# sterilizing FF culture cups



## CharValley (Feb 29, 2016)

I'm not sure if this is a good forum for this post but I wanted to pass on something that I have been doing for several years now that might help others with their culturing and reusing culture cups WITHOUT the mite carry over.

I had problems with culturing fruit flies and trying to reuse culture cups. I'm sure a lot of you have had this happen too. You start with strong cultures and after a few months of culturing your fruit fly cultures either seem to become weak in size and also a smaller and smaller hatch content.

I tried using mite paper, bleach wash on the cabinet surfaces but there always seemed to be an infestation of those dang tiny mites and I would have to buy new cultures and brand new culture cups, find a new cabinet space to start culturing again.

A couple years ago I started trying to sterilize the cups with each reuse and found that yes I could 'Steam" them in a microwave, wipe them down and try to clean the lids as thoroughly as possible but the mites just simply kept coming back.

I haven't found a way to keep them completely out of the culturing cycle but I did find a way to 'sterilize' the cups without all the prep of microwave and a way to safely clean the lids that always seemed to get so disgustingly dirty and clogged with the waste material from the fly cultures.

First I take and remove as much of the old culture material from the used cup, rinse out any loose material and flush the water. If you have enough cups and can wait until they have dried out you can easily empty the 'ruffy' into a plastic garbage bag so that you are not handling any of it.

I put the lids into the bottom of the sink and while filling the used cups up with hot water, I let the hot water run over the lids to soften up the discoloration on them. 

Once the cups are filled, the lids will often already start to look white again, I take the top off of a generic mouth wash bottle and using the top I fill it about half full and pour it into each culture cup that I am going to clean. I then pour a generous amount of the same mouth wash on to the lid so that it will cover the vent material on the lid.

I let each stand for about a day (probably doesn't need to be 24 hours but it seems to also clear hazing that might have happened to the cup too)

The lids will have almost dried by this time but if you run hot water over them again it will take a couple minutes and they will almost rinse clean, I take a individual paper towel and gently scrub each lid.

With the culture cups I dump the wash into the toilet and refill them with hot water rinsing most of the remaining solid material off the sides, dump that into the toilet and then with a clean paper towel I will hand wipe the entire cup making sure that I also wipe out the top of the cup around the rim to be sure nothing has collected under the rim. Once I have wiped the cup completely clean I rinse it with fresh water and let it drain dry and move to the next cup to be cleaned.

Once you do this you can immediately go into culturing. I have yet to have this method cause any visible issue with the subsequent fruit fly culture and the lids will look almost brand new with no damage to the vent material. 

You should be able to do this many many times with a culture cup and lid so you won't have to keep buying and throwing out cups. I've noticed a great improvement with my fruit fly cultures and the only 'negative' that I have run into is that after 5 or 6 cleanings the lid material can start to lift and/or the lid may crack along the outside edge (although I think this is more because of how I pull back a small opening to pour out flies for feeding)

Hope this helps, I might not have explained it in the easiest way but I think you can get my meaning.

All the best success with your cultures!
Charlie


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## ds51 (Oct 13, 2017)

Hope this helps, I might not have explained it in the easiest way but I think you can get my meaning.

All the best success with your cultures!
Charlie[/QUOTE]


that's a lot of messing about with the lids
I just take the cover of the lids
I use some pva glue and stick a new bit of tissue back on to the lids each time 
the cups I rinse out by hand then put them in the dish washer job done


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## Socratic Monologue (Apr 7, 2018)

Thanks for the write up!

I wash with soap and water, then sanitize in bleach water (1t/gal, same as I use to sanitize all the bowls in my rep room). Soap and water and bleach whiten the fabric lids fine. After drying, the cups, lids and excelsior get stored in the freezer until needed, and the media gets stored in the fridge.

I've not had good results with mite paper. I use a pan with a layer of DE, and put the cups on it.


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## Tinc Tank (Jun 29, 2019)

CharValley said:


> I had problems with culturing fruit flies and trying to reuse culture cups. I'm sure a lot of you have had this happen too. You start with strong cultures and after a few months of culturing your fruit fly cultures either seem to become weak in size and also a smaller and smaller hatch content.


This could also be due to the genetics of the flies. If you consistently select flies from cultures early on in the cycle, you can actually selectively breed for flies that aren't adapted to handle the conditions of that specific media. It may help to choose flies to start new cultures from different cultures of varying stages in their cycle.



CharValley said:


> I tried using mite paper, bleach wash on the cabinet surfaces but there always seemed to be an infestation of those dang tiny mites and I would have to buy new cultures and brand new culture cups, find a new cabinet space to start culturing again.


It might help to use culturing containers that are mite-proof themselves. There are no "mite-proof" containers on the market designed specifically for culturing fruit flies, but there are lids designed specifically for culturing mushrooms in Mason jars that will serve that purpose. The .3 micron filters are also designed to filter out other fungus spores in the air therefore eliminating contamination in the mushroom cultures. There are multiple benefits to these:
1) I had less mold growth due to the air filtering action of the lids.
2) The lids kept the culture humid therefore benefiting the flies and maggots and giving me awesome cultures.
3) The lid filters both ways. I estimate a 50% reduction in bad odor after using these.
4) They are reusable - they are rated to be autoclaved.
5) They are mite proof - eliminating the need for mite paper and reducing the possibility of developing an allergic reaction to the cultures through repeated exposure to the mites after handling the cultures with mites colonizing the outside of the container (therefore ruining the hobby for you).

Here is the thread where I learned about these:
https://www.dendroboard.com/forum/food-feeding/94348-3-micron-filters-why-how-where.html



CharValley said:


> I put the lids into the bottom of the sink and while filling the used cups up with hot water, I let the hot water run over the lids to soften up the discoloration on them.
> 
> Once the cups are filled, the lids will often already start to look white again, I take the top off of a generic mouth wash bottle and using the top I fill it about half full and pour it into each culture cup that I am going to clean. I then pour a generous amount of the same mouth wash on to the lid so that it will cover the vent material on the lid.
> 
> I let each stand for about a day (probably doesn't need to be 24 hours but it seems to also clear hazing that might have happened to the cup too)


Sounds like too much work. I just freeze an expired culture for 24 hours before disposing of the contents and rinsing it out. I then fill the container half way with water, place the lid back on, and microwave it until it reaches a rolling boil. The steam will sterilize the lid and the inside will kill any nasties. Easy, simple, and no questionable chemicals.


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## CharValley (Feb 29, 2016)

Thanks to everyone adding in additional advice and personal insight!

I actually was just wanting to relay a bit of stuff I picked up along the way in my own breeding and culturing over the years (started in 1980's).

Yes, the idea of using the dish washer is great, but yours must be better than the one I tried with as I kept getting soap residue on the cups and unless you rinse it out it will get into your media eventually and can spoil a good culture, as for gluing down new media screening.. tissue. If it needs to be replaced awesome but why discard it if all it needs is a little cleaning. The process I laid out only takes about 5-10 minutes to do half a dozen or so cups and lid and with a gently once over the lids and venting material look good as new without tearing.

As for the rest, I have over come the problem with the smaller culture hatch and physical size by simply sterilizing the cups as I explained. What I was referring to with the actual size getting smaller had to do with the mites getting into a culture and it seemed that when the mites got well established (before I started using mouth wash to sterilize the cups before re-using them) the fastest and usually smallest of the fruit flies were the ones that could survive long enough to rebreed.

I rarely experience any issue with my cultures now and I can easily make 6-8 months worth of breeding before I consider buying new stock from a vendor and the vendor's I usually use (one is no longer a 'vendor') selectively screen for mites before culturing, so I hardly ever have a full blown mite problem like I did in the beginning of my frog keeping days... ah to go back and buy lehmanni or histronicus for $25.00... but then again that is why they are nearly extinct in the wild now, so never mind! I just wish I had my frog room from the past and knew then what I know now about keeping and breeding them.

Thanks again everyone and if you have more hints, tips and/or suggestions for keeping our cultures healthy and our animals thriving please don't hesitate 

Charlie


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## salvz (Nov 3, 2014)

Socratic Monologue said:


> I've not had good results with mite paper. I use a pan with a layer of DE, and put the cups on it.


I am a firm believer in diatomaceous earth as well...never wears out and is harmless (make sure to use food-grade stuff).


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## Tinc Tank (Jun 29, 2019)

I also use the food-grade diatomaceous earth, but it isn't even needed since my fruit fly culturing containers are mite proof.


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## Ravage (Feb 5, 2016)

My method is pretty similar to many mentioned, but a bit different too, so I thought I'd add some thoughts.
When a culture has stopped producing I freeze it. For 3 days, that's the time it takes to get spores and mite eggs (below zero F). I let them thaw for 15 minutes, which is long enough to thaw the very outside of the gross mess inside the cups. I can then "spin" the whole wad out in on piece. The stuff that clings: I let the cups sit for a few days (I wash once a week or two). The remaining gross matter is dry and can be removed by knocking the container on a table. Throw the flakes away.
I wash the cups and lids by hand with plain dish soap. The key is to wear Playtex gloves and wash (and rinse) in as hot of water as possible. I wash the lids first and use a sponge/scrubbie to clean the mesh with the sponge and the top with the scrubbie side. This takes off the sharpie date/species label off quite well in super hot water. The gloves make this possible. 
Air dry and then spray with 70% isopropyl alcohol. a cheapo spray bottle works well. Get the surfaces wet with the rubbing alcohol, and then air dry again, stack and repeat. You can wipe off the alcohol with a tissue if you want but it should dry without residue.
91% isopropyl does *not* work better than 70% for surface sterilization, so save the buck a bottle. it's what I use with mushrooms and it works fine (easier on the skin as well).
I keep growing cultures in a completely different room than producing cultures (same with mushrooms, standard protocol) and haven't seen mites in quite some time.
Lots of good suggestions here, so I think the reader can pick and choose techniques that make sense for them.
Edit: I use micron filters for mushrooms in the jar stage, they are excellent but expensive, and likely overkill for fruit flies.


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## Dizzy Olde pharte (Jul 30, 2019)

There is such great info on this site!
Already use DE for worming my dogs, tata flakes for when I have zero energy to cook and half the other stuff from keeping snakes and other critters for more years than I can remember, so all to the good.

Just got to clear all the cr*p out of the lounge now to display to best advantage..... oh my..... still so much to do, so little time to do it in after my eyes finish bleeding from all the reading necessary!


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## Kmc (Jul 26, 2019)

Mites are fragile but mobile and they are easily eliminated from a surface even mechanically in hot soapy water. Its the ones that are not contacted, that happen to be located in a fissure of micro terrain wherever there is traction that gravitate back to resources to re infest. Especially the wayward gravid ladies.

Wiping down the area and floor has helped with other more ardent species. 

Interesting anecdote I once looked at O. natricis under magnification on a brand new stainless steel table and found they could not locomote properly.


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## npaull (May 8, 2005)

Jeez. So much rigamarole here. 

Dump the cup out in your compost bin or trash. 
Wash in the sink with the disposal on. Use your hand to get the gunk out. It’s mildly gross but utterly harmless and you’re washable. Don’t be a wuss. 
Fill cup halfway with hot water. Place top on loosely. Microwave until the water boils. Dump it out. 
Remake culture. You’re good to go. 
This has worked for me perfectly for over a decade. Zero mites. I’ve been using the same 6 plastic cups and fabric vented lids for years and years and years.


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## CharValley (Feb 29, 2016)

for years and years and years? You must have received the finest hand made plastic cups in history! Yeah I've tried the boiling water in culture cups, steaming the sides and lid... but some how the cups and lids tended to become brittle after a time and cracked, the fiber covering the vent holes would eventually tear, rip or peel and the adhesive would just give out and the fruit flies would eventually find a way out... which was awesome for the opportunity spiders that would hide around the frog room and usually made for a pain when they found their way into the rest of the house. Last thing I ever looked forward to was a complaining girlfriend about having fruit flies crawling on her!

I'm glad that this works for you Npaull but for the rest of us we have had, from time to time, infestations of mites, spoiled cultures and hungry frogs. 

The idea behind this thread is so that anyone starting to culture can find helpful information to help them avoid some of the issues we have experienced and for others to share what has worked well in the past to give our hobby some simple but effective ways to clean culture cups and lids, reduce any mite growth before it becomes a problem.

I tried steaming in the microwave, dishwasher, hand washing with bleach, mite paper and a number of 'home made' remedies suggested by friends and family over the years and what I had come to discover is that everyone finds what works for them and it isn't always the same thing for everyone else.

I let the cups dry out so that the "gunk" either drops out or lifts off with a simple hot water/mouth wash solution (at one time I was culturing 6 to 8 cups every other week, so letting them dry out during that time was not a wasted couple weeks) and the lid and fiber material comes clean with very little effort, very rarely rips or loosens and any micro debris and discoloration seems to be removed again with little or no actual effort.

But I have yet run into a solution that included being a "wuss" about the "gunk" in the culture cups.

I'm happy that microwaved boiling water cleans your cups works for you but for me it has lead to cracked and brittle cups and lids and escaped fruit flies. Maybe not for the first half dozen cleanings but I have had to replace any number of cups over the years, maybe I just don't microwave as well as you do.

So thank you for your input but please don't insult everyone else because you find that your way works and you think other ways are less worthy.

Charile

Oh and one other thing, washing it out in the sink with the disposal running? That works depending on the media you use for culturing, ie. the stuff that is needed for the flies and maggots to climb up on for hatching and 'adulting' but if you use "tuffy" or any type of 'straw' type material you better be sure you have removed it all before you get it into the disposal because if not even small pieces of that material will not only not get cut up but will eventually build up in the disposal and/or immediate drain from it and in a year... maybe two you will be wondering why your sink is always draining so slowly and why you had to call a plumber to fix the drain in the sink. I know because in the beginning I did wash them out over the sink and had to replace a disposal after pulling it myself because the blades stopped working, being wrapped up in a twine of straw material.


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## npaull (May 8, 2005)

Hey Charlie,

Sorry you were offended. Tone is hard to convey in text posts and I wasn’t trying to be negative. Don’t really see what I said to be offensive (the wuss comment wasn’t directed at anybody, tongue in cheek)... But yes the technique I described has worked for me for literally years with the same plastic cups. I’ve had a different more fragile plastic deform before but the cups I settled on seem to work just fine. Fabric lids too. The disposal isn’t an issue, I grab the excelsior and pull it out and it’s all gone before the cups get scrubbed. It’s simple and reliable and works well. Good luck!


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## Serafim (Mar 29, 2017)

I have never had mites bad enough i guess. I just scrub and soak cups and tops and keep everything in the freezer until used.


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## CharValley (Feb 29, 2016)

Thanks for the clarification Npaull 

You are right it is a hard thing to provide a simple personal comment, every day natural 'feeling', or just an innocent reflection without an unintentional inflection of tone. I have done this myself and couldn't get why what I said was taken so wrong. 

I will take all that on myself, it more than likely has to do with my own reading and 'implying' my mind set at the time I read it. I had a tough day and I probably should have taken a day or two before responding.. my bad and my apologies. But, thank you for making this clear and not responding with equal misunderstanding, so that anyone else who might read that as 'wussing' out when not wanting to clean out the wet stuff didn't also misread it. It wasn't my intention to insult you, if it came across that way only to express that taking a different approach to cleaning the cup wasn't necessarily 'wussing' out. (I guess it depends on when or where you grew up as to what that term might mean too 

Like I said, my own experience with the boiling water has left me with cups that would crack up the side making it useless unless you tape up the crack and that only works for so long before the cup just doesn't hold up to need and use. 

So we can add another tried and used method for cleaning out the culture cups.

Thank you for your input and clarification, again my apologies.

Charlie

I'm not a mite expert, although I've had my fair share of infestations and lost cultures to them but I believe where you live, the external climate as well as the internal room climate probably has a great deal to do with why some of us experience heavy mite problems and others seem blessed to rarely get them at all.


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## Kmc (Jul 26, 2019)

I do not think infestations originate from containers. As illustrated from the posts shared, the varied protocols applied by posters differ in personal preference, but all include adequate heat. If coverage is complete, kill temp is a conventional 130 F. With mites it is instant. There are oocysts eg, that require higher temps for a lengthier duration, and "true" sterilization - the elimination of all microorganisms, autoclave, long dwell high heat, or agent contact, a common duration point is 10 min.

The environment, media materials and the transferred seeding flies themselves (hitchhiking inadvertently in hairs) are an open link. 

It would be great if there was only one thing to control, and all it took was the perfect wash technique of the containers.


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