Contamination problems

[NatalieS]

I incorrectly used the term super-heated steam. Super-heated steam is actually dry steam and occurs when all the water has been removed from the system, and we're really talking about wet steam here.

Wet steam is by definition hotter than 100 C at sea-level, otherwise it would not be steam. Yes, the combined average temperature of the liquid/medium and the steam would tend towards 100 C because the steam would start giving away its heat energy to other surfaces while the liquid would continue to boil, but the steam itself would have to be above 100 C. The medium is also not just water - it contains sugars so it would increase the boiling point a little (though probably not by much).

Yes, a pressure cooker would allow both the vapour and the liquid to get hotter. As you say, the microwave is not sealed so you can't maintain the temperatures, but the microwave method still works. This is where I took my instruction from and it worked for me:

Orchid Flasking - Part 2 (2011) - YouTube

Maybe you can't technically sterilise the inside of the microwave, but no living organism would be able to survive for very long in a microwave anyway. Unless you have access to a clean room, everything you do to sterilise is just a best effort, and if it works, I say just go with it!

Edit: I stand corrected about the temperature of steam - it's not necessarily hotter than 100 C. However, a gram of steam contains more heat than a gram of boiling water, which is why steam burns can be so severe. I also learned that microwaves often super-heat water, which I was not aware of.

[Tsuchibuta]

The microwave method is heavily debated, but definitely is tried and true. Even phytotech labs lists it as a viable method to sterilize their media.

In terms of seed sterilization, see my other thread, I'm trying all kinds of things and it's still too early to tell what seems to be the best for the home hobbyist. (the air in the test tube shouldn't make a difference though as the bleach would sterilize that air by the time you sow the seeds)

[littlefrog]

An inexpensive pressure cooker is better in my opinion... Spores are tricky things. They don't have a lot of water in them, and the microwave is basically heating water and not much else. Microwaves might work, but I know a pressure cooker does. They aren't expensive.

Allow your flasks to cool in place. Do not move them until they are mostly cool. If you start moving things around hot: A) you risk burning the tar out of yourself, and that is no fun, B) as air inside the flask cools, it sucks in non-sterile air from the outside, and C) ... well, there is no C...

I've done a whole lot of tissue culture (cancer cells) and contamination is almost always due to poor initial sterilization (the autoclave isn't working) or poor technique. Usually the latter. Technique gets better with practice. You won't be perfect the first few dozen times, keep at it, you'll get there.

See if you can find some of the AOS Orchid Bulletin (I think it was still the bulletin) where Calcium hypochlorite was suggested as a sterilizing agent. It seems to work better and is less toxic to the seed.

[Mxptrsn]

Sorry to get back to you guys a little bit late. Thank you very much for all of your recommendations. I tried every recommendation you have given, pressure cooker, microwave and baking oven. And, to me, microwave sterilization doesn't really work for me. Contamination rate is really high. I don't know why. I went with 6 mins, but all of them got contaminated.

With pressure cooker, the contamination rate is 20%. Maybe my technique is still the major problem. But, in my experience, it's way better than microwave. I also tried using baking oven (150 degrees for 30 minutes), and I'm still waiting if there'd be any sign of contamination. Will keep you guys posted on this.

[Mxptrsn]

Originally Posted by NatalieS

I sterilised my seed using the method in this thread: http://www.orchidboard.com/community...y-seeding.html

I found this method very straight forward to follow and you are also essentially re-sterilising the surface of the medium when you sow the seeds. I think it's brilliant!

NatalieS, thank you so much for the link!! Very very much appreciated. The gaseous sterilization really works!! I tried to sow my dry seeds (spathoglottis) for 4 hours with pure 5.25% Sodium hypochlorite fume, and there hasn't been any sign of contamination.

Like you said, it's very straight forward and BRILLIANT!!!
100% agree with you!!

Anyway, I was wondering if one could use this method for the green pod though??

[Mxptrsn]

Originally Posted by Kyle

I recommend a pressure cooker.

For dry seed, I recommend a twenty four hour soak in a sugar solution, followed by a fifteen minute soak in ten percent bleach. The theory is that the sugar activates dormant spores and organisms in the seeds, making it easier for the bleach to work.

Thanks Kyle for the dry seed sterilization method. I'll definitely give it a try later. Thanks! =D

[Mxptrsn]

Originally Posted by NatalieS

Ray, you make a good point about the treatment of the flasks after removing them from the microwave. Re-reading Mxptrsn's post I missed the fact that he'd closed the flasks straight after putting them into the plastic bag sprayed with bleach. I used the same procedure (except I used sodium hypochlorite instead of bleach) and got zero contamination. However, I left my flasks slightly open while cooling in the sealed bag which would've caused the sodium hypochlorite to be sucked into them.

Some of the flasks I sowed the next day, some a few weeks later - zero contamination even in the later ones, so I'm confident the microwave method works.

Edit: Thinking about it a little more, I still think it's just down to heating the medium for long enough. Technically, the super-heated steam from the medium should sterilise the microwave and the atmosphere in the flasks.

When I think about what I'd done with regard to the procedure I went through, I think using pp containers may be the source of the problem. You know, when I transferred them from microwave to the pre-sprayed plastic bags (I sealed them when they cooled down a little bit in the plastic bags), the contaminants could have got in through the air first. So, that could be the source. Now I've tried to use baby food jars and pressure cooker, the contamination rate is significantly lesser than using pp containers.

I have also tried using baking oven, I'm still waiting to see if there's any sign of contamination. If it works, i'd say that it could be one of my sterilization alternatives.

[Mxptrsn]

Originally Posted by Discus

Considering that professional autoclaving happens for at least 15 minutes at what I imagine are far higher temperatures (usually around 121ºC) than you'll be generating in a few minutes in a microwave, I suspect much of your contamination stems either from too low a temperature or too short a treatment period or a combination of both of these things.

You may find this chart I just stumbled across on google of interest:
Autoclave Time Temperature Pressure Chart

Discus, you may be right on this. I can't know for sure either, since household microwave never indicates how high the temperature inside is. Anyway, thanks for your link. Very much appreciated.

[Mxptrsn]

Originally Posted by Tsuchibuta

The microwave method is heavily debated, but definitely is tried and true. Even phytotech labs lists it as a viable method to sterilize their media.

In terms of seed sterilization, see my other thread, I'm trying all kinds of things and it's still too early to tell what seems to be the best for the home hobbyist. (the air in the test tube shouldn't make a difference though as the bleach would sterilize that air by the time you sow the seeds)

Tsuchitbuta, I've just visited your blog and they're interesting and quite informative!! I've a couple of questions though. Do you find it sufficient, in terms of sterility rate, to use your flasking cabinet to sow your orchid seeds? I personally use glove box. But I really doubt if contaminants could get in any time when I try to open and close the caps of my baby food jars.
I use 70% ethanol to surface-sterilize my glove box. But, I really doubt if it's gonna be sterile enough.

[Mxptrsn]

Originally Posted by littlefrog

An inexpensive pressure cooker is better in my opinion... Spores are tricky things. They don't have a lot of water in them, and the microwave is basically heating water and not much else. Microwaves might work, but I know a pressure cooker does. They aren't expensive.

Allow your flasks to cool in place. Do not move them until they are mostly cool. If you start moving things around hot: A) you risk burning the tar out of yourself, and that is no fun, B) as air inside the flask cools, it sucks in non-sterile air from the outside, and C) ... well, there is no C...

I've done a whole lot of tissue culture (cancer cells) and contamination is almost always due to poor initial sterilization (the autoclave isn't working) or poor technique. Usually the latter. Technique gets better with practice. You won't be perfect the first few dozen times, keep at it, you'll get there.

See if you can find some of the AOS Orchid Bulletin (I think it was still the bulletin) where Calcium hypochlorite was suggested as a sterilizing agent. It seems to work better and is less toxic to the seed.

littlefrog, you're absolutely right about poor initial sterilization and the technique! I totally agree with you. Yeah, as you said, I've seen myself doing better day by day with more practices. Thanks very much.