# Baby steps seed prep



## Trithor (Oct 26, 2013)

I have started this thread in an attempt to answer questions about the basic steps of seed sterilization and rinsing as required for seed culture. I have tried a whole host of methods, but this is by far the easiest and most successful. It has an added benefit that due to the effective barrier, the process can be carried out in almost any situation, and does not require access to specialised equipment. If there is any interest I will make a more detailed step by step program, but to start, I think this should suffice?

The basic equipment needed is;
Packet of seed
Plastic disposable syringe
toothpick
face cotton swab
bottle of spray alcohol, (as distinct from that to be consumed during the process)
scissors
general purpose chlorine (unscented Jik or similar, basic household Sodium Hypochlorite)
..... and of course a glass of wine. Rose is a good early afternoon option. I can highly recommend a good dry shiraz rose for the task ahead.


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The bleach is prepared using 5ml bleach and 40ml tap water. (a lower or higher concentration may be required depending on what you aim to achieve with the bleach) The bleach is used to both sterilize and to 'etch' the seed. 

Cut a small square of 'face swab', about 7mm square. Using the tooth pick, press this square into the syringe nose. A bit of trial and error is needed at this point to get the correct fit. It must be secure, but not tight! A test draw on the plunger should leave the wad in the nose, but should not be too difficult to draw the plunger. (It will get much tighter when it gets wet)


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The cotton wad can be seen in the syringe nose. It is resting on a beaker of chlorine solution, and we are ready to start. Time to take an appreciative sip of wine!

Now remove the plunger from the syringe and pour a measure of seed into the syringe body. Replace the plunger and depress the plunger till it is near the bottom, but do not compress the seed. The whole sterilization process takes place from here on out inside the syringe, so can be conducted anywhere that is vaguely clean. Sterility is not needed.



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Now draw up about 1ml of chlorine solution, and about 2ml of air. Shake the syringe and contents for about 1 min. Now spray the outside of the syringe with alcohol spray and insert it into the sleeve from the syringe while still wet.
The syringe needs periodic shaking for about 10 to 15 minutes. While it is in the plastic sleeve, wet with alcohol, it will remain sterile.


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Now move your seed containing syringes into your flasking area (lam flow, glovebox, oven or other clean area)



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You will note that there is a scissors in the 'clean area' It is needed along with a forceps/tweezers to remove the cotton swab from the nose later in the process. At this point sterility is still not required as the seed is still isolated, but it makes a convenient time to move into the clean environment.



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Squirt out the chlorine solution into a suitable container. Now draw up a few ml of sterile water, shake the syringe, squeeze out the water, and draw up fresh sterile water. I find that three rinses is more than enough. When drawing up water, make sure that the tip of the syringe is in the mid column of water. Any spores and bacterial falling on the water will be held by the surface tension and will not be present in the short term in the mid water.

Draw up a final 1ml sterile water. Remove the cotton wad using forceps (at this point you may need to trim the plastic nose of the syringe to get to the cotton). Shake the syringe and squeeze a few drops of water with the suspended seed into the flasks. This is the only stage that requires a fairly sterile environment!



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Using this process I have been able to maintain 95% un-contaminated mother flasks.

I am sorry if my pictures are not illustrative enough, but it is difficult to perform the task and take pictures at the same time. (It did give me ample opportunity to take a few sips though)


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## Dane (Oct 26, 2013)

Have you tried polypropylene containers, I've found them to be allot quicker to sterilize like 3min for 3 containers ... I see you have a laminar flow cabinet... You're so lucky, I have to do mine in a glove box.


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## abax (Oct 27, 2013)

Fascinating process. I find following the narrative and the photos to be
very enlightening and easy to understand. Thank you!


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## JeanLux (Oct 27, 2013)

Very interesting process!!!!

Maybe I could assist once for refilling the wine glasses  !!!! Jean


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## Ben Belton (Oct 31, 2013)

I've never sown Paphs. Only Phals. I like THIS kind of syringe for dry seed sterilization. I do it a little different. 

What media do you use?

I'd like to try Paphs some day for fun. Someone is suppose to make me some pods sometime to play with.


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## Trithor (Nov 1, 2013)

Ben, I use half strength MS and a amino acid based medium, both with coconut water. I make two of each with each batch of seed. I find some do better on the one medium than on the other.


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## NYEric (Nov 1, 2013)

Interesting, thanks for sharing.


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## TyroneGenade (Nov 1, 2013)

Great little article. Mods, can we make this a sticky post?

Trithor, try put some clear plastic across the front of the laminar flow so you can look down but not exhale into the hood. This may cut your contamination down to 1%. Ideally, you need just enough space to put your hands in as well as put in flasks, bottles etc... But having such a wide open space provides a lot of opportunity for infection to be blown into the bottles etc... as you exhale.

Those plastic tubs work great for sowing and replating. When you start doing green-pods you will realize how small those little jars of yours are...


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## Trithor (Nov 1, 2013)

TyroneGenade said:


> Great little article. Mods, can we make this a sticky post?
> 
> Trithor, try put some clear plastic across the front of the laminar flow so you can look down but not exhale into the hood. This may cut your contamination down to 1%. Ideally, you need just enough space to put your hands in as well as put in flasks, bottles etc... But having such a wide open space provides a lot of opportunity for infection to be blown into the bottles etc... as you exhale.
> 
> Those plastic tubs work great for sowing and replating. When you start doing green-pods you will realize how small those little jars of yours are...



Hi Tyrone,
The contamination that I am referring to is the mother flasks that I prepared from the seed sent to me by JohnM. Of the twelve seed varieties, one proved to be impossible to sterilize. I tried firstly a 10min sterilization, all 4 flasks were contaminated (accounts for 8% contamination). On the second attempt I retried using 15min, 20min and 25min in 3 separate portions, but to no avail, I was unable to prevent the same orange fungal growth. All the rest of the seed have shown almost no contamination, so my current percentage is heavily skewed by that one seed batch.
I find this syringe method to be effective and very easy. I will keep posting as I carry on and change techniques and media. 
Thank you for your encouragement.


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## biothanasis (Nov 2, 2013)

Thank you for all the detailed info and photos!!! Very helpful!!!!


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## Brabantia (Nov 2, 2013)

@Thritor: when the seeds sterilisation is difficult Bob Hamilton signaled many years ago that a prior 24 hours presoak in a sugar solution of these reduce greatly the contamination problems.


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## Trithor (Nov 2, 2013)

Thank you, there is a certain logic to that. I assume that the soaking will cause germination of the fungal spores, and I am sure that germinating fungal spores are a lot more permeable to sterilizing agents than otherwise.


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## John M (Nov 4, 2013)

This is the first time that I've opened this thread. Very cool information and illustration. Thanks! I have only played with seed sowing at home, so far. I've never heard of sterilizing the seed the way you do. It looks like a very clever solution to the problem. Thanks for posting this tutorial!



Trithor said:


> Thank you, there is a certain logic to that. I assume that the soaking will cause germination of the fungal spores, and I am sure that germinating fungal spores are a lot more permeable to sterilizing agents than otherwise.


 I've heard of this before and it was done for exactly the reason you state. The germinated fungal spores are much easier to kill than dormant ones.


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## Ozpaph (Nov 4, 2013)

great thread. Thanks!


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## Clark (Nov 4, 2013)

Thanks for taking the time to put this together.


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## Trithor (Nov 4, 2013)

I am pleased that the post has met with a positive response. I posted the syringe method because it can be performed anywhere, (Dane), and only needs to be moved into the 'clean environment' just before inoculating the flasks. It makes sterilizing the seed easy, and means that a domestic oven can be used as a flasking chamber.


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## Leo Schordje (Nov 4, 2013)

definitely easier than the old mix bleach solution and seed in a vial method. 

Only problem with using the oven as my sterile chamber, I don't bend down well. 

''Getting old ain't for sissies, takes courage to get up in the morning and look in the mirrow'' - quote from Betty Davis.


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## Dane (Nov 8, 2013)

Trithor said:


> I am pleased that the post has met with a positive response. I posted the syringe method because it can be performed anywhere, (Dane), and only needs to be moved into the 'clean environment' just before inoculating the flasks. It makes sterilizing the seed easy, and means that a domestic oven can be used as a flasking chamber.



With the syringe method I've sown on a table top and have had 95% success  Just replating needs more sterility. I use the needle part of the syringe and put the cotton wool on that end so that when I finally suck up distilled water, I can just remove the needle and squirt the seeds straight into the flask by opening a corner of the flask and squiring the seed in, in a couple second reducing contamination by pro-longed exposure


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## chrismende (Nov 12, 2013)

This is terrific! Thanks for taking the trouble to make all the pictures. I have a strong urge to run into the kitchen and find a bottle of wine, now...


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## eteson (Nov 27, 2013)

Thanks a lot Trithor, very well illustrated! I use basically the same method and also works very fine for most part of the genus.


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## eaborne (Nov 27, 2013)

I will definitely have to give this method a try.


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## Trithor (Nov 28, 2013)

I have a 'lab weekend' planned. I am dead tired of repotting plants and am sure I can find enough to occupy me in the lab this weekend. I have promised a number of people after PM requests to post a series of brief notes on the whole process. So I will start with a note on media prep and autoclaving this weekend. (I can almost taste the wine)


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