Tetraselmis Culture (my first phyto culture attempt)

Tetraselmis Culture (my first phyto culture attempt)

Postby EasterEggs » Wed Mar 21, 2012 8:56 pm

I got a Tetraselmis culture from a fellow hobbyist (I have not microscope IDed it yet) on Sunday. Since I was unprepared to receive the phytoplankton on Sunday I did all I knew to do; I doubled the water volume with RODI saltwater (1.025), then split the culture into two 250 mL cultures and one 50 mL culture. I was (still am) waiting on a FAF order, so I didn't have any fertilizer, nor did I have my phyto station set up! I placed the small culture in the fridge with no lid, one 250 mL culture on the couch beside a window with a loose lid, and one in the fish room with an open top beside the sump with some air where it could get some light from the sump. I have since learned that an open top on phytoplankton is a big no no!

Monday after work I spent almost two hours running around looking for liquid Miracle Grow fertilizer. I picked up a couple of 13 watt T5 fixtures that can be daisy-chained together. They came with 3100K bulbs. I also picked up a small bottle of Kent Essential Elements. I made a liter of medium using RODI saltwater (1.020), 2.5 mL Miracle Grow, and 0.5 mL Essential Elements. I failed to sterilize this medium (didn't know any better). I added some medium to the main culture making it about 350 mL. I added some medium to the couch culture making it about 300 mL, and the fridge culture just got a squirt (10 mL?). I was alerted to the open tops on my cultures so I put plastic wrap over the cups.
Image



Tuesday morning the main culture looked awful; it had settled out really badly, so I turned the air up to almost a violent level. The couch culture having no air added settled a bit, but not as much as the main culture (weird?!). The fridge culture settled out quite a bit. At this point I figured both the main culture and fridge culture had both crashed, but the couch culture still seemed ok.
Image



This morning the main culture was thoroughly green, but had not really darkened at all. The couch culture had darkened a bit, and was not settled much at all. The fridge culture was still settled, and still light colored. I had hope that maybe I didn't crash the main culture after all.

I just checked on the cultures now and am delighted to report that the main culture has darkened quite a lot today! The couch culture has also darkened although not as much. The fridge culture still looks awful. I'm thinking I should transfer the main culture to a 2L pop bottle soon...I'm not sure if I should wait until tomorrow to let it darken more. Any input?
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby Suzy » Wed Mar 21, 2012 9:31 pm

Tetraselmis is my favorite alga! Once you get the hang of it, it grows easy peasy, IMO. I use a strong bubble, a slow roiling boil because the cells clump and die if I don't, which is weird for a motile alga!

Have you looked at it with a microscope?

I will let mine grow for at leat 5 days or so before I use it. I get nervous if the cells haven't consumed the fertiler.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby EasterEggs » Wed Mar 21, 2012 10:32 pm

Suzy, now you say it, the cells did clump along with settling. I have the air turned up quite high now, and it seems happy (for now!). I haven't looked at it under my microscope. My microscope is digital and I haven't reinstalled the software since reinstalling Windows awhile back. Is there a way to tell if the fertilizer has been consumed?
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby Suzy » Wed Mar 21, 2012 10:49 pm

Not that I know of, but you could run a nitrate test on it. I just waited until I was sure it was consumed.

My digital still has an eyepiece type viewfinder. You really need to see it move! It is hard to believe it is an alga!
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Tetraselmis Culture (my first phyto culture attempt)

Postby wooboodoo » Wed Mar 21, 2012 11:53 pm

Use as much air bubbles as you can for strong circulation, if you'll decide to move your culture to the bigger container use small power head algae need co2 for photosynteze process so air bubbles are fine but providing too much o2 to your algae and your culture need more time to grow. Light for 20 hours and shake your culture every day very strong.
I'll suggest to use minimum 1/2 gal plastic jars i got my from ebay so i can clean and reuse them.
I used to have my phytoplankton in 60 gal tank 48"x24"x12" with two 60w 3500K bulbs and big power heads.
Occasionally I'm also using Zin reactor but is pain in neck to not "infect" green water with Rotifers and crash whole culture.

http://www.noc.soton.ac.uk/soes/staff/t ... toco2.html

Looks like all cumulated co2 gonna stay in the system.

GL and regards,
mark
PS I'm sorry for my bad English I'm European.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby Suzy » Thu Mar 22, 2012 8:35 am

I've used a powerhead in a 5 gallon car boy before, when I wanted a lot of phyto. It worked ok. It really made making the water easy! Throw in some salt, some bleach and some fertilizer, wait over night and voila! Perfect culture!
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby EasterEggs » Thu Mar 22, 2012 10:39 am

Suzy, I went on You Tube a couple days ago and looked at videos of Tetraselmis and Nannochloropsis I couldn't believe how fast moving they are! I am told the color of my culture looks right for Tet, but no harm. The culture was given to me as Tet, so it should be Tet!

Mark, thanks for your input. :) I am going to move my main culture to a 2L pop bottle when it is has grown sufficiently. I plan to have at least two 2L bottles going. I'm practicing with Tet to learn good sterilization and culture techniques, and will move on to C-Iso when I feel comfortable. My end goal is to culture Apocyclops panamensis using C-Iso.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby Suzy » Thu Mar 22, 2012 4:58 pm

It could be Tetraselmis, but cultures can be contaminated, so you are going to want to get your scope running so you will know for sure. If you are going to all the work to grow it, you want to know it is what you think it is. You can't tell by the color, IMO. Plus, you wil want to see if a protozoans gets in, so you can restart it when you need to.

I use Diet Pepsi bottles. I think the name brand gives the extra ooomph it needs!

One word of advice? Dont make it too complicated. It can be easy and not so time consuming if you want.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby EasterEggs » Sun Mar 25, 2012 10:50 pm

Here is the culture this morning:
Image

I tried looking under my microscope under 185x magnification which wasn't enough to see the shape very well. This is the highest magnification that I could get the microscope to focus. The cells were definitely very active though! What magnification do I need to use to identify phytoplankton?

I did my first split today as sterile as I know. I mixed up the medium using RODI saltwater at 0.012, added MG and EE, microwaved for 8 minutes (will boil next time...oops). I dumped the pop out of a 2 liter pop bottle, rinsed well, added a few drops of bleach, shook it a few times, and rinsed real well. I poured the hot medium into the pop bottle and allowed it to cool for an hour or so. Then I poured 3/4 of the phyto culture into the pop bottle, added air and set it in front of the lights. I poured the remaining 1/4 of the culture into a newly sterilized glass, topped up with medium, added air, and set it in front of the lights.
Image

I checked every major pharmacy in the city looking for sodium thiosulphate today, and could not find any. One pharmacists suggest I look at a "compound pharmacy"...whatever that is.

Suzy, so far phytoplankton seems much less demanding than rotifers even. :D
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby EasterEggs » Wed Mar 28, 2012 11:52 am

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.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby Luis A M » Wed Mar 28, 2012 2:41 pm

The smallest air pump should be fine for one bottle.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby EasterEggs » Fri Mar 30, 2012 10:45 am

Luis, I am getting a lot of settling with a small pump on the 2L pop bottle. It works fine for the glass though. In my very limited experience so far, it seems the more air I add the faster it grows...?
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby Luis A M » Fri Mar 30, 2012 11:03 am

Indeed,but even the smallest pump gives a strong bubbling when serving just one bottle.You run a rigid air line to the bottom?.Perhaps the rubber diaphragm is punctured?.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby rayjay » Fri Mar 30, 2012 1:01 pm

It works a lot better if you have the bottle inverted so that a lot of settlement naturally falls into the cap where the air flow throws it back up again.
It means cutting the bottom out and using cling wrap to cover but it does work better.
Conversely you can just shake the bottle up a couple of times a day.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby EasterEggs » Fri Mar 30, 2012 10:23 pm

No Luis, I have 3 different small pumps and none of them seem to really be big enough to serve a 2L pop bottle. My larger Hagen pump should be able to serve 3-4 pop bottles though, so that should fix the problem. :D

Rayjay, yeah that would probably work better, but for now pop bottles are the easiest. I have some ideas for making a more permanent phyto station, but I have sooo many projects on the go right now!
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby rayjay » Sat Mar 31, 2012 12:13 am

Pop bottles are what I'm talking about.

http://www.angelfire.com/ab/rayjay/Hatcher.html
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby EasterEggs » Sat Mar 31, 2012 10:35 am

Yeah I know, I should have been more clear. I've used that style for Artemia before, but found it really messy. I also knocked one over once and that's when I gave it that style! Haha! I'm worried about contamination with a flimsy cling wrap covering too. I have my drinking glass backup culture covered with cling wrap and it drips salt spray sometimes. The upright pop bottles are much cleaner and easier. Thanks for the suggestion though. :)
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby rayjay » Sat Mar 31, 2012 10:50 am

If you are worried about contaminants of the cultures, what are you doing to sterilize the air going into them?
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby BaboonScience » Sat Mar 31, 2012 3:09 pm

Easy way to get sterile air into your culture.
Pack a short piece of tubing (2" should do") with cotton, the type that comes as cotton balls. You will need to draw the cotton out and pack it in tight. Place a short piece of stiff tubing at each end of the packed tube and then fit this between the air supply and the stiff tube in the algae culture.
Now, IMPORTANT, before you place the tube into the live culture, draw some alcohol (isopropyl will do for this) and then attach the line to the air supply. let it go for an hour or so or till the alcohol has entirely dissipated from the cotton in the packed tube. Then, swab the portion of line going into the algae culture with alcohol and drop the line into the culture.
The air that bubbles out of the line will be clean and sterile.
Hope that this helps.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby EasterEggs » Sun Apr 01, 2012 12:40 am

Thanks for posting John! I have actually already purchased the materials to make this air filter you describe as I read about it in another post. I bought 95% ethanol "rubbing alcohol compound" and cotton balls. I have a lot of projects on the go right now though...
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby CaptCrash » Sun Apr 01, 2012 8:58 am

BaboonScience wrote:Easy way to get sterile air into your culture.
Pack a short piece of tubing (2" should do") with cotton, the type that comes as cotton balls. You will need to draw the cotton out and pack it in tight. Place a short piece of stiff tubing at each end of the packed tube and then fit this between the air supply and the stiff tube in the algae culture.
Now, IMPORTANT, before you place the tube into the live culture, draw some alcohol (isopropyl will do for this) and then attach the line to the air supply. let it go for an hour or so or till the alcohol has entirely dissipated from the cotton in the packed tube. Then, swab the portion of line going into the algae culture with alcohol and drop the line into the culture.
The air that bubbles out of the line will be clean and sterile.
Hope that this helps.
John


John, I am assuming that once the air pump is running that the cotton will get dirty/not sterile, so how often should this procedure be repeated?
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby BaboonScience » Sun Apr 01, 2012 6:36 pm

Mr Crash
The cotton can be re-sterilized simply by drawing more alcohol through it, periodically. Once it is dry, it should be sterile once more. You can replace that cotton, periodically if you wish, but not absolutely necessary.
Repetition?
I would do the alcohol thing every time I revitalize the culture. It is a low cost means of ensuring that the air is clean.

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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby BaboonScience » Sun Apr 01, 2012 6:45 pm

Mr. Eggs
I believe that the air supply and water sterilization are the most important considerations in algae culture. I have provided a quick and reliable means of getting a relatively clean air supply. Let me know how it goes for you. I will try to trouble shoot.

Mr RayJay
I hope that this answers your concerns as well. Let me know.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby rayjay » Sun Apr 01, 2012 7:34 pm

Thanks. I was aware of the need for many cultures to have sterile air supply.
However, as all I grow is nanno, I haven't had any problem with open air containers.
I've been using 26g garbage pails and 20g rubbermaid storage containers, open topped, and no problem.
I can keep harvesting for a month or two before cleaning out the containers and replacing the culture and continuing on.
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Re: Tetraselmis Culture (my first phyto culture attempt)

Postby BaboonScience » Sun Apr 01, 2012 7:43 pm

OK, Rayjay
Nano is sort of like Thypus. It operates by it's own rules. Tet, Iso, Rhodo and most of the flagellates will be dominated by Nano if they are not protected from it. I think that the dark side of the force is Nano.
LOL
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