Culture Journal, Species: Isochrysis galbana (C-ISO)

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chuenwe
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Culture Journal, Species: Isochrysis galbana (C-ISO) - Thursday, August 18, 2011 1:32 PM
Culturing Journal DataSheet
This first post should be updated regularly to include new information as events take place or changes are made to your system

General
Species:  Isochrysis galbana (C.iso)
Species description:  Brown Phytoplankton
Culture source (link if possible):  http://www.seahorsesource.com/cgi-bin/shop/search.cgi?&category=Foods-Algaes
If algae, CCMP # (Optional): 
http://ccmp.bigelow.edu/
Culture Establishment Date:  8/18/2011
Continuation Date:  on going  

Culturing Vessel Details
Salinity:  1.018
Temperature:    Unregulated Basement Room Temperature (70-80s)
pH:  Untested.  Newly mixed Instant Ocean Salt.

Vessel description:  2L Soda bottle.  Right after I finish drinking the soda, I rinse it a few times and soak it with tap water for days until whenever I'm about to use it.  When I need it, I rinse it with tap water few more times then fill with RO water and salt.  I do not sterilize them.
Lighting description:  4 tubes of Home Depot 3' T-5 light combo. The color looks like 3000K.
Lighting cycle:  18 hours a day
Aeration description:  1 so fast like boiling and another 1 bubbling a bit slower at a rate that is is possible to count.

Methodologies
Split methodology: Pour out 1/2 of the bottle for feeding.  Pour another 500ml into a new bottle for splitting to another culture.
When I split and create a new culture, I only use new bottles for the new culture.  For the original dense culture bottle, after I pour out the amount that I use, I just refill it with salt water.  The original bottle will be keep re-using until there are stuff or film developing near the bottle neck, then I just trash the bottle (by putting them into the city recycle bin. Not really trashing it).  I do not clean or sterilize any them.

Culture medium description: 
Just poured the source culture into the salt water (already mixed with fertilizer) in the soda bottle.  Put on the drill cap and air tube.

Cell count: unknown


Reference links:  

Additional Information
(No Pictures or Videos in the Section Please)
Notes: 



You will be required to provide photographic evidence and as much detail as possible about your project in this thread.
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<message edited by chuenwe on Friday, September 9, 2011 1:42 PM>

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Thursday, August 18, 2011 8:09 PM
My bubbling rate for the nano and tetra are like boiling.  Should I use much lower bubbling rate for ISO? 
 

 
The light color ones are the new C-ISO culture.
<message edited by chuenwe on Thursday, August 18, 2011 10:07 PM>

JimWelsh
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Thursday, August 18, 2011 9:39 PM
I use a low boil for my Isochrysis.  Not a full roiling boil, but not a simmer either.  If in doubt, use more air rather than less, IMHO.

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Thursday, August 18, 2011 10:06 PM
Thank you!  Let me turn it back up a bit.

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Saturday, August 27, 2011 3:06 PM
It's been 7 days.  I'm going to split the bottom one either today or tomorrow.
 

 
 
 

JimWelsh
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Saturday, August 27, 2011 3:51 PM
I would wait a little longer.  For me, after about 10 days it usually gets a dark chocolate brown.  That's when I usually split.

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Saturday, August 27, 2011 5:06 PM
ah, ok.  Thank you.  Let me do that as well.  
 
When you split, do you usually split it into 2 bottle or 3?   
 

JimWelsh
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Saturday, August 27, 2011 5:12 PM
Each split is 500 ml of dense culture added to 1500 ml of fresh media.  I usually make 2 splits (1000 ml of dense culture used to make 2 x 500 ml splits) each time, but sometimes only 1.  The remaining 1000 - 1500 ml of dense culture gets fed to things.

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Saturday, August 27, 2011 5:38 PM
Ah, I see.  I'll do the same then.   
 

THEJRC
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Sunday, August 28, 2011 1:09 PM
Sorry to hijack the thread here but iso is my nemesis... Jim, you noted splitting oa 10 day schedule, is this your typical? I tend to stick with a rigid 8 day for most of my cultures allowing me to easily pipeline daily supply with 9 cultures (1 culture split each day and a 9 th culture maintained as a fallback.)

Given a 10 day schedule this means 11 cultures using my methodology. Have you done a shorter split cycle with any luck?
Pelagically yours,
~J      

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Sunday, August 28, 2011 1:27 PM
No problem at all.  I like to know as well.  I usually don't even do the split method.  I use 1/4-1/3 of it and refill it with fresh media.  Then I'll use it again when the color looks right, usually within just a few days.  This splitting method is totally new to me.
 
 

JimWelsh
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Sunday, August 28, 2011 1:48 PM
Yes, I have been able to split sooner with success.  My philosophy is to split near, but beforee, the end of the exponential growth phase.  Additionally, the cultures are at their peak nutritional value for feeding out.  To me, it just makes sense that the denser the culture I'm splitting (within reason), the healthier it is, and it will more likely be able to "win out" over any few contaminate cells that make it in.

THEJRC
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Sunday, August 28, 2011 1:49 PM
The scheduled splits I took from Hoff (a hero I guess) I remember early on harvesting based on color which worked well for nano but I had a lot of failure with other species. It was not until I started my regimented controls (as Randy Reed stated you have to run it as a factory) that I noticed my failure rate drop and success with other species work.

Sadly, with all the species I have done iso has always been the most difficult for me, I suspect temperature but also suspect longer times for the culture to reach log state. Jim cultures iso as second nature so I am of course always curious as to what he is doing different!
Pelagically yours,
~J      

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Wednesday, August 31, 2011 1:42 PM
I don't know how i count the date but few days ago I thought it was the 7th day but it was wrong...  lol
 
Today is already the 13th day.  The color is a little darker then the above picture.    Not dark chocolate brown at all.  I just splited the bottom bottle that had more starting culture to begin with.  1L went to feed stuff.  The rest of 1L went into 2 bottles and filled the bottle up with 1.018 clean salt water.

JimWelsh
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Wednesday, August 31, 2011 1:50 PM
You were correct to split today then.  Be patient.  It can take a while for the culture to acclimate to your environment and habits.  How are you measuring your F/2?
 

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Wednesday, August 31, 2011 1:54 PM
I premake clean salt water in lot of 2L bottles.  I put 15-20 drops of F/2 into one bottle that I'm about to use.
 

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Wednesday, August 31, 2011 2:16 PM
I took this picture after I split the bottom bottle.
 


chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Thursday, September 1, 2011 7:38 PM
1 day after the split.  Lot of dark brown color algae like stuff sticking on the bottle wall.  They are like hair algae waving around by the air bubbles.   Never had that before.
 

 
 

JimWelsh
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Sunday, September 4, 2011 1:58 PM
I have seen something like this before.  I've had cultures recover from this without issue.  I usually turn the air down a bit if I see this.  Now that it's been a few more days, how does this culture look? 

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Sunday, September 4, 2011 2:28 PM
It doesn't seem to have any different in the last few days.  I thought it got darker, but after I took a picture and compare to the one posted, they are about the same color.

Fishtal
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Sunday, September 4, 2011 3:02 PM
I wonder if the shape of the bottles has anything to do with this. You can see that the algae has settled in particular areas. Just a thought.
http://www.fishtalpropagations.com/#!home/mainPage
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chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Sunday, September 4, 2011 3:05 PM
The thing is, it never happens like that when I started the culture.  During the entire 13 days.

Fishtal
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Sunday, September 4, 2011 3:12 PM
Quote Originally Posted by chuenwe


I took this picture after I split the bottom bottle.



Did it only happen in the bottle on the right that wasn't full?
http://www.fishtalpropagations.com/#!home/mainPage
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chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Sunday, September 4, 2011 7:55 PM
No, actually, the bottle on the right that wasn't full doesn't have that kind of long swaying brown algae on the bottle.  Those look more like dusting.
 
It is the one on the top shelf, right in the middle and has super light brown color one that has the most of that long brown algae.  

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Wednesday, September 7, 2011 2:31 PM
Trying 2 different things on 2 different split atm.  In the upper shelf middle bottle with the lighter color, I changed the bottle with a Sunkist soda bottle.  It has straight wall all the way up.  Let's see if it makes any different.
 
On the upper shelf right most bottle, i used a 56um sieve to filter out all the crumbed up stuff.  The color became much lighter.  Let's see how it goes.
 


JimWelsh
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Wednesday, September 7, 2011 2:58 PM
What did you sterilize that sieve with before you poured your phyto through it?  I assume you used a funnel to guide the phyto into the bottle -- what did you sterilize that with before using it?  Was there an intermediate container, like a bucket or something involved?  If so, what did you do to sterilize it before using it?
 
Everything must be considered a potential source of contamination.  Please read Plankton Culture Manual by Hoff & Snell, pages 34-41 on Contamination and the various precautions against it.
 
I sterilize my culture bottles and media with a microwave.  I use foam stoppers, and leave them in until the very last second (literally).  I use the same pipette only for dosing the F/2, and am careful not to touch it to the mouth of the jar.  I take the stopper out only long enough to dose the F/2.  When I split, I have both the new media jar and the dense media jar side by side, and both are still stoppered.  I remove the stopper from each, and quickly pour the 500 ml of dense culture into the new media bottle, hopefully without splashing any (it all goes directly into the center of the mouth of the new jar -- the anti-drip rings on my media jars helps this a great deal).  I have the new airline and its stopper ready to go, or else I quickly recap the new jar with its solid stopper, use an alcohol wipe on the old airline, and then quickly switch the two.
 
I could take even greater precautions than I do, such as swabbing the mouths of the jars with alcohol, turning off all the fans in the room first, doing this transfer in a hood, etc., but find that the precautions that I take work well enough for me.  I think it is helpful to be paranoid about contamination, especially when working with phyto species that have a reputation for being "difficult".  With Nanno, you can basically "get away with murder", because it is such an easy phyto to culture, and is so strong and vigorous, it can shrug off contamination pretty easily.  Isochrysis is probably a bit more susceptible to contamination, and should probably be treated with more paranoia.
 
Just my $0.02.
 

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Wednesday, September 7, 2011 3:07 PM
Thanks for the tips.
 
I got a set of tools, sieve, funnel, and bucket, just for the phyto, well, actually, just for the ISO.  This is the first time doing it.  I only washed the sieve and funnel in tap water.  Air dry it and used it.
 
I got that culture manual when I first started culturing nano and tet back in may.  I read most of it that's related to what I need to do, but I didn't do any of the sterilization.  lol.  I know I should.  

chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Thursday, September 8, 2011 10:16 PM
Today is the 8th day after the first split.  Looked at them using a microscope and the culture looks dense and lively.  
 
The one on the right most that didn't look right to begin with and I used a sieve to filter out the crumbs, there are still iso in there.  Only handful of them in the view at 200x.  I'm going to give up on that bottle and start a new one on the next split tomorrow.
 


chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Friday, September 9, 2011 1:47 PM
I just looked at my darkest culture at 100x and lot of them are not moving.  Is this normal?
 


chuenwe
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Re:Culture Journal, Species: Isochrysis galbana (C-ISO) - Monday, September 12, 2011 9:07 AM
As of yesterday, the 2 lighter color culture that started crumbing up didn't look right.  Color seemed to have changed to a bit of greenish and seemed to be more clear.  Looking under the microscope and there are still some iso, but I didn't want to try to rescue it.  I dump them and split the darkest culture 60/40 into a new bottle and started a new culture.