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Offlineblgrnt1
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Spore Syringe and agar
    #27545060 - 11/15/21 11:47 PM (2 years, 2 months ago)

I am pretty sure I already know the answer to this but I am second guessing myself. I purchased some spore syringes and when I received them I decided to test them on some agar. I added a few drops of solution to a dish and where the drops landed are now growing green little dots. This means the syringe is contaminated? Right?  There are 5 spots, 3 of them are green in the middle with a white ring on the edges the other 2 spots are white. I know, dumb question. Thanks guys.


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OfflinePlazmotech
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Re: Spore Syringe and agar [Re: blgrnt1]
    #27545063 - 11/15/21 11:48 PM (2 years, 2 months ago)

Well, it could be the spore syringe, or it could be your sterile technique. You should probably streak your plate (not just drops) and try to isolate a clean bit of mycelium that way.

Never go straight spore to grain/pf cake/whatever, in my opinion!


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OfflineMastershakes
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Re: Spore Syringe and agar [Re: blgrnt1]
    #27545069 - 11/16/21 12:02 AM (2 years, 2 months ago)

I had luck when my spores were very contaminated just going straight to BRF cakes. I tried straight to grain and that is almost a guaranteed failure.

A lot of people seem to use BRF to get away from contaminates.

Useful link someone shared with me here:
https://www.shroomery.org/forums/showflat.php/Number/24806569


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OfflinePetMonkey
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Re: Spore Syringe and agar [Re: Mastershakes]
    #27545286 - 11/16/21 06:38 AM (2 years, 2 months ago)

Pictures would help us to confirm, but yes, it sounds like it's probably a contamination. How many plates did you do? You have to factor in the high chance of contamination in general, so the more plates you do, the more likely you are to get a few clean ones. That's been my experience, at least.


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OfflineA.k.aM
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Re: Spore Syringe and agar [Re: Mastershakes]
    #27545287 - 11/16/21 06:39 AM (2 years, 2 months ago)

What you’re describing is definitely a green mold.


But like they said it’s very easy to contaminate plates just by cracking them open if you’re not using a hood or SAB with good technique.


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Re: Spore Syringe and agar [Re: A.k.a]
    #27545304 - 11/16/21 06:52 AM (2 years, 2 months ago)

Quote:

A.k.a said:
What you’re describing is definitely a green mold.


But like they said it’s very easy to contaminate plates just by cracking them open if you’re not using a hood or SAB with good technique.



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Offlineblgrnt1
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Re: Spore Syringe and agar [Re: Tormato]
    #27546503 - 11/17/21 12:48 AM (2 years, 2 months ago)

Let me clarify, I am aware that there is contamination on my agar.  What I was asking is if that means the spore syringe is compromised.  I will tell you my procedure.

I use a SAB, I spray everything with 70% alcohol.  By everything I mean, myself, the inside and outside of the airbox, the petri dishes, packaging the syringe came in and once opened the syringe itself, and the torch and the gloves I wore.  I stacked the petri dishes on the left and will start with the top dish, when done I would move it to the bottom.  I shook the SS outside the box when it was in its packaging, then once inside the SAB opened it and wiped down with alcohol.  Then opened the needle package, took off the syringe cap and attached the needle.  I then flame sterilized the needle, put the torch down and squirted a few drops to cool the needle.  With the SS in my right hand I moved the top of the petri dish halfway off and added a few drops of the SS to the agar, while removing the needle from above the agar I simultaneously put the lid back on.  I then took the agar I just worked with and moved it to the bottom of the stack.  When I was done with all the dishes I wrapped each in a double layer of parafilm.

No, I only had enough plates to do one with each new SS I received.  I inoculated a total of 6 agar plates and this is the only one that has a contamination where the mycelium is starting to grow.  So I am making the assumption that the contamination got to the agar through the spores syringe. I was under the impression the way to check a SS for contamination was with agar, is my thinking wrong?  I appreciate all the responses.


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OfflineKizzle
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Re: Spore Syringe and agar [Re: blgrnt1]
    #27546566 - 11/17/21 02:36 AM (2 years, 2 months ago)

If it only appeared where you put the spore solution then it's very likely that syringe was contaminated.


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Invisiblerince
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Re: Spore Syringe and agar [Re: blgrnt1]
    #27546570 - 11/17/21 02:48 AM (2 years, 2 months ago)

Quote:

blgrnt1 said:
I spray everything with 70% alcohol.  By everything I mean, myself, the inside and outside of the airbox, the petri dishes, packaging the syringe came in and once opened the syringe itself, and the torch and the gloves I wore.




Don't spray the petri dishes or the syringe once opened if they came in sterile. Remember: Sterile > Disinfected, and alcohol only disinfects.


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OfflinePetMonkey
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Re: Spore Syringe and agar [Re: blgrnt1]
    #27546579 - 11/17/21 03:26 AM (2 years, 2 months ago)

Quote:

blgrnt1 said:
Let me clarify, I am aware that there is contamination on my agar.  What I was asking is if that means the spore syringe is compromised.  I will tell you my procedure.

I use a SAB, I spray everything with 70% alcohol.  By everything I mean, myself, the inside and outside of the airbox, the petri dishes, packaging the syringe came in and once opened the syringe itself, and the torch and the gloves I wore.  I stacked the petri dishes on the left and will start with the top dish, when done I would move it to the bottom.  I shook the SS outside the box when it was in its packaging, then once inside the SAB opened it and wiped down with alcohol.  Then opened the needle package, took off the syringe cap and attached the needle.  I then flame sterilized the needle, put the torch down and squirted a few drops to cool the needle.  With the SS in my right hand I moved the top of the petri dish halfway off and added a few drops of the SS to the agar, while removing the needle from above the agar I simultaneously put the lid back on.  I then took the agar I just worked with and moved it to the bottom of the stack.  When I was done with all the dishes I wrapped each in a double layer of parafilm.

No, I only had enough plates to do one with each new SS I received.  I inoculated a total of 6 agar plates and this is the only one that has a contamination where the mycelium is starting to grow.  So I am making the assumption that the contamination got to the agar through the spores syringe. I was under the impression the way to check a SS for contamination was with agar, is my thinking wrong?  I appreciate all the responses.




Just curious, did you wear Tyvek wrist sleeves or a clean long-sleeved shirt while doing this? During the summer I was doing some agar work with short sleeves (I still wore nitrile gloves and cleaned them with IPA), and was having huge contamination rates inoculating agar with both spore syringes and with spore prints. I started wearing long-sleeve cotton shirts and I've been having better luck the past few weeks, so part of me thinks I was getting dead skin cells or hair in my petri dishes by accident.

That being said, I see a lot of other people here doing SAB work with short sleeves and sometimes even no gloves at all, so I could be totally off...


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OfflineSpooff
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Re: Spore Syringe and agar [Re: PetMonkey]
    #27546622 - 11/17/21 05:18 AM (2 years, 2 months ago)

Quote:


Just curious, did you wear Tyvek wrist sleeves or a clean long-sleeved shirt while doing this? During the summer I was doing some agar work with short sleeves (I still wore nitrile gloves and cleaned them with IPA), and was having huge contamination rates inoculating agar with both spore syringes and with spore prints. I started wearing long-sleeve cotton shirts and I've been having better luck the past few weeks, so part of me thinks I was getting dead skin cells or hair in my petri dishes by accident.

That being said, I see a lot of other people here doing SAB work with short sleeves and sometimes even no gloves at all, so I could be totally off...




I think that comes down to paying attention to what you hover your hands/arms over. As far as i know, a SAB doesnt even have to be sanitized. Soapy water will work i think. Ive heard of people just spraying it down with water along with their arms and hands and using it while everything is still wet. It works by design. So as long as you dont hover, you shouldnt get contam from anything falling off your hands/arms.


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OfflineA.k.aM
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Re: Spore Syringe and agar [Re: Spooff] * 1
    #27546758 - 11/17/21 09:06 AM (2 years, 2 months ago)

Yeah lots of people work dry SAB too.


Try cutting a hole or divot in the middle of a plate and dripping a drop from the syringe into it.


Then if you get any contam outside the divot you know it’s not from the syringe.


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OfflineNossninja
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Re: Spore Syringe and agar [Re: A.k.a]
    #27546856 - 11/17/21 10:16 AM (2 years, 2 months ago)

"stacked the petri dishes on the left and will start with the top dish, when done I would move it to the bottom.  I shook the SS outside the box when it was in its packaging, then once inside the SAB opened it and wiped down with alcohol.  Then opened the needle package, took off the syringe cap and attached the needle.  I then flame sterilized the needle, put the torch down and squirted a few drops to cool the needle. "

Im no expert.

First off if the syringe came out of a sterile package, you should not need to hit it with a flame until after you do something with it.  On the video I watched about SAB work, it said to start with the bottom agar dish and then work up. Im not sure of why, but ive had good luck with it that way.


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Offlineblgrnt1
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Re: Spore Syringe and agar [Re: PetMonkey]
    #27547530 - 11/17/21 09:04 PM (2 years, 2 months ago)

You know what? I did not have any protection like that besides the gloves.  I do soak my entire arms and hands, then wait for hands to dry to get the gloves on then I spray all over again.  Maybe that will be something I take a look at.  Do you think they have them at amazon? I dont know why I asked, pretty easy to check myself.  Yes amazon has them  lol
Regarding the tyvek sleeves


Edited by blgrnt1 (11/18/21 01:39 AM)


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OfflineMushyMom
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Re: Spore Syringe and agar [Re: blgrnt1]
    #27547533 - 11/17/21 09:09 PM (2 years, 2 months ago)

I had a spore syringe and I used it for 5 plats.  Of the 5, 2 developed nasty green infections, 1 didn’t take and 2 showed normal growth.  I took 4 samples from one of the plates with normal growth and started 4 more plates - all of which are beautiful.  I’m not sure where the contam happened - it could have been my technique, but I was happy that I did get a couple clean plates. Can you make a couple plates from the syringe?


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Offlineblgrnt1
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Re: Spore Syringe and agar [Re: MushyMom]
    #27547687 - 11/18/21 01:51 AM (2 years, 2 months ago)

Yes, I can take more samples from the SS but if it is contaminated there is really no reason to do that, right?  Or can a contaminated SS produce healthy enough mycelium to transfer?  I am really only worried the entire SS in useless. 

What are your opinions on this, if you bought 5 premade agar plates and when you receive them one is already contaminated.  Would you expect the vendor to replace it, or even return your email regarding it?  Also, if the advertised product was different then what you received, how would you feel about it?  I bought the premade agar only because it was activated charcoal and I hadn't used it before, when I got the package it was regular agar.  Also some spore varieties I ordered were out of stock (completely understand, business owner myself) I was never asked about an interest in other varieties and was sent whatever the vendor wanted to send.  I would not even bring this up if somewhere on the site it said they would send what they could if my choices were not in stock, but it doesn't. I feel I should have been given a head up and maybe asked about backups.


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OfflinePetMonkey
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Re: Spore Syringe and agar [Re: Spooff]
    #27547713 - 11/18/21 02:56 AM (2 years, 2 months ago)

Quote:

Spooff said:
It works by design. So as long as you dont hover, you shouldnt get contam from anything falling off your hands/arms.




In theory, but not necessarily in practice. I've seen many posts on here from people claiming they had nonstop failure and contamination with an SAB, and it seemingly disappeared overnight when they started to use a laminar flow hood - that's kind of where I am right now.

I'm a neurotic OCD clean freak who compulsively washes his hands 50 times a day, and out of maybe 100 plates I've inoculated in my SAB, I've ended up with 5 good ones. I did a few transfers the other day from these plates, and over half my new plates have bacteria/mold on them. I'm still new at this and obviously there's a learning curve, but good lord. 90%+ failure rate makes me think there might be some other factor at play than my sterile technique.


Edited by PetMonkey (11/18/21 03:18 AM)


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OfflineMastershakes
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Re: Spore Syringe and agar [Re: PetMonkey]
    #27547808 - 11/18/21 05:29 AM (2 years, 2 months ago)

Quote:

PetMonkey said:
Quote:

Spooff said:
It works by design. So as long as you dont hover, you shouldnt get contam from anything falling off your hands/arms.




In theory, but not necessarily in practice. I've seen many posts on here from people claiming they had nonstop failure and contamination with an SAB, and it seemingly disappeared overnight when they started to use a laminar flow hood - that's kind of where I am right now.

I'm a neurotic OCD clean freak who compulsively washes his hands 50 times a day, and out of maybe 100 plates I've inoculated in my SAB, I've ended up with 5 good ones. I did a few transfers the other day from these plates, and over half my new plates have bacteria/mold on them. I'm still new at this and obviously there's a learning curve, but good lord. 90%+ failure rate makes me think there might be some other factor at play than my sterile technique.




You could do mock transfers.  for every 10 or so plates just take one and do all the steps, minus the mycelium on it.

I'm new to agar and I was having huge issues as well at the beginning. I was buying pre made Agar, and sterile petri dishes. About 80% of my plates would contam. Long story short I traced that back to bad agar (from the lab I was buying from).

Now when I do bulk pours (switched to ketchup cups), I leave them for a few days to catch any with contamination. When I am working, I use plates to test my sterile technique and instruments. (taking a swab I sterilized and throwing it in the agar cup to see if it tams).


--------------------
:mushroom2:My LAGM 2.022! :mushroom2:

Imagine 30 minutes after eating mushrooms. You feel something in your teeth and a nice flavorful chunk of re hydrated mushrooms falls out. THE FLAVOR.


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OfflinePetMonkey
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Re: Spore Syringe and agar [Re: Mastershakes]
    #27547839 - 11/18/21 06:21 AM (2 years, 2 months ago)

Quote:

Mastershakes said:
Quote:

PetMonkey said:
Quote:

Spooff said:
It works by design. So as long as you dont hover, you shouldnt get contam from anything falling off your hands/arms.




In theory, but not necessarily in practice. I've seen many posts on here from people claiming they had nonstop failure and contamination with an SAB, and it seemingly disappeared overnight when they started to use a laminar flow hood - that's kind of where I am right now.

I'm a neurotic OCD clean freak who compulsively washes his hands 50 times a day, and out of maybe 100 plates I've inoculated in my SAB, I've ended up with 5 good ones. I did a few transfers the other day from these plates, and over half my new plates have bacteria/mold on them. I'm still new at this and obviously there's a learning curve, but good lord. 90%+ failure rate makes me think there might be some other factor at play than my sterile technique.




You could do mock transfers.  for every 10 or so plates just take one and do all the steps, minus the mycelium on it.

I'm new to agar and I was having huge issues as well at the beginning. I was buying pre made Agar, and sterile petri dishes. About 80% of my plates would contam. Long story short I traced that back to bad agar (from the lab I was buying from).

Now when I do bulk pours (switched to ketchup cups), I leave them for a few days to catch any with contamination. When I am working, I use plates to test my sterile technique and instruments. (taking a swab I sterilized and throwing it in the agar cup to see if it tams).




When you say pre-made agar, are you talking about the agar powder or the pre-poured sterilized Petris you can purchase?

I find my contaminations start to show up after I do transfers, usually not before that.


Edited by PetMonkey (11/18/21 06:22 AM)


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OfflinePBJ710
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Re: Spore Syringe and agar [Re: PetMonkey]
    #27547851 - 11/18/21 06:37 AM (2 years, 2 months ago)

Quote:

PetMonkey said:
In theory, but not necessarily in practice. I've seen many posts on here from people claiming they had nonstop failure and contamination with an SAB, and it seemingly disappeared overnight when they started to use a laminar flow hood - that's kind of where I am right now.




You could be running into issues caused by the heat rising off the agar bottles causing the air in your SAB to no longer be still.  I noticed that I had seemingly random very unacceptible failure rates when I tried to keep a couple media bottles in the SAB while pouring plates.  Also, if the bottles are completely closed in the PC, they will suck in air when finally opened which can contaminate the entire batch of agar in that bottle.


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