Preventing contamination when culturing phytoplankton

[Elbereth]

I recently delved into the world of phytoplankton culturing. My first attempt with Nannochloropsis in 500ml plastic bottles started off well but stalled after 3 weeks: it hasn't crashed yet but it's not getting any greener. I did add F/2 fertilizer and it's bubbling to produce a nice rolling boil at the surface. So after doing some research, I suspect my cultures got contaminated either when I prepared the saltwater or when I split them. I'm planning to try culturing in glass jars instead so I can microwave/boil them to sterilize the medium but...

Would it worth using a brewing airlock (https://homebrewjunction.com/produc..._content=sag_organic&utm_campaign=sag_organic) in the vent hole/tubing (the one not connected to the air pump) to make sure no outside air gets into the culture? Or would that be overkill? Would it be sufficient to stuff it with some polyfill/filter floss?

I've read about different ways to sterilize the culture medium and equipment when splitting but I can't help but wonder: isn't it possible for culture vessels/equipment to get contaminated after they're sterilized and before they're sealed? So say I microwave the sealed culture vessel with the saltwater inside in a microwave. Won't I again be exposing the water to contamination when I pen the vessel to add F/2 and phyto inoculant? Or say I acid/chlorine soak the tubing, again isn't there a chance of contamination when it's rinsed off? Maybe I'm being too anal fearing contamination by airborne particles but if marine ich can be dispersed via air for several feet, wouldn't that also be true for other microscopic organisms that might adversely impact a phyto culture?

Any serious advice/tips would be much appreciated! Yes, I know some people get away with just rinsing their containers when splitting and I've seen some people even culture in open top containers. If you're one of those lucky people who doesn't have to sterilize when splitting or has a thriving exposed culture I'm happy for you but that doesn't seem to be working for me.

 

[68 Stang]

I utilize glass jars with a large opening. When I split the culture I pour the entire gallon in a clean glass jar container. Rinse the glass jar with tap water. I then squirt a bit of alcohol on a paper towel. I wipe the entire inside and let it air dry. I pour in a couple of cups of the culture back in with salt and RO water to achieve 1.020. and 1 mill of F2. Each week I follow this same process and have never lost a cultur. Hope that helps

 

[Elbereth]

Thanks for responding! Out of curiosity, do you transfer in a space that's far from any tanks? Because I wonder if maybe that's what I did wrong.

 

[68 Stang]

my tank is in the living room and I transfer in my kitchen.

 

[Elbereth]

I see. I'll note down wiping with alcohol. Thanks!

 

[Elbereth]

Anyone else want to share some tips/experiences?

 

[Pistondog]

3 weeks seems a long time for a culture. A week is max for me.

 

[DopamineKata]

Anyone else want to share some tips/experiences?

The Poseidon Reef Systems kit uses plastic bags inside the container. That way you start with something that's known to be clean from the start.

 

[CK00020]

I use old pretzel containers and got some bags that fit perfectly inside. It’s the same thing as the posidon jars but a little bigger.

 

[Elbereth]

3 weeks seems a long time for a culture. A week is max for me.

I've been splitting weekly.

 

[Elbereth]

I use old pretzel containers and got some bags that fit perfectly inside. It’s the same thing as the posidon jars but a little bigger.

That's a neat idea. Do you have to get a specific type of plastic bag since some plastics leach and aren't reef safe?
Might be a cool way to reuse milk bags actually!

 

[jx.reef]

I've been using fish shipping bags I found off of amazon

 

[CK00020]

Yes they are fish bags, also I bought a light strip and wrapped it around the container then got some black tape and went around it to keep light spill down

 

[68 Stang]

Keep it simple here is my setup

 

[Elbereth]

Keep it simple here is my setup

Is that cling wrap?

 

[68 Stang]

Is that cling wrap?

yes it is, the lid began rusting and concerned me. Tossed and been using cling wrap

 

[Elbereth]

yes it is, the lid began rusting and concerned me. Tossed and been using cling wrap

Nice solution! Thanks for the tip.

 

[Anthony Scholfield]

I recently delved into the world of phytoplankton culturing. My first attempt with Nannochloropsis in 500ml plastic bottles started off well but stalled after 3 weeks: it hasn't crashed yet but it's not getting any greener. I did add F/2 fertilizer and it's bubbling to produce a nice rolling boil at the surface. So after doing some research, I suspect my cultures got contaminated either when I prepared the saltwater or when I split them. I'm planning to try culturing in glass jars instead so I can microwave/boil them to sterilize the medium but...

Would it worth using a brewing airlock (https://homebrewjunction.com/produc..._content=sag_organic&utm_campaign=sag_organic) in the vent hole/tubing (the one not connected to the air pump) to make sure no outside air gets into the culture? Or would that be overkill? Would it be sufficient to stuff it with some polyfill/filter floss?

I've read about different ways to sterilize the culture medium and equipment when splitting but I can't help but wonder: isn't it possible for culture vessels/equipment to get contaminated after they're sterilized and before they're sealed? So say I microwave the sealed culture vessel with the saltwater inside in a microwave. Won't I again be exposing the water to contamination when I pen the vessel to add F/2 and phyto inoculant? Or say I acid/chlorine soak the tubing, again isn't there a chance of contamination when it's rinsed off? Maybe I'm being too anal fearing contamination by airborne particles but if marine ich can be dispersed via air for several feet, wouldn't that also be true for other microscopic organisms that might adversely impact a phyto culture?

Any serious advice/tips would be much appreciated! Yes, I know some people get away with just rinsing their containers when splitting and I've seen some people even culture in open top containers. If you're one of those lucky people who doesn't have to sterilize when splitting or has a thriving exposed culture I'm happy for you but that doesn't seem to be working for me.

I would agree with you sanitation is important but maybe youre going down the rabbit hole a bit haha. Youre not alone, i felt the same way when i started out!

I do take many percautions though. I use 70% rubbing alcohol to wipe down all surfaces and equipment between harvests. I rinse my equipment with hot water, dry with fresh paper towel then use the rubbing alcohol. Being sure the alcohol evaporates all the way before filling with fresh saltwater, phyto culture and F2.

I also use separate equipment for water making, syringes for testing water, ect. I have only phyto equipment. I also on occasion use a bleach solution to soak those things and let air dry 24hrs before using again. I also sanitize my storage containers with a bleach solution to ensure my back up cultures dont get contaminated.

A note on why 70% rubbing alcohol...it has more water in it so it evaporates slower staying on the surface longer, killing more things.

When using a bleach solution, always use cool/cold water as warm/hot water will render the bleach useless for sanitizing.

if youre on instagram i have a video series there all about all of this. Its @doubleadeejay....Look under the video tab of my page.

Also here is a youtube video that may be helpful. https://www.reef2reef.com/threads/just-a-couple-of-fellas-talking-bout-phytoplankton.889720/

 

[dricc]

I would agree with you sanitation is important but maybe youre going down the rabbit hole a bit haha. Youre not alone, i felt the same way when i started out!

I do take many percautions though. I use 70% rubbing alcohol to wipe down all surfaces and equipment between harvests. I rinse my equipment with hot water, dry with fresh paper towel then use the rubbing alcohol. Being sure the alcohol evaporates all the way before filling with fresh saltwater, phyto culture and F2.

I also use separate equipment for water making, syringes for testing water, ect. I have only phyto equipment. I also on occasion use a bleach solution to soak those things and let air dry 24hrs before using again. I also sanitize my storage containers with a bleach solution to ensure my back up cultures dont get contaminated.

A note on why 70% rubbing alcohol...it has more water in it so it evaporates slower staying on the surface longer, killing more things.

When using a bleach solution, always use cool/cold water as warm/hot water will render the bleach useless for sanitizing.

if youre on instagram i have a video series there all about all of this. Its @doubleadeejay....Look under the video tab of my page.

Also here is a youtube video that may be helpful. https://www.reef2reef.com/threads/just-a-couple-of-fellas-talking-bout-phytoplankton.889720/

Just watched your video before I found this thread. Very informative and well done. I suggest anyone looking to culture phyto to check out the video!

 

[Pistondog]

I would agree with you sanitation is important but maybe youre going down the rabbit hole a bit haha. Youre not alone, i felt the same way when i started out!

I do take many percautions though. I use 70% rubbing alcohol to wipe down all surfaces and equipment between harvests. I rinse my equipment with hot water, dry with fresh paper towel then use the rubbing alcohol. Being sure the alcohol evaporates all the way before filling with fresh saltwater, phyto culture and F2.

I also use separate equipment for water making, syringes for testing water, ect. I have only phyto equipment. I also on occasion use a bleach solution to soak those things and let air dry 24hrs before using again. I also sanitize my storage containers with a bleach solution to ensure my back up cultures dont get contaminated.

A note on why 70% rubbing alcohol...it has more water in it so it evaporates slower staying on the surface longer, killing more things.

When using a bleach solution, always use cool/cold water as warm/hot water will render the bleach useless for sanitizing.

if youre on instagram i have a video series there all about all of this. Its @doubleadeejay....Look under the video tab of my page.

Also here is a youtube video that may be helpful. https://www.reef2reef.com/threads/just-a-couple-of-fellas-talking-bout-phytoplankton.889720/

Swirl 99% alcohol around vessel including the cap, coating all surfaces.
Leave outside, capped with a small vent for a day, then rinse with saltwater before use.