# Lab work



## Trithor (Oct 20, 2013)

A rainy Sunday is a great day to spend in the lab. A few hours to while away till lunch time. A miserable rainy day calls for a big steak and a bottle of red wine
But while I wait, time to do a few mother flasks. 64 of them done today! I am starting to get slick on the process now, and find that I am working much more efficiently. 


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Just to show that I am having some success with germination. This is a mother flask of some seed that JohnM kindly sent me. I guess I am soon going to have to teach myself how to replate them. There are 16 mother flasks that look like this already.


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## SlipperKing (Oct 20, 2013)

You better start replating now!


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## biothanasis (Oct 20, 2013)

cool!!!!


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## Dido (Oct 20, 2013)

looks great congrats, 
I you have not enough space for all flask, you can send me some


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## eggshells (Oct 20, 2013)

Wow. Good luck on the replates.


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## Cheyenne (Oct 20, 2013)

So are you going to tell us what things you have there? Looks like you have been busy. I hope you are building another greenhouse too. Did you build a flow hood with a UV light or are you using a glove box or something?


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## abax (Oct 21, 2013)

Gary the mother flasks look wonderful! And all this time I thought you
were just playing around. ;>) Oh, how I wish I could get my little greedy
hands on some of your plants when the time comes.

Please explain your replating method for me.


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## Trithor (Oct 21, 2013)

Cheyenne said:


> So are you going to tell us what things you have there? Looks like you have been busy. I hope you are building another greenhouse too. Did you build a flow hood with a UV light or are you using a glove box or something?


JohnM kindly sent me a selection of seed to get started with. The fantastic thing is that it was a wide selection, so it has given me a chance to try a wide selection of methods and treatments. I portioned the seed into three equal portions. The first third I took with me to a local team who are studying mammalian tissue at the local university. We mixed up a batch of general purpose plant agar at half strength and they taught me the various aspects that I needed to know, such as media prep, sterile technique and plating techniques. Then the second third I tried in my own lab using 5 different media and the techniques which I had been taught. Then a 2 month interval to assess any results. I have now settled on 2 different media, and yesterday flasked the remaining portion of seed on the two selected media.
I purchased a small lab setup from a heritage plant nursery that had found it was not cost effective to propagate the quantities of plants they needed using tissue culture. So one of the items included was a small laminar flow.
I have since found a large lab that is closing their plant tissue culture division. They have three more laminars for sale (one of which is a six station beast!), ...... no I have not bought this equipment ......yet
The seed sent to me by John
Encyclia cordigera
Stanhopea embreei
Thunia marshalliana
Pot Little Toshie
Paph St Swithin
Phal philippinense
Phrag Hanna Popow
Stanhopea wardii
Cat gaskelliana alba x coerulea
Cat gaskelliana
Laelia lobata x Cat luddemanniana
Cat forbesii x Cat luddemaniana

And seed from my own greenhouse
Paph glaucophylum
Paph liemianum
Paph sanderianum
Paph venustum




abax said:


> Gary the mother flasks look wonderful! And all this time I thought you
> were just playing around. ;>) Oh, how I wish I could get my little greedy
> hands on some of your plants when the time comes.
> 
> Please explain your replating method for me.


Angela, I intend to try out two methods to see which works best for me. The first is the spoonula method (traditional) and the second is one they use in mammalian cell culture. I will post pics and descriptions later in the week when I give them a go. I first need to get some plastic tubs irradiated to sterilize them to make vented replates. The job for earlier this week, then the trial on my spread/replate flasks. I intend to do two stages of replate. Firstly a thinning spread flask into a vented container, and finally a proper replate into flasks of 5 and 20 plants. (The current plan in any case)


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## Ozpaph (Oct 21, 2013)

Looks like great fun.
Ill follow with keen interest.
Thanks.


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## Dane (Oct 21, 2013)

Awesome to see another South African doing flasking ... Just a tip, you really only need about 1cm of agar medium to germinate the seeds on. How did the phrag seeds go? Any success?

Dane


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## TyroneGenade (Oct 21, 2013)

Cool result. What sowing medium and sterilization methods are you using? JohnM sent me lots of seed as well but I never got a single one to germinate...


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## Trithor (Oct 21, 2013)

Dane said:


> Awesome to see another South African doing flasking ... Just a tip, you really only need about 1cm of agar medium to germinate the seeds on. How did the phrag seeds go? Any success?
> Dane


I have used around 1.5cm, some flasks have less, some have more. The cost of medium is low, so I do not try and save by making it stretch as far as possible. 
The phrag seed has germinated on one of the mediums only. That is one of the media that I have selected as my defaults going forward. 




TyroneGenade said:


> Cool result. What sowing medium and sterilization methods are you using? JohnM sent me lots of seed as well but I never got a single one to germinate...



I have tested three sterilization methods, sodium hypochlorite, saturated calcium hypochlorite and hydrogen peroxide. I have found the peroxide to be more of a pain in the butt than anything else and wont use it going forward. Both chlorine compounds seem to work equally well and the simplicity if buying an 'off the shelf' prep of sodium hypochlorite which works, makes it my current choice.
I used 5 germination mediums. Two of them are from large companies and have had moderate results, the other three are tweeked formulas which I had mixed specially for me to test (problem was I had to take 100liter batches in order for them to consider making them for me, so I now have culture media all the way up my 'ying-yang') Of my 3 tweeked media, the one has given me the best results so far, and another is easily equal to the lab order product, so these two are my current favourites. The third has proven to be a disaster up till now, but I will still try it as a replate medium, who knows.
Of the twelve seed batches from John, only one has proved impossible to sterilize, and as such I have no hope for that one. Of the rest, 5 have germinated, and another 3 are showing good 'swelling' even if there is no protocorm development as of yet. The remaining 3 are unchanged, but they are only 5 weeks down the line. I am considering splitting these mother flasks into two further groups and treat them to slightly different temperatures to see if that has any effect. 

It is still very early days for me, and I am sure that I will be changing many things in the coming months. One thing is for sure, .... this has the potential of getting badly out of control! Raising flasks is one thing, but potting them out and growing them might just be a very big problem (I have considered moving to Hawaii as I believe Limuhead buys a lot of flasks!)


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## Dane (Oct 21, 2013)

Which seed sowing method are you using, Dry seed or green pod method?


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## TyroneGenade (Oct 21, 2013)

How are you rinsing the chlorine from the seeds? I had great difficulty doing this and retaining sterility.

Are you using coconut water in any of the mixes?

If you are using Sigma Phytamax it is an EXCELLENT replate medium. Better than any of the others I have tried (various Western media, Knudson, MS-mixes etc...). Excellent root development---especially for Paphs.


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## abax (Oct 22, 2013)

All this sounds very complex and waaaay above my head, but I find it very
interesting. I know you'll do it well and bring us along with you. The
whole process humbles me when I think "hey, that's where my lovely Paph.
came from". Lots and lots of work between a lab and my greenhouse
bench.


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## Dane (Oct 25, 2013)

> I have used around 1.5cm, some flasks have less, some have more. The cost of medium is low, so I do not try and save by making it stretch as far as possible.
> The phrag seed has germinated on one of the mediums only. That is one of the media that I have selected as my defaults going forward.



Where do you get your medium from in SA at a low price?


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## NYEric (Oct 25, 2013)

Looking good. Now I'm kind of mad that my Toluminia pod never made it to you!


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## Trithor (Oct 25, 2013)

TyroneGenade said:


> How are you rinsing the chlorine from the seeds? I had great difficulty doing this and retaining sterility.
> 
> Are you using coconut water in any of the mixes?
> 
> If you are using Sigma Phytamax it is an EXCELLENT replate medium. Better than any of the others I have tried (various Western media, Knudson, MS-mixes etc...). Excellent root development---especially for Paphs.


Yes, I am using coconut water in all my germination mixes now. At first it was one of my 'test additives', but the results were improved across the board, so it is a standard now. I have not used any Sigma products up till now, but will certainly try phytomax after your recommendation.
I will start a thread of how I am doing my flasking and prep, as perhaps my learning will be informative to others as well. One of the things I will be sure to show is my sterilization and rinse process as I am very big on keeping it simple. 



Dane said:


> Where do you get your medium from in SA at a low price?


Dane, if I can help you in any way, drop me a mail and I will do my best to assist. I have been forced to buy in quantity to avoid the high prices, also I am fortunate tostill have access to our local university Biotech Lab. It kind of helps, guess that is about the only benefit of having graduated from Med School!



NYEric said:


> Looking good. Now I'm kind of mad that my Toluminia pod never made it to you!


You and me!  I was looking forward to receiving it.


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