# Saving a contaminated mother flask?



## gore42 (Nov 1, 2006)

I discovered this morning that one of my mother flasks has developed some nasty cultures that I wasn't intending to grow. So far, there are two big ugly spots of the stuff, but I assume that it will only be a matter of hours before they take over the whole flask.

Luckily, this was the last motherflask that I made with the pod... so there aren't very many seeds, and I still have several other motherflasks of this cross.

As you can see in the photo, I have a few protocorns that are beginning to develop, but this is _Paph parishii_, and they aren't especially fast growing. Normally, I'd just chuck the motherflask and use the other ones. Since this is one of my favorite species, though, I'd like to do what I can to save them.

For scale, this is the interior landscape of a babyfood jar. The two contaminated regions are about 2 cm across, total. Take a look: 







Sorry for the blurry photo... there was a bit of condensation on the side of the flask.



So, is there anything that I can do to save these? It seems pretty optimistic to thing that I'd have any luck just scooping out the areas that are visibly contaminated. Is there any anti-microbial that is safe enough to use in a flask?

Any suggestions would be appreciated... otherwise, I'll start experimenting in a couple of hours.

- Matt


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## Drorchid (Nov 1, 2006)

If I get mold or bacteria, and discover it at an early stage I try to scoop out the protocorms/seedlings that are far enough from the infected areas, and put them on fresh media. About 1/2 of them will still get moldy; but at least I am able to save some. Once I tried scooping out just the mold too, but that usually does not work, as in a few days the mold will usually grow back (as there are probaly spores present in the contaminted flask allready).

Good Luck!

Robert


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## gore42 (Nov 1, 2006)

Thanks Doc, I guess I'll give that a try. I'll let you all know how it works out 

- Matthew


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## littlefrog (Nov 1, 2006)

If you can get hold of Amphotericin D, that is a anti-fungal agent that seems to have no effect on plant (or mammalian) cells. We used to use it routinely in mammalian tissue culture. I tried some in replate medium for orchids when I was doing that sort of thing and it worked fine. In fact, the plants seemed to grow better... Didn't repeat it.

I think it was 1ml/L, but that was 15 years or so ago now. I'd put that in your replate medium in addition to avoiding the obviously contaminated bits.


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## paphjoint (Nov 1, 2006)

Try to burn out the mold with a magnifying lens - works in some cases


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## kentuckiense (Nov 1, 2006)

Matt, you can thank me later.


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## gore42 (Nov 1, 2006)

Thanks Rob -

I didn't have immediate access to Amphotercin D, so I had to go without it this time. Any suggestions about where I might get it in the future? It sounds like a controlled substance.

Once I was down in my lab and took of the lid of the motherflask, I realized that it was a lost cause. There was a little bit of liquid on top of the medium which was in contact with the contaminants and was also swishing across the top of the rest of the flask... so it was clear that everything was contaminated. So, I just trashed them.

Thanks again for your help Rob and Dr. Robert 

- Matthew Gore


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## Drorchid (Nov 1, 2006)

Kentuckiense.....That was funny:rollhappy: 

Sorry to hear that you had to trash the mother flask.

By the way Rob is also Dr. Rob.....(I am not the only Doc on this fourm)

Robert


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## Kyle (Nov 1, 2006)

I have had OK luck with scooping out the contaminate. Fungi is more difficult because of the spores. But with bacteria or yeast, I either resue the plants or protocoms, or scoop out the contaminant. 

At least you have more flasks.

Kyle


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## littlefrog (Nov 1, 2006)

Amphotericin D was a pretty typical tissue culture reagent, probably still is. I think we used to buy it from Sigma. You can probably find it from any reasonably decent tissue culture supply place. Not a controlled substance, as far as I know. Buying things from Sigma as an individual rather than a company was hit or miss. I had some luck because I was working at a university (still am) and used a university address, although it was charged to my own 'personal' account rather than the lab...

Rob (the other Dr. Rob... *grin*)


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## gore42 (Nov 1, 2006)

Hehe, I know that there are plenty of Doctors around here, I just needed a way to distinguish the Roberts, and thought I'd go with your nicks...

Anyway, thanks Rob, I'll check on Sigma. 

While I was trashing the first flask, I also made a few new ones. I had a second pod that I wasn't going to bother with, since it appeared that I was getting good germination on my first one, but with the contamination, i decided that I'd better use all the seed I can get. So, now I'm back ahead of the game (I hope), just a little behind schedule on some of them.

Thanks for all of your responses, everyone! Uri, that's a cool idea! If only I had a decent laser.... 

- Matthew


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## ScottMcC (Nov 1, 2006)

amphotericin is also used in people with severe fungal infections. tends to be reserved for pretty sick folks in pretty limited settings though.

there's obviously way too many doctors on this forum...we should start kicking them out, starting with the non-MDs, of course


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## littlefrog (Nov 1, 2006)

I think we should start kicking out the MDs, in order of specialty, cranial to caudal... So, your neurosurgeon goes first, then the ENT guys (Sorry Scott...), I guess the OB/GYNs go last? *grin* Maybe the proctologists?


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## Drorchid (Nov 1, 2006)

I think we should start kicking out the non-plant doctors first this is a plant forum afterall...

Robert


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## Heather (Nov 1, 2006)

Hey, hey, hey....no kicking anyone out! 
Remember? That's our MO here.


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## Marco (Nov 1, 2006)

Matt sorry to hear about the mold. Hope you can manage a couple of suvivors.


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## ScottMcC (Nov 2, 2006)

Heather said:


> Hey, hey, hey....no kicking anyone out!
> Remember? That's our MO here.



whatever Heather, we're gonna find a way to kick you out first. oke: 

now that'd be ironic, getting kicked off your own website.  

but back on topic, fungal contamination is bad news. you should probably do a very thorough cleaning of your entire lab area with something that's powerfully antifungal. It's been a few years since I did any tissue culture, but someone in our lab managed to contaminate our hood with some mold, and it took us months to finally get rid of it.


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## NYEric (Nov 2, 2006)

In the book 'Orchid Fever' reference is made to a grower in the vicinity of Paris, France using systemic pesticides and endangering his own health. Does anyone know the grower and the pesticides referred to?


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## kentuckiense (Nov 2, 2006)

I believe it was Xavier de Loubresse. Something like that. I know the first name is right, at least.


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## gore42 (Nov 2, 2006)

At Rick's suggestion, I contacted Troy Myers about this issue, and he had some helpful info. I thought that I'd pass it along here:

-----------------------------------------------

Matt,

It's true, excising a mold spot on dry-topped agar can sometimes work, 
but with a little liquid on top it's unlikely. Also, excising in a 
laminar flow hood is much less successful than in a still-air box (like 
some glove boxes) because spores get swirled around.

We also use a wet mother technique, so when this happens we excise the 
bad spot then empty the mother liquid and seeds/embryos/protocorms into 
a vial and rinse them with sterile water, let the plants settle to the 
bottom, aspirate most of the liquid, rinse again and repeat at least 3 
times. This cleans the protocorm exterior somewhat, but mostly highly 
dilutes any contaminated liquid. Then the protocorms are re-disinfected, 
as if sowing as seed again, and put into a new mother or farm flask, 
but we generally use a saturated calcium hypochlorite solution. That 
seems to work best in most cases. Sometimes it kills the plants, of course, 
but it often works. Sometimes the contamination recurs, too, of course.

-Troy


------------------------------------------------

Unfortunately, I was a little too hasty in chucking my parishii motherflask. Next time, though, I think that all of these suggestions will be helpful 

As Ever,
Matthew Gore


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## ScottMcC (Nov 5, 2006)

NYEric said:


> In the book 'Orchid Fever' reference is made to a grower in the vicinity of Paris, France using systemic pesticides and endangering his own health. Does anyone know the grower and the pesticides referred to?



Any of the organophosphates (such as malathion) have known toxicity to humans and can cause rather dramatic symptoms (excess salivation, tearing, urination, and defectation) along with cardiac difficulties and death.

here's a link for the medically inclined: http://www.emedicine.com/MED/topic1677.htm


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