Phytoplankton Culture

 

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Things you'll need:
 

• Live Phytoplankton Starter culture (You can get starter cultures from various sources such as Florida Aqua Farms, Reed Mariculture or even a local hobbyist)
•  Flexible Airline tubing
•  Rigid Airline tubing
•  Air pump
•  Culture containers (2L soda bottles work great. Drill a hole in the cap to accommodate the rigid airline tubing.  Collect some extra caps that remain undrilled.)
•  Fertilizer (Some people use Miracle-Gro but I prefer Guillards F/2 solution from Florida Aqua Farms.)
•  Clean culture medium (Saltwater with a specfic gravity of 1.015 with fertilizer added as per instructions.)
• Light (a simple shop light or aquarium fixture is fine.)
• Airline gang valves
• Ordinary, household, 5.5% bleach
• Dechlorinator (Some people use sodium thiosulfate for this, I use AquaSafe and it works fine.)
•  Funnel
• Plastic Bottle "crate" (The type that the soda bottles arrive at the store in.  I got mine from a local party store for free by simply asking.  These work great for holding the bottles... I've seen many people make elaborate contraptions to hold the bottles, these are perfect since that is what they were made for.)   
  

 

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Getting Started: 
 
Sterilize the 2L bottles with bleach and dechlorinate. 
 
If you are using a commercially available starter culture simply follow the directions provided. 
 
If you are starting with a culture from a local hobbyist pour the culture into a 2L bottle and add culture medium until you achieve a light green color.

Insert a rigid airline through the hole in the cap, attach to a pump via flex airline and a valve, and start the air  flow, not too much.  Leave the cap on loosely so air can escape from the bottle.

Set in front of the light and wait a week. The culture will get darker, and in about a week's time, you will have a nice dense culture that can then be propagated by pouring a third of it into a prepared bottle as above.  The two thirds remaining can be used to feed your pods, clams, filter feeders, brine shrimp, rotifers, or fish larvae (via the rotifers).  More often, I split the dense culture in half by pouring half of the dense culture into a fresh bottle and refilling both bottles with the culture medium and starting over.  I do this because I can't use 2/3 of the concentrate fast enough and it also makes for a shorter time for the culture to mature.  
   
   
   
 
About non-live phytoplankton preparations:

Concentrated, "non-live" phytoplankton or Instant Algae, can be purchased economically, and has many advantages over home grown live phytoplankton in some applications. For some breeders, live phyto is completely unnecessary, and they rely on purchased concentrated microalgae.  For more details on both types of phytoplankton, see the next page. 

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Suppliers of live cultures, culturing supplies, and concentrated phytoplankton:

www.florida-aqua-farms.com 
www.brineshrimpdirect.com

<message edited by Fishtal on Saturday, November 17, 2012 12:01 PM>

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

Edit:  I had these vids in the wrong thread.

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

Yes, I am culturing Nannochloropsis.
I do not use C02, only air.

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

only Nanno?
I`m cultivated Tetraselmis, 2 week. 7 days - low green color.
I use L1 medium DIY

I do also culture Tetraselmis but it is Nanno in the pics.  BTW, the rotifer videos belong in the rotifer thread, my mistake, they'll be moved.
 
I'm not familiar with the L1 medium.

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

Here's my T-Iso setup:



The bags hold around 6 liters when full.

How are you liking those bags Andy?

I use "Crystal Geyser" natural spring water bottles (though I doubt the natural alpine water contents).  These jugs are nice square 1 gallon jugs that fit nicely on my culture rack.  I run 4 across on each shelf (4 ft shelf) with three twin tube NO Flourescents mounted vertically on the wall.  Using adjustable shelving each one of these units will give me between 12 and 16 culture spaes (3 or 4 shelves).  and run pretty efficiently.  The bottles come cheaply (10 for $10) and I utilize the water in other areas.  By using the disposable bottles I pretty much avoid having to clean vessels altogether.  A bottle will be used on average 3 times before being thrown away (unless it fouls sooner).  I replace the rigid on average every 12th cycle.

On the subject of medium and Co2, I've bounced between a few mediums (and dependant on algae species will change) but have settled into the Guillards available in mass packs from FAF.  It's cheap and effective.  I run Full F strength on my Tetraselmis and Isochrysis, and just below full F on my Nannochloropsis.  I have long since removed my Co2 as I found it simply wasnt necessary for my cultures.  Split Nannochloropsis every 8 days, Isochrysis every 7th and Tetraselmis every 10th. 

Co2 typically raises growth rate too high in my realm and I find that splitting too often not only stresses the cultures but leaves me with a ton of unuseable culture.  My goal is to maintain direct live sources and not have bottles of algae sitting in the fridge.

Cultures are strained through a 250um seive for restarts / seeds, and a 20um sieve for harvests / feeds.  Occasionally I will "pelletize" samples prior to feeding using a microcentrifuge (this allows me to separate medium from the algal cells and will most likely prove to be more useful as my larvicology runs continue.).  The algae is centrifuged for approximately 2 minutes (or until visible separation occurs) the medium poured off, and the cells re-constituted using fresh saltwater.  I'm still working on this method to reduce cell damage but so far it's been an effective means of concentrating the cells in a cleaner culture (removal of the nutrients from the original medium).

and thus ends my nifty brain dump!

my racks look pretty much like this one (which sits behind my main system)

Pelagically yours,
~J      

I do like the bags, though I'm not generally a fan of plastic bags. But in this case since sterility is so important (as well as flow dynamics from crud buildup inside the container), I think they're good.

What do you do to minimize contamination when transferring seed stock to a new culture? I have the narrow bottle neck as you can see. I'm using a sterile funnel, but flow rate is fairly slow and pouring through a funnel creates a suction that's taking any micro-organisms from the air straight into the culture.

I just pour direct through my strainer, while it's not the best approach I dont seem to have too many problems.  I do turn my lighting exhaust fans and such off prior to working with cultures    

Pelagically yours,
~J      

Okay, thanks. So I won't worry too much then.

What size air pump do you guys use?  I have one from the skimmer to my nano that I never used.  It's got two ports and a dial, and says its rated at a pressure of .012MPa, and an output of 4.5L.min!  I'd like to get a setup that would do up to 7x 2L bottles and what I need for Rotifer cultures! My guess is I'd need alot bigger, but I just trying to get a reference.

I originally thought I'd need a bigger air pump too but I found that the cheap little ones do just fine. A couple of gang valves and some air line and you're all set.

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

Tal, I'm looking at the algae disks and fertilizers at Florida Aqua Farms.  I'm going to order Iso and Nanno.  I want to order the dry "Mass Pack" fertilizer (shipping charges to Canada).  I notice I can order "dry or moist" and with or without silicate.  Which do I want?  I notice other things listed too like vitamin c, trace packs, plant fuel... I don't need any of that do I?
 
FAF Micro and Macro Nutrients

Don't bother with the disks for Iso. Try to find a liquid starter elsewhere.

--Andy, the bucket man.
"Not to know the mandolin is to argue oneself unknown...." --Clara Lanza, 1886

Do NOT waste your money on Iso disks from FAF, in my opinion!  I know of nobody who has ever reported any success with them, and I have tried multiple times without any success, and numerous other people have also reported consistent failure with FAF Iso disks.
 
While it is possible to start Nanno from FAF disks, I've seen (and heard others report) that they have been contaminated with Tetraselmis.  The best starters, by far, are from Algagen via SeahorseSource.
 
Regarding the F/2 media, the "Mass Pack" is probably more than you will use in a lifetime.  I have a 1L bottle of the Micro Algae Grow that I have had for about 2 years now, and I still have about 1/4 of it left.  You need silicate if you are growing diatoms.  Neither Iso nor Nanno is a diatom.  If you do decide to start growing diatoms, you can just get some sodium metasilicate, or the "Liquid Silicate Solution" that FAF offers.

Whew!  Glad I asked!  Thanks guys. 
 
Jim, I wasn't aware AlgaGen did mono cultures.  I can't see these on their website, am I blind or is it by request only?  I realize the Mass Packs are a large amount of fert, but I also noticed that one Mass Pack is $20 and ships as 2.2 lbs where the liquid f/2 is $15 for 1L and ships 2.6 lbs.  May as well go for the mass pack.
 
However, I do have one pound each of Potassium nitrate, Monopotassium phosphate, Potassium sulfate, Iron chelate 10%, and Plantex CSM+B.  These are from a short dabbling with FW planted aquaria.  Any chance I could make my own fert with this stuff?

Get Algagen starters from SeahorseSource.com:  http://www.seahorsesource...;category=Foods-Algaes
 
You might be able to make adequate media with your chemicals, but Guillard's F/2 (Micro Algae Grow) is a proven medium that includes vitamins and trace minerals, and is very inexpensive.

I tried both Nanno and Iso from FAF. The Nanno did fine but the Iso didn't.

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

nobody gets ISO to grow from a disc. I know. I tried many times.

 
The trouble is duties and shipping Jim.  I need to order starter from SeahorseSource, fert from FAF, I need some bigger micron sieves (nesting set from BSD?)...ack, one thing from 3 different places.  Argh.  My last order from FAF was $90 in products and $65 in shipping and duties.     I can order AlgaGen and Reed products through my LFS though, so I try to get whatever I can from those two sources.

For the sieves, just buy the mesh and make your own.

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

And you can make Guillard with your NO3 and PO4 plus some aquarium trace and vitamins.

 
Ya?  I will look into this more.  It would be nice to use what I already have.  The Plantex CSM+B is a trace mix:
 
Total magnesium - water soluble chelated magnesium                1.5%
Copper - chelated                                                                    0.1%
Iron - chelated                                                                          7.0%
Manganese - chelated                                                              2.0%
Molybdenum                                                                           0.06%
Zinc - chelated                                                                         0.40%
Boron                                                                                      0.8%
EDTA - minimum content                                                        55%
 

 
I have been considering this.  Order mesh from Amazon maybe...?

he he...
 
https://ncma.bigelow.org/node/58
 
more suitably:
 
https://ncma.bigelow.org/node/79
 
I was playing with K medium for a while as the lower trace metal concentrations really lends itself to a cleaner more useable medium in regards to larviculture
 
https://ncma.bigelow.org/node/81
 

Pelagically yours,
~J      

Awesome, thanks Joe!  Those are not the exact same chemicals I have though dangit.  My chems are KH2PO4 (0-51-34), KNO3 (13.5-0-46.2), K2SO4 (0-0-50).  The chems listed in the f/2 recipe are all sodium based, not potassium based...I bet that makes a big difference.  I also notice they are mixing the recipes with natural seawater, not artificial seawater. Oh, and holy wowzers NCMA sure has a lot of strains available for purchase!  A little pricey though...

Mindy,you only need to buy sodium nitrate and phosphate,weigh 75 and 5 grams and make the stock solutions in destiled water.Piece of cake...

lol I dont buy the average alga from CCMP... errr now NCMA lol....  just the oddballs I like to play with
 
Plankton is sorta my thing...  as far as the water mixture I have not had a problem using most of the formulations with artificial seawater, in fact the only medium I've had issue with lately is with those provided by carolina biological supply, they simply hate artificial SW!!
 
The 75/5 recipe that Louis mentions also works well and it's actually an FAO guideline if your using sodium dihydrogen orthophosphate.  Bear in mind the Guillards formulation is meant to be very flexible and generic and may contain more trace than needed for many species (a lot of formulations are like this).
 
if formulating your own your going to find a fun experimental course... things like sodium metasilicate, Iron chloride, maganese chloride..... eh the list goes on... always fun!!  Just be ready to experiment and change formulations based on species.

Pelagically yours,
~J      

Hmm...maybe I'll just buy the Guillard's.  LOL.  I don't know where to buy these other chems from.  I just figured it would be as simple as mixing up some saltwater and adding some N, P, and K.  Apparently not...
 
Oh, and sorry I was AFK for so long without warning...puter got a REAL GOOD virus this time!

Sigma-Aldrich is one place to get the chemicals from, and you can get some very pure materials, but they are a bit pricey:  http://www.sigmaaldrich.c...=RS&N25=0&F=PR
 
I do wonder what quality/purity/grade you might need when making your own media, since having high level of some trace metals seems to matter. 
 
Since there are so many different DIY projects we can throw ourselves into in this hobby, I submit that experimentation with phyto media should be very low on the list!  I prefer to just buy Micro Algae Grow and be done with it.  It works.  Phyto culture is difficult enough for most people that I don't think that uncertainty about whether or not a DIY media is a source of potential problems should be introduced into the mix, IMHO.
 

I'm with Jim.  I live in Sigma's home town, and its cheaper to buy stuff from FAF.  Also, a lot less work.

Chemicals are best bought with major shopping.  Buying all from one supply house is not recommended unless you have bottomless pockets
 
Don't limit yourself to the major chemical houses either.

 
I second that!  But much like Jim Welsh pointed out, this hobby is full of DIY possibilities we can spend our time and dough on.  As with everything theres a ton of research, trial and error, and just plain time involved.  Buying prepared medium from FAF just makes sense.  That said I would check around locally and see if there are others interested, the Mass Packs are very cost effective  and you can easily split an order with multiple people (not sure how much medium you go through).
 
Then again, it's always good to realize that algae is not an american deal   there are algae banks all over the world you can hit up
 
http://www.phycol.ca/
 

Pelagically yours,
~J      

Thanks for the link Joe.  I didn't realize there were all these "phyco farms" around, nor did I know that this type of place would sell starter cultures to any ole person.  That particular place does have Iso, but not Nanno.  Which is ok because Iso seems to be the tough one to get anyway.  Their prices are the highest I have seen yet, but it would cost me that much in shipping to buy from the US anyway so I guess it doesn't really matter.  Apparently Canadian algae is more lucrative. 
 
Regarding the DIY fertilizer, I think you guys are right.  I didn't realize it would be so in-depth to mix the ferts so I will go ahead and buy the ferts from FAF.  They can be sent USPS so that won't be too bad for shipping.  When things have to be sent via courier that's when shipping gets pricey...brokerage fees are ridiculous (for UPS in particular).

google around for "algae banks" the link I posted was just the first thing that popped up under "canada algae bank".  There are tons of sources worldwide for many of the species we hobbyists want (iso, nanno, dunaliela, etc.) you'd be amazed what you can find when you break down the other uses and track along those lines.  i.e. biofuel hobbyists, university biology supplies, and so on. 
 
Algae banks are typically going to be your most expensive sources but also your most isolated and "axenic" cultures (very clean).  the biofuel sources are going to range in accuracy and cleanliness depending on how dedicated the supplier is (much like the traditional aquaculture / reef hobbyist sources).
 
I tend to only go to the algae banks for odd strains that arent too common, everything else typically comes from FAF or carolina biological supply as both sources are low cost here in the US.
 
Important to have your ducks in a row though before you order the algae itself, especially if your paying a premium to an algae bank or for customs.  I've gotten impatient and stupid more than once and lost a culture due to it showing up prior to me having my culture rack ready.  Scrambling last minute is always a bad thing!

Pelagically yours,
~J      

Thanks Joe, I do have to set up my rack first!  Maybe today or tomorrow....?

Could some of you please comment on temperature?  I see "temperature tolerant" referred to sometimes, but what is the preferred temperatures for Nanno, Tet, C-Iso, and T-Iso?
 
Fwiw, Googling found a fert recipe of 2 liter saltwater, 5 mL liquid Miracle Grow, and 1 mL Kent Essential Elements.  I'm using this as I await my FAF order to arrive. 

My Nanno cultures were kept at room temp so it varied a bit. The light was close too so that kept it a little warmer than ambient temp.

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

What is room temp in your fish room Tal?  I try to keep mine near 80F so my heaters don't have to work as hard.

It varies by season. 70's and up.

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