Culture Journal, Species: C- Isochrysis galbana

General
Species:  C- Isochrysis galbana
Species description:  brown motile phytoplankton
Culture source (link if possible):  kind gift of Jim Welsh , then Seahorse Source, produced by Algagen.
If algae, CCMP # (Optional):  1323
http://ccmp.bigelow.edu/
Culture Establishment Date:  8/14/2011 , then 10/17/2011 successful one
Continuation Date:  forever, hopefully , 90 days at 1/28/2012

Culturing Vessel Details
Salinity:  20 ppt
Temperature:  ambient 
pH:  8 ish

Vessel description: two liter soda bottle, drilled cap, rigid airline descending to bottom          
Lighting description:  5500K temp 18 watt fluorescent bulb
Lighting cycle:  14day/ 10 night
Aeration description: since 10/17, light aeration: couple of bubbles per second from open end of airline tubing at bottom of bottle.

Methodologies
Split methodology: pour 1/3 of dark culture into a 2/3 filled clean, freshly dechlorinated saltwater, bottle, add 1 ml f/2 fertilizer.

Culture medium description: 
Saltwater is filled to 2/3 of the top of the bottle, bleach is added at 1 ml/gallon. Shake, and store overnight.  Airline tubing, also similarly bleached, and drilled cap are assembled.  6% Na Thiosulfate, 0.7 ml/bottle, (tested for chlorine) and 1 ml f/2 fertilizer added. Dark culture added to top off the last 1/3 volume.         

Cell count:
   sorry, not known.

Reference links:  

Additional Information
(No Pictures or Videos in the Section Please)
Notes: 
Previous attempts at culturing this phytoplankton in summer have met with failure, as seemingly  the warm temperatures stress the algae, and it "goes green".
In initial attempt, I will try to grow the algae in a styrofoam cooler with a suspended light bulb, and come cold packs, recycled daily to the freezer. This attempt failed immediately.
In successful attempt, culture bottle was prepared, attached to air pump, and set in front of light at room temperature.  Initially, this culture appeared to crash, was taken off of air pump, but within a few days , appeared to recover. Brown culture grew well and quickly from this attempt, with no supplemental air.  Then was transferred to new bottle and propogated in classical way with air.  


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What a bad idea that was! Came home from work to a crashed 85F culture! Two freezer packs cannot compete with a fluorescent bulb…. Back to the drawing board.

It's almost sounding like getting a small fridge and setting the temp in the 65-70 mark is about the only way to go with T-iso and C-iso. I'm glad now that I didn't start a culture of it.

RLTW

180 Gallon Mixed Reef

Then I heard the voice of the Lord saying, "Whom shall I send? And who will go for us?" And I said, "Here am I. Send me!" Isaiah 6:8

I think one can culture it outside of a refrigerator, I just have had bad luck in summer.  I've ordered some more, and plan to start again.  This time without the styro box, as my basement temps have dropped.

i was going to mention a basement floor
 
another possibility maybe to have a fan blowing on it to dissipate some heat as well

I wonder if just putting the airpump in the fridge would lower the temp enough?

the induction of cold air could work but could also be a bit tough to regulate

Sounds interesting, but I'm not even sure it was the warm summer that caused it to crash.
This time it lasted 12 hours at basement temp, so we push on.

double post accident. pls delete

OK, I ordered a new starter from Seahorse source, and added half of it to a partially filled sterile soda bottle, added fertilizer, and bubbled air through it.  It was on a shelf with a compact fluorescent bulb pointing at it.  After about 4 days it "crashed", meaning it had turned clear with no brown algae visible.
 
While gazing in disgust at the disaster of my third and most expensive recent attempt to culture Isochrysis,  I noticed the bottle next to it.  The abandoned attempt #2.  It was the result of the second attempt.  With no air to this bottle, the added light from attempt #3 had caused a green tint to the liquid.  I decided to shift the airline from attempt #3 (clear) to attempt #2 (green), and just see if anything really grows.  Green does grow quite nicely under this regimen, however, I don't know what species I have.  
 
While gazing in awe at this meager accomplishment, I noticed that bottle from attempt #3.  The one that I had removed air from, but left on the shelf with the light.  It is turning brown! Go figure. 
 
I either have two color morphs of the same algae or two different kinds.  Do I dare restore air to attempt #3?
 
 

If I can draw any conclusions, it is this: 
It is best to start a new culture with no air, but light. Both bottles began under a no added air condition.

I split the green one and the brown one last weekend, added fertilizer and air to the green one.  I left the brown ones without air. Several days later the splits are almost as dark as the mother bottles.
Mother brown to the left, new green one in the middle, and new brown one on the right.

This is the mother green one:

 

Green ISO.Kathy?.Very unlikely...
Feral Chlorella or such,I guess.Get a scope!

I was going to start out with "Sadly, I am no longer employed where there are good microscopes." but I don't really feel sad at all. My own microscope only goes to 40x, so it is worthless to try to see algal cells. I hope that Santa Claus brings me a better microscope in late December.
 
I agree that the green culture is not likely Iso.  The brown culture is, though, as it is a recent purchase.
 
Is chlorella worth growing? Ill look it up.

It is underestimated,as everything that comes free But in Japan they make algae paste with it,with the addition of missing HUFA and such.IME copepods can be raised on it.
Check for large, round non motile cells.It grows in FW as well.

It would be nice to know what it is, since it is so easy to grow.  I'll have to see if my local plant experts can help. 
 
Luis, have you tried plastic bags for algae growth? If so, do you like that kind of vessel?

Sure,I use 10 L bags.Check my old sticky 

Iso did not thrive, though it hung on for a while.  I've recently purchased some fresh f/2 Guillard's, and restarted a bottle.  It is slowly getting more brown, and I am hopeful.  It may have been my ancient fertilizer was the problem all along.

i think it was my ancient fertilizer, as  the new fertilizer is making some fine golden brown iso.  It's also 63F in my basement; much cooler than the 79F of last summer.  
I've split it once, yesterday, and I'm feeding remainder of the older bottle to my A. panamensis.  This copepod seems to do well on live Iso.  I'll also order some parvocalanus to see if I can keep it alive this time, as long as the ISO continues to grow.

I have a fan on mine. Not sure that's enough, cause it's all in an 80F room.

RLTW

180 Gallon Mixed Reef

Then I heard the voice of the Lord saying, "Whom shall I send? And who will go for us?" And I said, "Here am I. Send me!" Isaiah 6:8

Perhaps temperature is not the problem, but I don't know that since both temperature and freshness of fertilizer are changed in my setting.  
 
Iso seems darker every day.  We'll call 90 days at 1/28/2012, since that will be a continuous culture with no crashes.  Then we'll have to see if I can keep the culture going over the summer of 2012.

Here's my beautiful golden brown:

Nice color.  Is that before or after a split?

I know, right? It's a much prettier color than I remember T -Isochrysis being, but this is C-Iso...It's before the split. I know it's a little thin, but I am trying to split while the culture is in log phase.  So I wait for a dramatic color intensifying change, and then I split.

Just FYI, that's slightly darker than my Iso cultures look *after* I split them.

Yeah, I have the feeling I could let them go longer, but I'm anxious to keep this batch growing, and I don't want to chance that I'll hit plateau growth phase.  I'm sure my natural laziness will override this concern within a short while, however, and I'll have darker cultures to work with, soon.

Actually, I've been looking at so many things with my new scope that I am not sure, but I think this is C. Iso.  Not all cells are swimming, though… Click on video:
 

Looks like my Isochrysis does with my new scope, but mine is a dark brown T-Iso culture, and more dense with more cells moving, even though it was about 3 weeks old!  Still, this looks like a viable Iso culture to me.  Keep up the good work with the Iso!  Try to see if you can get it to a darker color before splitting.  Mine usually takes a good 10 days or so before it's ready to split (at least).  Although Iso is not supposed to require silicates, I found that it would get dark and ready to split in fewer days -- more like 6 days or so -- if I added silicate to the medium (like I do for diatoms).  If you have any sodium metasilicate, you might try that and see how it works for you.

Thanks Jim, I don't have any of that.  Where do you get it?

FAF:  http://florida-aqua-farms...=MICRO_MACRO_NUTRIENTS
 
One pound is a lifetime supply for a hobbyist.  Be very careful not to breathe the dust!  I mix up 6.2 grams in 250 ml of RO/DI water at a time, and then put 2 ml of that solution into each 2 liter media jar when I prepare the media for diatoms, and like I said, Iso seems to like it, too.  If it gets thick towards the end of the 250 ml supply, I just discard and mix up a fresh solution.

I split the pictured bottle yesterday, the fifth of January, 13 days after the split.  It was the color of weak coffee, or strong tea. Here's videos:
40 x

 
100x

 
400x

 
I'm not happy with the resolution at 400x. Can't seem to get any kind of focus….
 
 

In my experience, 400x is just about useless for what we do.  I'm looking to replace my 40x objective with a 1x objective so I would have 10x, 40x and 100x magnifications.  That would be much more usefull, in my opinion.
 
By the way -- I'm backing away from adding silicate to Iso as being a good idea.  Twice now, after I started adding silicate to my Iso media, I started growing green Iso.  I know correlation does not prove causation, but just sayin'.....
 

Sometimes it does, though.  My Iso seems to be doing better just with the new fertilizer, so I am thinking that was the problem all along.

It's dark tea again.  I think I'll split tomorrow.  That's 7 days this time.

i didn't get to it.  Perhaps tomorrow.  Gotta find some bottles.

I did split it from the above post.
 
Here's the 90 day culture.  I just took the picture:

 
Finally….

I got lazy and didn't split the culture until it was quite far gone.  Still, it looked like it was growning fine, a lovely golden brown, and then one morning, it was green! I'll check under the microscope later.

Well, that old culture crashed long ago, and I'm at it again!
 
Today I risked my life in extreme winter weather to pick up this culture from Fedex.  I always ask them to hold at the FEDEX when I order live things.  It's a good idea when the weather is bad, so the box isn't driven around all day in the back of an unheated truck.
 
This time, inspired by the Hawaii video presentation, I hope to be culturing at 30ppt.
 
My Algagen culture (Seahorse Source) came in at 40ppt, so I'm starting them out at that salinity.
 
 I need to get a new air pump.  My ancient ones are so weak, I need 2 of them to power two 2-liter bottles.

check out Kathy's Clowns, llc website:
http://kathysclowns.com
Captive bred clownfish and more
(Wholesale to the trade.)

I've been back culturing Isochrysis again, as I've found it is like magic with copepods. Pods culture very well with a light dosing of live Iso. Recently I expanded my Iso cultures to 8 gallons, so I can use one gallon a day for cultures and propagations.
I'm using 1 gallon jars from Wallmart and undercabinet T5 light strips, 2 per shelf.
 

check out Kathy's Clowns, llc website:
http://kathysclowns.com
Captive bred clownfish and more
(Wholesale to the trade.)

A gallon a day of Isochrysis is a LOT of phytoplankton!  And, yes, it is magic, isn't it?