Contamination problems

[Mxptrsn]

Ok I think I have a couple of questions with regard to contamination.

A couple of days ago, I sterilized my homestyle media for orchid seed flasking. I used microwave and sterilized it for 3 minutes. After that, I took them out and immediately put them in a pre-sprayed (with regular household bleach and no dilution) plastic bag and sealed them asap. The question is I used PP vessels and when I sterilized them in microwave, I just closed the cap loosely so that the heat generated would not deform the vessels. After cooling down, I then close the caps tightly. And, some of the media got contaminated. Any advice?

When it comes to orchid seed sowing, is it ok if I don't drill a hole and plaster it with micropore tape to allow gas exchange? What about baby food jars? What's the better and cheaper way?

I used organic medium formula:
2 g balanced fertilizer (NPK: 20.20.20)
7 g Agar
500 ml coconut water
500 ml distilled water
4 g activated carbon
20 g sugar
What do you think for orchid seed sowing media? Any suggestion?

Thanks very much in advance for your help.

Mxptrsn

[Tsuchibuta]

I have tried that method as well, and plan to do more testing, but perhaps you need to do more than 3 mins. Like 5 mins or so. I found in my first batch I did 4.5 mins and the second batch I did 6 mins, the 6 min batch has had no contamination but the 4.5 had about a 20% contamination rate.

[NatalieS]

I've only made flasks once but I also used the microwave method and didn't get any contamination. I didn't heat the medium for longer than 5 minutes, but I would agree with Tsuchibuta and say that 6 minutes would definitely be enough time in any microwave.

The only difference for me was that I didn't use bleach to spray the inside of the plastic bag, I used undiluted sodium hypochlorite (Milton). I don't think that would really make a difference though.

[Mxptrsn]

Thanks very much Tsuchibuta and NatalieS.

I think I'll go with 6 mins, you know, just to be safe. And, there're another two possible contamination problems, again, besides the media sterilization method.

Yesterday, I tried to sterilize my dry orchid seeds (spathoglottis plicata) with 1:10 bleach for 10 minutes without a drop of dish detergent and today I've found that the fungus has had a wild party inside the media!! GOSH... I tossed them all just now.

I think the possible problems would be the air bubbles during seed sterilization with syringe and the sterilized distilled water. Yeah, I microwaved the distilled water for 3 minutes as well. There's a big chance that it could be the major source of contamination.

Anyway, Tsuchibuta and NatalieS, how did you sterilize your orchid seeds (dry ones)? Did you have any problem with air bubbles inside the syringe? I found it really hard to get rid of those air bubbles which might be another possible source of contamination.

Thanks very much, Tsuchibuta and NatalieS.

Mxptrsn

[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!

[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.

[Ray]

I haven't done much flasking, and have always used a pressure cooker, but if you're getting contamination, the microwave must be the source, or you're taking them out too soon.

In a pressure cooker, the entire interior becomes sterilized, so when to jars cool and the tops get sucked closed, there is nothing to be drawn in. The microwave does nothing to kill anything in its atmosphere, so whatever is present can be drawn into the flask upon cooling.

[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.

[Ray]

Just an FYI, sodium hypochlorite is the active ingredient in household bleach.

I really don't see how you would get enough steam inside of a microwave to sterilize it. For one, microwaves are not sealed and air tight. Secondly, the laws of physics tells us that until 100% of the liquid is turned to water vapor, the temperature of both is fixed at 100°C, and at that point your flasks would be full of powder.

[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