Limiting infection when reflasking - how

[s1214215]

Hi Everyone

I have been using a glove box to reflask and flask seed - how I dream of a laminar flow unit. I still get contamination no matter how careful or over-zealous I am with disinfecting.

I have read a link suggesting the use of hydrogen peroxide when potting seedlings. I wonder, could we use 3% Hydrogen Peroxide when reflasking. I am sure many of you have had flasks sent in the mail, arriving looking like a fruit salad or at best agar all over the leaves. If you dont want to pot up, then you reflask, but that for me has so far been hit and miss.

Another possibility is antibiotics - I just found this link for PPM PPM: A Powerful Tool to Prevent or Eliminate Microbial Contamination in Plant Tissue Culture

Now, some countries prohibit the use of antibiotics in flasks. I also know that some species cant take this particular disinfectant.

How do members here reflask and make sure you dont get infections.


Brett

[Ray]

Assuming you are sterilizing the medium and flasks sufficiently, the likely issue is insufficient sterilization of the glove box itself. Chlorine bleach is your friend in that regard.

Make up a solution of 100 ml household laundry bleach (5% sodium hypochlorite is typical) per liter. Put some in a bowl for tools, the rest in a spray bottle, both inside the box before closing it up.

Before opening up the flasks, thoroughly spray the entire inside surface of the box and its contents.

[s1214215]

Thanks Ray

I am using Detoll disinfectant rather than bleach as I find the bleach to choking. I know of other people using disinfectants to spray with.

I leave my flasks for a week or more to check there is no contamination before I flask. I seem to have little issue flasking seed. It is reflasking seedlings that is my problem. All the seedlings I have done so far have been seedlings from flasks sent in the mail. I wonder if this is were they are being contaminated.

Today I got 2 flasks from the USA after 5 days in the mail and used 3% hydrogen peroxide for one flask to wash the agar off the seedlings and 1.5% on the other flask. I will see how this goes. I only breifly rinsed the seedlings in it to get them clean.

I am quite careful sterilising the glove box. So I am not sure why this is still occuring. I think a laminar flow unit is not far away for me

Brett

[Royal]

Originally Posted by s1214215

Hi Everyone

I have been using a glove box to reflask and flask seed - how I dream of a laminar flow unit. I still get contamination no matter how careful or over-zealous I am with disinfecting.

If this statement is true, then Rays advice is good. I use alcohol as a spray because I don't like bleach either, but if I kept getting contamination I'd go back to the bleach as the proven standard. Detoll may work, but if it isn't you have to know when to move on.

Assuming it's not your disinfection protocol, sometimes flasks contain latent contamination (probably more often that we realize). Although a flask may look "clean" there is a strong likelyhood that there is some contaminant in there just waiting for a good opportunity to grow. The rough trip in the mail might just be that opportunity. It is also possible that delivery via airplane could subject the flask to such varying pressures that the flask either sucked in air, pushed air out, or both.

Here's what I'd try: Do a first rinse in plain water to remove most of the agar. Then, do a rinse in a weak bleach solution (you don't have to spray it, just a small dish). Then, do a third rinse in the peroxide and into the flask. Hope some of this helps. Good luck.

[s1214215]

Thanks Royal

So far the flask I did the wash with 3% hydrogen peroxide are clean of any infections. Still, it is only day 2. Fingers crossed that they make it ok.

Brett

[Bloomin_Aussie]

Hi Brett, I'm curious about how the reflasks went. Any update?

[s1214215]

Hi

I have a laminar flow now. It has made a huge difference. I dont get the amount of infections I used to.

I still get the odd one with seed or stems, but you can't disinfect them all perfectly. Sometimes stems work when sterilised a second time when infections if caught early.

As for importing flasks, it is still touch and go, but where I had little success before, now I find I am having successes. I always wash the agar off using 3% hydrogen peroxide before replating them.

Sometimes the seedlings get a little damaged by it, but they in most cases make a come back with new growths. Without this treatment I find infection rates rise a lot.

Protocorms by post work quite well with little infection too. These I never wash, but put straight into new flasks.

Brett

[s1214215]

My Bulbo Ipanamense from Troy Meyers. I accidentally knocked it over tonight and had to reset it. It was good in a way, as I was able to check the old media and it was fresh.

You can see some leaves did not like the hydrogen peroxide 3%, but new growth is emerging after 3 weeks

Brett

[Bloomin_Aussie]

That's great news. Why do you think you weren't so successful with the glove box? Because it wasn't air tight?

[kavanaru]

Why don't you try this method instead of the glove box? Lotte & Thomas Orchids

I used to work this way at the university during my thesis, as we got the laminar flow hoods infected with a fungi, and it took about two months to clean them. The contamination rate was minimal using the method described above. The only extra I used to do, was spraying everything around (table, walls, pots, etc) with alcohol before starting to work (but be carefull if you will use an open fire! let teh alcohol evaporate before starting any fire!)