nannochloropsis phytoplankton culture (questions)

[phyto4life]

So far I started culturing nannochloropsis phytoplankton indoors using the petri dish method inwhich will be used to add directly to my saltwater tank that is at 1.024

I added a little less then 1/4 cup of rodi water that was boiled in the microwave for 1 min then I let it cool to room temp and added it to the petri dish then placed it under 2 normal output plant light's for 24 hours

where do I go from here?

I read that I should use the q-tip and add the entire dish to 1 liter of water under a gentle rolling boil and leave for 12-24 hours

my question is do I add this to fresh water or saltwater?

because ultimately I want the culture to be saltwater @ 1.020 ppm

and should I give the algae a break for a time period between adding the dish that was being lit for 24 hours to be lit in the 1L with flow for 16 hours?

I'll have more question's as I go

[hillbillyreefer]

I started out doing it that way too. Lasted for about a month. Now when I restart cultures I just swab the top of the petri dish, and swish the Qtip around in the nsw, in a clean container with fert. Turn on the lights and bubble the crap out of it. I use saltwater of around 1.017 for the batches, some of the water evaporates as the brew matures. I've never tested it when it's done, never worried about it. When I need to harvest I turn off the air for a day or two and let it settle. Pour off the top 1/2 or so, collect the rest for use, fill the container back up with nsw, fertilize and turn the air back on. Haven't had a culture crash yet, been growing for almost 2 years. I have on a couple of occasions shut down and replaced all the equipment and restarted though. The phyto is grown in my basement which is cold around 15 C. ATM I run an 18 hr lights on cycle. I have run with 24 hr on and haven't seen any detriment to the cultures. I'm know this isn't the recommended method of culturing, but it works for me and my lazy style.

Now to keep rotifers going, haven't had much luck with them, lol.

[phyto4life]

I posted early. tomorrow at 3pm I will have the petri dish with water under light for 24 hour's

from here you say to add it straight to a salinity of around 1.017 ppm with fertilizer and bubble the heck out of it in which my pump can do because it is set to bubble a 1 gallon

my temp is probably around 70-75F

my question is how much fertilizer and what amount of pre mixed saltwater/ liter/gallon etc to use right away?

the bottle from f.a.f liquid says 1/4 tsp for 1 G @ f/2 level and double that for full strength

should I go full strength fertilizer right away?

and the petri dish says 1 liter for the entire dish to inoculate should I go more or the recommended amount at first right away?

ultimately I'll be doing 1G cultures x 8

I have a second dish so i'm open to what ever suggestions

[hillbillyreefer]

I posted early. tomorrow at 3pm I will have the petri dish with water under light for 24 hour's

from here you say to add it straight to a salinity of around 1.017 ppm with fertilizer and bubble the heck out of it in which my pump can do because it is set to bubble a 1 gallon

That is what I do.

my temp is probably around 70-75F

I don't think temp is a large factor, with higher temps you may get faster growth. I'm not sure, never worried about it, researched it or experimented.

my question is how much fertilizer and what amount of pre mixed saltwater/ liter/gallon etc to use right away?

the bottle from f.a.f liquid says 1/4 tsp for 1 G @ f/2 level and double that for full strength

should I go full strength fertilizer right away?

I would, it should help establish the best culture you possibly can.

and the petri dish says 1 liter for the entire dish to inoculate should I go more or the recommended amount at first right away?

I would dilute it, cultures are expensive. They will multiply rapidly. My qtip swab starts take about 3 weeks to become useful feed. When I start them they look like perfectly clear water.

ultimately I'll be doing 1G cultures x 8

I have a second dish so i'm open to what ever suggestions

Start as many jugs as you want with the first dish. Save the second one for an emergency. If you have multiple cultures of the same species going it is highly unlikely you will lose them all to a crash at the same time. You can always restart a jug with phyto from another culture and still have an emergency backup dish. It really isn't difficult. I was overwhelmed by the info when I started too. It really is as easy as you want to make it.

I forgot to mention, I use miracle grow for fertilizer. The one with the smallest first and second numbers I can find. I started out using 5 ml in 2l of culture now I use 3 ml and haven't seen much difference in the cultures under the microscope. I haven't noticed any problems with this fertilizer in the tanks. I have been toying with the idea of filtering the phyto out of the culture water, and putting it into NSW but haven't done this yet.

Hope that helps.

[phyto4life]

thanks

I'll start 2 x 1G going with 1/4 tsp of fertilizer and i'm thinking of splitting it after 4 day's

after that when I split them again I might leave them a little longer and add more fertilizer

do you use a density stick and if so do u know the density I would be going for that will leave vary little fertilizer with the max amount of phyto cell's?

or a shade of green?

[hillbillyreefer]

I have enough cultures going that I run them until I need them. Usually 3 or more weeks. They are deep green in that time. I don't have a density meter. If I'm really curious I'll look at them under the microscope. Someday I might pickup a density meter, but I much prefer to keep things simple. I've too many other things going on to worry about phyto too much.

[phyto4life]

everything seems to be doing good and growing

it's good to know on how dense and dark green a culture can get

[rayjay]

I don't use the disk's any more, I buy liquid starter from ReefCrew.
While I grow a lot of nanno, I no longer use it for my tank. Instead, I feed rotifers to my tank about an hour or more after lights out and see better results, especially with corals like the acans.
I no longer sterilize the water I use for phyto culture but when I did, I used to add about 8 to 10 drops of bleach to a 2L pop bottle, fill with tap water, and let sit for at least 24 hours. Then, I add ClorAm-X (or any dechlorinator) until the chlorine is neutralized and then dump half into another sterilized bottle and top both bottles up with full strength nanno that is already cultured.
It was too much hassel to keep sterilizing the water at the volumes I use now so I just take my chances on crashes and if it happens, just restart.
CULTURING PHYTO THE WAY I DO IT NOW ( http://www.angelfire.com/ab/rayjay/phyto.html)

[phyto4life]

Thanks Rayjay good info

I'm using 1/2 tsp in a 1G of fertilizer but just started it with half a disc and will split the culture when it get's greener in 4 day's then bump up the fertalizer to 1 tsp hoping to go 6 days before splitting on a regular schedule

should I be aiming for 2 tsp in a 1G in the long run?

how long before I split?

[shrimpchips]

Do you guys refertilize after a certain point or number of days? Right now I'm only adding ferts in whenever I do a split.

[rayjay]

Thanks Rayjay good info

I'm using 1/2 tsp in a 1G of fertilizer but just started it with half a disc and will split the culture when it get's greener in 4 day's then bump up the fertalizer to 1 tsp hoping to go 6 days before splitting on a regular schedule

should I be aiming for 2 tsp in a 1G in the long run?

how long before I split?

What kind of fertilizer are you using? I don't use MG as the FAF f/2 is economical and doesn't have all the extras that don't get used up by the phyto, and thus getting dumped into the tank.
For f/2, I use 1ml for every 2.5L of culture but simplify it to 1ml per 2 litre bottle for my stock cultures because they will never be used in a tank, only to start new larger cultures.
The problem I have with people using MG, or with overdosing f/2 is that we can't test for expired nutrients because our colour based tests are rendered pretty well useless by the phyto colours.
If you have nutrient that is NOT used up, then it will be added to the tank when you add phyto to the tank, and that fuels nuisance algae in the tank.
For f/2, I go by the recommended dosing and find that it lasts my cultures 8 to 10 days when my density gets to 3.5 cm on the density stick and is ready for harvest.
Occasionally I find a culture going a bit lighter and will add a bit of f/2 to see if it can be salvaged. Works sometimes, but I make sure that I let it go for enough days to cover what I've added.
If you split sooner than needed, you increase the chances of a crash as I've found that the stronger the culture at any time, the less likely it is to crash. Not a guarantee though as bacteria can invade and wipe out anyway.
If I were to dose 2 teaspoons, that would be about 10ml of f/2 so it would do for 25L or approximately 5 1/2gCDN or 6 1/2gUS.

[phyto4life]

I'm using a new bottle of F.A.F liquid f/2 fertilizer

I'll stick with this over miracle grow for the same reason due to adding it directly to a reef tank and I'm only culturing 6-8G of phyto a week

my brother-in-law has a bottle of FAF liquid f/2 that is 6 months old and used once should I risk using it because it will probably be another 3 month's or so before I use up the new bottle I just bought?

do you store the liquid fertilizer in the fridge or is it ok to leave it in a cool dry place?

8-10 day's to leave culture to a density of 3.5 cm on the density stick using 1/4-1/2 tsp of liquid f/2 fertilizer

does this sound close to what I should be aiming for?

do you turn off air when you measure the density?

do you use 16 hours of light?

[rayjay]

I light 24/7.
To measure the density I let it sit until the surface is completely motionless and I shine a 30 million candle power light on the stick and read the centimeters where I can no longer discern the spot on the stick.
Time to get to a density of 3.5 will vary with starting point and light duration and light intensity.
If the light isn't sufficient to produce the required density in about 8 days, 10 at most, I pour into another sterilized container through a 53 micron mesh and put back under the light source again.
If I don't clean out the container, any settlement can cause a crash before the culture gets to where I want it to be.
I buy mass packs that make four litres of f/2 and I store it in the refrigerator at 36°F. It has a limited shelf life according to FAF but I have gone over the limit quite a bit and many times, without any noticeable problem.

[phyto4life]

cool I'll put the fertilizer in the fridge and not use the old fertilizer

I'm not sure if the 1 liter I bought has a date on it. if not how long approx in the fridge would it last?



this is the unit I hooked up it has dual normal output approx 6500K-8000k but closer to 6500K plant/aquarium light from grow lux I believe

http://i458.photobucket.com/albums/qq303/clintos08/Picture153.jpg

is this weak light?

the light is 3" from the container and the container's are 1G

what is the best way to clean a 53 micron screen the one I have is used, look's clean but regular water goes through really slow is this normal?

I just used regular warm tap water on full blast is this enough? or is there something to soak it in?

[rayjay]

I forget how long FAF says it's good for, I think 4 months.
However, I've had some for six months with no problem.
Lighting looks just fine if those lamps are 80w or higher output each.
(8 ft strip?)
Are those 1g US or Imperial measure, or possibly 4L glass jars?
They are great for nurseries for raising reidi seahorse fry if they are.
Where did you get them?
As for cleaning the screen, I guess it depends on what material it's made of.
I have nylon and cloth types. The cloth I put in bleach water for about 10 minutes and then rinse well, the nylon one I put in vinegar for 10 minutes, rinse, put in bleach a couple of minutes, and then rinse well.

[phyto4life]

the culture container's are plastic 1G U.S from walmart for $4 only thing about them I find is there is a slight grove on one side so a person can pick them up on the sides but seems ok so far no trapped large bubbles or anything besides really small micro bubbles that seem to vanish every once and a while

the light's are 40 watt 4" normal output

this is the screen it feel's like nylon:

http://i458.photobucket.com/albums/qq303/clintos08/Picture014.jpg


what kind of bleach? from where?

when I go to clean the plastic containers/plastic pails/spoons etc what should I use to clean/sanitize it?


the culture water:

I'm using rodi and boiling the water after wards is the boiling necessary?
is there anything else to be done before hand

[rayjay]

Too bad on the bottles as I would like to have more than the 12 glass ones I have now.
Where in Walmart were the plastic ones?

Your mesh may not be clogged but because it is so fine it sometimes doesn't allow water to flow easy, right off the bat.
I usually run warm tap water over the bottom and run my fingers over the mesh to "tin" it. This allows water to make better contact with the fine mesh so that it will flow through better.
It won't take much large particle phyto to plug it up though, and running your finger over the bottom of the mesh when it slows up, can help to get it flowing a little better again. Finger has to be clean and sterile if you're doing it with phyto that you intend to continue culturing, but not if you are just harvesting to use.
I do a ten minute bleach every once in a while if I can see a build up on the mesh.
I use the cheap bleach I buy at Walmart in the 5L container, with no additives or perfumes.
To sterilize the equipment, I use IO pails with tap water and about a cup of bleach in them, and let the equipment sit in it at least overnight.
As for culture water, that depends on who is doing it, the quality of the water or just personal preference.
I use tap water and no longer sterilize the culture water but grow more of it to compensate for the crashes. (it's a hassle to sterilize very large volumes)
However, I keep 4 two litre bottles of culture stock solution that I do sterilize the water as I described earlier, with 8 to 10 drops of bleach in a two litre bottle and let sit for a day before neutralizing the bleach with ClorAm-X.
For both my stock and my using cultures, I like now to only harvest one half the culture and replace half new water as I find fewer crashes which makes up for not harvesting as much.
There are a lot of ways people grow phyto and my way works for me but I don't say it's the best or that it is the right way for you.
It started much different but over the years I change methods as I increased the volumes and got older and lazier.

[phyto4life]

I got the containers from the tupperware section in walmart

thanks for all the help. the phyto seem's to be coming along nicely

[PoonTang]

There is a good podcast of growing phyto and rotti over on talkingreef.com

[phyto4life]

thanks, I just checked it out, there is a good podcast on there to about harpacticoids