Culture Journal, Species: Tetraselmis

General
Species:  Tetraselmis
Species description:  Green motile alga (I think?)
Culture source (link if possible):  fellow hobbyist gave me about a 1 1/2 cup starter
If algae, CCMP # (Optional): 
http://ccmp.bigelow.edu/
Culture Establishment Date:  Mar 18, 2012
Continuation Date:  Jun 18, 2012

Culturing Vessel Details
Salinity:  1.020
Temperature:   Ambient temperature is 78-80F in the culture cabinet.
pH:  Not measured

Vessel description:  Drinking glasses for now.  Will buy some 2L soda bottles (I don't normally drink it).
Lighting description:  2x 13 watt T5 fluorescent 3100K
Lighting cycle:  18 hours on, 6 hours off
Aeration description:   Almost violent.  Will make an inline air filter asap.

Methodologies
Split methodology:   I'm told to split it every 10 days.
Culture medium description:  Waiting for Guillard's f/2 to arrive from FAF.  Currently using 2 liters RODI saltwater, 5 mL Miracle Grow liquid, 1 mL Kent Essential Elements.
Cell count:
Reference links:  

Additional Information

Notes:  This is my very first try culturing phytoplankton, so PLEASE make suggestions if I'm not doing something right!

Yesterday when I received the 1 1/2 cup starter I dripped in some fresh RODI saltwater to double the water volume. I didn't have any fertilizer so I didn't add any. I split the culture into 3; 50 mL into the fridge, 250 mL in a window sill (no air), and 250 mL near my sump where the Chaeto lamp coul shine on it a bit.  I wasn't prepared to receive the phyto yesterday!
 
So today I bought some Miracle Grow Liquid and Kent Essential Elements while I wait for the Guillard's f/2 to arrive from FAF.  My Googling seems to say that I should make a medium by adding 5 mL MG and 1 mL EE to 2 liters of RODI saltwater (1.020).  Then add the phyto I have now and fill up the rest of the 2 liter pop bottle with the medium.  Or should I just double the water volume with medium every 10 days?  Someone please advise.
 

If it were me, I'd do the following:
 
-1) (Hindsight):  Treat the culture like something you are trying to keep as sterile as possible.  You didn't mention having sterilized the "fresh RODI saltwater" you dripped in.  Re-read Hoff & Snell's Microalgae chapter, and in particular the part about sources of Contamination, Prevention, and Treatment of Culture Water (Pages 34-36).  Also, not sure why you dripped it.  Phyto generally does not need acclimation the same way larger organisms do.
 
1)  I'd take the 50 ml out of the fridge.  My experience is that refrigerating phyto (especially motile phyto) tends to make it settle out quickly.  Backup stock cultures should be kept at room temp, and in indirect ambient light.
 
2)  I'd do the "double every X days" approach, but I wouldn't base it on the number of days, more like the density of the culture.  Once it has darkened noticably, double it again, until you are up to 2000 ml.
 
Tetraselmis is pretty hardy -- you'll probably be OK, but I'd suggest being a little more paranoid about contamination in the future.
 
EDIT:  Just looked at the image.  Yikes!  Container open to the air.  Yikes!
 

Mindy,I would drop it in your soda bottle (in case you drank it all) and fill half of it with Cl treated new 1.010 ASW.When you get the fertilzer from FAF,add 1 ml and when you see it getting greener,fill it up.
Easy!

-1)  I did not sterilize the fresh RODI saltwater.  The thought didn't cross my mind.  I will read Hoff & Snell's chapter on microalgae...never thought of that.  I need to get some sodium thiosulphate.  I just made an order to FAF, but figured shipping on sodium thio is probably more than the sodium thio so I will (hopefully) find a local source.
 
1) Ok.  I have a backup culture sitting in the back of my couch near a window.  It is kind of covered with a lid.
 
2) I thought phyto was supposed to follow a set day schedule for splitting...?  Not so much by color.  Hmm.  Ok, this will take some getting used to then.
 
Open air is fixed...Tal was exclaiming about that too!    I covered it with plastic wrap until I can get some pop bottles.  I forgot to do that today, and spent 1 1/2 hours just finding liquid Miracle Grow.  Ack.
 
I sure hope Tetraselmis is hardy...it's my first go culturing phyto!  This is my practice phyto until I can get my hands on some Isochrysis.  From what I understand, I have to learn very good sterilization techniques in order to be successful with Iso, so this should be good practice for me.  The guy I got this Tet starter from claimed to get success from FAF's Iso algae disks not once, but twice.  He crashed his Iso culture recently though, so he's ordering some more Iso disks.  If he gets it going again he will share with me.  Thanks JimW!
 
Luis, I don't have any soda bottles!  I will get some tomorrow.  1.010 you say, eh?  I just doubled the water volume with the Miracle Grow and Essential Elements medium.  It's at 1.020 right now.  I hope that's ok for now until I get set up properly.

I hope this practice run goes well for you, but if it doesn't, just chalk it up to experience.  Don't get discouraged, and do try a second time.  Keeping phyto cultures clean enough for hobbyist use isn't that hard -- it's just a matter of establishing good habits early on.  I think that many people fail with species besides Nannochloropsis because they don't take the contamination issues seriously enough.

I will take it seriously Jim.  I want to be good at culturing Isochrysis, so this is a needed first step.  I just need to learn those good habits and then use them. 

And if you want to be on the safe side,make stock cultures!http://www.marinebreeder.org/forums/viewtopic.php?f=145&t=324

Luis,
When you sterilize by microwave, do you include the fertilizer in the medium?

Yes,and it works,despite the possible heat damage to the vitamins.If you start with sterile inoculums,you keep everything sterile.

Thanks for the link Luis.  I'm not sure what you mean by "stock cultures", but I'm assuming your link will inform me! 
 
The alga really settled out overnight, so I'm assuming I need much more airflow, almost violent.  So I turned it up this morning, and still had a bit of settling tonight, but much better.
 
Here is the culture first thing this morning:

You´re welcome Mindy!.Hope the link will be helpful.
Coul you check your algae with a microscope?.Some TET are non motile and drop to the botom like stones.

I will try to check Luis. I have 100x handy, but I would have to install some programs to get my USB microscope working again (250x). Checking You Tube for videos I see Tetraselmis is really fast moving!  Holy mackerel, I was expecting a few twitches here and there...

Mindy,
 
What Luis means by "Stock Cultures" is basically locally preserved versions of what you would think of as "Starter Cultures" that you would usually purchase elsewhere.  They might be called "Insurance Cultures" or "Backup Cultures" or "Plan B Cultures".  They are held in reserve, in case your primary cultures go south.
 
I held off earlier commenting on your most recent pictures.  I know I was pretty harsh ("tough love" kind of harsh) on you earlier in this thread about contamination.  Quite frankly, the appearance of your cultures is alarming to me.  I fear that either (a) your culture has been contaminated beyond recovery, or (b) your culture has, for whatever reason, crashed.
 
If (a) is true, then it is time to arrange for a second shipment of a Tetraselmis starter from the original source.  Another option is to try to work with one of the other cultures you mentioned (the refrigerated version, or the other 250 mL version).
 
If (b) is true, then there is hope that there will be enough healthy cells surviving to keep the culture going.
 
Have you ever worked with Nannochloropsis?  It might be another good candidate for a "trainer" phyto.  The FAF Nanno disks have a pretty good reputation as successful starters.  Just a thought.

Jim is right,only that I start new stock cultures every 15 days and I keep 3 flasks  for each alga.The old culture,45 days old, goes for a soda bottle which is labeled as pure and true ID,and I start new bottles and bags from this one.So bad or contaminated cultures go to a dead end.Yes,it is kinda buying starters twice a month.

So essentially I have "stock cultures" seeing as I have 3 mini cultures, but my stock cultures are definitely not as pure as Luis describes.  Of course I have no idea how pure this culture is to begin with, nor do I really know if it really is Tetraselmis.  I know I can't tell the difference.  I will try to get a video on my USB microscope and hopefully get a positive ID.
 
JimW, I don't mind if you are harsh (although I didn't see it that way).  You do have to understand that I was totally unprepared to receive the starter both in station preparation and in research!  I was afraid I had already crashed the culture and was disappointed I did it so freaking fast!  So please continue with the "harshness"!  Kick my butt into proper protocol! 
 
The 250 mL culture on the back of my couch near the window has darkened up quite a bit and interestingly it has not really settled much at all (so it must be motile, right?).  This morning the main culture was thoroughly green now that I had turned up the air, but had not really darkened much.  I was delighted to come home to a much darker culture today!  Whereabouts is this culture on the darkness scale in relation to splitting/diluting?  I want to make sure it isn't stressed out when I move it to the 2L pop bottle.  Maybe I should do this tomorrow or maybe tonight?

So...this ethanol air filter as desribed by BaboonScience (and JimW), "an air filter made from 3/4" PVC (2" section) and 2 end caps with holes drilled into their centers (cheap air line nipples used for in and out). Filter is cotton packed and the entire thing filled with ethanol (see the drug store not the liquor store) and purged for at least 2 hours."
 
How tight do I pack the cotton?  I assume the cotton is what is doing the filtering and the ethanol sterilizes everything?  By "purging" I assume this means to push air through the cotton until it is thoroughly dried before using the filter?

Nicer looking Tetraselmis!  Check it out with a scope (you have one, yes?) to make sure you see lots of oval, motile cells, just to be sure of what you're growing.  The color does look right for Tet, though -- it is a little bit more of a bluish green than Nanno, which is a rather pure green -- leaning towards neither blue nor yellow.
 
You pack the filter tight, tight, tight.  Don't worry, the air will still get through.  And yes, the purging is to dry the ethanol before use.  The ethanol should be the 100% denatured stuff (unless you know where to get everclear!  Woot!).
 

Yeah, I have to get it under my scope.  I'm just really busy (distracted) right now because hubby is home for a week (he works out of province).
 
I added some more (microwaved) medium lastnight so the glass is almost full now.  When I get to the point I want to feed this to something, like say my reef tank, how do I know the fertilizer is used up? 

Guillard's, when used at F/2 strength, is only appx. 55 PPM of nitrate and 3.4 PPM of phosphate.  Let's assume that NONE of it was used up, and you are pouring straight F/2 into your tank.  If you have a 100 gallon tank, and pour 1 liter of fresh F/2 into your tank, you are adding appx. 0.14 PPM of nitrate and 0.009 PPM of phosphate to your overall tank concentrations of these compounds.
 
Now, let's assume that the algae has consumed 90% of the nutrients, and 10% remain in solution.  Then divide those numbers by 10.
 
Just because the algae has consumed the nutrients and converted them into proteins, etc., that make up the algae, that doesn't mean they have disappeared.  You are still adding the same amount of nitrogen and phosphorus to the tank, just in a different form.  A certain amount of the nutrients will be consumed by corals, copepods, etc., and assimilated into their tissues, but a significant number of the algae cells will also probably die and decompose, releasing these nutrients back into solution. 
 
Of course, this depends on many factors, like whether you are running UV or a protein skimmer (both of which you should shut off, of course, when dosing live phyto).  The UV will help break down the phyto cells, speeding the release of the nutrients into the water column, while the protein skimmer will tend to extract the cells from the water and leave them in the collection cup.  You get the idea.  I'm just trying to point out that the F/2 has not disappeared -- it has just changed form.
 
Hope this helps!

This got me thinking a bit further, and asking myself the question, "How does the amount of nitrogen I am putting into the tank with a liter of phytoplankton compare to the amount of nitrogen I am putting into the tank with a cube of frozen food?"
 
The nitrate in 1 liter of F/2 media, expressed as absolute milligrams (and not a concentration), is appx. 55 milligrams.
 
The typical cube of frozen food is approximately 3 grams in wet weight.  Typical frozen food contains between 5 and 15 percent of the wet weight of protein, so for the purposes of this exercise, let's call it 10%.  Protein contains, on average, 16% nitrogen.  That means that the typical cube of frozen food contains 3 g * 10% * 16% = 48 mg of nitrogen.  The molecular weight of the nitrate ion is 62.44, and the atomic weight of nitrogen is 14, so that means that the 48 mg of nitrogen would convert into 48 * 62.44 / 14 = 214 mg of nitrate.
 
1 typical cube of frozen = 214 mg of nitrate.
1 liter of F/2 = 55 mg of nitrate.
 
So, adding 1 cube of typical frozen food adds roughly 4 times the potential amount of nitrate as 1 liter of F/2 media.  This is assuming, of course, that all of the nitrogen in each ends up being ultimately converted into nitrate.

Weird, I replied to this and even saw my post!  Now it is gone!
 
I said thanks for posting the math, Jim!  That makes a lot of sense.  I understand that nitrogen going into the tank in any form is still nitrogen.  It's the same as when nuisance algae is growing and it dies off.  You're better off removing it than killing it.  The math regarding the fertilizer used for phyto is so miniscule though that it really doesn't seem to have any threat at all.

I think last time I bought Sodium thiosulphate I bought it at a drug store, so I'm going to see if I can find some there today as well as some ethanol to make a filter.
 
I think I better split it today, it sure is dark!  Like soup...

Definitely time to split.  Got that microscope fired up yet?

I'm working on firing up the microscope this morning.
 
Right now I'm working on a sterile split, and wondering...how long do I microwave the medium for?  I can't find reference. 
 
EDIT:  I microwaved 2 liters of medium for 8 minutes which made it quite warm, but not hot.  I'm not sure if it is supposed to boil or maybe just the microwaves have a sterilizing effect...?
 
I just took a video and am uploading to You Tube.  Unfortunately the best I can do is 185x magnification which doesn't look like enough to ID it.  What magnification do you guys use for phyto?  1000x?
 
EDIT 2:  Ok, so upon searching I find that ethanol is what is in isopropyl rubbing alcohol.  I have 70% isopropyl rubbing alcohol.  Is this sufficient to make a cotton filter with?  Do I just use "cotton balls"?

I microwave until the media is 185F / 85C.  I find that the media does boil in the microwave, even though it is not up to "boiling temperature".  I microwave 2 x 2-liter bottles that each contain 1500 ml of media for 22:22 in an 1100 watt microwave, and that does the trick for me.
 
Isopropyl rubbing alcohol contains isopropyl alcohol, not ethanol.  It is probably good enough to kill off the things in the cotton, but the 30% water will also probably take longer to evaporate.  I used 100% denatured ethanol from the drug store.  Denatured ethanol contains ethanol and a little bit of nasty-tasting things that make it so nobody wants to drink it.

Ok, thanks for clarifying the microwaving strategy and the ethanol. Google must be wrong about isopropyl alcohol being ethanol.  Ha!
 
Here is my first split, done as sterile as I know how.    No air filter yet though.

So I went to the pharmacy and asked for the purest ethanol they have.  I was given a bottle that says "Rubbing Alcohol Compound".  Medicinal ingredient: 95% Ethanol Anhydrous. Mfr. Standard.  Non-medicinal ingredients (alphabetical order): Camphor, Diethyl Benzyl Benzoate, and Diethyl Phthalate.  It was like $3.  Is this the right stuff?
 
I had absolutely no luck finding sodium thiosulphate...maybe I should have included it in my FAF order!  I figured I could buy it locally...

That's funny. I thought Rubbing Alcohol was Isopropanol, not Ethanol.  If you want ethanol, use Vodka.

Ethanol has 2 carbon atoms, Propanol has 3, and isopropanol or isopropyl alcohol has 3 carbons in a V configuration, with the OH alcohol part attached to the carbon in the middle. I majored in Chemistry, but this is high school stuff.  I'm surprised at Google….
 
http://en.wikipedia.org/wiki/Isopropyl_alcohol

The "Rubbing Alcohol Compound" doesn't mention isopropyl at all, and lists "ethanol" as 95%.  Funny you mention it Kathy, I have been reading some vodka dosing articles lately and have seen it referred to as "ethanol dosing" on several occasions.   I'm just wondering if this 95% ethanol "Rubbing Alcohol Compound" will be sufficient for an air filter for my phyto?  Or maybe soaking the cotton in vodka is good enough?  Haha!
 
Wikipedia says, "Rubbing alcohol, USP / B.P. is a liquid prepared and used primarily for topical application. It is prepared from a special denatured alcohol solution and contains approximately 70 percent by volume of pure, concentrated ethanol (ethyl alcohol) or isopropyl alcohol (isopropanol).[1] Individual manufacturers can use their own "formulation standards" in which the ethanol content usually ranges from 70-99% v/v.[2] In Ireland and the UK, the equivalent skin preparation is surgical spirit, which is always an ethyl alcohol-isopropyl alcohol mixture."  http://en.wikipedia.org/wiki/Rubbing_alcohol
 
Which makes me think that "rubbing alcohol" could be isopropyl or ethanol.
 
Wiki also says, "Denatured alcohol or methylated spirits is ethanol that has additives to make it more poisonous or unpalatable and, thus, undrinkable. In some cases it is also dyed."   http://en.wikipedia.org/wiki/Denatured_alcohol

I think Rubbing alcohol COULD be Ethanol, I've just never encountered it as such. I would be more comfortable using something that is less toxic generally than whatever they are putting in rubbing alcohol to make it unpalatable.  (I do know of an alcoholic who drank Listerine until it nearly killed him, in an effort to remain inebriated without smelling of alcohol, and alerting his long suffering wife to his fall from sobriety. )  I only wanted to make it clear that isopropanol is not ethanol.  For the purposes of sterilizing cotton for an air filter, I would use ethanol, vodka is probably cheapest.  Not sure what those other things in rubbing alcohol would do to the phyto.

I think a concern here is not only using something that will sterilize the cotton, but also something that will evaporate away quickly.  My concern about using Vodka (40% ethanol and 60% water) is that the water will persist for some time, perhaps encouraging the growth of bacteria, fungi, etc., in the cotton.  Using 100% alcohol  (denatured or not), whether ethanol or isopropyl (or even methanol, for that matter!) will greatly decrease the chance of lingering water in the cotton allowing the very same microbes we are trying to filter out to flourish.
 
Between vodka and 70% isopropyl, I'd go with the 70% isopropyl.  For me, it was very easy to go to the nearest drug store and get 100% denatured ethanol.
 
EDIT:  You know, in practical terms, you could probably dispense with the ethanol altogether, and just run air through the filter for 24-48 hours to "flush" out any microbes hiding in the cotton.  If you pack it tight enough, this is probably a non-issue, and the ethanol is just an extra, but not absolutely necessary, precaution.  I would fear a soggy wet filter from residual water more than I would one that was dry, and had had no alcohol treatment at all. 
 

Thanks for the input Kathy, I was also worried about the other ingredients in the 95% ethanol.
 
Jim, what about the 95% ethanol rubbing alcohol?  Would you take the 70% isopropyl over the 95% ethanol?
 
You bring up a good point about soggy cotton Jim, I could easily do an experiment packing a filter, soaking in vodka or isopropyl, or the ethanol, let it run and then take it apart in an hour or so and see if it is dry or not.

I need a bigger air pump on the pop bottle.  I guess it is time to hook up the Coralife Super Luft SL-65 pump in the broodstock room.  That way I can steal the bigger Hagen pump for the phyto station.
 
I'm going to do what I always like to do...I'm going to crash one of the cultures.  I will split the cup culture tonight (it's dark again already), and will leave it in front of the lights until it crashes.  I like to test cultures that are new to me so I can learn where the crash point is and if I am able to bring a culture back from a partial crash.  I think these are good things to know.

So pop bottle #1 was start on March 25th so it is 9 days old today and has been very dark (can't see through it) for several days now.  This morning it suddenly settled out real bad so that I could just barely kind of see through it.  I assume this is the start of a crash?
 
I poured a third of the bottle down the drain (no empty pop bottles), and topped back up with sterile medium.  I will get more pop bottles today so I don't have to waste the phyto when I'm trying to ramp up!  That was annoying dumping it down the drain although now I think about it I probably could have saved it in a bucket for the day.  Argh.
 
In the future, when a phyto culture settles out like that (assuming this is the start of a crash), should I only split the phyto that is still motile?  Should I toss the bottom of the bottle that settled?

These cultures are still just plugging along.  I find sometimes after I split the new culture fails to darken and falls out of the water column in a week or so.  I just chuck it out and make a new one.  I need to purchase some more lights and get another shelf going.  I currently have (2) 24" 13W T5 on the one shelf, and I'm wondering if (1) T5 on each shelf would be enough?

The Tetraselmis has been having a tough go lately (not darkening, kind of yellowing a bit)  since the temps outside have warmed up into the mid 20s (Celcius).  I checked temps in the phyto closet at the warmest part of the day (lights were off) and noted 86F.  Ok, that's a tad warm!  I had the lighting cycle reversed to daylight thinking that would keep temps down in there, but it didn't.  So now I switched back to daylight light cycle and I'm opening the door of the closet while the lights are on and that is keeping temps closer to 81-83F.  I also moved the phyto bottles 6" away from the lights instead of the 2" they were.  The phyto is finally starting to darken again.

Don't let fear and common sense stop you! =]

That darkening didn't last long, the Tet started to lighten/yellow a tad again.  I haven't had to split in over a month, so it must be barely hanging on.  Today, I split the cultures to freshen them up and put them through a 23 micron sieve.  I got some yellowish "silt" (critters or deposits I'm not sure) in the sieve.  Hopefully these cultures revive soon!

Don't let fear and common sense stop you! =]

There was still no revival of the Tetraselmis, and I haven't had a culture darken in at least 6-10 weeks.  So I added a fan to the phyto shelf.  I've also been trying to keep the fish room a few degrees cooler.  With these changes I have been able to keep the bottles of Tet just under 80F.  I added the fan to the right hand side of the shelf, and you can see in the photo that the Tet is (finally!) darkening with the right culture the best looking, and the left culture still very light.  The one on the left is how these cultures have looked for the last 6-10 weeks!  I now moved the fan to the middle of the shelf where it can blow directly on the cultures.  I'm hoping I have finally found the solution!

Don't let fear and common sense stop you! =]