Minimum Requirements for Flask Sterility

[Royal]

Hi Everybody!

I'd love to hear from all you mad scientists what was the least amount of precautions taken while maintaining sterile conditions. Anybody try flasking or replating without a glove box or laminar flow hood? Anybody use a glove box that is not air-tight?

Just curious...

[Royal]

Anyone???

[Des]

I've sown seed in the centre of my hob with all four plates switched on medium heat and i've sterilized bottles in the microwave . Also i have sterilized bottles and seed using H2O2 .It all works but you do have some failures .It is ok to use these methods if you are just experimenting . but if you are commercial you really need the correct equipment. If you want I can e-mail some diy methods to you .
Regards Des

[Royal]

Thanks Des,

This is exactly what I'm looking for. I have made a crude glovebox that works, but it is so clumsy to use. I was looking for a few "home made" techniques that require a little less set up time, and hopefully a little less chlorine fumes.

[Des]

Try not to use cl2 if you can. it will wreck your mucus membranes

[Royal]

Now you tell me! What should I use?

[Des]

I use 10% hydrogen peroxide I wear gloves and safety specs though. It does not give off like chlorine , but can burn you ,so be careful

[Des]

RoyalOrchids _ You might like to try this idea i had recently Make up your flasks with your agar sterilize in an autoclave or pressure cooker or microwave , I then take the green pod and coat it with latex , let it dry. I then take a large syringe, take up a small amount of sterile distilled water into the syringe and inject it into the seedpod . (the seed within the pod is sterile) Then suck the water and some seed into the syringe and inject into the mother flask !! I am still waiting to see if there will be any contamination , so far not a sign of any problems

[maitaman]

You can use far less sterility procedure with some media than with others. Agar is an easily contaminable medium base. There are some non-sterile catalayze methods, but none are particularly effective.
If you avoid air movement and direct contamination, you can do it. With cornstarch or rice starch you can use a cardboard box and merely avoid touching anything with anything but the sterilized tools.

[korxi]

You can also use the syringe method of sowing.

Firstly you use a jar with a metal lid, like jam or baby food.
In this jar you drill a hole and "plug" it with silicone. Let this dry for a day or so before you make your medium and sterilize.

When you are going to sow do the following:

You'll need:
1. syringe and scalpel
2. small amount of destilled, RO or rain water in a small cup or glass
3. 5 grams of sugar (aprox. 0.2 Oz)
4. 1 deciliter of destilled, RO or rain water (aprox. 0.45 cups)

Dissolve the 5 grams of sugar in the 1 deciliter of water.

Cut open you seed pod, and scrape out the amount of seed you want to sow into the smalle glass with the distilled/RO/Rain water, the rest can be saved in the fridge.
Shake the mix until the seeds fall to the bottom.

Now get out your syringe(s) and suck up the seeds (no needle on yet). Let the seeds fall to the bottom by holding the syringe vertical. Now gently push out the water, leaving the seeds in the syringe.

Now suck up some of the sugar water you prepared earlier, and leave the syringe for 12-24 hours.
This step is nessesary to wake up any spores or bacteria that might be in their "resting" state, since they can be VERY resistant to even high temperatures when not actively growing.

After waiting, get out the rest of the needed stuff:
5. jars/flasks with sterlized medium that have rested for 2 weeks
6. needles for the syringes
7. your two syringes with seeds in sugarwater (rested for 12 hours here)
8. 3% peroxide sollution and a small glass

Now you push out the sugar water as you did with the ordinary water earlier.

Now to the sterilization part!:

You suck up some peroxide from the small glass. A lot of small bubbles will start forming, and you need to get those out!
To du this you put your finger in front of the tip of the syringe and start compressing the sollution with the piston, this is followed by a negative pressure by pulling the piston (be careful here! Once I pulled to hard on one syringe and spilled all the seeds in it.. IM SO GLAD I HAD TWO! otherwise it would be 9 months of waiting wasted..). While you make the negative pressure tap the syringe with your nail, to make the bubbles gather in the top where they can be pushed out.
This is done every 5 minutes for half an hour. Then you are done sterilizing!

You now take out you jars/flasks and sterilize the silicone plug with a bit of rubbing alcohol. Attatch the needle to the syringe
(if this needle is not sterile, before using it, you should suck some rubbing alcohol through it, and wipe the outside with alcohol to)
Now stick the needle through the silicone plug, and push out as many seeds in each jar as you like and tilt the jars/flasks to disperse the seeds on the medium. There is no problem in sowing the seeds floating in peroxide since this turns in to water and oxygen, but try to limit it as much as possible.
Between each jar you can again sterilize the needle by wiping it with rubbing alcohol - just to be on the safe side .
Each hole you punched in the silicone plugs, should now be sealed by either a piece of tape or a small amount of silicone.

The pictures are:
1. split open seed pod
2. seeds sucked up with water
3. water pushed out
4. Sugar water sucked up, left for 12 hours like this
5. Peroxide sucked up
6. Sowing (here you can also see the silicone plug made in the lid)

Hope this helps

Christian