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PostPosted: Thu Oct 25, 2012 12:18 pm 
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Hi All, This is going to be in a similar fashion to CricketTerry.

I thought I would start a new thread to keep things in order if you like, instead of keep hijacking Terrys thread.

Hopefully it will progress with good information to help us all at different levels, like Terry has.
I have got to say also from a personal point of view it is good to look back and see what has happened, so like a diary, this is one reason I use Facebook, looking through your timeline is very interesting!
https://www.facebook.com/#!/www.koolorchids.co.uk

I will post pics, good and bad.

Below is the basic protocol I am following, with the video series on hand and pdf printed out it is a lot less daunting.
Flasking is a lot of messing about and fiddly, so having patients is lesson 1!! If you haven't got that then doesn't bother.......
Never the less, it is all interesting stuff if you don't actually do flasking.
To get started here are good links to information that I have found incredibly useful.
Frank Tromble - videos are great and he is extremely helpful http://youtu.be/YbGd9ycGwi0

Carol Stiff also knows her tissue culture and has sent me a Pdf work shop handout which I am sure she will send you if you join the Home Tissue Culture Group - Its FREE http://hometissueculture.org/ Franks video link is available here too.

I am at the stage of having jars with sterile agar and 3 mother flasks from greenpod under lights, one of the mother flasks has contamination, so I am going in there to scoop it out, just for fun and to learn!

That's all for now

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PostPosted: Thu Oct 25, 2012 12:53 pm 
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Best of luck on your flasking adventure... will be interested in how you get on.

Not had any success with scooping out contamination in a flask, tried several times, it always spreads for me, :cry: , let us know if you manage it.


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PostPosted: Thu Oct 25, 2012 1:29 pm 
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crystalorchid wrote:
Best of luck on your flasking adventure... will be interested in how you get on.

Not had any success with scooping out contamination in a flask, tried several times, it always spreads for me, :cry: , let us know if you manage it.



Seeing how quickly it grows in only one night I can see why! Being as it is a learning process for me I have got to give it a go, IF I get half decent at this I am sure I will just discard the contaminated ones which hopefully will be few.

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PostPosted: Thu Oct 25, 2012 5:53 pm 
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I have just set my clean box up, gone in to the contaminated jar and took a large scoop of agar/ contamination out with a spoon. Whilst the lid was off the jar I did a spray of isopropynol, my thinking was anything going in and already in will be hit.

Not expecting anything, but to learn.

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PostPosted: Mon Oct 29, 2012 10:04 pm 
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Here is my growing success so far! Contamination in all three flasks so 100% failure, the positive news is all 6 flasks I made up by using the microwave sterilizing method are still sterile.

It is very simple to do, you basically prepare your media and put in to flasks as normal, put the flasks in the microwave with the lids loose. Heat them up until the media starts to bubble/boil, then heat for an extra minute, let them cool for a few minutes then tighten the lids.
Some people put the flasks in a bag to cool, this bag is sprayed inside with a bleach solution, this idea is to prevent contamination when air is drawn in to the flask by the cooling process.

I skipped this part as I wanted to try the simplest and most hassle free method first, any way it has worked so far without this part, remember not to put metal lids etc in to a microwave!.

I am buying a flow hood soon! More information will follow...


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PostPosted: Wed Nov 28, 2012 11:21 pm 
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Hi Folks,
My Standard hepafilter flow hood arrived today and thus far it looks a very good bit of kit, very clean and basic in design, the main hepa filter on the front really is quality with a aluminium edge. The blower at the back looks robust too.

I obviously have ditched the open box and closed box way of flasking and hope this flow hood works out well, I am going to use this hood as it is in the open as in the picture.
I left open a flask in the flow hood work area for 15 minutes for a contamination test, hopefully this will not contaminate :? .

As you can see from the pictures space in limited under the stairs but I had a good rearrange and now it is workable, just! The shelf will hold about 50 flasks, the aim is to produce my own hybrids and remake some others so I have a trickle of sales plants every year.

A waiting game now to see if contamination raises its ugly head.

I purchased this flow hood from http://www.standardhepafilter.com the site now has other tissue culture products amongst the flow hood complete kits or bundles.


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PostPosted: Thu Nov 29, 2012 11:39 am 
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Keith are you making a hood for this? As the air that is being sucked into the unit will have to come from the front as you have it enclosed in a small area, which means that you envelope of clean air is going to be very small.

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Last edited by neil on Thu Nov 29, 2012 12:30 pm, edited 1 time in total.

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PostPosted: Thu Nov 29, 2012 12:07 pm 
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Keep going Keith! It will be great when you sort out the contamination problems. Its good to see all of your pics.

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PostPosted: Thu Nov 29, 2012 9:07 pm 
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neil wrote:
Keith are you making a hood for this? As the air that is being sucked into the unit will have to come from the front as you have it enclosed in a small area, which means that you envelope of clean air is going to be very small.


Hi Neil, No, I am going to try it as it is. Completely basic as it is then adapt if need be, there is an air vent below at the bottom/back which comes from the hallway.

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PostPosted: Thu Nov 29, 2012 9:15 pm 
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OK, I hope it goes well with you. Since I bought my cabinet I have only the odd plate/ jar get contamination.

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PostPosted: Thu Nov 29, 2012 9:57 pm 
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neil wrote:
OK, I hope it goes well with you. Since I bought my cabinet I have only the odd plate/ jar get contamination.


I do too Neil :crazy: , I may put a hood on the front but my philosophy is to try basic then adapt. I have been told this flow hood has good results without any other adaptions, I firstly have to trust this and learn I guess.

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PostPosted: Fri Nov 30, 2012 7:30 pm 
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Looks like a nice set up to me. Good idea to try it without a hood; if you get contamination the hood will solve that.
So much easier than a glove box, good luck and keep us posted on how it goes....


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PostPosted: Sun Dec 30, 2012 6:19 pm 
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Just had my first try with dry seed with the flow hood, the seed pod I found in my greenhouse and am sure it was off M. coriacea - it is only a practice so that is irrelevant really.

Even though it should have been very easy, I made it more difficult by making a newbie mistake I guess! Read on...........

First the flow hood went on and I sterilized the area with a spray of isopropyl alcohol.
Washed my hands for 20 seconds, put on gloves, then sprayed the ready media flasks and put them in the flow area.

I then prepared and sterilized the seed - I used a 5ml syringe/needle(pink), between the syringe and needle I made a filter out of kitchen paper towel about the size of a postage stamp and folded in half. The filter is to prevent the loss of seed, sorry if that is obvious! I placed the seed in to the syringe (maybe a pinch or two with forceps). I then sucked hydrogen peroxide(3%) in to the syringe and then agitated for a few minutes. I removed some of the peroxide from the syringe until I was left with about 3 ml and left for 30 minutes at room temperature. I sprayed the syringe and my hands with isopropynol and put it in the flow area with the two flasks.

I timed the 30 minutes.
After this period I sprayed my hands again before putting them in to the work area, removed the filter from the syringe, removed a lid from one of the flasks and squirted some of the seed in to the flask and put the lid back on. Did this again for the other flask without removing my hands from the work area, with practice the removing of lid could be done with one hand.
That is basically all I did and when written down looks more complicated than reality.
The flasks went straight under white light where the peroxide will dissipate and hopefully seed germinate.

Now for the bit where I have learnt from this first experience and why I made it more difficult!

I basically think I put too much of the seed/peroxide in to the first flask, call it a mind lapse or just inexperience and a learning curve. I kind of panicked and tried to remove some of the fluid with the syringe, which resulted in some of the seed being stuck on to the inside of the flask and me dropping the lid!
So that part of my protocol got messy! and something I am sure I won't do in the future.
I will also aim for about 1ml per flask next time and also be extra careful at this point.

It is a waiting game now and hopefully I won't get contamination. If I do I try again!

No doubt I will be hovering over these two flasks like a expectant dad!

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PostPosted: Sun Dec 30, 2012 7:38 pm 
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I have also gone through some similar learning experiences with flasking, but it does get easier each time as you get used to the working space and techniques, everything becomes more familiar. I remember the first flasking felt like I had two left hands and everything got in the way and I put way too much seed into the flasks.

I found the flask lids got in my way so what I do now is put a piece of kitchen roll in the laminar flow cabinet and spray it with 50% bleach solution so it is well sodden. I then put the lids face down on it when working on a flask. Don't seem to get contamination that way so its now what i do. Then I don't need to hold onto the lid and i throw the kitchen roll after every few flasks.

When I started I have been able to use my own green pods where the seeds are sterile as the pod has not opened. I have not much experience with seed sterilising but have been trying recently. I have tried two methods, a syringe with cotton wool wrapped inside a small piece of tights, donated by my wife. :thumbup:

The cotton wool is pushed to the end of the syringe and seed put into the open end of the syringe. I then used 10% bleach, domestos, as the sterilising agent, agitating the syringe for 10 mins. Then I draw sterile water into the syringe three or four times to remove as much sterilising agent from the seeds. I dont seem to lose too much seed with this method, then using sterile tweesers I pull out the cotton and dab it in the flasks. I did about 8 flasks like this with no contamination but it is too early to see if any protocorns have developed.

The other method in tried was with filter paper folded into a small envelope and stapled closed. This was then put into 10% bleach to sterilise the seeds. I left for 6 mins this time agitating the envelope, wonder if this time is too short? This seems a better method in terms of very little loss of seeds. I found opening the envelope tricky and got contamination in a couple of flasks.

Not tried peroxide as a sterilising agent for the seeds, does it perform better than bleach? I use isopropyl alcohol to sterilise tools and 50% bleach spray for flasks and the cabinet.


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PostPosted: Sun Dec 30, 2012 8:42 pm 
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using hydrogen peroxide eliminates the need to rinse as the peroxide breaks down in to water and oxygen, so is a one step procedure, unlike household bleach which you have to rinse a few times. The flasks do need to go straight under light to aid the peroxide breakdown.

This is my first time so cannot really comment on methods through experience, but my aim is to try the easiest and most time effective method I can first, then adapt if need be.

I am hoping my work area is sterile with the flow hood, seed is sterile with this peroxide method which leaves my routine and whether seed is viable!

I did check the seed under a microscope and think they were viable.

The piece of paper towel between cap and syringe worked really well and the standard hepa filter flow hood was a dream, and obviously effortless compared to a box.

Just to add I use 70% isopropynol alcohol in a spray bottle to sterilize the work area, everything going in to the work area, including my latex gloved hands. This evaporates very quickly and I cannot imagine a better way to sterilize, but I could be wrong.
I also wear a dust mask and take extreme caution when using isopropynol as it is flammable.

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PostPosted: Mon Dec 31, 2012 8:04 pm 
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Just a quick post to add that the liquid or peroxide in the flask has all dispersed and now the seeds are nicely visible, instead of floating in a lake! I am surprised how quickly that has happened and won't worry so much about it in the future, with hindsight I wouldn't have worried and tried to remove the liquid, which may have resulted in contaminating the flask. That is learning! Just hoping I get some sprouts!!!!!!!!! Won't be long before I am getting the magnifier out :lol:

I am already itching to get sowing again :jumpy: , but I must be patient 8-)

Someone advised me to practice with mock flasks with a sugar solution in the flask instead of agar to improve my technique, of course I haven't done that yet :whistle: etc

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PostPosted: Mon Jan 21, 2013 11:20 pm 
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Hi Folks, Just an update with the two flasks I sown, I am thrilled to be able to report that the two flasks with Masdevallia seed sown have no contamination! This proves I have sterilized the seed and standard hepa filter flow hoods work, I love this unitā€¦. I used the flow hood as it is without any enclosed work area, but I am going to put cling film over the front of the hepa filter to protect it from unwanted dust and dirt when not in use.
No seed has germinated yet but who knows it still may do.

I know I haven't done much sowing yet but that result is pretty exciting and conclusive for me, one test flask and two sown with seed and no contamination. Compared to two previous attempts with a clean box and 100% contamination!

I cannot recommend this flow hood enough and the price isn't going to blow your budget. Buy others or build one yourself and it will, I am convinced. I have been the guinea pig so go ahead and treat yourself!
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Last edited by Masdyman on Tue Jan 22, 2013 11:31 pm, edited 1 time in total.

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PostPosted: Tue Jan 22, 2013 8:13 am 
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Its so good to hear your great news Keith. I will keep my fingers crossed for germination now.

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PostPosted: Tue Jan 22, 2013 9:47 pm 
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cricketerry wrote:
Its so good to hear your great news Keith. I will keep my fingers crossed for germination now.


Thanks Terry, It is steps and hurdles isn't it!

What I need is seed that someone has sown successfully, then obviously this will remove another question mark.

I looked at the seed with a magnifier and can see the sack but not whether there are embryos or not, they are not very dark if they are fertile so am am thinking they aren't fertile!

Once I see fertile seed under a scope I guess I will just know..

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PostPosted: Sun Mar 03, 2013 10:03 pm 
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Hi Folks, My flasks still have no contamination, I have sown another couple of Masdevallia flasks since my last post and just done a couple of flasks of a Paph cross tonight.

Still no green stuff though! Since found out I could get germination after six months so still hope I will get some growth.

I looked at the Paph seeds under microscope and definitely think these are fertile as I am sure I could see greenish embryo's.
We will see!

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