It's not easy being green (Phytoplankton Q and A)

Alright everyone,
 
Andy has asked that I help out with discussion in regards to phytoplankton culture as it is more of a strong suit for me (that and I love chattin about it).  I know there are a ton of questions, answers, basics, and well.... detail devils if you will.  In any case feel free to bring in questions and I will try to find the answers to the best of my ability.  I really hope that some of you will share your own observations as well because there really is no right or wrong way of doing things!  Many of you know my interests and background but for those of you who do not I'll give you a quick intro:
 
My name is Joe (and I work in a button factory....)
 
I have around 6 years of experience in the marine aquarium hobby with the majority of it (5 years) spent ignoring the aquarium and spending most of my time playing with plankton.  I truly love plankton, algae, copepods, rotifers, cilates, bacteria... it amazes me how much life is in such a small package.  Living inland I have had to deal with obvious problems when trying to find new species to play with but I have learned to make due ordering plankters from educational supply shops, hobbyist supply shops, and spending a great deal of time emailing and calling people begging for specimens.  In no specific order and not complete list, some of the species I have played with are:
 
Algae:
Nannochloropsis
Dunaliella
Gymnodinium (various)
Periodinium (various)
Isochrysis
Tetraselmis
Chaetocerous
Oxyrhiis
Rhodomonas (one of my personal favorites)
Chlamydymonas
 
Zooplankters:
Tigriopus californicus
Tigriopus japonicus
Parvocalanus sp.
Pseudocalanus (various)
Tisbe
Acartia tonsa
Oncea venusta
Mysidopsis bahia (now americamysis)
Gammarids (various)
Brachionis
Pseudomma sp.
Siriella affinis
Siriella aequiremis
Heteromysis sp.
Australomysis
 
While it may seem like a very long list, I find myself wanting to try more and more species.  Do bear in mind I am by no means an expert and do experience quite a large rate of failure in my cultures.  Each failure results in me examining what I did and hopefully improving my methods and skill (though it does not always work that way).
 
I will hopefully be taking part in this workshop more to learn as well, as I have very limited experience with Apocyclops and cannot wait to see what the future holds!!  When things settle down (just got back from the workshop and have a lot on my plate) I will try and post up a general primer on algal culture and some images of my culture stations to illustrate how I keep the green stuff green, the red stuff red, and the brown stuff brown.  Until then please feel free to post questions and I will attempt to answer them the best I can!
 
Pelagicly yours,
 
~J
 

Pelagically yours,
~J      

Hi Joe,
 
Just a few questions. 
 
I'll be recycling soda bottles to culture the phyto in, which method(s) can I use to make sure they are sterile? 
 
Once I get my cultures going I will want to keep a back-up crash recovery sample, how much should I keep in the fridge and how long is the shelf life on the sample?  Does it vary by species?
 
Last one for now, how much air flow should I be pumping into my culture vessels?  Again, does this also vary by species?
 
Thanks,
Joshua
 

 
I typically do a very good hot water rinse and sometimes a vinegar soak followed by a drying and then a 30 second microwave run.  If you can get your hands on sodium thiosulfate (available at a ton of places including florida aqua farms) you can do a bleach soak and wash followed by a rinse in the thiosulfate mixture.  *MOST* people do this as it works on a commercial scale at relatively low cost.  This works well for cleaning and re-use as well (you may get 3 or 4 culture runs per bottle before they foul, you can wash and re-use nearly indefinately with a brush and this method)
 

This does vary by species however it is safe to suggest that one can get 10-12 days of good (<70% viability) cell life with most species.  Bear in mind most of the benefits of using live include the nutrient uptake of the live cells as well as it's ability to continue to reproduce in the target culture until eaten / out competed.  By refridgerating we put these cells in a stasis of sorts and reduce the effectiveness of these functions.
 

 
This varies widely by species... motile alga require much less aeration to keep the cells in suspension wheras non motiles such as nanno require more.  *HOWEVER* we also have to worry about gas turnover within the culture.  Since most cultures are done in jugs and vessels with low surface to air ratio's higher aeration is typically required.  I do a bubble count... 3 per second or 90 per minute is my start point and I adjust from there.  Always best to run multiple cultures and observe.  Eventually you will establish a *knack* for setting bubble rate.  Another real good way is to set the bubbles on one side of the culture and watch it rotate through the vessel.  60 rotations per minute for Nanno is probably the fastest you will want, whereas Iso you will want a little bit less (I set at around 40).  This I have found is probably the best way to set bubble rates.... though it is an undocumented art (Hoff mentions it but not in detail).
 

eny time!

Pelagically yours,
~J      

Thanks, Joe!

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

Here's something that's always been a mystery to me. How much of the F2 do you add to the nanno culture each day?
 
My example.
I just started a new culture to be sure it's 100% pure. It's right at a week old.  I've followed all of FAF directions and have split them into two small containers of maybe a quart each. I have rolling bubbles for the air. Not to fast. I've been adding around .5cc every day into each. Is this to much? To little?

RLTW

180 Gallon Mixed Reef

Then I heard the voice of the Lord saying, "Whom shall I send? And who will go for us?" And I said, "Here am I. Send me!" Isaiah 6:8

Here is what I do:
 
First, for clarification, I use FAF Micro Algae Grow.  The label says to use 1.45 ml per gallon of medium for F/2 strength (NOTE:  There is a typo on the label of the bottle I have that says "4 quarts = 7.8 L", but aside from that, the math otherwise works out).  I assume that when I split my phyto culture, the nutrients in the old medium is all used up.  So, I need to dose the new medium with enough F/2 for the total volume of the fresh medium PLUS the amount of innoculant I am using.  Here's an example (and what I regularly do):
 
I use 2 liter borosilicate media bottles.  I fill them with 1200 ml of used tank water, and then add 300 ml of RO water to bring the total volume up to 1500 ml.  I then microwave that for appx. 12 1/2 minutes in a 1100 watt microwave (enough to get the medium up to 185 F / 85 C) and then let it cool to room temperature.  I will be adding 500 ml of an active, dark culture to bring the total volume up to the 2000 ml, so I add 0.75 ml of Micro Algae Grow.  Here's my math:
 
1.45 ml / gallon of Micro Algae Grow = F/2 strength.
There are appx. 3.78 liters / gallon.
I am using 2 liters of media.
Therefore, 1.45 * ( 2 / 3.78 ) = appx. 0.767 ml, (which I round to 0.75).
 
I use a graduated pipette, and truth be told, am happy if I get the level somewhere between the 0.7 and 0.8 lines.
 
I ignore any residual Nitrate or other nutrients that may be present in my tank water.  Depending on the amount of Nitrate in the tank water, the amount of additional Nitrate can be significant, compared to the F/2 Nitrate level.  Per the CCMP site, Guillard's F/2 media has 1.0 ml/L of a stock solution that has 75 g/L of Sodium Nitrate. The Sodium Nitrate has a molecular mass of 84.9949. The Nitrate ion has a molecular mass of 62.0049. So, 62.0049 / 84.9949 of the 75 g is Nitrate. That means that the amount of Nitrate in fresh F/2 Media is 75 * (62.0049/84.9949) / 1000 / 1000 = 0.0000547, which is 54.7 PPM.
 
Hope that helps!