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Thread: Culturing Phytoplankton

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    Master Reefer THEJRC's Avatar
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    Quote Originally Posted by V View Post
    I few of us have done some killer posts on phytoplankton keeping , can we get procedure on to these pages please
    Procedures for Phytoplankton culture – Generic culture methodology
    This procedure assumes use of seed cultures in disc form from Florida Aqua Farms and Guillards F formulation “Micro Algae Grow” also provided from Florida Aqua Farms. This procedure is written as generic as possible with aims at general culturing of species such as Nannochloropsis, Isochrysis, and Tetraselmis. Adjustments to procedure for varying species, culture sizes, and environments will most likely be desired.
    Topic One: Culture Medium Preparation
    For the sake of this article, culture medium is described as prepared salt water with the addition of Guillards formulation at any strength.
    It is best to prepare culture medium immediately prior to starting / splitting cultures, while some keep prepared culture medium on hand in storage containers there is substantial increase in contaminate possibility as medium sits. This method allows for preparation at different strengths as needed.
    Use RO (or better) water and desired salt, mix to 1.018-1.024 specific gravity or 23-28 ppt. salinity, it is important to remember that the salinity is a better measure of salt content as specific gravity measurements are affected by temperature. Once salt water is prepared store in vessel that can easily be tapped such as a water jug with a petcock, you can prepare this ahead of time, it is merely the addition of fertilizer (guillards) that we wish to avoid until the last minute.
    When preparing culture medium for starter cultures I recommend using full F strength, this is achieved by adding micro algae grow to your culture at 2ml per liter. For split cultures, use F/2 strength achieved by mixing 1ml per liter. If you expect a longer split time, or are running CO2 injection you may want to mix at full F strength to avoid depletion of nutrients in the culture.
    Utilize a syringe to add the Guillards directly to the culture vessel and be careful to avoid allowing the syringe to touch the culture or culture vessel as cross contamination will occur. When culturing multiple species, it is a good practice to utilize multiple syringes and formula sources (one for each species) to further avoid cross contamination.
    ~J


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    Master Reefer THEJRC's Avatar
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    Topic Two: Culture Vessels
    Culture vessels can play a large role in success of species growth. When starting cultures it is best to utilize a smaller vessel (8-16 ounces) and through splits slowly build up to the targeted final vessel. For this document I will explain my vessel methodology and recommend that everyone examine their own needs and tune to suit. I utilize disposable vessels to avoid the need to sanitize and clean them. If you wish to utilize re-usable vessels I recommend examining the sterilization processes documented by Rob Weatherly at the Talking Reef. Talkingreef (coincidentally this whole document is being written due to a thread posted there so you are probably already here!).
    For starter cultures I utilize soda or water bottles in the 8-16 ounce range, after draining / drinking the contents I wash the bottle thoroughly using Alconox soap (available in powder form at Florida Aqua Farms, it is important to note that I use the Alconox soap to clean all utensils and have had excellent luck with it. I use a ¼” drill bit to put a hole in the cap of the vessel for air supply (more details under Air Supply Considerations).
    At the first “split” date (8-12 days after culture start) I move the entire contents of the cultures to a 1 Liter soda bottle prepared following the same procedures. For splits from the 1 Liter I move to either a 3 Liter or 1 Gallon water jug (available at the grocery stores with water of course for around $1 each) which I have adopted as my standard culture vessel size. If larger cultures are needed I will move from a 1 gallon water jug to a 3 gallon carboy, and from the 3 gallon carboy to a 5 gallon carboy. For most the 1 gallon or 3 Liter vessels will work fine unless a great amount of culture is needed. I will re-use vessels for 3-5 splits before discarding and replacing dependant on how fouled they are.
    After medium has reached its final “grow out” vessel during splits I will harvest approximately two thirds of the culture and replace with the same amount of prepared salt water. Guillards is added to the culture after the refill and prior to capping and placing back on the rack.
    ~J


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    Topic Three: Air Supply Considerations
    A great deal of success or failure will hinge on the air supplied to aerate the vessels. Most algae’s cultured are non motile and will not stay in suspension in still waters. With the advent of introducing air bubbles into the water we also allow ourselves a great possibility to introduce contaminates that may kill or compete with the algae for nutrients so it is important to have as clean of a supply as possible. I typically follow the following procedure.
    My air pump is placed above the culture vessels; this helps to avoid “bleed back” of air or water in the event of a pump outage, one way “duck” valves are also utilized as a second method of bleed back prevention. Separate pumps and manifolds are used for each species of plankter cultured to help isolate cultures from each other and avoid cross contamination. Small airline filters (available at any pet/aquarium shop) are installed either at the main supply to the manifold or before each culture (dependent on need) to help filter out larger contaminations pulled in from the air.
    I replace the rigid tubing used in each vessel every two vessels or if excessive fouling of the tubing has occurred. Airlines are replaced in full every 6 months and should a crash occur from the same feed line twice in a row that line will be replaced.
    To avoid using too much (or too little) air, watch and “time” your cultures turnover rate. Do this by tilting your rigid tubing to one end of the vessel and count how many revolutions your water does within 15 seconds, multiply that by 4 and you now have your turnover per minute rate. I like to keep this between 60 and 90.
    ~J


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    Topic Four: Lighting
    Lighting as well will play a huge role, without delving into spectrum and multiple species needing multiple spectrums I will throw my general considerations out as one could easily spend a lifetime delving into what specific Kelvin each species of algae prefers.
    I vertically light my cultures; this allows me to set up quite a large amount of cultures in a compact space. I use four 4 ft fixtures mounted vertically to the wall in an even spread behind a 4 foot shelf unit. This gives me 4 shelves to set cultures on with each shelf having a standard capacity of 4-6 culture vessels. For lights I use normal output T8 florescent bulbs in the 5400 Kelvin spectrum. I have been known to change spectrums for other species however have found that this is the most versatile (and easily obtained) bulb. Keep cultures a minimum of 2” away from bulbs to avoid heat problems. Light cycle is another arguable variable but I have found a simple 16 on 8 off cycle has been most versatile and productive.
    ~J


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    Topic Five: Split Timing
    Split timing is pretty simple to figure out but can be a thorn in the side for many. The goal here is to keep cultures as dense as possible without allowing them to outcompete themselves. I will avoid a lecture here and recommend that anyone serious about culturing should read the Plankton Culture Manual released by Florida Aqua Farms as it delves into the theories and ideas of keeping cultures in “logarithmic” state for higher nutritional value and cell counts.
    When starting cultures, split between 8 and 12 days, it is fine to give the culture a little extra time at this stage. For subsequent splits it is very important to keep as rigid and repeatable schedule as possible to avoid shocking the cultures. Subsequent splits should typically be performed every 8 days, if extended time between a particular split is necessary it is a good idea to add a small additional amount of Guillards to keep the culture from depleting nutrients.
    While many preserve harvested cultures through refrigeration or freezing I prefer to utilize the culture as soon as possible to maintain its viability.
    ~J


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    Broad level: Step by Step guide and additional information

    1. Prepare appropriate amount of saltwater to 1.019
    2. Put a few drops of the saltwater in the culture starter Petri dish and set for 12-24 hours
    3. Fill small (8-16 ounce) vessel with saltwater and add approximately 1ml of Micro Algae Grow
    4. Use sterile swab to swab culture from dish into vessel
    5. Set vessel on culture rack with air supply at 60-90 circulations per minute
    6. Run 16 on /8 off light cycle for culture, visually inspecting color daily
    7. On day 10, transfer culture to 1 liter bottle, adding 1ml of Micro Algae Grow and fresh salt water to top off
    8. Allow culture to grow for an additional 8 days
    9. Transfer from 1 Liter bottle to 1 gallon bottle, adding 3.75ml (4ml is fine) of micro algae grow and topping off with fresh saltwater
    10. Let grow for 8 more days
    11. Harvest approximately two thirds of the culture, refilling the vessel with fresh salt water and adding 3.75ml of micro algae grow
    12. Repeat steps 10 and 11, carefully monitoring vessel, replace vessel every 5 splits or when fouling occurs, replace rigid tubing every time vessel is replaced.
    I know it’s long winded and probably glosses over a ton of missed stuff but that’s the gist of how I run my cultures. I do perform some advanced tasks on other cultures such as CO2 injections and working with other strange species with different temperature / lighting / fertilizer requirements but those topics are beyond the scope of this document. If you wish to learn more I would recommend the following (which I use as constant references):
    The Plankton Culture Manual – Book by Frank Hoff and Terry Snell from Florida Aqua Farms
    For sources of other algae’s check out the University of Texas algae bank UTEX - The Culture Collection of Algae
    For other supplies (including more algae cultures) head to Carolina Biological Supply Carolina Biological Supply: Science Supplies, AP Kits, Chemistry Supplies, Microscopes
    For other culture medium formulas, more cultures, as well as a whole wealth of information take a good look at the Provasoli-Guillard National Center for Culture of Marine Phytoplankton http://ccmp.bigelow.org

    and my apologies for the thread spam he he!
    ~J


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    Nice bud, very nice.

    Read every word, & if i was starting out, id be able to follow it no problems



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    Master Reefer THEJRC's Avatar
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    eh you asked for it... it's your fault...

    The cool thing is, if I ever get a wild hair I can edit it and clean it up someday...

    hrmmm... someday
    ~J


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    sunny d polyp (02-22-2010)

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    ha-ha, still weirder than I though!



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    Apprentice sunny d polyp's Avatar
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    JRC, my rodi's are doing much better since taking your advice and using synthetic water @ 1.020 and innoculating with .04ml of sodium hypo. I have 4/2 liter vessells and remove 800ml's of roti's every other day from each vessell. I'm up to 2/gals of nano, 3/gals of tet and 3/gals os T.iso. I want to try Acartia again now that I'm having better luck with roti's, all the culturing has turned into a sub hobby if that makes any since.
    Also, a person who gets phyto from me wanted to know why he couldn't add phyto every day instead of every other day as long as he only adds half the dose that is recommended daily (he adds 1 cup per 55/gal every other day and has a 210).
    I may be setting up a 180 soon because the 75/gal is busting at the seems and I have no more room. Please let me know your recomendations on phyto additions every day and what would be a good size tank for Acartia (5/gals)?
    Don't sweat the small stuff...

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    Master Reefer THEJRC's Avatar
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    as far as phyto additions every day it's a salt to taste type of question.... I've been known to go through phases where I toss gallons of the stuff into my system at a time, you just have to realize the potential ups and downs. Consider this as a scenario... without regarding tank size or filtration or any of the milliion other factors... if I were to add microalgae to my tank each day upping my addition incrementally over time (so for instance 100ml the first week, 200ml second week, 300ml third and so on) eventually I hit a critical mass of how much I can actually add (for various reasons, water quality, production, etc.) Lets just say critical mass is 1 liter per day... now... my tank and it's inhabitants have slowly adjusted to the new food source meaning many of the critters that feed on microalgae have begun to spring up in population to the degree that there is sufficient food.

    What would happen if I were to suddenly stop feeding microalgae? What happens to the starved population? is it possible for a die off from such consequences to be massive enough to damage water quality worse? And also consider, what happens to the nutrients that the live algaes are feeding on?

    So the reality is as far as adding microalgae (or anything else) to your tank the real answer is... what ever you are comfortable with maintaining. Bear in mind that the nutrients we add by way of fertilizer is also adding many trace items that should probably not be in our tanks. Though, the typical water change process helps alleviate a lot of this risk.


    odd way to answer the question I know... but in the realm of dosing phyto there isnt any "phyto test kit" so it's hipshot!
    ~J


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    Phyto additions...

    Good analogy and it would make since that if the fauna was not increasing exponentially in relation to the increased additions of phyto sooner or later it would hit the proverbial breaking point, like nutrient saturation.

    My next question would be, how much phyto would I have to add and how often (I know there had to be a study on this somewhere) to keep a decent rodi population going in my fuges, do they eat anything else (marine snow?) or would they parish with their high metabolism from starvation?

    I talked to you about Acartia T last year and you stated they are among your favorite copes, would they be better suited for my fuges because of what they eat? If I could keep a decent pop of them going in the fuges I wouldn't have to worry about culturing them externally.

    Thx for your help JRC.
    Don't sweat the small stuff...

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    heh I just noticed this was sticky... I made a sticky thread I'm so proud

    thank god I'm so cool I can deflate my ego at will, otherwise it'd be tough to get through doors.

    on feeding studies there are a ton!! A plethora of plethora's even, the problem is the studies are done counting cells per ml and then worked up so unless your prepared to spend a lot of time with a microscope and a hemocytometer... well your in hipshot mode once again.

    on the question of rotifers eating other things there has been many many successful runs using yeast based foods, ground rice, egg yolk, you name it!! Hoffs "Plankton Culture Manual" goes into many of these. I have been known to use brewers yeast from time to time and they take it quite well. The downside is that water fouling tends to occur pretty quickly with this method. Moving on to the Copepods... well Acartia are rarely found "thriving" in marine aquaria, their appetites are huge and they consume a LOT of microalgae. They will also take some other foods (going back to the yeast) as well as feed on some detritus but we need to look at where the species spends most of it's time. Calanoids spend all their time swimming in the water column so detritus unless in suspension (think in terms of bacterioplankton here) is a non food group. Harpacticoids like T. californicus spend time on surfaces so more detritus feeding. Acartia tonsa however is fairly easily cultured!! lesse here...

    Producing Acartia tonsa

    should get you started... while vague it should give you a real good idea! one of the real nice things about Acartia is that it lays resting eggs... very nice feature for aquaculture!

    in edit, forgot to mention but the original discovery of Acartia tonsa was in the north sea, which also hosts a lower temperature. The species has of course dispersed to many other areas and adapted to warmer waters. This should be considered a point to ponder when looking at temperate vs. tropic species. Where the initial specimens for culture were harvested makes a large difference!
    Last edited by THEJRC; 02-22-2010 at 10:58 PM.
    ~J


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    Unfortunately temp was my demise with tigriopus, I'm lucky to keep my phyto cultures below 83/85F and that proved fatal to the tiggers. Maybe I should be content with my roti culture and quit being so copepod greedy, hehe.
    Thx again JRC.
    Don't sweat the small stuff...

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    haha, yup, your finally sticky. welcome to the mile high club bud!



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    Master Reefer THEJRC's Avatar
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    hah just a mile? I'm going down, out here on the other side of the planet we keep reefs at 6800ft for you metric types... a mile is only 5280 so 1609 meters vs. 2072.... give or take a few points...

    hows the air down there!
    ~J


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    Apprentice sunny d polyp's Avatar
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    Has anyone here ever used the 40lb bags of Morton salt (pool) for roti's, brine shrimp or phyto, I'm converting our pool from chlorine to salt and it's cheap. I was going to make up a culture of the salt and ro/di and add whatever (cal, mg and buffer) to set it to the right parameters and try it, what do you think or is this not a good idea?
    Don't sweat the small stuff...

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    I would pull down a listing of whats in the salt as far as trace minerals and such, at first thought I would caution against it however it may be a viable alternative. Bear in mind with roti's and brine as well as most larvae less trace (especially metals) is better. As far as phyto, most of the available salts on the market are formulated based on old data with algae as the target species (interesting thought huh). Your getting deeper into chemistry at this point, which can make the whole culture thought much much more difficult.
    ~J


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    Quote Originally Posted by THEJRC View Post

    hows the air down there!

    Pretty good, thanks for asking, we have better quality due to the "other" island nations being swallowed up by the rising sea levels



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