How long does a culture last?

wildeone · 06-04-2006, 09:46 PM

I ordered a 250ml culture to start phyto growing in 2 Qt jars. The gentleman at www.seahorsesource.com said 250ml would start about 30 gallons. Is that about right?

Can i store 2/3 of the culture for later use?

And if so what is the best way to store it?

Thanks!

**AlgaGen** · 06-04-2006, 11:52 PM

What spp did you order? The amount used in an inoculation depends on the system..one big issue being light intensity. We use alot of light and so use heavier inoc volumes. The guys at seahorse probably use that much, they test everything before making any recommendations..they are good people.

The 250mL starter is hi-density and will go along way. Some spp store in the refrig better than others. Ultimately, I would recommend putting it in the system and using it. If you do not use all of it right away, I would recommend (again depending on species) aerating it and weekly taking volume out and replacing it with an equal volume and new nutrients.

What some people do is take the initial inoc and put it onto nutrient agar plates, which will store in the refrig for months. But storing liquid cultures (live and remaining healthy) in the refrig for any period of time can be tricky.

If there is anything that is not clear or other questions, please let me know.
thanks

wildeone · 06-05-2006, 10:16 PM

Sorry for the lack of information. I ordered the Nannochloropsis oculata. I guess maybe I jumped a little to soon on my order as I am a lil unclear on a couple of things. BUT I am a quick learner and I can see you are VERY willing to give me a hand so here is what I have so far.

I have setup four two quart rubbermaid containers with straight RO water. I know I need to get the right specific gravity of sw in there, I just need to look up the right SG level (I think 1.019) that I need, Rob mentioned the appropriate level in his video.

Now I have a 48" standard flourescent light that I plan to put behind the containers for light and each container is hooked to an air line hose.

I just don't know how much I should put in the first container to get started. I also am not sure but I have read here that Miracle Grow Liguid will work as a nutrient. What do you recommend as a nutrient?

Last but not leastI think that there is a way to check the density of the culture to gauge maturity. If that is true, can you tell me what you would recommend to check the density?

**AlgaGen** · 06-05-2006, 11:09 PM

W-,

I went through this before but am not sure how to access it so...Miracle gro is a terrestrial plant food. It will work for marine organisms but there may a difference in trace elements. For instance too much copper, a common trace metal in both terrestrial and marine nutrients is detrimental to corals. The typical media is called f/2 and was developed by Dr. Guillard specifically for marine phytoplankton and there are variants for specific species. We use hydroponic nutrients because they dissolve well and we cut back even further on certain trace metals. We get very good growth. I would be willing to send you some if you cover shipping..we can go over details if you send me a message.

As far as a starting inoc..there is an optimal number of cells to put into a new system and that depends on the species. In general if you add 1/10 by volume that would work very well. The cultures we sell are very dense and so less is needed. Regardless, I would definitely tinge the water so that there is a definite green color but still somewhat translucent. I think Rob covered it nicely in his video.

In theory algal growth follows a bell shaped curve. Initially there is an adjustment period, depending on strength and quality of the inoc, light intensity, water quality, etc that can last an hour to a day or more. If this adjustment period lasts longer than a day (no growth observed), it might be best to start over. The adjustment period is called the lag phase. The culture will then enter into an exponential phase where growth is steep, cell health is great and lots of good lipids and proteins are being produced by the cells. As the population gets heavier and nutrients become limited, growth slows and enters a stationary phase. Depletion of nutrients and overcrowding can cause a culture crash (so can ciliates, etc..). The way to determine the cell cycle is by counting with a hemocytometer under a microscope and plotting cell# against days (time). Quick 'n Dirty, one can assume under optimal conditions that lag lasts a couple of hours then expo phase initiates. A culture may get 1-2 days of this fast optimal growth and by day 3-4 growth slows but you will have a nice looking dark culture. By day 5 you can be into stationary phase. Stat phase is characterized by slow growth and in many cases the production of lipids, neutral lipids, that are not AS good for critters as the PUFA/Hufas associated with healthy younger cells. If you are asking when would be a good time to harvest? I would recommend day 3-4 in an optimally performing system...depending on the species.

I hope this helps.

wildeone · 06-06-2006, 08:03 AM

Wow, great information. I appreciate all the help, and the nutrient offer. I will PM you right away as my culture will be here tomorrow.

Thanks!

wildeone · 06-06-2006, 08:35 PM

So, by my math I am using 2QT containers.
2 qt = about 1.9 liters.
1.9 liters = 1900 ml.
1/10 by volume I am assiming is a 10:1 ratio so 1/10 of 1900 ml is 190ml.

So if I understand this I need to add 190ml for my starting inoc. That seems like alot since seahorse source said they use 250ml to make 30 gallons, even with better light intensity. I am not questiong your answer, I just want to be sure I am doing my math right and understand what you are telling me.

Thanks!

BTW I think I found the link to what you were referring to earlier. Phytoplankton

**AlgaGen** · 06-07-2006, 02:28 AM

A rule of thumb is to inoc 1/10 by volume (with typical starter) but our starters are fairly thick and so you can use less...going on SHS's experience alot less : ). We use about 400mL to inoc 60L but our light is intense. I think your math is right, there is a bit of flex based on the system and starter density. You can follow SHS or inoc a few containers at diff densities and see what grows best. What I should say is that for all species there is a specific cell number/mL that is optimal for inoculation purposes. If you hit less the culture will not grow as well and if you go over then you potentially waste inoculum but not hurt the culture. So you perform a cell count on the inoc and get a cell density per mL, then do the math to figure out the dilution and how many mLs are required to hit that magic number.

Sorry for any confusion

wildeone · 06-08-2006, 08:43 PM

Quote:

If you are asking when would be a good time to harvest? I would recommend day 3-4 in an optimally performing system...depending on the species.

When you say harvest, is that harvesting to feed the tank, harvesting to split the culture, or both?

**AlgaGen** · 06-10-2006, 01:37 PM

Both. If you look at harvesting as a way of keeping your culture healthiest, fast growing, etc you will constantly be taking product out of your system and meanwhile keeping your culture in a realtively healthy state in it s growth cycle.

If you inoc litely, then 3-4 days from start with nanno may be too soon, perhaps 1 week would be better, but after that first harvest and refill I would imagine every 3-4 days would work well. There is some room to play here.

Let me know how it goes.

wildeone · 06-20-2006, 09:43 PM

Just wanted to let you know that all seems to be going well with the cultures I am growing. I am producing about 2 gallons a week and everyything is going well. Iam going to get some of you decapsulated Brine Shrimp and get started on that phase next. Thanks!