This problem is familiar to my laboratory. We had mite contamination in cultures stored at room temperature, but not in refrigerator. No mite contamination was observed at 4-7C in refrigerator. To my experience dermatophytic fungi on agar slants can be stored at this temperature up to 12 months and remain viable. We have a deal with species of animal origin such as Trichophyton mentagrophytes, Tr. verrucosum, Microsporum canis, M. gypseum and some others. Another option for you I guess is the tubes with screwthread caps to prevent mite contamination.

Dear Roman Ovchinnikov

Thanks for your comment

This microscopic image of mite in lactophenol stain

Transfer a small piece of growing hyphal perimeter to new media, keep your plates parafilmed in plastic bags with moth balls, thoroughly decontaminate your work space!

I have had issues with mites in basidiomycete cultures (saprobes and wood decay fungi). A way to recover cultures already infested with mites might be to flood the plate culture with mineral oil, then decant oil again, and cut out some pieces of medium/mycelium and transfer to new medium. As the culture starts growing into new medium, cut out a tiny piece of this non-oily new outgrowth and transfer again. The smaller the pieces transfered the better as there are also eggs in the medium.

To prevent mites better stack the cultures in trays. Under the trays, have Petri plate halves with mineral oil (2, or 1 in each corner), and a glass beaker flipped around sitting in the middle of each. You place the tray onto this. On the buttom inside of the tray, either miticide (acaricide)-drenched paper or again mineral oil. In this pool of mineral oil, again empty Petri plates. You stack your cultures on top of that. If there is an ongoing mite infestation, place Kimwipes drenched with some miticide between them. Really thickly wrap plates with parafilm. Often check plates for tell-tale slimy tracks on medium or lids. Contamination with other fungi starting from the rim is another sign.

Mites can be a real problem to get rid of. It would be best to discard your cultures and decontaminate the lab. One problem with transferring hyphae from a contaminated sample is the potential of transferring mite eggs. In the event that you decide to try a transfer to clean agar it would be wise to select your hyphal tips under the microscope. Seal with para film and store in with moth balls.

When decontaminating it is important that the entire lab be done not just your bench area. Place moth balls to discourage mites but it would be good to find where the mites came from and put in measures to minimize their return once you have decontaminated the lab.

Good luck.

From time to time we have the same problem with mites. We use Ivermectin as supplement in our solid media. If you transfer your fungus at least two times to ivermectin plates, you get rid of these nasty little athropods.

Dear Jessie Uehling & Eric Kuhnert

Are mothballs and Ivermectin harmful for Fungi ?

and َAre they have bad effect for fungi for example killing fungi in medium?

Thanks for comments.

Prevention is better than Saving after infestation - follow Dirk's suggestion. Frequent checks . Remove cultures / plates that are old.

Long term storage can attract mites. Once spotted in your plates , the whole storage area needs to be decontaminated. If in an incubator remove all plates and increase temperatures to above 80oC for a couple of days. Then thoroughly clean with 70% ethanol and repeat high temperature for a 2-3 days.

It is near impossible to save infested plates. Save the non infested plates - ' walking tracks' on lids of Petri plates / walls of tubes( do check under microscope,too - to confirm free from eggs). Culture as suggested by Katherine. Add miticide agent into your plates.

Dear Javad,

in our experience some of the fungi tend to grow slower. However the majority grows without visual negative effects. In less than 1% of all cases the fungus isnt growing at all.

All the best

Eric

Thanks for all comments.

For yeast and other filamentous cultures excessive dilution plating is sometimes useful to get rid of mites and nematodes.

If someone know how to get rid of mites in lab, without affecting the cultures, I think it is worth a Nobel prize :-)

Prevention:

- check all your cultures that you intend to store

- double parafilm sealing is good, but we sometimes use also plastic tape used by electricians to isolate wires; this is virtually inpenetrable by mites and does not leave sticky spots after you remove it

- keep your lab clear of old cultures left and forgotten somewhere, clean surfaces by ethanol

- distribute moth balls or tablets with agaricide to boxes with your cultures and observe regularly your fungi that they are not affected by these chemicals; if so, remove the chemicals

- if you have agar slants with cotton plugs, you may immerse the plugs in mercury chloride, which keeps mites also away (but makes the plugs generally dangerous also for you)

If mites appear:

- immediatelly separate infected dishes/slants

- if your fungi survive freezing (-18°C), put the cultures in a freezer; mites die, fungi can be subcultured

- if you subculture infected cultures, do it carefully under dissecting microscope to be sure that you donť pick mite or an egg, but eggs are easily visible, so that you should not do it

- antoher problem with mites is fungal contamination that is carried by mites, e.g. your cultures may be contaminated by Aspergillus, because they came from its culture in this case, the re-isolation of your cultures is almost impossible

- clear boxes or incubators by ethanol; an old-fashioned and very effective (though drastic and dangerous method for high-tech incubators) is desinfection by formaldehyde vapours overnight.

- the heating to +80°C as already suggested is also a useful trick, providing that your incubator can reach these temperatures.

For faster growing fungi you can try to do hyphal tip transfer using a stereo microscope and (of course) avoid picking the mites.

Fungi are very good at penetrating structures. For plant pathogens, one way is to put the aplug of the culture on sterilized cellophane (without lacquer) on an agar with antibacterial antibiotics (to remove accompanying bacteria the mite have dragged into the plate). The fungus hyphae penetrates quickly through the cellophane using cellulases and leaves the mites behind. Then it is just to peel of the cellophane and cut out the agar where the fungus have penetrated and move that to a new plate. It should be done as soon as fungus have penetrated. I have not worked with dermatophytes but I imagine they should be good at growing through collagen membranes so the same technique might work.

To avoid the problem completely we routinely put plates in polypropylene bags and melt seal the bags. If the polypropylene is thin enough (0.05 mm) oxygen and carbon dioxide can pass through and it also keeps the plates from drying so they can be kept in room temperature for up to a year.

Best wishes

I have had this problem once before and I was able to clean up my affected cultures by preparing PDA with Novobiocin (for bacteria) and Kelthane which is a miticide. You will notice that your cultures will not grow very well but all you want is for the mycelium to grow a reasonable distance away from the transfer plug and then replate on to non-ammended media of your choice. Good luck.

I also suffered from the same problem. I had to through the cultures. The major problem is that they move from one plate to another and contaminates the cultures with Aspergillus.

I agree with the method of olsson good luck

i think the best way to remove mite from culture, just hyphal tip and transferring to water agar and isolate again on PDA,then reseal the fungi plates again with para-film and use plastic tape.

Thank for your comments..

https://www.youtube.com/channel/UCe19LucHIFI0fCAiI2vEvBQ\

Try the methods on this video.  I don't know if they will help, but we are going to try them.

Marie Langham

The control of mites is not easy. Read " An introduction to INDUSTRIAL MYCOLOGY by George Smith, Adward Arnold (publishers) Ltd, London, page 284. You will know what COMMONWEALTH MYCOLOGICAL INSTITUTE, Kew, do to control mites.

They use fumigants p-dichlorobenzene and crude kerosine. It is easy to use, infested cultures simply being put in an airtight box with some crystals of fumigant and left over night.

The best way of treating infested cultures is to purify by plating out, the old cultures being then killed off by heat.

Stephan, could you please recommend how much Kelthane you add to your media?

I WORK WITH MITES TOO AND IS VERY DIFICULT THIS DESCONTAMINATION; PLEASE READ A PAPER OF satiko Uehara ON REVISTA DE MEDICINA TROPICAL, REGARDS .

Sorry, I completely missed the question on the concentration of Kelthane to use. If I recall I used the recommended amount of Kelthane on the product label; 250g/100 US gallons (this is the low rate) for crop surface sprays which I calculated as 250g added to 378.5L. If you ammend 1L Potato Dextrose agar with Kelthane (after autoclaving but before pouring) you would add the equivalent of 0.66g of Kelthane per Litre of PDA. I washed the Kelthane into the molten PDA with a small amount of 70% ethanol and make sure that you have a stir bar in your media flask (before autoclaving) and stir the media to suspend the Kelthane before pouring into plates. This worked for me.

Some tips to prevent mite contamination of fungal cultures.

http://www.westerdijkinstitute.nl/BioloMICSNews.aspx?Rec=5646

We have found that a quick way to retrieve mite contaminated cultures is by freezing at -20 for at least 24 hours as outlined in this guide: (http://www.fgsc.net/neurosporaprotocols/How%20to%20deal%20with%20mite%20infections%20and%20to%20minimize%20their%20occurrence%20final.pdf )

And then thoroughly cleaning and decontaminating the lab.

Hi, Dear Samantha Jenkins.

Thanks a lot for your comments.

HI

I am experiencing contamination in Fusarium and Trichoderma sp. I suspect it is a mite/nematode contamination since all other contaminations are ruled out by using relevant additives. These are isolates from soil and we often identify by sequencing them.

1. can anyone share a picture of contaminated fungal plate by mites or nematode?

2. or recommend any literature about how to identify if it is a mite contamination in a microscope and also by naked eyes. Thanks in advance.

Vijayalakshmi Gunasekharan,

See my posting of February, 2015.

Mites are visible to naked eye. You can use hand lens. Purify the culture by plating. Discard the old cultures and heat the plates.

See: Article Effect of Abamectin on Fungal Growth and Its Efficacy as a M...