Culture Journal, Species: [Dunaliella tertiolecta]

General
Species:  Dunaliella tertiolecta
Species description:  "Cells permanently green, radially symmetrical, ellipsoidal, oval or rarely ovate or pyriform. Cells 5-18 µm in length (mean 9.412.4 m), 4.514.0 m wide (mean 7.1-8.2 µm)." - http://www.algaebase.org/search/species/detail/?species_id=51616
Culture source (link if possible):  http://florida-aqua-farms../shop/microalgae-disks/
If algae, CCMP # (Optional): 
http://ccmp.bigelow.edu/
Culture Establishment Date: 
Continuation Date: 

Culturing Vessel Details
Salinity:  1.028
Temperature:    Ambient, around 75-78F
pH:  Not Measured
Vessel description:  500ml canning jar
Lighting description:  3 Lights of America Linkable Fluorescent Undercabinet Portable Lights
Lighting cycle:  22 hours on, 2 hours off
Aeration description:  Turbulent

Methodologies
Split methodology:
Every day, 25-50% split per culture
Culture medium description: 

1. Add 2x Guillards F/2 formula per 100ml of culture water  
2. Heat in microwave until 212F has been achieved 
3. Allow cooling until room temperature 
4. Split algae into containers 
5. Run airline into culture with a mild-turbulent flow 
6. Have warm lights on the culture running at 22 hours on, 2 hours off 
7. Split every day or two, or until the jar has a light green tint to it 
8. Repeat 2-6 for every split 
Cell count:
 (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.

I am going to try and culture this, partially just to see how hard/how it is done, and also to try and feed to my copepods too. The only true "culturing" information I could find on it was a paper entitled: Culture of the Marine Phytoplankter, Dunaliella tertiolecta, with Light-Dark Cycles. Within this paper, it gave me a "basic" way to culture it. Essentially what it had said was that A.) A "Guillard 'F' culture medium was employed throughout." I'm fairly certain that can just be called our Guillard F2 formula, but please note me if those are two different things. B.) Autoclaving everything that holds or touches algae. I do not have the tools to autoclave my materials, so I will be using a water sterilizing method and using freshly cleaned containers throughout the culture. C.) The dilution of the culture as done at the end of each "Dark Period" (aka a few minutes or so before the lights came on) to around 50,000 cells/ml. I will try my best to get a moderately accurate density, I have a FAF Microalgae Density Stick to measure it with. D.) The "best" way to culture it was on a 24 hour light cycle. I'm not sure how I feel about this, I may just try different cultures, one with the full 24 hours, and one with around 20 hours or so of light. E.) Their lights were around 550-600 m µ, which I'm not sure what that means so if anyone cares to tell me what m µ is I would be very grateful! I will be ordering the culture in the next couple of days, and working on from there.
 
I think I will try some different containers with these. I will do one batch of 24 hour with a "round" mason jar (Later on I will post the exact size of it) and a more cylindrical container and see how those results vary day by day. As far as split cycle goes, it is starting to sound like a split should be done every day, which will get tedious at times, but I will get through it. 

Nice to see you working on something unusual and interesting, Zach!

A)  Guillard F/2 is simply Guillard F at 1/2 strength.  For F strength, just use twice as much F/2, and voila, you have F.

B)  Just do what you have already been doing with other phytoplankton cultures, and be as clean and sterile, and cautious about contamination as possible.

C)  Get a hemocytometer, and learn how to use it.  Meanwhile, your Secchi stick will do just fine.

D)  Knock yourself out.

E)  The 550-600 millimicrons is an indication of the main wavelength in the light used (although the abstract of the paper I read said 580-590 millimicrons).  That wavelength corresponds to a very yellow-orange color, indicating a pretty low color temperature.  I recommend that you use 6500K lights, although that wavelength would actually tend to suggest something even more warm white (lower Kelvin) than the 6500K.

Good luck with them!

Thanks Jim! Ah ok, that makes sense on the F/2. I will get a hemocytometer as soon as I can. I'll run by home depot in the next couple of days and see if I can find any bulbs that would best fit that description.
 
Thanks again Jim!

Awesome to see you doing this.  Have you started this up yet?   I wonder how this culture actually starts from the disk.  Will it be like the nanno disk that starts fine, or will it be like the Iso disk where it is actually nearly impossible for people to get a culture going off of it?  I am actually waiting to see how it works for you before I order some replacements for the ones my dog ate...

Hey shannpeach! I believe that it will be arriving tomorrow, since they only ship on Thursdays or something like that. One question for you guys, that I probably should have asked from the beginning. So when doing a culture from a disk (I've only done their starter culture in a liquid form, probably should have requested that but too late now) how do you guys start them up? Do you just stick the container in the bottom of your first culture container then after it has grown, take it out?

They will include really detailed instructions with the starter.  From what I remember, you put a bit of water on the disk and leave it with the lid on for a day or something, then use a sterile Q tip thing (which they usually send) to get the algae off the plate.  Then put it in your culture flask.  
 
Or something along those lines.  It's pretty easy...when it works lol

Sounds easy enough lol, if it turns out to crash, I'll get another culture and this time put in a request for a liquid form of the algae, I believe that they do that for most all algaes.

So shannpeach, apparently my dad (the man whom had actually ordered the algae for me) DID actually order it to come in a liquid form from them, so should be smooth sailing

Well that's good news for you, but bad news for me!  hahaha  So much for you being my guinea pig...  
 
I'll still be following along to see how this goes

Shannpeach, what were you going to try and culture it for? I'm mainly doing this to see how my tisbes do on it as a food source, not sure how well it would be benificial for us culturing it since around 30% or so of its body mass is oil.

There are some invert related breeding papers(urchins, sea hares, etc) that used this species so I thought it may be a good culture to have handy for those sorts of projects if I decide to work on them.  

Ah! I may have to try this now too That is if my ceriths decide if they ever want to spawn again lol.

Well.... Still has not arrived yet Going to contact them today and see what's going on...
 

Well, time for some exciting news! Culture JUST arrived in the mail today, think they just lost our last order or something. First off, HOLY COW THESE GUYS ARE TINY! I got a hemocytometer, as you recommended Jim, and these guys are not that motile, so they are fairly easy to count. First pic is of the culture it came in from FAF. Few pics afterwards is of it under my Celestron 44340, at 100x (for some reason my celestron doesn't read 400x that well, anyone else have that problem?) I'll try and see if I can convince my science teacher to let me use his dissection microscope to see if it helps.
Mother Culture from FAF:


At 40x:

 
At 100x:

  
Also, as I am updating this now, I'm still working on setting up the algae and will put up more of what the first "trail" of this species is. 

Also, I know the cells don't look right, which has me a tad worried. this species is suppost to be "tear-drop" shaped instead of circular, so I'll play around with it under the microscope and see if I can get that 400x to atleast give me cell shape

So, onto experiment #1:
 
Seeing as to how these guys explode overnight, (seen in Culture of the Marine Phytoplankter, Dunaliella tertiolecta, with Light-Dark Cycles) I took a chance and split the culture into three mason jars, all of which have fresh water I boiled earlier (I treated the water with Jim's microwave-kill-everything-method, see my Isochrysis journal for more detail) for 45 minutes, since it was only three jars. Now, the experiment itself. I labeled each jar 1, 2, and 3. Jar 1 has a mild aeration, just enough to get a small current through, ~10-15 bubbles per second. Jar 2 has a VERY rapid aeration, more of a turbulent water. Jar 3 has a very calm aeration, bubble per second. I'm going to leave them be for today after this, since the explode overnight I'll check them in the morning and see how they are doing.
 

Well, didn't get the "explosion" I was hoping to get. They are still alive, just not very dense. This could have been because they only had around 5-6 hours of light, then they went out. I'll be putting them on a separate timer today set at 22hrs on, 2 hours off. Hopefully that should escalate the growth more. I didn't see that much if a difference in cell numbers between 1, 2, or 3, but again, there wasn't much growth in the first place.

Well, today went MUCH better with the culture. I set the lights so that they went off around noonish, and back on at 2pm, so that there were 22 hrs on, and I got back home in time to see how it resulted. And there was a TON of growth! As you can see in the photos, Jar 2 had the best growth. Because of this, I'll be using turbulent water throughout the culture. Next experiment, how many days until split? Essentially, one jar split every day, one jar every two, one every three, and one every four days. These splits won't happen til tomorrow morning, due to the need to microwave the jars (which I totally forgot to do today!) So far though, I've managed not to crash it, GOOD SIGN!
 


Also, I know I already mentioned earlier about how they should be split every day, but I notice that this culture is not as dark as when it arrived to me, so I'm curious

Well, played around with the hemocytometer to calculate cell density, and got for chamber 1: 330 cells, chamber 2: 260 cells, chamber 3: 267 cells, and chamber 4 with 307. I averaged that amount, (average was 291) and did that to the 10^4, which was 2.91 x10^6 cells/ml, or 2,910,000 cells/ml. Which means a ROUGH estimate of how many cells in the container would be around 8,261,665,000 cells in the entire container, each mason jar was filled to about 12 cups (measurements on the side,) convert that to ml, 2,839.06, then multiplied 2,839.06 * 2,910,000. Jim, can you double-check my calculations on that? I'm fairly certain I did them correctly, but I just want to be sure. Also, although those may seem like big numbers, it is no where near as dense as when I first got it, proving the point of how these guys need more time.

Since you asked......

Zach, your math is overall good, except that you are using too many significant figures in your calculations that you almost certainly cannot support with facts.

For example:  Your "about 12 cups", when converted to mL, well, yeah, sure, if I Google "convert 12 cups to ml", then I, too, get an answer of 2839.06.  However, in order to use such a precise number, then you would HAVE to be able to measure your cups to within 0.01 ml = 0.0000423 cups accuracy.  Are you really asserting that (A) the measurement markings on the side of the jar are that accurate, and (B) that you can visually discern the meniscus with that accuracy, and (C) that the meniscus was exactly on the mark on the side of the jar to that degree of accuracy (never mind the effects of temperature and barometric pressure on the volume of the liquid)?  I very seriously doubt all three, and you would need all three to support using a number like 2839.06 in your calculations.  Since you used "about 12 cups" in reference to a VERY coarse measurement marking, then you can probably honestly claim just 2 significant figures, *maybe* 3, in the volume measurement, regardless which units you are using.  So, re-doing the math, I get:  2840 * 2,910,000 = 8,260,000,000 or even 8,300,000,000 (notice that even though the answer on the calculator when you multiply 2840 * 2,910,000 is 8,264,400,000, you still don't get any "free" significant figures out of the deal.  Your numbers are only as accurate as the accuracy of the least accurate number(s) you are starting with).

Let me give you another example.  As I write this, I am sipping a glass of wine.  That glass has a diameter at the mouth of 6.3 cm, which is the most accurate measurement I can make with the tool available to me (a metric ruler with mm divisions).  If I wish to calculate the circumference of said glass, would you agree that I can say with any real integrity that the circumference is 19.7920337176 cm (the answer I get when I Google "what is 6.3 times pi")?  I hope that you would agree that, considering the relatively coarse units of my original diameter measurement (6.3 cm), at best, I can only claim to know the circumference to within two significant figures as well.  Accordingly, I can only honestly estimate the circumference to be 20 cm, even though I might be able to estimate pi to many, many decimal places.  The extra significant figures in the value of pi I use do not make up for the relatively inaccurate measurement of the diameter my calculation is based upon.
 
Other than that, your math looks great!
 

Thanks for the refresher on sigfigs Jim! Never really understood them in chemistry since my teacher flew through them in a day. I'll be sure that any more cell counts I do, I'll use the right significant figures! Also, as I mentioned before, I'll be splitting this afternoon, once I get back from school. I think I will still keep the 3 cultures, since I don't wanna make too much algae with nothing to do with it. 

Also, on another note, EXPLOSION last night. The lights do not come back on until after I leave for school, thank you daylight savings... ANYWAYS, did another cell count. Let's see if I do my math correctly this time:
 
Section 1: 1,088 cells
Section 2: 1,007 cells
Section 3: 984 cells
Section 4: 1,046 cells
Avg cell count: 1,031.25 cells
1,031.25x10^4 = 1.03125x10^7 cells/ml, or 10,312,500 cells/ml
Sig Figs Allowed: 2
New Cell Density: 10,300,000 cells/ml or 1.03x10^7
Total Cells: 2840 x 10,300,000 = 29,000,000,000 cells, or 2.9x10^10
 
As you can see from the data above, this morning (the two hour dark period,) had a TREMENDOUS amount of growth, in a short little two-hour light/dark period. I don't think I actually need to do the lighting test, seeing as how these guys are doing great on 22 hours. I also believe I have a standard culturing guide for these guys, and they are fairly easy and very fast growing. I'll do some nutritional tests in a few weeks when I grow enough. Also, would it make sense if I'm using them to culture them in a larger container and just top-off the water with freshly-microwaved water instead of generating a new culture? (*next experiment that I will be doing once I find a good container*)
 
Culture Guide:
1. Add 2x Guillards F/2 formula per 100ml of culture water 
2. Heat in microwave until 212F has been achieved
3. Allow cooling until room temperature
4. Split algae into containers
5. Run airline into culture with a mild-turbulent flow
6. Have warm lights on the culture running at 22 hours on, 2 hours off
7. Split every day or two, or until the jar has a light green tint to it
8. Repeat 2-6 for every split

Well, might as well update this again. I didn't split a culture for two weeks to see what happened. It got VERY green, almost to the point of where it looked black. Tried splitting it yesterday morning, and this morning it looked like a healthy green culture. These guys can clearly take some laziness by their masters!

Culture is still doing good, and now that the species data was approved, time for some reports (thanks Matt!) I'll get some photos today for the continuation report.

So, I still need to get those continuation photos for the report. Would just a picture of the jar with the algae in it do the job? My Celestron 44340 broke, and the replacement will be here sometime, I have no idea when since it is on back order apparently!

Micro pics are best but get what you can.

http://www.fishtalpropagations.com/#!home/mainPage
"Making captive breeding easier."

Ok just got the microscope in yesterday, I'll have the photos up by the end of the weekend

Does this Dun. turn red at any point, like heam. does when stressed?  I know Dun. salina makes a red pond... 
 

SF Bay... ponds where SF Bay Brands used to harvest artemia before the landswap between the state and Cargil.

No, I have not experienced any red Dunalia at all. When it crashes and stressed, the algae just sticks to the walls and the rest of the culture is either clear or yellowish (Iso is on a floor above it, but haven't noticed any contamination when the culture is doing well.)

Well, we're not sure if we have Tetraselmis or Dunaliella, so unless I can borrow a microscope from my school to actually figure it out, I'm going to order a new culture.