# to kill chytrid, but not plants



## Rain_Frog (Apr 27, 2004)

Anyone have a safe method to sterilize plant roots of chytrid without killing the plant?

I've noticed that bleach will kill anacharis, hornwort, and java moss easily at low concentrations.


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## zBrinks (Jul 16, 2006)

A local zoo I'm working with treats all their plants with metroniadazole before using them in amphibian exhibits. I'll post details when I'm there next and get a chance to chat with the vet.


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## Rain_Frog (Apr 27, 2004)

interesting. Over the counter metro from a fish store, or stuff that you get prescribed by say, Dr. Frye?


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## Rain_Frog (Apr 27, 2004)

Anyone know how effective the hydrogen peroxide / vinegar method is for chytrid and mycobacterium spores? The ASN handbook mentions it.


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## bobberly1 (Jul 16, 2008)

I'd try hydrogen peroxide, there was a good article on sterilizing plants in an old Tropical Fish Hobbyist issue that mentioned that.


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## Dane (Aug 19, 2004)

I believe that a 1% solution has been recommended in other Chytrid disinfection protocols, and I've been able to get java, riccia, and some other touchy things through a few minutes in the solution without becoming inviable. It will take a few weeks for the plant to resume growth, but it can be done.


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## Rain_Frog (Apr 27, 2004)

It's been a while since I've had chemistry.

How do I make a 1% solution of sodium hypochlorite (chlorox) from a bottle that has a 6% concentration?


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## Roadrunner (Mar 6, 2004)

I`m not a vet but metronidazole only works in an animals body, not in the viv or on plants, from my understanding. If the plants can take 90f or above for a day. 
Recommended Protocols for Disinfection of Chytrid on Surfaces


Table 1: Disinfection strategies suitable for killing Batrachochytrium dendrobatidis and ranaviruses in field studies. Where concentrations and time are given, these are minimum shown to be effective. For B. dendrobatidis based on Berger (2001) and Johnson et al (2003) and for ranaviruses on Langdon (1989) and Miocevic et al (1993).

Purpose	Disinfection	Concentration	Time	Pathogen killed
Disinfecting surgical equipment and other instruments (e.g. scales)	Ethanol	70%	1 min	B. dendrobatidis
Ranaviruses
Vircon	1 mg/ml	1 min	B. dendrobatidis
Ranaviruses
Benzalkonium chloride	1 mg/ml	1 min	B. dendrobatidis

Disinfecting collection equipment and containers	Sodium hypochlorite (bleach)	1%	1 min	B. dendrobatidis

Sodium hypochlorite (bleach)	4%	15 min	Ranaviruses
Didecyl dimethyl ammonium chloride	1:1000 dilution	0.5 min	B. dendrobatidis

Complete drying 3 hours or greater	B. dendrobatidis
Heat	60 C	5 min	B. dendrobatidis
15 min	Ranaviruses
Heat	37 C	4 hours	B. dendrobatidis
Sterilizing UV light 1 min	Ranaviruses only
Disinfecting footwear	Sodium hypochlorite (bleach)	1%	1 min	B. dendrobatidis
Sodium hypochlorite (bleach)	4%	15 min	Ranaviruses
Didecyl dimethyl ammonium chloride	1:1000 dilution	1 min	B. dendrobatidis
Complete drying 3 hours or greater	B. dendrobatidis
Disinfecting cloth Hot wash	60 C or greater	5 min	B. dendrobatidis
(e.g. bags, clothes) 15 min	Ranaviruses



Reprinted from:
HYGIENE PROTOCOL FOR HANDLING AMPHIBIANS IN FIELD STUDIES
Speare R1, Berger L1, Skerratt LF2, Alford R3, Mendez D1, Cashins S3, Kenyon N3, Hauselberger K3, Rowley J3
Amphibian Diseases Group, James Cook University, Townsville 4811, Australia.
1 School of Public Health and Tropical Medicine
2 School of Biomedical Sciences
3 School of Tropical Biology
8 October 2004


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## Rain_Frog (Apr 27, 2004)

aaron, I have read the Johnson article, but I do not know how to make a 1% solution of sodium hypochlorite from a 6% solution (standard clorox).


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## Roadrunner (Mar 6, 2004)

At 6% to reduce it to 1% you should add 5X the quantity. 1 shot to 5 shots.
I just figured heat may be safer w/ tropical plants. I hate the residue it leaves.


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## kristy55303 (Apr 27, 2008)

curiosity struck. would the researched lamisial at treatment done on frogs done for ten days as same for the frogs work on plants? 
kristy


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## bobberly1 (Jul 16, 2008)

She also mentioned Potassium permagenate, if I got that right.


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## zBrinks (Jul 16, 2006)

I called a keeper friend at the Binder Park Zoo - he'll talk to the vet and email me a summary of the plant protocol sometime over the next week. The vet did mention using metro for a 5 day treatment of plants to eliminate the chance of introducing chytrid to an exhibit I'm constructing for them.

I'll post when I hear back.


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## Rain_Frog (Apr 27, 2004)

Are you positive about that dilution, Aaron? I remember you need to factor in the moles of sodium hypochlorite. It'd be nice if Bill Heath (a chemist) could check this post out.

Kristi, I was pondering that earlier, but not sure how that would work. Just because we can use it for frogs when they're sick doesn't mean we can use it to sterilize plants.


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## Roadrunner (Mar 6, 2004)

I`m no chemist, your talking over me.
My better half, a biochemist says 6% is 6g/100mil, if you want 1g / 100 mil you`d have to dilute w/ 5 x the volume or 500 mil to get 6g/600 mil


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## Roadrunner (Mar 6, 2004)

Rain_Frog said:


> Are you positive about that dilution, Aaron? I remember you need to factor in the moles of sodium hypochlorite. It'd be nice if Bill Heath (a chemist) could check this post out.
> 
> Kristi, I was pondering that earlier, but not sure how that would work. Just because we can use it for frogs when they're sick doesn't mean we can use it to sterilize plants.


As w/ spraying objects other than frogs w/ metronidazole.
Is metro your talking about a disinfectant? Metronidazole is a dog antibiotic I believe. I`ve seen it referred to about spraying tanks w/ it on a euro site. Dr frye said it would be useless used outside an animal since it works w/in the animals.


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## zBrinks (Jul 16, 2006)

Turns out the keeper misspoke when I talked to him last. He just texted me that the zoo uses itrachonazole to combat chytrid. 
I've used metro to treat various animals for protozoan infections - thought it was a little odd when it was mentioned in conjunction with disinfection.

Here's some info I googled:
Itraconazole


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## Roadrunner (Mar 6, 2004)

I lostr the link when I had to agree to the terms. Unless the zoo is made of money they probably don`t use it to sterilize stuff. it`s $345 for a small bottle worth. Sorry I couldn`t read the itraconazole info.


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## zBrinks (Jul 16, 2006)

If you agree to the terms, then paste the link in your browser, it will show up. Expensive stuff, the zoo must be doing better than I thought


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## kristy55303 (Apr 27, 2008)

i happened to recieve some free itraconazole tabs...for a lack of better word to use...they look like and are the same size as a church communion bread piece that some religions do. anyhow it looks like that. i have five. I had two sick frogs and the chytrid scare did scare me and we assumed it was that.....but turned out definately not. the results came back neg. from john wood for my whole collection. we never used them. I did hear the lamisil at treatment is safe for frogs...whether it would work for plants i dont know. maube a soak for ten days and good rinse and repeat just like frogs? interesting thread. however, i do believe itraconazole has shown to have negative effects on metatmorphs and very glad i didnt do it. especially since they are chytrid free. just my ol self being overcautious as usual. anyways, would the itracon. cause these effect and be soaked up into the plant tissues and harm our frogs when introduced into a viv then? Curiosity still. great thread. especially since some of the java moss comes from malaysia-chytrid known....and some come from private aquarists that could possibly house in the same tank the oh so famous carrier xenopus laervis ....its too bad they have gotten bad rep as well. 
kristy


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## nish07 (Mar 16, 2008)

Outside of bleach, the only thing I could suggest would be drying the stuff out for an extended period and/or putting it in the oven at 100 degrees for 10 hours or so. If I can't bleach it, I'm not so sure I want to use it (which is why I still can't figure out how I'm going to put riccia in my tanks).

-Nish


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## Rain_Frog (Apr 27, 2004)

Kristi, you can't desolve itraconazole in water unless you buffer it-- one reason why itraconazole is a difficult medication to administer for the home frog keeper.

Yesterday, I took out my plants in two established vivs and put them in a rubbermaid bin for a "growout" tank.

So, I can take cuttings and offshoots and experiment with bleach and other things without killing the entire plant.

Since the tanks are going to house something very rare and hard to find, I'd rather not take chances with these frogs.


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## zBrinks (Jul 16, 2006)

Rain_Frog said:


> Since the tanks are going to house something very rare and hard to find


 After that, you need to tell us what it is


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## Rain_Frog (Apr 27, 2004)

> After that, you need to tell us what it is



Nope-- at least for now


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## Frogtofall (Feb 16, 2006)

He did the same damn thing in the chat room!


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## Ed (Sep 19, 2004)

Rain_Frog said:


> Kristi, you can't desolve itraconazole in water unless you buffer it-- one reason why itraconazole is a difficult medication to administer for the home frog keeper.


Buffering it doesn't change the solubility of the itraconazole. (It simply keeps the acidity of the drug from harming the frogs.) It is pretty insoluble in buffered or unbuffered water and and really just forms a suspension. 

Is the concern only about chytrid? If so then the following can be considered for sensitive plants.. at this time while it has been theorized it has to my knowledge not been shown to have a resistant resting spore form and the zoospore is known to be sensitive to a number of disinfectants as well as temperature. 

While you cannot use it to treat infected frogs to attempt to cure chytrid, disinfectant strength benzalkonium chloride is effective against the zoospore. see Fungicidal effects of chemical disinfectants, UV light, desiccation and heat on the amphibian chytrid Batrachochytrium dendrobatidis. | Johnson, M. L., Berger, L., Philips, L., Speare, R. | Diseases of Aquatic Organisms | Inter-Research 

You are going to have the same problem regardless of the disinfectant used which is ensuring that it penetrates all of the potential areas of the plant. 

If you are that concerned about it, then you should disinfect the plants and then grow them under clean conditions where they do not touch and then take cuttings of those plants. 

Ed


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## Rain_Frog (Apr 27, 2004)

http://homes.jcu.edu.au/school/phtm/PHTM/frogs/papers/johnson-2003b.pdf

After studying this article again, I have an idea now:

What about submerging plants in water that is kept continuously at temps 37 C (for four hours) or 32 C (for 96 hours).

I have incubated roach eggs with a RENA submersible heater hooked up to a temperature controller, since the heater itself is not very good at controlling the proper temperature in small bodies of water.

This could help solve the problem of killing java moss and other aquatic plants that are harmed by bleach.

Also, I'm thinking about trying a 5% sodium chloride solution.


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## nish07 (Mar 16, 2008)

The only problem is (and much more likely the problem than chytrid in moss) that it still can contain protozoans (coccidians). These won't go away with bleach and the coccidians won't go away unless you dry them out completely.

-Nish



Rain_Frog said:


> http://homes.jcu.edu.au/school/phtm/PHTM/frogs/papers/johnson-2003b.pdf
> 
> After studying this article again, I have an idea now:
> 
> ...


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## kristy55303 (Apr 27, 2008)

i could be wrong but i dont even think drying kills all types of coccidia? please correct me if i am wrong but believe it can actually hang out for quite a long period of time in say a dry tank for example.

I forgot to clarify earlier in the discussion that the itraconazole disks i have can simply be diluted/dissolved in frog safe water as per instructions from a dr. russell poulter from new zealand who has some plublications on the effects of chloremphenicol on chytrid fungus , so in fact i would have way more of it than i could ever need if i ever used them. I think the directions were to dilute in 2 liters and i have for sure 5 disks and maybe ten. off topic sort of but on as i have been out of the discussion for some time.

kristy


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## nish07 (Mar 16, 2008)

Needs moisture to live.

-Nish


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## Ed (Sep 19, 2004)

kristy55303 said:


> i could be wrong but i dont even think drying kills all types of coccidia? please correct me if i am wrong but believe it can actually hang out for quite a long period of time in say a dry tank for example.
> 
> I forgot to clarify earlier in the discussion that the itraconazole disks i have can simply be diluted/dissolved in frog safe water as per instructions from a dr. russell poulter from new zealand who has some plublications on the effects of chloremphenicol on chytrid fungus , so in fact i would have way more of it than i could ever need if i ever used them. I think the directions were to dilute in 2 liters and i have for sure 5 disks and maybe ten. off topic sort of but on as i have been out of the discussion for some time.
> 
> kristy


It depends on what the author considers a coccidian. Some references include cryptosporidia as a coccidian and drying does not kill cryptosporidia. 

Itraconazole doesn't really dissolve in water. The best you can get is a suspension and the water needs to be buffered to prevent mortality from the low pH of the itraconazole. 

Ed


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## kristy55303 (Apr 27, 2008)

correct. that was the coccidian i was referring to. A nasty often seen in reptiles i believe. 

I agree ed on the itraconazole.All I have are instructions to go off of sent from a new zealand scientist. I know i wont be using it more than likely anyhow. But in my med drawer i guess just in case

edit: ed how would i go about buffering the water....never hurts to learn something new for those disks i have just in case.


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## Ed (Sep 19, 2004)

kristy55303 said:


> edit: ed how would i go about buffering the water....never hurts to learn something new for those disks i have just in case.


Use Amphibian Ringers for the solution. It is buffered and will hold the pH in a safe range. 
This is what we did when we quarantined Atelopus zeteki and Bufo baxteri. 


Ed


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## kristy55303 (Apr 27, 2008)

Ed said:


> Use Amphibian Ringers for the solution. It is buffered and will hold the pH in a safe range.
> This is what we did when we quarantined Atelopus zeteki and Bufo baxteri.
> 
> 
> Ed


perfect. i always keep a couple liters on hand. after i have inserted the disk into those liters, how long would the itraconazole be effective by chance. sparked my curiosity on the subject. thanks again for all the advice. kristy

edit: i must have given you good rep somewhere. wont let me add anymore. just wanted to thank you for all the great help and advice. i need to spread more rep before adding i guess.


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## Ed (Sep 19, 2004)

kristy55303 said:


> perfect. i always keep a couple liters on hand. after i have inserted the disk into those liters, how long would the itraconazole be effective by chance. sparked my curiosity on the subject. thanks again for all the advice. kristy
> 
> edit: i must have given you good rep somewhere. wont let me add anymore. just wanted to thank you for all the great help and advice. i need to spread more rep before adding i guess.


Itraconazole is light sensitive so any solution needs to be kept in the dark. We made up new solutions daily from the stock solution. I forget what the stock solution is made from... 

Thanks for the vote of confidence

Ed


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## kristy55303 (Apr 27, 2008)

welcome lol
what would that shelf life be by chance ed?
kristy


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## Ed (Sep 19, 2004)

I have to get ahold of the vets at work and ask them. I haven't run into them for a few days. 

Ed


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## kristy55303 (Apr 27, 2008)

Ed said:


> I have to get ahold of the vets at work and ask them. I haven't run into them for a few days.
> 
> Ed


i can be patient. well i'll do my best it would be nice to know when you have a chance for future reference. thanks a million,
kristy


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## Rain_Frog (Apr 27, 2004)

back to the original topic  I am using a 5% sodium chloride solution now. The heat idea killed the plants, even at 92 degrees F.

So far, sodium chloride seems to be not affect the plants as much, but I'm still in the early phases of experimenting.


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## zBrinks (Jul 16, 2006)

Does anyone know if Physan 20 will kill chytrid?

Josh's Frogs - Physan 16 oz. - Disinfectant


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## james67 (Jun 28, 2008)

UV???
let me start by saying that i admittedly have not read all posts contained in this topic, so please excuse any material that may be repetitive. 

i was just doing some reading and from what i found bleach, potassium permanganate (which is what i use even on mosses with no noticeable ill effects to the frogs or plants), heat, ultraviolet exposure, and drying all have the ability (under laboratory conditions) to kill Batrachochytrium dendrobatidis. i have heard recently that a zoo (cant remember which) killed chytrid in hellbender tanks by slowly (over the course of a few weeks) raising the temp to just over 90 degrees F.

success with moss may be truly attainable only if one were to grow moss either for the purpose of drying and collecting spores which can be dried (completely) killing the chytrid, and introduced into a sterile agar filled tank, etc. or by purchasing already harvested dried spores. (which limits the ability to use most species since very few are commonly offered for sale in this form) and again introducing to a clean environment. 

just some thoughts
james


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## tclipse (Sep 19, 2009)

james67 said:


> UV???
> let me start by saying that i admittedly have not read all posts contained in this topic, so please excuse any material that may be repetitive.
> 
> i was just doing some reading and from what i found bleach, potassium permanganate (which is what i use even on mosses with no noticeable ill effects to the frogs or plants), heat, *ultraviolet exposure*, and drying all have the ability (under laboratory conditions) to kill Batrachochytrium dendrobatidis. i have heard recently that a zoo (cant remember which) killed chytrid in hellbender tanks by slowly (over the course of a few weeks) raising the temp to just over 90 degrees F.
> ...


(sorry for the zombie thread)

If this is the case (depending on the timeframe & intensity needed) couldn't tough-to-clean plants such as riccia simply be submerged in a tank with a UV filter for a few days? I have a few of these and used them on my tanks with great results.. even parasite-prone fish like the electric blue jack dempsey were 100% clean for as long as I used them. The EBJD in tanks without them had probably a 90% infection (and 80% or so mortality) rate by a year old. Given, these were different pathogens/microbes/parasites/whatevers, it seems feasible that the light could be powerful enough to deal with chytrid as well.

The ones I have are rated for 40gph... theoretically, placing a patch of riccia and the filter in a 2.5 gallon tank for several days could do well to get rid of it? With the filter pumping 16 times the tank volume per hour, it seems within reason that a few days' time would ensure safe eradication (assuming a given fungal spore is killed within the first few passes inside the filter... from my experience with other parasites/pathogens/algaes and these filters the likelihood is high). 

Also, tanks with a UV filter under the false bottom might then be able to create a *nearly* chytrid-free environment. I'm assuming that some might still be present in the substrate... but my best guess would be that the speed of its propagation (and implicitly, the speed of intake by the frogs) would be slowed down substantially.

Again, this is all speculation based on the effectiveness I've seen from UV filters firsthand, coupled with the notion (a reference would be nice) that UV exposure can destroy the fungus. It sounds good on paper, anyways. At the very least, it could handle many other harmful pathogens in the water feature.. if only slightly helpful for PDF's, it seems like a nice security blanket for semi-aquatic species. The one issue with chytrid is that I don't know of any adhesive properties it might have that could allow it to stay stuck on the plants.


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## Ed (Sep 19, 2004)

How is the UV getting into the areas shaded by the bends and twists of the plant material? 


Unlike Ick and some other parasites, which fall off of the host and are then developing on the substrate for a few days before becoming freeswimming again, the active zoospores move until they contact a substrate and then they attach to the substrate. It them stays stuck on the substrate. Also the fungus can be spread by frog to frog contact or (untested yet) by tadpole on tadpole contact. 


Ed


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## tclipse (Sep 19, 2009)

Yeah, like I said I wasn't sure if chytrid had that ability, but if it did.... the idea becomes essentially null and void. 

It will still take out many other harmful pathogens so I still feel it could a positive, but that's a shame. It'd be interesting to see if there were any change in the number of bacterial infection and parasite cases (again, mostly with semi-aquatics) in tanks with vs. without UV sterilizers. I know it works for fish, one would think that it could for frogs. Not totally necessary, but not a bad insurance policy either IMO.


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