Best tips for sterile phyto?

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Hello,

For all of you that run phyto cultures for long periods of time, could you list your steps as to what you do to keep everything sterile. I think my issue was that my bottles of f/2 got contaminated or my salt mix did (that is my main guess). Also, does anyone know what exactly bacteria (or other microbes) do that crash a culture?
 

Jay Hemdal

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Hello,

For all of you that run phyto cultures for long periods of time, could you list your steps as to what you do to keep everything sterile. I think my issue was that my bottles of f/2 got contaminated or my salt mix did (that is my main guess). Also, does anyone know what exactly bacteria (or other microbes) do that crash a culture?

I've had protozoans crash a lot of my phytoplankton cultures. f2 Gulliard's can get contaminated, so I try to mix that up fresh more frequently, to try to avoid that. Another trick might be to chlorinate and then dechlorinate your culture media prior to inoculation. I try really hard for a "xeric" culture, but contamination still does happen. My "fix" has always been to culture phyto in a number of 2 liter soda bottles, and then, if one crashes, I just dump it, and sub-culture new bottles from the good ones. Note: I only grow phyto in order to culture Brachionus, so it is possible that I avoid long-term issues because the rotifers get to the algae before it truly crashes on me.

Jay
 

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I add 10 ml bleach to a 5 gallon carboy with fresh saltwater and let it stand for at least an hour. Then, under heavy aeration I add the f/2 fertilizer (using syringes that were sterilized with boiling water) and let it mix for at least 10 minutes. Only then I add heat sterilized Sodium Thiosulfate solution to neutralize the bleach and inoculate the water with a clen culture starter (using sterilized syringes).
Also, make sure you put a 0.2μm filter on the airline going into the culture.
 

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For me, antibiotics have worked so far when cyano takes over the culture, but it hasn't proved to be a long- term solution. Experts claim that routine checking the culture with a microscope and centrifugation are the best way to separate pure strains but I have no experience in how to proceed. On Amazon, some basic lab centrifuges are offered for less than $100.
 

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For me, antibiotics have worked so far when cyano takes over the culture, but it hasn't proved to be a long- term solution. Experts claim that routine checking the culture with a microscope and centrifugation are the best way to separate pure strains but I have no experience in how to proceed. On Amazon, some basic lab centrifuges are offered for less than $100.
What antibiotics do you use, and where can I get some? Just had a tetraselmis culture turn into cyano. It's becoming my nemisis.
 

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I add 10 ml bleach to a 5 gallon carboy with fresh saltwater and let it stand for at least an hour. Then, under heavy aeration I add the f/2 fertilizer (using syringes that were sterilized with boiling water) and let it mix for at least 10 minutes. Only then I add heat sterilized Sodium Thiosulfate solution to neutralize the bleach and inoculate the water with a clen culture starter (using sterilized syringes).
Also, make sure you put a 0.2μm filter on the airline going into the culture.
I hadn't thought of the f/2, wonder if that's my problem. I was worried bleach would react some way with f/2, so I've been adding f/2 after neutralizing.

I recently started using UV to neutralize the bleach, wonder if that would mess with f/2?
 
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What antibiotics do you use, and where can I get some? Just had a tetraselmis culture turn into cyano. It's becoming my nemisis.


I personally would not use an antibiotic, as we there is a bit of a crisis of people creating superbugs by frequent and improper use of antibiotics for whatever they are doing.
 

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I add 10 ml bleach to a 5 gallon carboy with fresh saltwater and let it stand for at least an hour. Then, under heavy aeration I add the f/2 fertilizer (using syringes that were sterilized with boiling water) and let it mix for at least 10 minutes. Only then I add heat sterilized Sodium Thiosulfate solution to neutralize the bleach and inoculate the water with a clen culture starter (using sterilized syringes).
Also, make sure you put a 0.2μm filter on the airline going into the culture.

Heat sterilized sodium thiosulfate? Is this something that you buy or do yourself? How are you transferring the sterilized water in the carboy to the phyto containers?
 

fryman

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Heat sterilized sodium thiosulfate? Is this something that you buy or do yourself? How are you transferring the sterilized water in the carboy to the phyto containers?
Not sure how he does it but I just use a gas range. Bring rodi to a boil then mix w/ sodium thiosulfate. I also have autoclavable bottles that I've used, they can be placed in a large steel pot that I bring to a boil. I leave the caps loose for safety and add extra water to account for evaporation. Takes some trial and error.
 

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What’s the sodium thiosulfate used for? Anyone have a link for the stuff they use?
 

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What’s the sodium thiosulfate used for? Anyone have a link for the stuff they use?
It's used to inactivate bleach. In short, first use 1-2 ml bleach per liter water to sanitize the culture media (water, salt, and f/2). After 12-24 hrs use sodium thiosulfate to make the water safe for phyto. Then we can add phyto for grow out.

I make a solution of 17 grams sodium thiosulfate in 500ml water. I have something like this:
Pool Dechlorinator Sodium Thiosulfate Pentahydrate 4 oz by Cesco Solutions - Premium Chlorine Neutralizer for Pools, Aquarium, Pond - Technical-Grade Chlorine Remover for Hot Tubs - Bulk Package https://a.co/d/f7irCRw

1ml of this solution will inactivate 4ml bleach.
 

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It's used to inactivate bleach. In short, first use 1-2 ml bleach per liter water to sanitize the culture media (water, salt, and f/2). After 12-24 hrs use sodium thiosulfate to make the water safe for phyto. Then we can add phyto for grow out.

I make a solution of 17 grams sodium thiosulfate in 500ml water. I have something like this:
Pool Dechlorinator Sodium Thiosulfate Pentahydrate 4 oz by Cesco Solutions - Premium Chlorine Neutralizer for Pools, Aquarium, Pond - Technical-Grade Chlorine Remover for Hot Tubs - Bulk Package https://a.co/d/f7irCRw

1ml of this solution will inactivate 4ml bleach.
So it's safe to mix f/2 with the bleach? Do you know if f/2 can be mixed and stored long before use (like a month)? I ask because I plan to mix NSW in a 5g carboy, which should last me about a month if I'm splitting my cultures on a weekly basis.
 

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So it's safe to mix f/2 with the bleach? Do you know if f/2 can be mixed and stored long before use (like a month)? I ask because I plan to mix NSW in a 5g carboy, which should last me about a month if I'm splitting my cultures on a weekly basis.
I used to add f/2 after sodium thiosulfate because I wasn't sure if bleach affected the fertilizer in some way. I had persistent synechococcus contamination issues. I even bought new f/2 and still had the problem. So now I sanitize everything w/bleach (water, salt, & f/2) and then use sodium thiosulfate or UV to inactivate the bleach. I've not had the problem since doing so, and phyto grows out so I assume f/2 is ok with bleach.

So yes I believe you can mix f/2 with bleach, however bleach will break down over time. I don't think it's recommended to store sanitized media. Not really sure, it could have some shelf life but I've not tried more than a day.
 

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It's used to inactivate bleach. In short, first use 1-2 ml bleach per liter water to sanitize the culture media (water, salt, and f/2). After 12-24 hrs use sodium thiosulfate to make the water safe for phyto. Then we can add phyto for grow out.

I make a solution of 17 grams sodium thiosulfate in 500ml water. I have something like this:
Pool Dechlorinator Sodium Thiosulfate Pentahydrate 4 oz by Cesco Solutions - Premium Chlorine Neutralizer for Pools, Aquarium, Pond - Technical-Grade Chlorine Remover for Hot Tubs - Bulk Package https://a.co/d/f7irCRw

1ml of this solution will inactivate 4ml bleach.
It took 8ml of this solution to dechlorinate 1ml of bleach (clorox) for me. Wonder if not mixing in the f/2 had something to do with it. Very odd though.
 

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Like Jay, I also use 2L bottles. I have eight of them and harvest on an 8 day schedule.
I fill a Brute trash can with RODI water and add dry pool chlorine. Every day I add enough sodium thiosulfate to neutralize 1.5 L.

A few things that help me avoid crashes are I always wash my hands first, I spray my funnel and work area with isopropyl alcohol and use large cup hooks to hang the aeration tube. Every now and then a bottle goes clear but no big deal.

I started out 2 or 3 years ago with Tetra and Nanno. The Tetra cultures all crashed inside a year but the Nanno keeps chugging along.
 

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It's used to inactivate bleach. In short, first use 1-2 ml bleach per liter water to sanitize the culture media (water, salt, and f/2). After 12-24 hrs use sodium thiosulfate to make the water safe for phyto. Then we can add phyto for grow out.

I make a solution of 17 grams sodium thiosulfate in 500ml water. I have something like this:
Pool Dechlorinator Sodium Thiosulfate Pentahydrate 4 oz by Cesco Solutions - Premium Chlorine Neutralizer for Pools, Aquarium, Pond - Technical-Grade Chlorine Remover for Hot Tubs - Bulk Package https://a.co/d/f7irCRw

1ml of this solution will inactivate 4ml bleach.

Where did you find the ratio of bleach to thiosulfate? I'm having a heck of a time finding the actual ratios. My bleach is a 7.5% solution so I mixed a 7.5% (37.5g to 500mL RODI) of sodium thiosulfate and I was planning on mixing 2.5mL thiosulfate to each 1 mL of bleach but I'm concerned I may have misunderstood the reaction when I was trying to find the answer looking at your ratios.
 

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Where did you find the ratio of bleach to thiosulfate? I'm having a heck of a time finding the actual ratios. My bleach is a 7.5% solution so I mixed a 7.5% (37.5g to 500mL RODI) of sodium thiosulfate and I was planning on mixing 2.5mL thiosulfate to each 1 mL of bleach but I'm concerned I may have misunderstood the reaction when I was trying to find the answer looking at your ratios.
Sorry it was some time ago and I don't have the source anymore.

IIRC there is a safety factor, and having excess sodium thiosulphate isn't usually a problem. You can buy chlorine strips and just check it empirically. The caveat is to get test strips with low limit of detection. There are strips intended for pools or drinking water that will indicate no chlorine at low levels that are still lethal to phyto.

Nowadays I prefer using UV.
 

fryman

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I make a solution of 17 grams sodium thiosulfate in 500ml water. I have something like this:
Pool Dechlorinator Sodium Thiosulfate Pentahydrate 4 oz by Cesco Solutions - Premium Chlorine Neutralizer for Pools, Aquarium, Pond - Technical-Grade Chlorine Remover for Hot Tubs - Bulk Package https://a.co/d/f7irCRw

1ml of this solution will inactivate 4ml bleach.
Sorry that is wrong. 17g/500 ml was a typo, should be 17g per 50ml to make a 4:1 solution. My bad.

I stopped making the 4:1 solution because it was easier to use 1:1. So, 9 g of the sodium thiosulphate crystals from amazon (listed above) dissolved in 100ml rodi (or 45g sodium thiosulphate in 500ml rodi). This is a 1:1 mix (i.e. 1ml this solution to neutralize 1ml bleach).

I may have came up with this ratio via trial-and-error, using chlorine test strips. I don't remember, it's just what I have written on the bottle I use to make this sodium thiosulphate solution.
 

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