Culture Journal, Species: Isochrysis galbana

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waldend
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Culture Journal, Species: Isochrysis galbana - Tuesday, July 29, 2014 11:29 AM

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
Species description: Motile brown microalgae
Culture source (link if possible): Kathyl
If algae, CCMP # (Optional):
[link=http://ccmp.bigelow.edu/]http://ccmp.bigelow.edu/[/link]
Culture Establishment Date: 8/6/2015 
Continuation Date: 6/28/2017

Culturing Vessel Details
Salinity: 1.023 SG
Temperature: Ambient Room Temp (65°F in winter - 72°F in summer)
pH:Not monitored

Vessel description: Master cultures are 0.5 gallon mason jars.  In-use cultures are 1.5 beverage dispensers
Lighting description: LED fixture 
Lighting cycle: 14 on, 10 off
Aeration description:  steady stream of bubbles 

Methodologies
Split methodology: Master - 50% split to in-use jar or fed and the other 50% to the new master

Culture medium description:  Master - heat sterilized newly mixed ASW at 1.023 SG.  In-use is fresh mixed ASW then sterilized with 1ml bleach for 24 hours and then neutralized with equal part sodium thiosulfate.  1.023 SG   For F/2 I use about 3-4ml per gallon of water.  

Cell count: Not known but color ranges from green tea at split to coffee at maturation
(if known)

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.
If your thread does not contain detailed enough photos and information the MBI Council will not be able to approve your reports.


<message edited by waldend on Thursday, June 29, 2017 10:24 AM>

waldend
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Re:Culture Journal, Species: Isochrysis galbana - Tuesday, July 29, 2014 11:12 PM
Cultures started. Picture from 7/24. Iso is 2 jars on right. 2nd from right started on 7/21, end jar started 7/24.




Same 2 jars on 7/28. Left appears same as it did within a few days of starting. Jar on right is taking a greenish hue and there is green algae on bottom of jar. Will be dumping this jar as it appears I contaminated with my Tet culture or a wild micro algae.


waldend
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Re:Culture Journal, Species: Isochrysis galbana - Monday, August 04, 2014 10:24 AM
Due to concerns with cultures not getting as dark as others I have seen I am starting a new jar. This time I used plastic ex-mayo jar as I was concerned the thick canning jar glass was blocking too much light. Due to not being able to boil the plastic I used .5ml of bleach and then added .75ml of Prime (all I had on hand) to neutralize the chlorine. This jar was started from my original split which is continuing to stay brown but is not getting any darker.



Here is the remainder of the original culture. No green on the bottom so I am assuming it still pure.



waldend
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Re:Culture Journal, Species: Isochrysis galbana - Monday, August 04, 2014 10:37 AM
Concerns and questions -

1. On my original split which isn't getting coffee colored, more of a green tea I use a T8 bulb. On two additional cultures I am using a T12 fixture. One was from original split and the other was from remaining iso in the bottle from the conference. I had been keeping it in the fridge and it was brown but stressed. Would the lighting be limiting and stressing the cultures?

2. As you can see both cultures below are green or turning green. I grabbed the original split and started the more brown one on a separate air system across the room. The second jar was started from the stressed culture and is completely green. Do stressed cultures turn green? I boiled the heck out of the water and really would not expect
them to be contaminated because the original split is not appearing to be and I didn't even handle the Tet jar when I split these.





waldend
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Re:Culture Journal, Species: Isochrysis galbana - Monday, August 04, 2014 10:40 AM
As a note for the new plastic jar culture I also turned the fixture vertical so that the jar is receiving more light along the entire length of the jar.

Will inoculating a new culture with too little algae cause it to stress and/or not flourish?

Lrood
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Re:Culture Journal, Species: Isochrysis galbana - Tuesday, August 05, 2014 4:52 PM
Daniel,
I've read that having too little inoculate can cause a culture to struggle and allow contaminants to take over. It sounds like you thoroughly sterilized the culture media, but there could have been some chlorella or tet cells in the inoculate. I lost my last t-iso to some sort of green algae. The current one you gave me is fairing much better, so let me know if you need some.

Someone here also had a contaminated iso culture that they "purified" by letting it sit without aeration, with the concept that non-motile algae settles to the bottom, and the motile iso could then be decanted off the top (at least I think I read that in this forum).
~~~~~~~~~~~~~
Ken

waldend
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Re:Culture Journal, Species: Isochrysis galbana - Tuesday, August 05, 2014 4:59 PM
Thanks for the info Ken. I will do a full split of the original tonight then. I had been stingy with the algae for fear of using up my clean culture. I will just do it and have no fear!

KathyL
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Re:Culture Journal, Species: Isochrysis galbana - Wednesday, August 06, 2014 9:38 AM
Putting Isochrysis in the fridge is really not a good idea.  It doesn't like cool temps, and will start dying. It is unlike Nannochloropsis which will tolerate cold quite well.  I keep Iso out of the fridge, and use it up as fast as I grow it.
 
If you started later cultures from refrigerated stock, that is probably why they did not do well.  Just a thought.
Owner of Kathy's Clowns, LLC
 
Yes, we have no more clownfish.  
We have no more clownfish today!

waldend
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Re:Culture Journal, Species: Isochrysis galbana - Wednesday, August 06, 2014 6:10 PM
Thanks Kathy. The last picture of the green culture was started from a refrigerated sample. I should have researched that - your advice will pay off as I had planned on saving a portion of each split in the fridge to feed my Parvo with. I don't have anything to raise at this point so I will have way more algae than copepods.

KathyL
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Re:Culture Journal, Species: Isochrysis galbana - Thursday, August 07, 2014 8:03 AM
Not a bad place to be.
Owner of Kathy's Clowns, LLC
 
Yes, we have no more clownfish.  
We have no more clownfish today!

waldend
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Re:Culture Journal, Species: Isochrysis galbana - Friday, August 08, 2014 3:41 PM
Well, the plastic container was a fail. It stayed the same color for several days then suddenly went clear. I won't be using Prime anymore either. I got a bottle of sodium thiosulfate at the pool store so I will be switching to a chlorination sterilization over the microwave.

Below is the first true spilt of the phyto cultures. I used a full 25%-30% of the mature culture for the new start. I also added a second small light fixture to the opposite side so illumination should be much better. I am hoping for that nice chocolate color for the Iso.



Lrood
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Re:Culture Journal, Species: Isochrysis galbana - Tuesday, August 19, 2014 7:09 PM
Daniel, how are these cultures coming along?
~~~~~~~~~~~~~
Ken

waldend
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Re:Culture Journal, Species: Isochrysis galbana - Wednesday, August 20, 2014 12:07 AM
Hi Ken.

Doing ok so far. The split above darkened up to the same light tea color and no green. So while no chocolate color at least I am keeping it going. Now I need to ramp up to multiple cultures to make it easier and safer to feed the Parvo I have too.

waldend
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Re:Culture Journal, Species: Isochrysis galbana - Friday, August 29, 2014 11:29 PM
This iso is tricky stuff. My Tet just cruises along but the iso....well I am learning a lot. I split 50% of my culture into a 1/2gallon plastic container. The jars were a pain to pour and I didn't have lids for them. Bad news is the new split now has a greenish tint to it. Luckily I had not used the other half of original to feed as planned so I now added it to a new culture of about a quart volume. I think the larger volume diluted my start too much on the 1/2g. The other factor is with the increased outdoor temps the room they are in has increased about 5deg too. The room stays about 76 or so now. I may struggle with the iso until it cools off again. The picture of the 1/2g is on right and my clean on left. The green is slight but definitely there.


Lrood
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Re:Culture Journal, Species: Isochrysis galbana - Sunday, November 09, 2014 5:38 PM
Hi Daniel, how is the iso doing?
~~~~~~~~~~~~~
Ken

waldend
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Re:Culture Journal, Species: Isochrysis galbana - Tuesday, June 13, 2017 4:50 PM
After listening to Andrew Rhyne's info on microalgae culture at the MBI conference I overhauled the way I do my Iso and now have good, consistent results.   
I now use heat to sterilize 0.5 gal mason jars to maintain a "master" culture.  I never open or mess with this master except to pour half of it once mature into the new master.    I also now maintain 1.5 gal beverage dispensers for an in-use culture.   I think the key here is that with the spigot for taking out what I need I never open this culture either unless its to add more water so it stays contamination free as well.    Due to the volume, and the fact that I don't have a container to microwave more than 1 qt of water at a time, I use chlorine for sterilization of this water.  I then use sodium thiosulfate to neutralize the chlorine after 24 hours.    While I am doing any maintenance on the master or in-use I am very careful to hopefully not contaminate it, going so far as to disconnect the air feed and take the bottles to a different room to work.  I wipe everything off with alcohol prior to removing any lids or lines and keep a rag soaked with alcohol in my hands at all times to help prevent accidental contamination.  I wipe and then wipe some more!  I have poured my F/2 into a smaller container to prevent  losing an entire bottle if for some reason I would need to get rid of that batch.  The pipette I use to add the F/2 I wipe with alcohol before and after use and suck some alcohol up each time after use as well and store I store the pipette in a baggie.   This focus on cleanliness and focus on anything that will touch and could contaminate the Iso has really turned the corner for me.  

Lrood
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Re:Culture Journal, Species: Isochrysis galbana - Tuesday, June 13, 2017 9:17 PM
Good to hear the cultures are doing well. Your fastidiousness pays off. The culture I got from you was awesome, and continues to thrive through multiple splits. Thanks again!
~~~~~~~~~~~~~
Ken

waldend
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Re:Culture Journal, Species: Isochrysis galbana - Thursday, June 29, 2017 1:42 PM
Here is a pic of my in-use from yesterday for continuation report.