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PeeWee2000
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[*] posted on 22-3-2014 at 16:45
Aseptic Culture Transfer


Recently I obtained a fungal culture and was informed that if I wish to keep this culture for any length of time I will need to transfer it from time to time (every two to four weeks). I myself have not had much experience in biology outside of highschool and was wondering if anybody else here has any tricks of the trade to add to what I've learned. After I've got a good idea about the whole procedure I'd like to repost a solid guide that details the entire process.

Here's what the culture looks like:

IMG_1743.jpg - 58kB IMG_1748.jpg - 82kB

My plan is to use premade Potato Dextrose Agar (PDA) to make the broth growth medium. After thats prepared then pour it on pyrex petri dishes to be sterilized in a pressure cooker then transfer the mold from plate to plate in a positive pressure ozone sterilized glove bag (ghetto glove box) with a small wire loop.

Heres a more comprehensive step by step layout for each process.

Potato Dextrose Agar broth preparation
1. Boil ~1L (distilled?) water on the stove
2. Add ~40g Potato Dextrose Agar powder.
3. Mix for one to two minutes while boiling to completely dissolve.
4. Allow to cool slightly, not enough to solidify.

PDA plate preparation
1. Pour thin layer of still warm broth into petri dish.
2. Cover Petri dish, wrap in aluminum foil and autoclave in pressure cooker for at least 15 minutes.
3. Allow to cool until solidified

Aseptic culture transfer
1. Unwrap and place both petri dishes and flame sterilized transfer wire in the glove bag and inflate with ozone pump and allow to sit for at least 30 minutes.
2. Turn off ozone generator and pump just enough air to keep the bag inflated and allow to sit for another ~10 minutes.
3. Unseal petri dish to be transferred and open prepared PDA plate.
4. Very lightly scrape the surface of the culture with the transfer wire and then very lightly scrape the surface of the premade agar plate.
5. Recap the new culture plate and seal with (electrical?) tape.
6. Place freshly prepared culture and store it in the fridge until next transfer.

The only things I am unsure about are: wether or not 10 minutes is enough time for the ozone in the glove bag to dissapate to a safe level that it will not kill the mold and wether or not I should let the agar solidify before performing the transfer (pretty sure that you're supposed to let it solidify). Please share your thoughts on this!!

And on a side note the room I will be using for this procedure is a small room with no airflow in my basement thats had two ionic breezes sitting in for about a week now to minimize any airborne contaminants to start out with.

Heres some of the sources that I gathered this information from and the original PDA preparation instructions.

http://www.sciencebuddies.org/science-fair-projects/project_...
http://mcb.berkeley.edu/labs/krantz/protocols/agar_plates.pd...
http://www.youtube.com/watch?v=WgJ75pIWIDI

Preparation: Mix 39.0 grams of the medium in one Liter of purified water until evenly dispersed. Heat with repeated stirring and boil for one minute to dissolve completely. Distribute and autoclave at 121.0°C for 15 minutes. If desired, the pH of the medium can be adjusted to 3.5 ± 0.1 prior to pouring plates by the addition of 14 mL of sterile 10% tartaric acid to melted sterile medium cooled to 45°-50.0°C. Avoid reheating medium after the addition of the tartaric acid solution.




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Etaoin Shrdlu
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[*] posted on 22-3-2014 at 17:19


If you're just going to pump ordinary air in again anyway, why are you re-sterilizing the tools which I assume have only come into contact with air after being flame sterilized? Are you going to run the air through a filter fine enough to remove contaminants? I'd just flame-sterilize and perform the transfer in a place without dust being kicked around, personally. If your air is filthy you're probably SOL for other reasons.

Yes, let the agar solidify.

EDIT: Out of curiosity, what's the fungus?

[Edited on 3-23-2014 by Etaoin Shrdlu]
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PeeWee2000
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[*] posted on 23-3-2014 at 15:01


Thats a good point but I was thinking the fresh air would get rid of any remaining ozone and the remaining ozone would be enough to kill any newly introduced bacteria. And the air is quite clean to my knowledge, I might just be overthinking it but I figured it couldnt hurt to do as much as possible to clean it even more. But thinking about how much simpler it would be to just be quick and concise with the transfer in open air makes the idea sound more and more attractive.



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jwpa17
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[*] posted on 25-3-2014 at 15:32


Is there a reason you feel it necessary to use ozone?

In my lab, we use fungus as a source of a particular enzyme. To maintain plates, we work in an area with minimal air turbulance. Assuming you have sterilized PDA plates. Wipe down an area of about 3' x 4' with 70% ethanol to sanitize it. Light a bunsen burner, place in the center of the sanitized area. Hold a transfer loop in the flame until the wire glows red, moving the wire so the entire surface (of the wire) is heated to red. Open the source culture plate. Touch the hot loop to a bare portion of the plate to cool it, then touch to the source culture. Close the source culture plate. Open the PDA plate, and touch the contaminated loop to the PDA surface. Close the PDA plate. Flame the contaminated loop. Turn off the flame, and wipe the surface with 70% ethanol again.

From the time you open the plates until the time you close them, don't talk to anyone. Work efficiently and quickly. Don't set the lids onto the bench and don't touch any surface which will contact the media.

There's a nice book on microbiology for chemists which has information on sterilization, manipulation, and storage of microorganism - I don't have the title here, but I'll try to remember to post it tomorrow. Also, you don't have to propagate your cultures; there are several ways to store them for long periods. In fact, serial propagation is usually frowned upon as it leads to genetic drift.

In summary, I think your ozone idea is unnecessary.
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PeeWee2000
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[*] posted on 28-3-2014 at 09:18


Not at all I was just being very cautious about this whole thing as I have little experience in the area and really don't want to mess this up. And thanks for the tip I did a quick search and found this:

http://www.shroomery.org/8509/Resurrecting-a-Better-Method-f...

Seems to be a decent guide, I'll do some more research today and I have a face shield that I can wear during the transfer to help prevent breathing on the cultures. And thank you very much for the input!




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Chemosynthesis
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[*] posted on 28-3-2014 at 09:43


I don't think I want to know what kind of fungus you're using, but one of the biggest sources of contamination in most cell culture is from your arms/hands. You have a lot of dead skin and microbiota on your clothing or arms, and gloves only reach so far. There are goofy tyvek sleeves you can buy, but they are hardly necessary if you're cognizant of your technique.
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essbee
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[*] posted on 28-3-2014 at 09:50
pure magic................


An interesting and perhaps safer way to 'expand your horizons' using the interesting chemicals produced by fungi....years ago, when I had hair, we collected our fungi growing wild in the fields and so were open to munching on the occasional mushroom which may not have been 'magic' in the way we had hoped.
I'm sure all the correct ones were perfectly safe, but the mistakes led to all kinds of illnesses, so it became prudent to only eat what you had picked yourself. This grow your own does seem safer, again, you know what you have so only risk overdosing ( if actually possible....I have anecdotal evidence that 1000 liberty caps sends you to a place where 'out of body viewing' is possible?? really!) and that is easily avoided.
I myself have degrees in Biochemistry and Biotechnology and commend you on your enquiring mind, the shroom site seems to cover all you need to know, just beware of your own mood when taking unknown ( to you I mean) chemicals, just as easy to have a really bad time as it is to have a good one.
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PeeWee2000
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[*] posted on 28-3-2014 at 22:36


Ha good to know essbee, however this isnt a mushroom spore though and nor do I plan to ingest anything that is produced by this fungus but I do plan on growing some cool mushrooms in the near future http://www.ebay.com/itm/Glow-in-the-dark-mushroom-Panellus-s...

I appreciate the insight into the mushroom world but would like to keep this thread more geared twoard simply culturing mold of any sort in general! And also thanks for the praise its always nice to have well educated people make me feel like I'm smart :D

The reason I am leaving this fungus unnamed is because I am unsure it is really what it was advertised to be as of right now, and am not 100% sure of the legality surrounding it, but from what I've read it may be/is classified as a poison.

And Chemo I have a face shield, gloves, and sleeves that I use for doing anything that involves strong acids bases etc so I will put them to good use for this as well!



[Edited on 29-3-2014 by PeeWee2000]




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[*] posted on 28-3-2014 at 22:56


Awesome. Also, I read the longterm storage link. We use glycerol stock in liquid nitrogen or neg 80C freezers for most biological sample storage (cell lines, bacteria, etc.), so that is what I would recommend to avoid large passage numbers with genetic drift as mentioned above. You eventually allow for selection of gene populations best suited for petri dishes rather than environmentally relevance, to an extent.

For stock solutions, you can use a short chain alcohol and any enclosure, including a supported garbage bag, as a really cheap glovebox. Most hobbiests seem to use a HEPA filter in an enclosure as a glovebox, or even buikd their own laminar flow hoods (squirrel cage fan can be pricey). Even with glycerol as your stock, it is probably best to chill your sample in the fridge for a few minutes before sticking in a freezer, in my opinion. I generally go fridge, commercial freezer, neg 80. When thawing, thaw on ice.
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[*] posted on 29-3-2014 at 00:35


Can't you make your fungus to form spores? If stored cool and dry they should viable longer then you could wish for.
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PeeWee2000
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[*] posted on 29-3-2014 at 22:49


Indeed I can Tsjerk and indeed I will for a master sample, thanks for the idea. I had planned on propagating this fungus naturally but didnt even think about collecting spore prints! Should be interesting as the fruiting body is quite small.



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[*] posted on 30-4-2014 at 19:15


Yes, spores make things a lot easier. We usually grow our cultures on carrot agar to induce good sporulation. Harvest them by putting sterile water on the plate, then sucking it up. We've had such spore suspensions last 3-4 months before we discard them. And we usually discard them simply because I get nervous about contamination - the cultures still grow.
In fact, you can store spores in milk on silica in an ordinary freezer for years. Before -80 freezers, it was the standard method of storing filamentous fungal cultures, along with simply putting sterile mineral oil onto a culture-tube slant of the specimen.
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[*] posted on 24-8-2014 at 16:18


Aseptic transfer is pretty easy. You can whip up a simple aseptic hood out of an old box even!
Aside from your hands, the next most common source of contamination in cultures is dust falling down.
To do this, you just need to turn a box on its side. If you spray the inside of the box with 95% isopropyl alcohol, let it sit for a little, and turn off the air conditioning, the inside of the box is essentially sterile.
To prevent contamination as much as possible in this kind of setup, when transferring the culture, you only have to lift the lid of the cultures enough to put your transfer implement in the culture vessel. Never put your hands over the culture vessels, and spray your hands down with alcohol as well.

I've got this experience from plant tissue culture experiments that I performed at home. For my last batch of cultures, I only got contamination from explants.

Once you get used to it, it is fairly simple. I suggest practicing first with empty culture vessels, and incubating them to check for contamination.

If you are going to be doing a LOT of sterile transfers, it may be worth building a laminar flow hood, which essentially guarantees sterility.
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[*] posted on 3-9-2014 at 20:01


if you don't have experience with sterile technique definitely read up on it and I would steer away from trying to make your own PDA.
Start with something easy and very dependable malt. back the sugar content off though and make the culture work for it. Use 82% iso for wipe downs of surfaces, and sterilize your medium in a flask, not in the plates. you end up with alot of spillage otherwise. sterilize all your tools, keep everything wrapped in foil and always in the order of cleanest away from you and dirtiest closest to you.
It's important to visualize moving in one direction to keep yourself from cross contaminating. over cooking the medium will also insure failure
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Actinium
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[*] posted on 6-9-2014 at 15:43


What kind of Fungus is it?
If its the Psilocybe type the acidic medium you propose will not be suitable.
Look into using sweet potatos as a substitue, I've havd great success.
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Chemosynthesis
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[*] posted on 7-9-2014 at 05:31


Quote: Originally posted by Actinium  
What kind of Fungus is it?
If its the Psilocybe type the acidic medium you propose will not be suitable.
Look into using sweet potatos as a substitue, I've havd great success.


Quote: Originally posted by PeeWee2000  
this isnt a mushroom spore though and nor do I plan to ingest anything that is produced by this fungus but I do plan on growing some cool mushrooms in the near future http://www.ebay.com/itm/Glow-in-the-dark-mushroom-Panellus-s... I appreciate the insight into the mushroom world but would like to keep this thread more geared twoard simply culturing mold of any sort in general! And also thanks for the praise its always nice to have well educated people make me feel like I'm smart :D The reason I am leaving this fungus unnamed is because I am unsure it is really what it was advertised to be as of right now, and am not 100% sure of the legality surrounding it, but from what I've read it may be/is classified as a poison.


Why did you bump a thread you last posted in 3 months ago with a double post when it's not relevant and Google disagrees with you? Google Psilocybe and potato dextrose agar. Plenty of hits.

[Edited on 7-9-2014 by Chemosynthesis]
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