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Old 06-26-2008, 12:22 AM
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Home Seed Flasking

I have noticed several posts here on doing your own flasking. At the risk of drawing some major cannon fire, I decided to do this anyway. First of all, I am not a major hybridizer and, at my age, it has been recommended to me not even to buy green bananas. Much of what I have to say has been said in the various post so please forgive any sensed plagiarizing.
1. First of all, I think you will find that purchasing ready made media should be considered a viable alternative to "rolling your own". You can purchase already sterilized media from some companies. Also, if you just have to get your hands wet and mess up the kitchen, at least consider purchasing the media as a ready mixed powder. When I was doing my own flasking, I purchased most of my media from Sigmaaldrich Chemical Co. I finally settled on two; Vacin & Went and their Phytomax. Its been so long I can't remember everything, but I think it came without gelling agent and sugar. Ordinary table sugar will suffice for the sugar and you can buy agar from the same company as the media. They used to also have a product called Phytogel that can replace the agar.

2. When making up the media, if you are using agar, remember that agar will not melt until the temperature reaches 100 C (212 F) but once it melts, it will not gel until the temperature drops to ~ 46 C. One should use distilled water (like for irons) to make the media (note I did NOT say bottled water). Lots of measuring cups today have metric markings on them.Add about 5% more water than called for (this will ~replace the water lost in sterilization). Containers are your choice, but I prefer ones that will lay flat on the table. Dispense the media to make a layer 1/2" to 1" deep. Remember to loosen the caps or stoppers or remove completely and cover the flask with aluminum foil (if you choose this method you must sterilize the caps separately) prior to sterilization. Sterilize in a pressure cooker at 15# or pressure (121 c or 250 F) for 15 minutes. If you do not have a pressure cooker, try cycling them in a microwave until they reach a rapid boil and repeat 3 times, 10 to 20 minute rest period between cycles.

4. Immediately after sterilizing, lay the containers on a solid surface and cover with a towel that has been boiled in water for ~ 10 minutes (put on some disposable gloves and wring out the towel first). Once the media has cooled and solidified, remove the towel and immediately tighten caps.

5. It is much easier to work at home with dry seed (it also has an advantage in that if the pod parent was virused you are much less likely to transmit the virus). When you notice that the pod is beginning to turn yellow, watch carefully and (hopefully) just before the pod splits, open and remove the seed by gently cutting off one end and tapping the pod over a sheet of clean paper. Fold up the paper so the seed will not get out and place in a tupperware continer (I usually added some of the silica gel used to dry flowers to the container). Let sit in a dark, dry, place at room temperature for at least 20 - 30 days.

6. Make a passive "hood" by cutting out the side or end of a cardboard box which has had the flaps removed from two sides. Place a towel on a flat surface (one that will stand up to things like bleach, alcohol etc.) and turn the box over it so the other open end faces a wall (in other words just so the open end in not turned up or sitting on the towel). Soak the towel with a antimicrobial liquid such as 3% hydrogen peroxide, 70% isopropyl alcohol (remember, it's flammable!!!), a good quantanary ammonium salt etc. Items that need to be sterilized may be wrapped up in foil and sterilized in the oven @ 350 to 400 F for 3 hours.

7. Spray the inside of the box with a sanitizing solution such as mentioned in #6 above. Move the presterilized flasks containing the media under the box. Remove a sterilized glass container from the protective foil and add a small bit of dry orchid seed (a little smaller than a wooden match head). Add sufficient 3% hydrogen peroxide to the container to cover the seed. Unwrap an iced tea spoon and stir the peroxide/seed mixture then cover with a piece of sterilized foil. Let stand at room temperature for a period of 20 - 30 minutes. Remove the cap/stopper from the media flask and using a new sterilized iced tea spoon, scoop up some of the seed/peroxide mixture. Add it to the flask and tilt the flask back and forth until the surface of the media is covered. Stopper tightly and place in a shaded spot in a greenhouse or better yet, under fluorescent light fixture.

8. A better alternative to all the above is to make friends (and I mean VERY good friends with your local vet. Most likely, they will have laminar flow hoods and autoclaves that they may allow you to use which will make the task MUCH simpler. Try bribing them with a nice orchid plant of two!!

9. Best alternative, find a good flasking service and send the seed pods off to them.
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Old 06-26-2008, 01:04 AM
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Jerry that is a lot of good info. I don't think I'd ever attempt it but good to know nevertheless. I'll take your last advice and send it to someone else to do the hard work.
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Old 06-26-2008, 01:38 AM
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Thanks, that's a good read. How successful is this method?
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Old 06-26-2008, 03:46 AM
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Very good advice.

A couple of points of discussion, on the topic of media, many flasking labs will sell the home TC'er ready made media and realistically it isn't that expensive. I probably wouldn't bother to go to great efforts to try and purchase media through Sigma and similar chemical/biotech companies. They may have been more lenient in the past but currently most will only send reagents to approved laboratories. While culture media may seem harmless these companies also sell potential explosives, toxins, narcotics, etc so rather than making their sales staff decide what appropriate for Joe Sixpack to buy they normally opt not to sell anything to him at all. They also don't take to kindly to lab workers purchasing material for home use should they find out. There are a few companies that specialise in lab supplies for schools etc who carry flasking supplies and are more willing to sell hobbiests reagents.

Regarding containers be aware that not all containers handle heat equally well and a lot of plastic containers found around the home aren't particularly thermostable. If you're using containers with lids, any warping of plastic containers can result in a poor seal with the lid leading to the potential to introduce contaminants. Glass jars with metal lids are usually fine although those without much thread tend not to seal as well. If you're not going to use jars I'd recommend commercially produced tissue culture flasks (usually available where you buy the media).

Pressure cookers make good substitutes for autoclaves. However, microwaving media is not as effective in sterilising media. Autoclaves (and pressure cookers) use pressure to raise the boiling point of the media well above 100oC to kill bacteria, fungi and most importantly their spores. Microwaving will not achieve temperatures above ~100oC. While boiling may kill off most microbes many spores are quite tough and may not be effectively inactivated by simple boiling. Microwaving may work for you but don't be surprised if it doesn't.

RE: the cardboard box, I've never heard of that method but no doubt it works. An old aquarium turned on its side and the opening covered with a curtain of plastic sheeting or bleach soaked towel is as a more commonly used transfer chamber amongst home TC'ers. While I guess the box is disposable, using an aquarium has the benefits of easier surface sterilisation and being able to see what you're doing more easily. Personally, I have access to a laminar flow hoods so I don't use either approach.
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Old 06-26-2008, 08:52 AM
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Interesting info. Thanks !!
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Old 06-26-2008, 10:31 AM
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Jerry, it's very good information for the first time flask user. I totally agree that first time sowing users needs to buy commercial medium
from good labs. Don't try to make your own. Some people found a weird formula on the web such as honey & starch and try to sow orchid seeds. I don't say that it doesn't work but there is less chance you get seedlings and good seedlings. Try a well known formula that many people have been using so you get more chance to be successful as well as to get more confidence.
You can buy medium from flasking labs and from well known companies such as Sigma, Phytotech, Caisson (in US). Sigma is expensive but Phytotech and Caisson are not. I found many medium, hormones and chemicals of them are less expensive than retail sellers. If you are serious about seeds sowing and tissue culture you may want to buy from them not from flasking labs. Why? Take a look at the local fertilizer and brand name fertilizer. With local fertilizer, you can see flakes, grain, and powder. What's happen? The first teaspoon you could get all flakes and second teaspoon you could get all powder so your medium change from time to time. How can homemade companies mix an extremely amount of minor elements and make sure that they distribute well in mixture. That is one of several technical issues of small flasking lab medium. You don't want to take pills that one has zero milligram penicillin or B12 and another pill has 1000 mg.
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Old 06-26-2008, 08:55 PM
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I see your point but premixed media should be measured out precisely enough that each container contains a uniform amount of each chemical (I don't know of anyone who combines media salts and divides it up while still dry - this is really bad laboratory practice). Premixed media is designed to be made up as one batch. If you are only using it a spoonful at a time, there is no guarantee of consistency in the media. This is true of media sold by flasking labs as well as that sold by the chemical companies. The only reason Sigma's product has an even particle size is because they use a fine grade agarose. The containers of media are still as unevenly dispersed as that from the flasking labs and still needs to be made up as a single batch.

If anyone hasn't been scared off flasking the Orchid Seed Project has some good information. If you have access to a good analytical balance the site also has a number of established media formulations if you want to make your own (I take no responsibility for loss of limbs while using some of the nitrate components). Be aware that buying the components to make your own media is only cheaper than buying pre-made media if you're planning on doing thousands of flasks.
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Old 06-27-2008, 12:48 AM
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Brookn, it's surprising how successful this can be if one knows something about sterile technique. Like Andrew, I worked all my working life for companies that had autoclaves (some big enough to park your car in) laminar flow hoods, balances, etc. However, some companies frown at employees who sign security sheets at 1 AM upon entering the plant and don't leave until 5-6 PM later that day. So, sometimes you just have to be creative. I really never kept track of the success rate by either method, but I don't think I ever had a contaminated flask when I worked under the hood. Can't say the same about home but imagine it was around 60% successful as far as mold contamination is concerned.
Andrew brings up a very good point. Most of the plastic containers around the kitchen are not meant to be heated to the temperatures you will achieve in a pressure cooker and maybe not even the microwave. I preferred to use either Pyrex milk bottles sealed with rubber stoppers with one hole thru them that I very tightly packed with cotton. I sometimes used glass RX bottles that I could buy at a local pharmacy. I don't know if you can do that today, seems like everything is plastic these days.
You may well be correct Andrew about microwaving. However, you would be amazed these days how many surgical instruments are sterilized via microwave. Most mold spores do not desiccate very well (one of the reasons I leave the seed in a Tupperware container with silica gel). Even the spores have some water and protein in them and the protein (and especially lipids) heats much faster than much of the surrounding material. I have personally never sterilized media in the microwave, but have talked to many that have and they seemed to be successful more often than not. Most home areas do not have an area clean enough not to have spores in the air and when you remove the bottles from either pressure cooker or microwave, they will draw in air from the outside. Many people do not realize that this is often a major source of contamination. In any case while I may not completely agree that it is impossible to sterilize media in the microwave, if I can find a pressure cooker guess which one I would use!!
Having been involved in mammalian cell culture for over 40 years, I must disagree that a complete medium can not be made as a homogeneous dry powder. Bacteriological media have been prepared this way for years! Also much of the mammalian cell culture media is made the same way. It is really amazing just how fine those ball mills of today can grind!!
TC, one major problem with buying the individual chemicals individually is that most homes do not have proper storage facilities for them. Some of the small diet scales today are probably sensitive enough to weigh out complete media (it really isn't rocket science) but I doubt you could get the accuracy needed with individual components. I just can't imagine anyone trying to weigh materials that are very hygroscopic this way (anyone who has had to weigh out small amounts of Choline Chloride knows what I am talking about).
Anyway, I still think that unless one has had some experience in working under sterile conditions and has minimal equipment at their disposal should spend their money in buying postage to mail off the pod. However, if one just has to try, as TC and Andrew have pointed out, at least use a good flasking media and not some witches concoction. I really shudder when I read some of the "formulas"?? being contemplated!!
Let me give you one reason why you may want to use an outside flasking service. First, you need to check around and make sure that the lab is considered to be one that knows what they are doing. I don't know how many of you have seen Phrag Silver Eagle, but the hybridizer collected a pod from the first cross and had it flasked. There have been at least 4 quality awards from this cross. Up until a few months ago, the hybridizer has remade this cross a number of times with absolutely no success. Had they used all the seed from the first successful cross in experimenting with home recipes and home flasking, the cross may never have been seen!
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Old 06-27-2008, 01:00 PM
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Quote:
Originally Posted by Andrew View Post
....Premixed media is designed to be made up as one batch. If you are only using it a spoonful at a time, there is no guarantee of consistency in the media.....
I agree that what you said is right but not true, not the way to do business. Do you think drug companies measure chemicals and make one pill at a time. Do you go to labs and they measure and make one batch for you, lol. They mix chemical/drug in a batch to make hundred thousands pills at a time then divide mixture to make a pill. They mix hundred pounds of chemical to make medium powder and then divide; put them into bags or bottles and sell them to you. Even if you buy MS for10 liters you don't use them all at a time. You may only use few teaspoons to make medium for few mother flasks. It's is the technical issue we are talking about. Anyway, I could be totally wrong. Who know that your labs could do business like that. Never know never tell. Cheers.
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Old 08-06-2008, 09:54 AM
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I tried once but all turns into fluffy mess. Hahahahahaha.
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Old 08-06-2008, 08:21 PM
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This is why I'm choosing to send my seed to a flasker! This is so facinating. I wish I worked at a facility that had all the equipment, etc. I would love to do this for a living.
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Old 08-06-2008, 09:03 PM
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Fascinating read! I have to say...my hat is off to all of you who flask and/or who have done flasking.
Personally, I would never have the patience for it...I choose to let someone else do all that (whew!) work.
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Old 08-06-2008, 09:46 PM
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Quote:
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Fascinating read! I have to say...my hat is off to all of you who flask and/or who have done flasking.
Personally, I would never have the patience for it...I choose to let someone else do all that (whew!) work.
I made the same conclusion after trying that out.
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