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Cleaning up dirty spores


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#1 peacefrog

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Posted 22 February 2017 - 06:35 AM

About 2 weeks ago, I noticed that some of my spores were getting pretty old without being replaced. So I decided to get on it and regrow them. They have all germinated but were very dirty and needed to be cleaned up very badly. I thought I would put a quick post of how I clean up dirty agar plates for anyone interested.

The 3 types were Psilocybe cubensis Treasure Coast, PES Amazon and PES Hawaiian. My Golden Teacher spores did not need to be regrown, as I have an older culture that I almost killed, but have brought it back to life.

TC plate:
IMG_0741.JPG

Bacterial as hell but some good healthy growth in the center of all that bacteria. So I very carefully used the back side of my X-Acto blade to scrape some growth to transfer:
IMG_0742.JPG IMG_0754.JPG

Microbe uses his inoculating loop to do this, which is a great idea, but I have always done it this way. I also take just a tiny bit of agar with it just to help the transfer not possibly dry out before it takes hold on the new agar plate. Transferred 1 piece to several awaiting agar jars:
IMG_0748.JPG

Another procedure would be to pour warm agar before it solidifies over this dish. From my reading, this "sandwich" tek will allow the healthy mycelium to grow upwards leaving the bacteria behind. I've just never tried it, but it sounds very feasible. Possibly some other growers who have experience with the sandwich tek will chime in.

PES HI:
IMG_0744.JPG

Once again, it is badly contaminated with bacteria. But this one is a little easier to deal with, as there isn't bacteria on all sides of the healthy growth. I transferd from as far away from the contamination as possible and actually cut into the agar like one would do for any isolation.
IMG_0746.JPG

PES AM:
IMG_0750.JPG

This one was interesting. No bacteria but it had some white mold satellite colonies. The contaminated areas are a little off on color and appearance. So once again, I transferred from as far away as possible and transferd to several plates:
IMG_0616.JPG IMG_0615.JPG

All are recovering as we speak and will most likely need to be transferred at least once more even if they appear clean to the naked eye, before taking them to grain, LI or LC. I will update this when possible.

And as always, happy growing!
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#2 tailsmcsnails

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Posted 22 February 2017 - 09:43 AM

About 2 weeks ago, I noticed that some of my spores were getting pretty old without being replaced. So I decided to get on it and regrow them. They have all germinated but were very dirty and needed to be cleaned up very badly. I thought I would put a quick post of how I clean up dirty agar plates for anyone interested.
!

Cool post. Can I ask a very rookie question? Can you back up one or two steps from 'I noticed that some of my spores were getting old'? I'm guessing that means you looked at the dates on your collection of stored spores?

Then you took your ageing spore prints or whatever and inoculated the agar seen above?

Everything you said was perfectly clear I'm just so new to this that I'm struggling to fit it into the part of the picture that I (sort of) understand.

At the end of your process you will grow fruit from your cleaned-up cultures and then store spores from those fruit?

Edited by tailsmcsnails, 22 February 2017 - 09:43 AM.

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#3 CatsAndBats

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Posted 22 February 2017 - 09:51 AM

 

About 2 weeks ago, I noticed that some of my spores were getting pretty old without being replaced. So I decided to get on it and regrow them. They have all germinated but were very dirty and needed to be cleaned up very badly. I thought I would put a quick post of how I clean up dirty agar plates for anyone interested.
!

Cool post. Can I ask a very rookie question? Can you back up one or two steps from 'I noticed that some of my spores were getting old'? I'm guessing that means you looked at the dates on your collection of stored spores?

Then you took your ageing spore prints or whatever and inoculated the agar seen above?

Everything you said was perfectly clear I'm just so new to this that I'm struggling to fit it into the part of the picture that I (sort of) understand.

At the end of your process you will grow fruit from your cleaned-up cultures and then store spores from those fruit?

 

 

 

Agar work is whatever you want it to be. :biggrin:


Edited by catattack, 22 February 2017 - 09:51 AM.

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#4 Jeepster

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Posted 22 February 2017 - 09:52 AM

AGAR RULES!!!
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#5 peacefrog

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Posted 22 February 2017 - 10:24 AM

Yes ask away.

I was going to start some spores from a print a great member sent me and as I was going through my collection, I noticed the dates of when they were printed. Almost 4 years old.

And yes I will grow them out after they are cleaned up and get fresh prints.

Agar is a wonderful tool, and while not completly necessary to grow mushrooms, is very valuable and recommended by me 100 percent.
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#6 Arathu

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Posted 22 February 2017 - 11:07 AM

Excellent post peacefrog! Excellent! Speaking of which I need to go get some more agar soon.......payday hopefully...........

 

Agar rules for sure and it is a nice medium to work on without wasting time on generating contaminated grains......

 

 

A


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#7 tailsmcsnails

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Posted 22 February 2017 - 05:47 PM

Yes ask away.

I was going to start some spores from a print a great member sent me and as I was going through my collection, I noticed the dates of when they were printed. Almost 4 years old.

And yes I will grow them out after they are cleaned up and get fresh prints.

Agar is a wonderful tool, and while not completly necessary to grow mushrooms, is very valuable and recommended by me 100 percent.

ok so next rookie question- the prints were contaminated?  Is that a thing?  I would have thought that a clean print would stay clean in storage?


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#8 MLBjammer

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Posted 22 February 2017 - 06:23 PM

No print is really ever sterile. And when you inoculate with a loop on agar, whatever is in that swipe of print will show up on the agar.

Great thread, PF. We'll add this to your gallery of good learning.
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#9 dead_diver

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Posted 22 February 2017 - 07:25 PM

One method of cleaning up spores is by dilution. I'll try to explain the concept as best as I can. I'm going to make the numbers small so they are easy to handle and get an idea of what I'm trying to say.
Say you have one syringe with 99 good spores in it and one contaminated spore. If you use that entire syringe to inoculate one jar you are guaranteed to get a contaminated spore in the jar :(
But if you dilute that syringe and use it to make 100 syringes you will have 99 contamination free syringes and only one contaminated syringe :)
The odds are now in your favor.

Edited by dead_diver, 22 February 2017 - 07:34 PM.

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#10 tailsmcsnails

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Posted 22 February 2017 - 07:39 PM

but what if you start with 90 good spores and 10 contaminant spores and you get unlucky and end up wit 10 contaminated syringes?

 

(I'm not deliberately being contrary I promise)


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#11 peacefrog

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Posted 22 February 2017 - 07:50 PM

Thank you for the kind words, Arathu and Jammer.

Tails,
Like jammer said, no print is completely sterile. I do not print in sterile conditions, but am very clean about the process. But sterility while spore printing isn't that important to me, as I always go to agar first with them, and can clean the growth up very easily. Most of my fresh prints will germinate very cleanly, but those were going on 4 years old, stored in a ziplock bag, inside another bag and have been neglected for too long. It just happens sometimes and that's the beauty of agar IMO. If you get clean growth anywhere on the plate, you can clean the growth up 98% of the time. Do many plates and you will increase your success of getting clean growth exponentially. I have germinated 5 year old spores with no contamination and have germinated 1 month old spores that were dirty. It just happens sometimes.
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#12 dead_diver

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Posted 22 February 2017 - 07:54 PM

You still have 90 good syringes vs one bad syringe :). The more you dilute it the better.
If you keep diluting a solution by 50% you won't have to do it too many times before you have an extremely dilute solution. It is actually possible to isolate individual spores this way.

Edited by dead_diver, 22 February 2017 - 07:56 PM.

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#13 dead_diver

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Posted 22 February 2017 - 08:09 PM

Here's an explanation of serial dilution as it relates to microbiology.
http://www.biotopics...obes/tech3.html
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#14 tailsmcsnails

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Posted 22 February 2017 - 08:22 PM

Thank you for the kind words, Arathu and Jammer.

Tails,
Like jammer said, no print is completely sterile. I do not print in sterile conditions, but am very clean about the process. But sterility while spore printing isn't that important to me, as I always go to agar first with them, and can clean the growth up very easily. Most of my fresh prints will germinate very cleanly, but those were going on 4 years old, stored in a ziplock bag, inside another bag and have been neglected for too long. It just happens sometimes and that's the beauty of agar IMO. If you get clean growth anywhere on the plate, you can clean the growth up 98% of the time. Do many plates and you will increase your success of getting clean growth exponentially. I have germinated 5 year old spores with no contamination and have germinated 1 month old spores that were dirty. It just happens sometimes.

wow,before this thread I had read a lot of agar stuff and kind of had it pegged as an "advanced and optional" step.  Now it seems more or less essential (after my first "derp I'm pouring spores onto BRF cakes" excursion anyway).  

 

I did wonder about spore prints- seems they could only ever be clean but not sterile.  Another piece of the puzzle has fumbled into place for me.  thanks for the patient sharing!!

 

Sorry to derail your thread but maybe someone else would wonder the same thing.


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#15 peacefrog

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Posted 23 February 2017 - 06:02 AM

No need to apologize, you have asked good questions that will surely help growers and that's what this this thread is all about.

Most newer growers think that agar is advanced, but it's really easy once you get past the stigma and try it for yourself. I started with agar and am glad I did. For me, and my style of growing, it just helps so much. That being said, there are many great growers who either don't use agar or very seldom do. Jammer uses very good sterile technique while printing and makes spore syringes a lot of times. He then uses that to inoculate grain with great consistent results. For me, I would suck at that. I haven't made a spore syringe in many years. It's whatever works best for you.

It's just a good tool to have in your arsenal IMHO, even if you don't always use it. Sometimes you will greatly benifit from using it. For instance, starting prints from collected outdoor specimens, or in the case that I found myself in, old dirty spores.

It also opens up many doors for you as a cultivator. Cloning, isolating and long term storage to name a few.

Good luck and please ask as many questions as you would like in this thread. We are all here to help and learn.
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#16 CatsAndBats

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Posted 23 February 2017 - 08:28 AM

@tails, it's really quite easy. Plus it affords one more time in one's clean area, which helps build confidence and muscle memory. Plus if you fuck up, you see it in a day or so. Bacterium and yeasts show up mighty quick!

 

Join us!

 

This post has most of the links that I learned from:

 

https://mycotopia.ne...r-30/?p=1303967

 

You can read my older one which shows how the members here guide a neophyte from clueless to an "authority" in less than a year:

 

https://mycotopia.ne...ome-agar-again/

 

 

 

 

 

 

Sorry @peacefrog, back to your beloved expertise! :bow:


Edited by catattack, 23 February 2017 - 08:29 AM.

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#17 tailsmcsnails

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Posted 23 February 2017 - 04:15 PM

@tails, it's really quite easy. Plus it affords one more time in one's clean area, which helps build confidence and muscle memory. Plus if you fuck up, you see it in a day or so. Bacterium and yeasts show up mighty quick!

Join us!

This post has most of the links that I learned from:

https://mycotopia.ne...r-30/?p=1303967

You can read my older one which shows how the members here guide a neophyte from clueless to an "authority" in less than a year:

https://mycotopia.ne...ome-agar-again/






Sorry @peacefrog, back to your beloved expertise!

Thanks. Will check all those out. I ordered all the agar bits n pieces a while ago because I knew it was something I would be doing sooner or later. This thread is great and I am following the froggy's agar adventure avidly.
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#18 MLBjammer

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Posted 25 February 2017 - 06:50 AM

Tails, you cannot go wrong with anything Froggie posts.  He is wise and helpful frog, lol.


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#19 Sidestreet

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Posted 25 February 2017 - 04:46 PM

archive material to agar!  :cool:  Thanks Peacefrog.  How are they doing?


Edited by Sidestreet, 25 February 2017 - 04:46 PM.

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#20 peacefrog

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Posted 25 February 2017 - 08:14 PM

Thank you for the kind words, guys.

All plates are doing as expected. Growing well but still showing some contamination. Really starting to dig into the agar now so in a few more days, another transfer.

The Treasure Coast plates, which were completly encircled by bacteria are growing with the most vigor and cleanest growth out of all.

Pics to follow in a few days.
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