How do I know if I’m adding too much f/2 to my phyto?

[csb123]

I’ve been culturing nannochloropsis for a few months. I’ve finally got my culture to not crash by improving my hygiene.

Currently I’m adding 12ml f/2 to culture a gallon (1 gallon of phyto plus 1 gallon salt water.) The culture doesn’t get anymore dense after about 5-6 days, but I’m concerned that there may be excess f/2, before I put it in the refrigerator.

Your opinions are appreciated!

 

[ichthyogeek]

So....12 mL f/2 to culture 2 gallons of phyto? OR 12 mL f/2 to culture 1/2 gallon of new salt water added to 1/2 gallon of phyto culture (1 gallon total)?

Something that you could do to test if you have excess f/2, would be to thin out the culture and see if it gets denser again. I don't know if Nanno does the whole allelopathy thing, but I know plants do. By taking out water, you would (theoretically) be taking out any allelopathic chemicals the Nanno is producing, and diluting the excess chemicals with more water. Although there're also other factors that will also change (light penetration into culture, etc.).

You can also test to see if you're adding too much/not enough f/2. When I was working with Rhodomonas, there was a "set point" where past a certain level of f/2, there were diminishing returns in terms of algal density. A horizontal asymptote, if that makes sense. With the Rhodo, I started off with standard fertilization of the lab (85 uL f/2 added to 300 mL total volume). I then set up an experiment of varying only the level of fertilization (127 uL added to 450 mL Total Volume vs. 169 uL added to 450 mL Total volume) and took note of what the cell counts were with a hemocytometer. So you could try seeing if 9 mL of f/2 added to the culture produces similar results as 12 mL of f/2 added to the culture.

Granted, without some sort of fancy gadget to determine the actual levels of nutrients, you won't be able to accurately determine if you have an overly large amount of f/2 present or not, and will have to rely on secondary tests (does algae grow more even when diluted? does changing the amount of fertilizer affect the growth times/density?) to determine if you have or not.

What makes you concerned about there being excess f/2 in the culture?

 

[csb123]

So....12 mL f/2 to culture 2 gallons of phyto? OR 12 mL f/2 to culture 1/2 gallon of new salt water added to 1/2 gallon of phyto culture (1 gallon total)?

Something that you could do to test if you have excess f/2, would be to thin out the culture and see if it gets denser again. I don't know if Nanno does the whole allelopathy thing, but I know plants do. By taking out water, you would (theoretically) be taking out any allelopathic chemicals the Nanno is producing, and diluting the excess chemicals with more water. Although there're also other factors that will also change (light penetration into culture, etc.).

You can also test to see if you're adding too much/not enough f/2. When I was working with Rhodomonas, there was a "set point" where past a certain level of f/2, there were diminishing returns in terms of algal density. A horizontal asymptote, if that makes sense. With the Rhodo, I started off with standard fertilization of the lab (85 uL f/2 added to 300 mL total volume). I then set up an experiment of varying only the level of fertilization (127 uL added to 450 mL Total Volume vs. 169 uL added to 450 mL Total volume) and took note of what the cell counts were with a hemocytometer. So you could try seeing if 9 mL of f/2 added to the culture produces similar results as 12 mL of f/2 added to the culture.

Granted, without some sort of fancy gadget to determine the actual levels of nutrients, you won't be able to accurately determine if you have an overly large amount of f/2 present or not, and will have to rely on secondary tests (does algae grow more even when diluted? does changing the amount of fertilizer affect the growth times/density?) to determine if you have or not.

What makes you concerned about there being excess f/2 in the culture?

I add 1 gallon clean saltwater to 1 gallon of culture.

I’m only concerned about nutrients. I’m riding a fine line of heavy protein in, heavy out, for my corals.

 

[flampton]

Why can’t you run the culture through a sieve and test the supernatent with the hobby kits? My guess is that will ballpark you on nitrate and phosphate. Then compare to the f/2 concentration and you can estimate whether you're using too much

 

[ichthyogeek]

Nanno is a really, really tiny microalgae (I think somewhere around 1-5 uM wide?). The standard sieves available would not work, and using tiny sieves would require using something like a vacuum flask to avoid wasting time, something not everybody has access to.

OP could maybe try fashioning a modified whirligig centrifuge if they really wanted to get at the supernatant. But even with commercial centrifuges, It's really hard to condense all the cells. I tried isolating Rhodomonas for the purpose of attempting to freeze it and see if I could reconstitute it and bring it back to cultures, but without a large (i.e. holds 500 mL tubes) centrifuge, it's nigh impossible to do that as well.

 

[flampton]

I know they're small but this is definitely doable.. Let's say if you go cheesecloth you can easily lock the majority up using five six layers or more properly layered.

Why would you need to spin that many cells down for freezing? That seems odd. Especially if your cultures were saturated could have just added in some glycerol or DMSO, dry ice ethanol bath and throw it in the -80 or liq N2 easy peasy!

 

[flampton]

And if lazy just throw culture in fridge and let settle. The majority will fall out eventually. Haven't looked into it but could also possibly add a floculent

 

[ichthyogeek]

I know they're small but this is definitely doable.. Let's say if you go cheesecloth you can easily lock the majority up using five six layers or more properly layered.

Why would you need to spin that many cells down for freezing? That seems odd. Especially if your cultures were saturated could have just added in some glycerol or DMSO, dry ice ethanol bath and throw it in the -80 or liq N2 easy peasy!

I remain skeptical about sieving out the microalgae, especially with cheese cloth. Hmmm...guess I'll have to try your method out and see if it'll work. The outdoor tub has some sort of microalgae (either synechoccus or Nanno), and I'm supposed to move the fish in tomorrow anyways, so we'll see...

Because the protocol I was using was to make sure that ice crystals didn't form and puncture the cells, and I wanted to be able to get a viable culture from the cells later post-freezing. Wasn't able to see if it worked. I wonder if they're still in the -80....

 

[flampton]

Next time run a quick experiment with just adding cryoprotectant to the culture and snap freeze with liqN2 or dry ice ethanol bath. This will limit ice crystal formation. Then when you pull the tube for culturing warm in a 37degC bath to get the culture past the danger zone as quick as possible.

You won't get the solution anywhere near sterile by passing through the cloth but you'll get it clean enough for a hobby test kit.