So I read a lot of research papers on TC because I was wanting to try it. I don't have a pressure cooker that is large enough OR a microwave OR an autoclave. Clearly I'd spend a lot of time dating the pressure cooker or I wouldn't be able to do TC. Or so I thought.
Then a large number of seeds I'd ordered arrived....
I came across some papers describing chemical sterilization of media in tissue culture using bleach as a part of efforts to enable low cost tissue culture that could be used in more rural communities, without laminar flow hood, for cultivation and propagation of potato, sugarcane, etc. There are several papers describing various agents tested for sterilizing different explants. The results with bleach caught my eye, mostly because I have bleach and the results seemed to be better than a lot of other alternatives they tried. Not listing any single procedure mostly because what I ended up doing didn't *exactly* match any of the research papers.
I actually have very accurate weighing scales and such, but given that I was following methods to make TC accessible in rudimentary situations, I figured I'd also attempt doing things by volume while I was at it.
The process I followed:
Media: 1/2 strength M&S salts + 4 spoons of sugar + 8g of agar + 200ml of coconut water + water to make 1 liter approx (one thing led to another and I think it was closer to 1.25 liters.
Boil while stirring on medium to low flame in an open pot. Turn off when boiled for a minute or two. Solution is slightly brown (probably impurities from sugar and agar)
Prepare TC containers: I used disposable transparent plastic containers with lids. Immersed in approx 10% solution by volume of bleach. Take out just before using, do not rinse, drain on clean surface.
Just before using = enough time to drain slightly, but not dry.
Back to media:Let cool. When cool enough to comfortably touch outside of the steel utensil, (50-60 degrees C I imagine) add one spoon of bleach. Solution turns clear-white. All that amber-brown tinge from the sugar (presumably) is gone.
Dispense media into TC containers - TC containers are not fully dry. Allow to set. Doesn't take too long, start working with seeds.
Used variation with psyllium husk to set the media in addition to agar. Worked well, but the media set really fast and I suspect is too thick.
Sterilizing seeds: My method was quick and dirty - used a plastic tea strainer and a small bowl filled with bleach solution. 5-10% or so. Plastic strainer should sit comfortably on the rim of the bowl, so that seeds in it are immersed in the bleach.
Place strainer in bowl, add seeds. Dip a couple of times (think tea bag), let sit - maybe for a minute or so. Less for seeds that were really delicate. I eyeballed it to something like "do they seem well soaked with the bleach solution? Ok good, take out". Rinsed with solution of hydrogen peroxide while in strainer - just held the strainer over the sink and ran the solution from a bottle with a nozzle over the seeds - about 10% solution of 6% hydrogen peroxide.
Use tweezers to grab "tails" of seeds and place onto solid media. I placed them well initially, all nice and separate. Later I was too exhausted and I had too many seeds and there were clumps and what not - I will probably regret these if they are the only ones to end up contaminated). Place lid (also not dry) and close firmly. Noting here that the containers seem fairly air tight.
The containers are kept in a relatively bright part of my normal bedroom.
3 days in, so far, so good. But I suspect contamination and stuff usually starts later.
Sterile technique issues:
Space was never really fully sterile. Worked on kitchen counter wiped clean with bleach solution. Heated medium on cooking gas in open pot right next to dinner (but it was cold and not actively boiling and sending vapour into the air).
Didn't sterilize tweezers or anything other than occasional dip in bleach while retrieving seeds. That said, I didn't get them exceptionally dirty either, like touching them with my fingers or putting on dirty surface, etc.
May have wiped tweezers on shorts to remove media stuck to it. Same old shorts I wiped hands wet with bleach solution on. This was toward the end - we'll see if that created problems, I've noted the containers that came after it.
This probably is a HUGE test of how well chemical sterilization works.
TC technique issues:
Used coconut water, since one of the papers said an advantage of chemical sterilization was that you could use things that could lose nutrients with autoclaving - no idea of contents and will probably vary from coconut to coconut
Have weighing scales, but used domestic measures like spoonful of sugar, etc. This is reliable for reproducing in my home, but may create problems to publish technique. Your spoons may vary, for example. (But if it works, I can probably measure out from my spoons and give rough weights)
Fan switched off, working surface wiped with bleach and no discernable "flow" of air in room, but no laminar flow hood or other protection from contaminants either. Containers were damp with bleach solution, but left open while the agar set and were covered only after seeds were put into them.
I suspect the pH of the media is going to drop once the bleach wears out. I tried to factor this in by keeping the media pH a bit on the high side 6.5-ish, but I'll test the pH of the first jar to be found with contaminants.
Results so far:
The agar seems to be a lot more solid than I anticipated. Must use less next time.
I put them in the containers on 25th. Today is 28th. So far no sign of contamination or anything growing - seed or fungus. I suspect the fun will start once the effect of the bleach wears off - either with fungus or my blood pressure in excitement.
I am not actually expecting a lot of success here, but hoping for enough observations to tweak this technique to arrive at something that works "well enough" - or give in and buy a pressure cooker (unlikely) or return to my sphagnum moss (which I've sowed seeds into anyway).
I'm almost wary of calling this technique TC given the relatively careless way I worked compared with the descriptions I read.
Learnings:
1 liter is a hell of a lot of media requiring a hell of a lot of containers requiring a hell of a lot of space. Luckily I had a hell of a lot of seeds, so ok, but as an experimental process, I'd probably have been better off with a much smaller quantity. Contamination in 4 cultures is just as learning an experience as contamination in 25 cultures
And if there is no contamination, I have no idea how I'll grow that many seeds further. But I suppose I will have plenty of material for the next learning curve.
This probably worked even to this small extent because I was putting seeds into TC. I will have to be more precise with quantities of bleach if I am working with growing tissue - like if this thing works (I'm defining "works" as seeing germination before fungus in even one container) and if I end up having to take seedlings out of one container and into fresh media. Excess bleach will fry them, I suspect. Being conservative with bleach will automatically require at least more effort to improve (maybe I should say begin) sterile technique.
I suspect, if I don't actually kill these, for transplanting them to new media, I'll skip the bleach in the medium or keep it really low and only sterilize containers with it and pressure cook the media. I may also make some kind of an enclosed space (was thinking of sticking an air purifier into a window cut out into a surface sterilizable box/compartment)
Regardless, however this pans out, I'll update here, just so anyone else thinking of these crazy shortcuts knows what happened.
Please don't consider this to be a protocol anyone should follow, unless you're fine killing all your seeds as well or unless I report wild success and revolutionize TC
Then a large number of seeds I'd ordered arrived....
I came across some papers describing chemical sterilization of media in tissue culture using bleach as a part of efforts to enable low cost tissue culture that could be used in more rural communities, without laminar flow hood, for cultivation and propagation of potato, sugarcane, etc. There are several papers describing various agents tested for sterilizing different explants. The results with bleach caught my eye, mostly because I have bleach and the results seemed to be better than a lot of other alternatives they tried. Not listing any single procedure mostly because what I ended up doing didn't *exactly* match any of the research papers.
I actually have very accurate weighing scales and such, but given that I was following methods to make TC accessible in rudimentary situations, I figured I'd also attempt doing things by volume while I was at it.
The process I followed:
Media: 1/2 strength M&S salts + 4 spoons of sugar + 8g of agar + 200ml of coconut water + water to make 1 liter approx (one thing led to another and I think it was closer to 1.25 liters.
Boil while stirring on medium to low flame in an open pot. Turn off when boiled for a minute or two. Solution is slightly brown (probably impurities from sugar and agar)
Prepare TC containers: I used disposable transparent plastic containers with lids. Immersed in approx 10% solution by volume of bleach. Take out just before using, do not rinse, drain on clean surface.
Just before using = enough time to drain slightly, but not dry.
Back to media:Let cool. When cool enough to comfortably touch outside of the steel utensil, (50-60 degrees C I imagine) add one spoon of bleach. Solution turns clear-white. All that amber-brown tinge from the sugar (presumably) is gone.
Dispense media into TC containers - TC containers are not fully dry. Allow to set. Doesn't take too long, start working with seeds.
Used variation with psyllium husk to set the media in addition to agar. Worked well, but the media set really fast and I suspect is too thick.
Sterilizing seeds: My method was quick and dirty - used a plastic tea strainer and a small bowl filled with bleach solution. 5-10% or so. Plastic strainer should sit comfortably on the rim of the bowl, so that seeds in it are immersed in the bleach.
Place strainer in bowl, add seeds. Dip a couple of times (think tea bag), let sit - maybe for a minute or so. Less for seeds that were really delicate. I eyeballed it to something like "do they seem well soaked with the bleach solution? Ok good, take out". Rinsed with solution of hydrogen peroxide while in strainer - just held the strainer over the sink and ran the solution from a bottle with a nozzle over the seeds - about 10% solution of 6% hydrogen peroxide.
Use tweezers to grab "tails" of seeds and place onto solid media. I placed them well initially, all nice and separate. Later I was too exhausted and I had too many seeds and there were clumps and what not - I will probably regret these if they are the only ones to end up contaminated). Place lid (also not dry) and close firmly. Noting here that the containers seem fairly air tight.
The containers are kept in a relatively bright part of my normal bedroom.
3 days in, so far, so good. But I suspect contamination and stuff usually starts later.
Sterile technique issues:
Space was never really fully sterile. Worked on kitchen counter wiped clean with bleach solution. Heated medium on cooking gas in open pot right next to dinner (but it was cold and not actively boiling and sending vapour into the air).
Didn't sterilize tweezers or anything other than occasional dip in bleach while retrieving seeds. That said, I didn't get them exceptionally dirty either, like touching them with my fingers or putting on dirty surface, etc.
May have wiped tweezers on shorts to remove media stuck to it. Same old shorts I wiped hands wet with bleach solution on. This was toward the end - we'll see if that created problems, I've noted the containers that came after it.
This probably is a HUGE test of how well chemical sterilization works.
TC technique issues:
Used coconut water, since one of the papers said an advantage of chemical sterilization was that you could use things that could lose nutrients with autoclaving - no idea of contents and will probably vary from coconut to coconut
Have weighing scales, but used domestic measures like spoonful of sugar, etc. This is reliable for reproducing in my home, but may create problems to publish technique. Your spoons may vary, for example. (But if it works, I can probably measure out from my spoons and give rough weights)
Fan switched off, working surface wiped with bleach and no discernable "flow" of air in room, but no laminar flow hood or other protection from contaminants either. Containers were damp with bleach solution, but left open while the agar set and were covered only after seeds were put into them.
I suspect the pH of the media is going to drop once the bleach wears out. I tried to factor this in by keeping the media pH a bit on the high side 6.5-ish, but I'll test the pH of the first jar to be found with contaminants.
Results so far:
The agar seems to be a lot more solid than I anticipated. Must use less next time.
I put them in the containers on 25th. Today is 28th. So far no sign of contamination or anything growing - seed or fungus. I suspect the fun will start once the effect of the bleach wears off - either with fungus or my blood pressure in excitement.
I am not actually expecting a lot of success here, but hoping for enough observations to tweak this technique to arrive at something that works "well enough" - or give in and buy a pressure cooker (unlikely) or return to my sphagnum moss (which I've sowed seeds into anyway).
I'm almost wary of calling this technique TC given the relatively careless way I worked compared with the descriptions I read.
Learnings:
1 liter is a hell of a lot of media requiring a hell of a lot of containers requiring a hell of a lot of space. Luckily I had a hell of a lot of seeds, so ok, but as an experimental process, I'd probably have been better off with a much smaller quantity. Contamination in 4 cultures is just as learning an experience as contamination in 25 cultures
And if there is no contamination, I have no idea how I'll grow that many seeds further. But I suppose I will have plenty of material for the next learning curve.
This probably worked even to this small extent because I was putting seeds into TC. I will have to be more precise with quantities of bleach if I am working with growing tissue - like if this thing works (I'm defining "works" as seeing germination before fungus in even one container) and if I end up having to take seedlings out of one container and into fresh media. Excess bleach will fry them, I suspect. Being conservative with bleach will automatically require at least more effort to improve (maybe I should say begin) sterile technique.
I suspect, if I don't actually kill these, for transplanting them to new media, I'll skip the bleach in the medium or keep it really low and only sterilize containers with it and pressure cook the media. I may also make some kind of an enclosed space (was thinking of sticking an air purifier into a window cut out into a surface sterilizable box/compartment)
Regardless, however this pans out, I'll update here, just so anyone else thinking of these crazy shortcuts knows what happened.
Please don't consider this to be a protocol anyone should follow, unless you're fine killing all your seeds as well or unless I report wild success and revolutionize TC
Last edited:
