Successful freezing/storage of algae

Successful freezing/storage of algae

Postby Luis A M » Fri Jan 22, 2010 10:36 pm

This will be our first project.To find out if we can freeze our phyto cultures keeping them alive.It will be very useful if it can be done.We could store lots of phyto for later needs,and we wont need to keep running stock or other cultures until we need them.
While algae from the arctic area have been shown to endure freezing,this might not be possible with our tropical species.
What to do?.
Put a small volume of phyto culture into the freezer for one week and then check if it is alive after thawing.
How do we know algae are living?
If we chose motile algae,like TET,ISO,PAV,and they are seen moving,they are obviouly alive.
If they dont move or if we are freezing non motile algae like NAN,CHLO,CHAE,we must start a culture using the thawed algae as an innoculum.In this case we must stress the measures to avoid contaminating the culture with other algae,resulting in false positive results.



Este va a ser nuestro 1er proyecto.Descubrir si podemos congelar nuestros cultivos de fito,manteniendolos vivos.Sera muy util si se puede hacer.Podriamos almacenar cantidades de fito para necesidades futuras,sin tener que mantener cultivos activos.
Si bien se ha comprobado que algas de regiones frias pueden sobrevivir la congelacion,esto puede no ser el caso con nuestras especies tropicales
Que hacer?
Poner un pequenio volumen de cultivo en el freezer y chequear a la semana si siguen vivos al descongelarlos.
Como sabemos si las algas estan vivas?
Si hemos elegido algas con motilidad,como TET,T ISO,PAV,y vemos que se mueven,estan obviamente vivas.
Si no se mueven,o estamos usando algas inmoviles,como NAN,CHLO,CHAE,debemos iniciar un cultivo,usando las algas descongeladas como inoculo.
En este caso,debemos extremar las medidas para evitar contaminar los cultivos con otras algas,resultando en falsos positivos.
Last edited by Amie on Thu Oct 23, 2014 1:06 am, edited 1 time in total.
Reason: updated subject
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Re: Survival of frozen algae.

Postby JimWelsh » Fri Jan 22, 2010 11:11 pm

It just so happened that I put 2 x 100mL samples each of Nanno, T-Iso, and Tetraselmis into the freezer last night, with this question in mind! Will follow through and keep you all posted.



Justo anoche puse en el freezer 2 muestras de 100 ml de T.ISO,TET y NAN,con este tema en mente.Voy a mantenerlos informados.
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Re: Survival of frozen algae.

Postby Jhawkor » Fri Feb 05, 2010 11:40 pm

Any updates on this guys? btw we need more research projects!
-Landon-

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Re: Survival of frozen algae.

Postby JimWelsh » Sat Feb 06, 2010 8:34 pm

I did not try to culture my T-Iso or Tetraselmis after thawing, but there were NO motile cells. I even placed them in bright, indirect light for a couple of days to see if they might "wake up" after a while, but no. Cultures stayed flat as a pancake on the bottom, too, unless shaken. I'd have attempted culturing them, but am planning a vacation right now, and will be leaving soon, so experiments will have to wait until after I return later this month.

Did anybody else attempt the frozen phyto experiment? If so, what happened?

I agree, we need more research projects! Any ideas?
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Re: Survival of frozen algae.

Postby KathyL » Sat Feb 06, 2010 11:15 pm

perhaps these experiments can by tried with some glycerol in the water, to see if they will survive in the freezer if they don't freeze?
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Re: Survival of frozen algae.

Postby Luis A M » Sun Feb 07, 2010 1:25 am

Jim,while motile cells are obviously alive,non motile should not be declared dead until they fail to grow in culture.I have a TET strain from Karen that are non motile and precipitate soon to the bottom,yet they are alive and a very hardy strain.
Kathy,that would be another experiment,cryopreserving.
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Re: Survival of frozen algae.

Postby rangereefer » Mon Mar 08, 2010 5:57 pm

Doesn't the act of freezing the algae essentially shred the cells walls with microscopic shards of ice?
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Re: Survival of frozen algae.

Postby JimWelsh » Mon Mar 08, 2010 6:59 pm

rangereefer wrote:Doesn't the act of freezing the algae essentially shred the cells walls with microscopic shards of ice?


Conventional wisdom says yes, freezing would very effectively kill the cells.

There are, however, anecdotal reports of T-Iso surviving freezing and re-thawing. Nannochloropsis is supposed to be able to survive freezing and rethawing due to its thicker cell walls. The purpose of this Research Project is to attempt to confirm or deny the theory that frozen phyto, and in particular non-Nannochloropsis species of phyto, can survive freezing and re-thawing, and be cultured afterward. If it turns out to be possible to culture T-Iso after freezing, that would be very useful for keeping stock culture backups.

I have recently taken a new sample of dense T-Iso and left it in the freezer for a week. I thawed it yesterday, and am now attempting to culture it. Once again, I could see no motile cells under the microscope after thawing. Time will tell if I will be able to culture the thawed sample. I'll keep you all posted.
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Re: Survival of frozen algae.

Postby JimWelsh » Sat Mar 13, 2010 2:06 pm

It's been a full 5 days now since I started trying to culture the frozen/thawed T-Iso. The frozen/thawed sample was 125ml. I innoculated 1800ml of F/2 with it, and have given it moderate air and moderate light. Nothing growing. I'll give it a few more days before I totally give up.

I have some Tetraselmis that has been in the freezer for a week, and will attempt to culture it as soon as the T-Iso experiment is done.
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Re: Survival of frozen algae.

Postby Luis A M » Sat Mar 13, 2010 9:30 pm

I´m back from vacations and will run some trials.Forgot to tell about another way to find if a culture of non motile algae is alive or not.Exposed to light,live algae consume all available CO2,launching pH to very high values. :wink:
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Re: Survival of frozen algae.

Postby JimWelsh » Fri Mar 19, 2010 2:47 am

10 Days now given to the T-Iso frozen/thawed culture. F/2 culture water is still clear. Looking under the microscope, I do see a few very small, very fast-moving organisms, but they are too small to be T-Iso. They are more along the lines of 2-4 microns in size. I have given up on this frozen/thawed T-Iso culture as NOT viable. Next is the same test with Tetraselmis.
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Re: Survival of frozen algae.

Postby JimWelsh » Sat Sep 11, 2010 1:17 am

I don't have any news on frozen phyto, but I do have some interesting news about another method of culture storage.

Literally a year ago, I put about 100 ml of T-Iso culture into a capped test tube, and have held that in my fish room at ambient light and temperature. About 5 days ago, I examined the contents of the test tube (there was a bunch of cells settled, but also still a brownish tint to the supernatant), and found weak but still motile cells! I added that 100 ml to another 150 ml of sterile F/2 culture media, and have been giving it moderate light and air for the last 5 days. That 250 ml is now a dark golden brown!

It appears that merely storing T-Iso at room temperature in a sealed container is sufficient to maintain it for many months. I don't know to what degree the ambient light was an important factor, but the stored test tube got no special lighting, though it was not in the dark the whole time, either.

T-Iso cultures I've put in the fridge seem to settle out to a clear supernatant very quickly.
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Re: Survival of frozen algae.

Postby BaboonScience » Tue Sep 14, 2010 8:16 pm

Does this mean that storing cultures at room temperature is actually better than the refrigeration method that I have been using. This could save me alot of heartache when cultures crash! I am going to have to try this.

On the other hand, after getting the algae room finished, I finally have ample cultures of Tet, Nano and Iso to play with. I think that I can concentrate these and try the freezer method, finally. Has anyone tried adding an antifreeze to the culture, like a small amount of glycerin?
John
"The exact contrary of what is generally believed is often the truth" Jean De La Bruyère (1645-1696)
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Re: Survival of frozen algae.

Postby KathyL » Mon Dec 20, 2010 6:33 am

Interesting topic.
On the cryopreservation idea, here is a paper:
Cryopreservation of marine microalgae and potential toxicity of cryoprotectants to the primary steps of the aquacultural food chain

I. Tzovenisa, , , G. Triantaphyllidisb, X. Naihongc, E. Chatzinikolaoua, K. Papadopouloua, G. Xouria and T. Tafasa

Suggests that 1% DMSO would work.

I am going to try this if I ever get some isochrysis going again.
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Re: Survival of frozen algae.

Postby Luis A M » Fri Apr 27, 2012 4:10 pm

Some recent news in another forum makes me remove this project from the freezer :D
http://reefcentral.com/forums/showthrea ... st20176218
Gresham,of Reed enterprises,sustains that while most algae can not be frozen and returned to life,it can be done with Nannochloropsis.He says that in fact,he and their phycologyst do it all the time,as well as some of their customers,starting cultures with IA algae pastes containing NAN.
So I tried to use NAN 3600 as a culture starter,as described in that thread,but failed completely.
I encourage readers having access to IA pastes to try this simple experiment and comment here. 8)
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Re: Survival of frozen algae.

Postby fla2341 » Thu May 17, 2012 1:09 am

Luis, The method I used for the freezing and revival of nanno was very simple. I cultured several bottles of Nanno and placed them in the fridge overnight. After that had settled out i siphoned off the clear water and combined the concentrated algae into one bottle. I placed that bottle into the freezer. I have found that Nanno will restart reliably IF it's only been frozen for up to 3 months after that it's hit or miss. Place it in the fridge and warm up what you want to use to the ambient temp. Use 5ml-10ml to inoculate a starter culture. The bottle is only half full of concentrated nanno when placed into the freezer.

How in the world do you include your avatar or post a pic here? I've tried and failed. I have a pic of the Resealable glass bottle I use but can't get it on here.
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Re: Survival of frozen algae.

Postby Luis A M » Fri May 18, 2012 12:44 am

Welcome! :D
Your experience is noteworthy,and the 1st "neutral" confirmation that algae (at least NAN) can be frozen and returned to life.Previously we had two people related to the algae business,who said that it could be done,but didn´t provide details and our initial attempts all failed.
So it will be our next move that we try to follow your protocol and match your result.Then we could try TET which,according to Randy R.,can be frozen without cell destruction,which happens to other algal groups.
My last test with NAN paste,involved a several years old product,which could explain the failure,according to your finding.
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Re: Survival of frozen algae.

Postby fla2341 » Sun Aug 05, 2012 2:11 pm

Luis,

Any luck replicating my success? I have just restarted nanno from a frozen sample I placed in my freezer which is 2 months old ( I couldn't go the full 3 mo because the Fridge/Freezer died last Friday). Right now it's 7 days old. I started out with 1 gallon boiled water sg 26 and split it into glass 1 gallon jars. I added 10ml of thawed algae to each jar and 3/4-1ml of F/2 fertilizer, supplied air and I've got light green tinted water. Both show the same results.
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Re: Survival of frozen algae.

Postby Luis A M » Mon Aug 06, 2012 3:28 pm

No,Chris,I have been insisting with the commercial frozen pastes,as some posted experiences pointed that they could be brought back to life.This time I tried "Rotifer Grow Plus" which seems mostly NAN,but with other algal species blended in.
Again results were negative.It will be interesting if other people try to start cultures with other brands of frozen commercial algae.
Now I will definitely try freezing/thawing my live algal cultures,starting with NAN :wink:
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