Flasking 101 - A how-to guide

[10010100102]

The handbook PROPAGATION OF CANADA'S ORCHIDS Methods and Issues by Marilyn H.S. Light has these recipies:

Oats Agar (for isolation of fungal symbionts and for symbiotic germination)

2.5 g oats (commercial rolled oats)
12 g agar powder
1000 ml deionized water

Water Agar (for symbiotic germination of Spiranthes sp., etc.)

8 g agar powder
1000 ml deionized water

Preperation is as for any other medium. Adjust ph as needed.

I'm not sure how well these recipies work as I've never actually attempted the symbiotic method. From what I've read, isolating the fungus can be tricky as other contaminants tend to overwhelm the culture before the desired symbioint. Once you're able to get a culture of the fungus you want going, you can take a piece of it and inoculate other flasks with the fungus for sowing. Again, I can't speak for the effectiveness of this method as I've never tried it.

[10010100102]

Originally Posted by Zozzl

I have been wondering about trying H202 because I am concerning about the sensitivity of some of my seeds to the clorox and because rinsing them is tricky for me. Thanks for all the great info you guy!

Pat

About rinsing, I've actually heard that it's not neccesary. My orchid society had a presentation a little while ago from Marilyn H.S. Light on growing from seed. She is quite succesful with this and has registered a number of her own hybrids, and is also a local authority on the conservation of orchids in the wild.

On the subject of sterilising seed, she said that with a 1:9 bleach to water (10%) solution the amount of bleach that actually remains on the seed is negligeable, and so rinsing isn't neccesary.

On a different topic (hopefuly to get the ball rolling again), I see no-one's said anything about using rubbing alcohol to sterilise the inside of a glove box... I've used both 70 and 99.9% isopropyl rubbing alchohol with good results. I like using it instead of bleach because while the alcohol evaporates, bleach doesn't and leaves everything wet. Also, alcohol won't make the hair on your arms go blonde, if you're worried about that sort of thing . On the other hand, I find the alcohol fumes much stronger when you finally open up the box.

[Royal]

Thanks for the input, Joseph. Good info. I use alcohol too, I think I may have mentioned it briefly. I agree that it is way better than bleach - hands down.

I've had some good results without a rinse (I use the same bleach concentration). It's just that I get batches with little or no germination, and I want to eliminate bleach as a variable. I know it can kill sensitive seed, and I know it has to have an effect on pH. I've done a few batches recently with an extra rinse. We'll see if it makes any difference.

[King_of_orchid_growing:)]

Originally Posted by 10010100102

The handbook PROPAGATION OF CANADA'S ORCHIDS Methods and Issues by Marilyn H.S. Light has these recipies:



Preperation is as for any other medium. Adjust ph as needed.

I'm not sure how well these recipies work as I've never actually attempted the symbiotic method. From what I've read, isolating the fungus can be tricky as other contaminants tend to overwhelm the culture before the desired symbioint. Once you're able to get a culture of the fungus you want going, you can take a piece of it and inoculate other flasks with the fungus for sowing. Again, I can't speak for the effectiveness of this method as I've never tried it.

Symbiotic methods of sowing terrestrial orchids are pretty effective if you can isolate the proper fungus. While I've only attempted symbiotic in-vitro seed sowing once (incorrectly), I know of others who've done it repeatedly with much success.

Isolating fungus is actually not a one step process like the article leads you to believe (I love how that quote made it sound soooo simple when it's not). I only know that you have to get a root cutting and isolate different fungi from it. Obtaining a pure culture may take multiple tries. Once some of the fungi have been isolated, you have a variety of fungi to choose from, which will make sense when I say the following...

If you don't already know which fungus will actually germinate the seeds of the species of orchid you intend to sow, it will require some trial and error before you find the right one.

After the media has been prepared and the seeds have been properly sterilized and have been sown onto the medium, then you would inoculate the batch.

Even with my "clarification", this process sounds easy. But don't be fooled.

There are very few articles on this and some of them aren't in English (luckily there's google translator).

[gixrj18]

Originally Posted by 10010100102

About rinsing, I've actually heard that it's not neccesary. My orchid society had a presentation a little while ago from Marilyn H.S. Light on growing from seed. She is quite succesful with this and has registered a number of her own hybrids, and is also a local authority on the conservation of orchids in the wild.

On the subject of sterilising seed, she said that with a 1:9 bleach to water (10%) solution the amount of bleach that actually remains on the seed is negligeable, and so rinsing isn't neccesary.

On a different topic (hopefuly to get the ball rolling again), I see no-one's said anything about using rubbing alcohol to sterilise the inside of a glove box... I've used both 70 and 99.9% isopropyl rubbing alchohol with good results. I like using it instead of bleach because while the alcohol evaporates, bleach doesn't and leaves everything wet. Also, alcohol won't make the hair on your arms go blonde, if you're worried about that sort of thing . On the other hand, I find the alcohol fumes much stronger when you finally open up the box.

I wipe the box and gloves with alcohol, then spray it down with 25% bleach solution.

[King_of_orchid_growing:)]

Originally Posted by RoyalOrchids

Pat, Some people make little packets of seed sealed in a filter paper. The whole thing is sanitized and rinsed as one, then cut open and the seeds scraped out. Is this what you are talking about King?

AJ Hicks sells the Whatman glassine filters, they shouldn't disintegrate in the bleach like the coffee filters. He also sells autoclaveable wash bottles and filter funnels. If using these filters, there is no need for agar in the germination media and all you'd need is a thin layer on which to float the filter. I've never used filters, so I'm speculating now too.

Alrighty guys! I just got my new filter discs and filter funnel in the mail a couple days ago. And yesterday I was sowing some seeds with them.

Man, this stuff cuts my work down drastically! The only deal is the filter papers can get clogged up by the seeds if you've got a lot per packet. My recommendation is to get 2 or 3 of these filter funnels just in case, in order to cut down on the time you spend having to wait for the liquid to drain out.

You also can minimize losing seeds by having multiple filter funnels in play at once. I was getting tired and started making mistakes like letting the filter funnel overflow or spilling the seed and liquid mixture into the work area, because the filter paper got clogged from too many seeds.

If you're gonna use this method, I also recommend using tweezers to handle the filter paper.

Instead of dropping the whole filter paper into the flask (which could cause problems with contamination). Just hold the filter paper over the mouth of the flask and spray the seeds off with 3% hydrogen peroxide or 70% isopropyl alcohol. The seeds will get in there.

Yeah, I also spray 70% isopropyl alcohol into the work box and all the things I put inside. The alcohol does evaporate, but the vapors are noxious. I intend to get a face mask, so I don't have to breathe those fumes in.

Another thing people didn't mention is using gloves. For people using glove boxes you guys already use gloves. But for those just using a plastic box not modified into a makeshift glovebox, using gloves is very important.

Alcohol tends to dry out your skin if used extensively on a regular basis. Although, I believe people on the OB are intelligent enough to understand this, I think it is good to have reminders of how flammable alcohol is. So no fires or bunsen burners around the work area (obviously no smoking either).

Hydrogen peroxide is a bleaching agent as well as an antiseptic agent, and according to some sources on the web (ie. Wikipedia), can cause vitiligo if used extensively on a regular basis. Plus it leaves a smelly sour residual odor (bad if you're on a date ).

Same goes for handling bleach as above for hydrogen peroxide (except bleach leaves a smelly chlorine filled swimming pool odor).

[King_of_orchid_growing:)]

Out of curiosity. How many of you all soak your seeds in sugar water prior to sowing?

[Royal]

Originally Posted by King_of_orchid_growing:)

Alrighty guys! I just got my new filter discs and filter funnel in the mail a couple days ago. And yesterday I was sowing some seeds with them.

Man, this stuff cuts my work down drastically!

Great to know. Are they set up to hold the filter flat, or are they like mini separatory funnels that require you to fold the filter into a cone? I guess a flat orientation would require a vacuum - Just wondering. Also, did you get them from OSP? Did you get the wash-bottle too? I've been wondering how large it is. He give the volume (500 ml) but will it fit in my tiny p-cooker?

[Royal]

Originally Posted by King_of_orchid_growing:)

Out of curiosity. How many of you all soak your seeds in sugar water prior to sowing?

Never used sugar, but I've soaked in plain water overnight. Really helps them get saturated allowing the sterilant to fully infiltrate the testa. I've never had an issue with contamination if I can get the seed to sink.

[King_of_orchid_growing:)]

Originally Posted by RoyalOrchids

Great to know. Are they set up to hold the filter flat, or are they like mini separatory funnels that require you to fold the filter into a cone? I guess a flat orientation would require a vacuum - Just wondering. Also, did you get them from OSP? Did you get the wash-bottle too? I've been wondering how large it is. He give the volume (500 ml) but will it fit in my tiny p-cooker?

The filter paper is a small 1" disc. You gotta be careful not to put two pieces of filter paper into the funnel. It's easy to do if you're not checking because of the way they're packed.

The funnel itself is a little taller than 3 1/2". There are markings on it that show the volume it can handle in mL.

You lay the filter disc flat onto a flat circular plastic sieve with a thin pipe for drainage attached to it. You can unscrew the main body of the filter funnel from the sieve.