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Culture Journal, Species: Tetraselmis
Monday, March 19, 2012 9:07 PM
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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: 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!
<message edited by EasterEggs on Tuesday, July 10, 2012 5:28 PM>
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Re:Culture Journal, Species: Tetraselmis
Monday, March 19, 2012 9:22 PM
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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. 
<message edited by EasterEggs on Monday, March 19, 2012 9:36 PM>
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Re:Culture Journal, Species: Tetraselmis
Monday, March 19, 2012 9:56 PM
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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!
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Re:Culture Journal, Species: Tetraselmis
Monday, March 19, 2012 10:34 PM
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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! 
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Re:Culture Journal, Species: Tetraselmis
Monday, March 19, 2012 10:35 PM
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-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.
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Re:Culture Journal, Species: Tetraselmis
Monday, March 19, 2012 10:44 PM
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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.
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Re:Culture Journal, Species: Tetraselmis
Monday, March 19, 2012 10:50 PM
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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.
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Re:Culture Journal, Species: Tetraselmis
Monday, March 19, 2012 10:54 PM
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Re:Culture Journal, Species: Tetraselmis
Tuesday, March 20, 2012 10:41 AM
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Luis,
When you sterilize by microwave, do you include the fertilizer in the medium?
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Re:Culture Journal, Species: Tetraselmis
Tuesday, March 20, 2012 11:49 AM
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 Originally Posted by KathyL
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.
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Re:Culture Journal, Species: Tetraselmis
Tuesday, March 20, 2012 8:07 PM
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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: 
<message edited by EasterEggs on Tuesday, March 20, 2012 8:26 PM>
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Re:Culture Journal, Species: Tetraselmis
Tuesday, March 20, 2012 9:57 PM
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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.
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Re:Culture Journal, Species: Tetraselmis
Tuesday, March 20, 2012 10:33 PM
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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...
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Re:Culture Journal, Species: Tetraselmis
Wednesday, March 21, 2012 2:47 AM
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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.
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Re:Culture Journal, Species: Tetraselmis
Wednesday, March 21, 2012 10:36 AM
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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.
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Re:Culture Journal, Species: Tetraselmis
Wednesday, March 21, 2012 6:34 PM
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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? 
<message edited by EasterEggs on Wednesday, March 21, 2012 7:06 PM>
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Re:Culture Journal, Species: Tetraselmis
Wednesday, March 21, 2012 7:17 PM
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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?
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Re:Culture Journal, Species: Tetraselmis
Wednesday, March 21, 2012 8:14 PM
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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!).
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Re:Culture Journal, Species: Tetraselmis
Thursday, March 22, 2012 8:19 AM
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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?
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Re:Culture Journal, Species: Tetraselmis
Thursday, March 22, 2012 10:10 AM
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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!
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