the fridge. It would be awesome if I could find a way to keep them live for a week or two so that I could use it live to feed the parvo little by little until I get the new harvest.

 

[Reefer Dan]

How do you guys store isochrysis?
I culture them and store them in the fridge but they seem to die when kept in the fridge. It would be awesome if I could find a way to keep them live for a week or two so that I could use it live to feed the parvo little by little until I get the new harvest.

I’ve been experimenting for this same purpose. I keep my room temp at 70-74F, the bottle of iso (I also mix chaetoceros with it) will last about a max of 10-11 days on my counter with mixing every time I walk by it. Obviously degrades over time, but the sniff test is a decent indicator (not that iso smells good in general). Visually noticed at day 10 with my last experiment.

 

[DaJMasta]

Same for myself, I don't refridgerate as some live species don't seem to do well with it.

 

[Reefer Dan]

Same for myself, I don't refridgerate as some live species don't seem to do well with it.

Do you do that with all your species or just iso? I’ve thought about trying it with some of my others like rhodo.

 

[DaJMasta]

I do for mine, but not everything likes it. I think the best case storage would be to keep them aerated and at least somewhat lit, but a lot of stuff stores well at room temperature and I've read some studies saying refrigeration can decrease nutritional content at least somewhat, though it's probably still far better than not feeding it. I've kept close to a dozen strains of algaes and not all of them store well - either in or out of the fridge - there are some that leave a residue in containers that's annoying to clean up, and there are some that die pretty quickly in the fridge, specifically.

Right now I keep isochrysis, chaetoceros, and tetraselmis at room temperature and blended together after they are harvested, and I'm currently getting some of the stink and dregs but because of an extra (bright forest green) algae that grows on the surface of the chaetoceros containers. Before that contamination in the cultures, it would keep more than a week without any significant smell, you just have to shake it up before using it because tetraselmis settles to the bottom pretty quickly without agitation.

 

[Levinson]

I do for mine, but not everything likes it. I think the best case storage would be to keep them aerated and at least somewhat lit, but a lot of stuff stores well at room temperature and I've read some studies saying refrigeration can decrease nutritional content at least somewhat, though it's probably still far better than not feeding it. I've kept close to a dozen strains of algaes and not all of them store well - either in or out of the fridge - there are some that leave a residue in containers that's annoying to clean up, and there are some that die pretty quickly in the fridge, specifically.

Right now I keep isochrysis, chaetoceros, and tetraselmis at room temperature and blended together after they are harvested, and I'm currently getting some of the stink and dregs but because of an extra (bright forest green) algae that grows on the surface of the chaetoceros containers. Before that contamination in the cultures, it would keep more than a week without any significant smell, you just have to shake it up before using it because tetraselmis settles to the bottom pretty quickly without agitation.

Thanks for the info.
Do you keep it sealed (air tight)?
The room temp here this time of the year is quite hot so I won't store it in my room for the time being (tried it and went bad in just a few days at about 29~32 celcius, it might've been because I had it in a sealed bottle though. Culturing in that temp hasn't been an issue so far) but I will try it once the temp goes down.

 

[DaJMasta]

I don't seal my containers. I'm not sure it makes a huge difference when their metabolism goes way down (no intense light), but at least in theory, they'll stay alive longer with some gas exchange at the surface (limited though it may be with a bottleneck.)

 

[themastr]

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).

Hey Guys

Wondering if anyone can send me some Iso culture? I’m in Alberta, Canada and cannot get anything here. I’ve tried algae research and supply paid $100 to ship it here and it arrived dead. I will pay for the Iso culture and the shipping. Please let me know and thanks in advance.

 

[Nate’sTanks]

It sounds like my basic methods are a bit different than yours, but here's a rundown of mine:
1 gallon cultures, take about 1/6 to 1/8 of the previous culture at harvest to start the next one
Add ~5mL of f/2 (I'm using Fritz two part, so one full pipette squeeze of each)
Fill with 30ppt fresh seawater
Aerate heavily (I try to keep it below what can be heard rumbling nearby, but more air means an ultimately darker culture)
Currently a 17h photoperiod a couple inches from a Stingray 2 light (I've run 24h photoperiods no problem, but don't need the growth rate and appreciate some darkness in the room).

Takes about 6 days to mature, but as I haven't needed it as much, I tend to let it go longer - not a great habit as it makes it easier for other things to take hold.

For cleaning/sterilization, I add an inch or two of fresh water and then a jigger of bleach to the used container, put its top on, and shake it around. I typically leave it for a day, shake it again, rinse throroughly, and let it dry. I use a dry container and lid for each new culture. I use pipettes to dose the fertilizer, so those get dried, then filled with bleach water, drained, filled with fresh water, drained, dried, and then put back in the cup of them. If a pipette touches the side of a culture container when adding fertilizer, it's immediately replaced, and I keep cultures separate when refilling with seawater to reduce chance of splashing.

Been going this way for more than a year now, letting it bubble away in my kitchen. The current batch has a benthic bright green algae as a contaminant that makes it more effort to clean up after, so I've just started a replacement batch's grow in period. The green algae definitely grows slower than the isochrysis, so I imagine if I harvested at 6-7 days instead of the 8-10 I'm averaging, it would lessen with each batch and be better managed.

I arrived at the fertilizer amount by slowly increasing it and seeing if it had any impact on culture opacity, then backing off a step at the end. With high aeration and that much fertilizer, my cultures are basically opaque in the jug, and are probably close to americano coffee opacity.

Are you doing 5 ml of each or total? I was a tad confused by your 1 pipette of each.