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Testing for LC contamination on agar vs Grain?


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#21 jkdeth

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Posted 20 May 2022 - 01:21 AM

Personally I think sterile is sterile, but I seldom use my agar immediately. And I like doing as much as I can when I run it, so most of the time, my agar is at least 5 days old, sometimes weeks. Now, I get my share of contamination but I haven't had agar show contamination prior to use, unless I obviously screwed up the process, like forgetting to wrap a plate, or forgetting the tape on a pp5 container.

Oh, those plastic mason lids, I wrap those as well. They cant be 100 percent trusted.
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#22 ShortcutSlim

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Posted 20 May 2022 - 01:45 AM

 

I forgot aout the edit time limit. :blush:   @FunnyFamer  . I hunted down the video that PGT did that discussed the LME and Peptone. I watched PGT's video,but didn't think to pause to see what brand he was using. I looked at Amazon,but there were quite a few different Malt Extracts offered and I didn't know if the differences were important or not..

 

Thanks to @sandman for sharing the LME I was looking for.

 

I redid the agar, and as suggested,the condensation was greatly reduced so:

 

Do I need to set the jars right side up and allow some time for any potential contamination to show before testing my LC?

 

Will that small amount of condensation be a problem of any kind?

 

Thx.

 

The contam rate has dropped so much that I pretty much use it after it's cooled down but that being said it's still not a bad idea to let it sit for about 5 days before working with them. I'm not very particular about the LME I use so anything used for beer making will be fine. Don't go overboard on your LME purchase as it is very hygroscopic, I've had powder turn into little tar balls in a matter of seconds, it's really challenging working with it during times of high humidity. A while back I got some empty freezer pop bags to store shroom powder in and they are a better container than the bag it comes in so I transferred the powder. It's still challenging but easier to store and break up the rocks that form over time. Those bags are designed to exclude air and humidity so I use what I need, roll up the end and use alittle binder clip to close it off.
 

 

I understand the humidity issue. I've lived it myself,but it is something I do consider. We don't get much humidity where we are now,but I always have getting out of here on my mind. I appreciate the mention of watching the humidity with the LME. If I could buy and 8oz bag or smaller,I would. I'll never go through a pound I'm sure. I don't know anyone else in the "hobby" locally,or I'd so the halfsies.lol

 

Personally I think sterile is sterile, but I seldom use my agar immediately. And I like doing as much as I can when I run it, so most of the time, my agar is at least 5 days old, sometimes weeks. Now, I get my share of contamination but I haven't had agar show contamination prior to use, unless I obviously screwed up the process, like forgetting to wrap a plate, or forgetting the tape on a pp5 container.

Oh, those plastic mason lids, I wrap those as well. They cant be 100 percent trusted.

 

Good grief. I never considered wrapping the lids. Now I'll be stressing over that. :eek:   I think I'll wait for a few days to test my LC. I know the world is on the edge of it's seat,but they'll just have to wait for the results. :biggrin:

 

Thanks for the help guys. Much appreciated. I'll only get better...hopefully.
 



#23 PerseidTraveler

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Posted 20 May 2022 - 08:12 AM

I use a premixed MAE...last pour was about 3 months ago, did 30 plates and 2 tam'd about a week later. I have about 10 left which are perfectly sterile. I usually lose plates to contamination at that rate, but I feel the visibility on a petri dish is so much better than in jars. I started agar using no pour in jars, and I had no tam issues, but I just felt like I could SEE the mycelium better on petri. Regardless of which method you go with, you will be pleased with agar.
*edit* just saw you've already chosen your method, maybe I should've read the whole conversation before adding my two cents lol

Edited by PerseidTraveler, 20 May 2022 - 08:21 AM.

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#24 FunnyFarmer

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Posted 20 May 2022 - 06:38 PM

I use a premixed MAE...last pour was about 3 months ago, did 30 plates and 2 tam'd about a week later. I have about 10 left which are perfectly sterile. I usually lose plates to contamination at that rate, but I feel the visibility on a petri dish is so much better than in jars. I started agar using no pour in jars, and I had no tam issues, but I just felt like I could SEE the mycelium better on petri. Regardless of which method you go with, you will be pleased with agar.
*edit* just saw you've already chosen your method, maybe I should've read the whole conversation before adding my two cents lol

I'd have to agree with you about the visibility when using petri dishes but I'm willing to deal with the tradeoff for the increased versatility of the jars. The other problem with the jars is getting an agar sample from the bottom, not fun... If only they could make a petri dish with a twist clear plastic lid that you could sterilize over and over I'd be a happy camper.


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#25 PerseidTraveler

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Posted 21 May 2022 - 06:57 AM

FF, I have seen glass petri dishes with heavy glass lids, but they were ungodly expensive...like $19/each or something like that...I'll see if I can find them again and send you a link. They look badass, but I'm not spending $1,000 on petri dishes I might be able to re use if I don't break them lol

#26 ShortcutSlim

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Posted 29 May 2022 - 01:03 PM

I use a premixed MAE...last pour was about 3 months ago, did 30 plates and 2 tam'd about a week later. I have about 10 left which are perfectly sterile. I usually lose plates to contamination at that rate, but I feel the visibility on a petri dish is so much better than in jars. I started agar using no pour in jars, and I had no tam issues, but I just felt like I could SEE the mycelium better on petri. Regardless of which method you go with, you will be pleased with agar.
*edit* just saw you've already chosen your method, maybe I should've read the whole conversation before adding my two cents lol

 

So this touches on my next question. How long do you wait before you can declare a sample clean with a high degree of confidence? I redid my agar,and then dropped some LC on the agar a couple days ago. I see white growth already, but I don't really know how long until I can declare it clean. (assuming any of the samples are indeed,clean)
 

 

On edit: I did a search and read that contamination should show between 3-5 days and certainly after a week.It did suggest that contamination could still be present but undetected,which isn't exactly comforting to read. I should imagine that maybe after a week you would have a higher likelihood that the LC was safe to use but not 100%.


Edited by ShortcutSlim, 29 May 2022 - 01:29 PM.


#27 FunnyFarmer

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Posted 30 May 2022 - 07:16 AM

The hardest contam to spot is the clear, look for a slightly raised shinier appearance to the agar. The vast number of contams will show up in 5 or so days after starting the test.

#28 ShortcutSlim

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Posted 31 May 2022 - 08:01 PM

IMG_0205.JPG IMG_0205.JPG IMG_0206.JPG IMG_0207.JPG IMG_0208.JPG IMG_0209.JPG

 

Well, we've got some examples of contamination here. I don't know what they are, but I'm relatively sure that all of these are a no-go. These attached pics are 4 jars. I did a total of 8,so I have 4 jars that are clean. Sad to say the 4 jars are 2 varieties that I really wanted to run next.  I did make LC from these 2 varieties previously that have not been tested. I plan on tossing the contaminated agar and then doing the test again with the jars I did some time back.With any luck,I'll get at least 1 of the 4 to be clean.

 

I can't expect to have gotten 100% clean on my first try,so it's ok I guess. This is the learning process everyone goes thru. :(

 

No exceptions for Uncle Shortcut.  :)

 

Edit: On further reflection,it seems odd to me that the first 4 jars were clean,and the last 4 were contaminated.I'm not saying it isn't possible...just odd. Maybe it was technique, although I was very careful. I do believe I will retest the varieties that are suspect,and go from there.


Edited by ShortcutSlim, 31 May 2022 - 09:38 PM.


#29 ShortcutSlim

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Posted 13 June 2022 - 02:28 AM

So,I re-tested the jars that showed contamination. One of the jars is for sure contaminated,so it's out. I had 1 jar that shows a spot about the size if a needle point that is bright red. I assume that's contam,as I can't see where red would come from,but again,I have very little experience. I do have a couple jars that are showing  minimal growth. When drawing up solution to test,is there mycelium suspended in the solution,or is mostly concentrated in the mycelium clump? I tried to shake the jars pretty well,but I simply could not see well enough to determine whether I got any mycelium when I drew up the solution into the syringe.I would think that is why I'm not getting a lot of growth on the agar ?


Edited by ShortcutSlim, 13 June 2022 - 02:31 AM.


#30 rockyfungus

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Posted 13 June 2022 - 07:08 AM

How was the LC acquired first off?


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#31 ShortcutSlim

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Posted 13 June 2022 - 12:54 PM

How was the LC acquired first off?

I was afraid somebody would ask me that. They are from spore syringes. Gotta run what I brung.  :)


Edited by ShortcutSlim, 13 June 2022 - 12:55 PM.


#32 rockyfungus

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Posted 13 June 2022 - 06:43 PM

Are you saying you injected a spore syringe into a liquid media to produce your own LC?
That's a huge no no and will lead to nothing but contamination.


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#33 ShortcutSlim

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Posted 14 June 2022 - 12:40 AM

Are you saying you injected a spore syringe into a liquid media to produce your own LC?
 

Yes. I realize that many have said it's not the best avenue to follow,and then again, I have read some positive accounts.In my enthusiasm I just jumped in with both feet hoping the water was deep enough. I do see that I have some jars that look to be good at least for now. I probably should really have bought prints and gone from there,but mistakes will be made. :ohmy:

 

I'm really just trying to get by for now.



#34 rockyfungus

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Posted 14 June 2022 - 01:10 AM

Have you had success? If yes, what's the point of using LC?

If no success you need to step way back and look at PF tek.

 

Yotube and reddit are some of the worst cultivation advice you can find. That abbreviation is a big purveyor of shit along with willymyco. Perhaps there's some nuggets of truth, but the overall things I see are going to net you heartache and loss of money.


Edited by rockyfungus, 14 June 2022 - 01:25 AM.

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#35 ShortcutSlim

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Posted 14 June 2022 - 12:56 PM

  what's the point of using LC?

 

 

 I figured if LC was faster at colonization, then that is the tek I would want to employ. If I deviated from established tek,it was only because I figured it was worth a try as I'm not getting any younger. :wink:


Edited by ShortcutSlim, 14 June 2022 - 01:04 PM.





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