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Offlinemushroomboy
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Syringe contam?
    #27800765 - 05/31/22 07:03 PM (2 years, 7 months ago)

I don’t have pictures, so I’ll describe the situation as best can. Which, isn’t hard. :frown:

So I’ve been having contamination issues. To combat this I made a liquid culture, I knew there was a clump from the original syringe (already germinated).

I watched and kept track of said puff, as it was the largest. When I took that puff and used it, it ended up turning grey.

This is all done under a hepa hood/box. I KNOW this was the original big mass in the syringe. The LQ has areas of good mycelium, I used part of it in some jars. A few have white and a few have grey.

Should I contact seller? The LQ was the first thing I did. I may have saved some but it’ll be ugly re-transfers. Or my only other solution is going to be try and save the other syringe that’s also got growth.

I’m currently doing a test of my hood. First is I did home made agar, letting the dishes dry in the hood. I also did agar in a jar. I’ve used a few of my home made agar as well as the jar agar. If those work I’m skeptical of my hood being the issue.

I’ve also just successfully cloned wild golden oysters and have mycelium off blue plugs in another agar. Both doing extremely well. As well as agar plates in either my to be FC or hood not getting contams I didn’t put there.

Outside of contact seller, what to do?

Edited by mushroomboy (05/31/22 07:04 PM)

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OfflineWeavieWonder
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Re: Syringe contam? [Re: mushroomboy] * 1
    #27800779 - 05/31/22 07:24 PM (2 years, 7 months ago)

Quote:

mushroomboy said:

So I’ve been having contamination issues. To combat this I made a liquid culture, I knew there was a clump from the original syringe (already germinated).

The LQ has areas of good mycelium




If you're having contamination issues, making an LC is not going to help with that. It sounds like you may be deficient in your understanding of how to make a clean inoculant. Work flow should be as follows:

spores --> agar --> sterile growth medium of your choice. The medium can be grain, LC, brf, ect.

What is LQ and puff?
Don't contact the vendor. Although no one's product is always perfect, it is reasonable to say that the source of contamination is usually the cultivator.

Edited by WeavieWonder (05/31/22 07:26 PM)

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OfflinePBJ710
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Re: Syringe contam? [Re: WeavieWonder] * 1
    #27800801 - 05/31/22 07:56 PM (2 years, 7 months ago)

:whathesaid:

You need to clean your culture before you expand it otherwise the latent contams will expand faster than the mycelium can.

By your usage of terms, I'm guessing you have some kind of turbulent air box and not an actual flow hood which is likely causing alot of your issues.  Use a properly made SAB with decent sterile technique and your contam rates should drop significantly.

If the LC in your gallery is what you're referring to, it's in really bad shape.  Looks really bacterial from the cloudiness and the growth in it does not look healthy.  IME cultures that float to the top or stick to the glass are usually molds.

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Offlinemushroomboy
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~ [Re: PBJ710]
    #27800806 - 05/31/22 08:08 PM (2 years, 7 months ago)

I did an agar culture with the original syringe, and an LC.  I haven't had success on either with this.  I'm doing a 2 more agars rn, to verify.  But I've had horrible luck getting agar with this vendor.  Horrible luck.

I've read up, drag the syringe, doing one drop.  I've had things in the hood for weeks without contams in filters.  I'm using self sealing syringe points.

I have gone through 2 syringes through this place. I'm using pre-steralized syringe tips.  So that shouldn't be my problem, and if the hood is good.  I should be able to take out the plug, put in the steril syringe and use.

I iodine swab all my injection points and let them dry in the hood. This has had massive amounts of redundancy. I've probably gone through 5 agar (commercial bought) with this vendor.

I assure you, there is massive amounts of precaution.

edit: I haven't drag/dropped on the same agar plate each technique was a different plate.

edit2: First, also I have easy access to brewers iodine in bulk.  I can iodione bathe anything.  Steralization in that aspect is easy.

To the post above, these are all first attempts with a vendor bought syringe.

Edit3: I have made 3 LQs from the same vendor and with the same results on agar. This last was specifically PE#6, so I've kept track.  RN I've got Blue Meaninies on agar, in a week I'll see how that's doing but I have a feeling the other syringe of PE#6 won't work well.


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Check my bio!

Edited by mushroomboy (05/31/22 08:14 PM)

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OfflineB Traven
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Re: ~ [Re: mushroomboy] * 1
    #27800816 - 05/31/22 08:29 PM (2 years, 7 months ago)

Ain't shit to contact the vendor about, those spores are for microscopy use only.

Assume that you have some things to sort out on your end, and figure out what they are.

I know one thing: I don't care how supposedly sterile something is, I want it glowing red before it touches the agar. I want the agar to sizzle. I want the first drop of spore solution to be a spurt of steam.

I'm really starting to like swiping prints with an inoculating loop and transferring that to agar.


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InvisibleMysticMycologist
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Re: ~ [Re: mushroomboy]
    #27800818 - 05/31/22 08:32 PM (2 years, 7 months ago)

The spores are dirty. All spore syringes are dirty. You have to make multiple transfers on agar to obtain a clean culture. Once you are sure it’s clean, then make LC from if you need to use LC, or just go to grain.

Also, what kind of hood do you have? Did you make it?


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Offlinemushroomboy
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Re: ~ [Re: MysticMycologist]
    #27801927 - 06/01/22 08:11 PM (2 years, 7 months ago)

Quote:

MysticMycologist said:
The spores are dirty. All spore syringes are dirty. You have to make multiple transfers on agar to obtain a clean culture. Once you are sure it’s clean, then make LC from if you need to use LC, or just go to grain.

Also, what kind of hood do you have? Did you make it?




this was the best reply.  so i should assume dirty and clean up with agar.  New lesson learned.

So from now on, i should spore to agar then re-assess. thank you. because  i've done wild to agar now.  with minimal contams. We took golden oysters from live to agar. Which was successfull with minimal contams. 

I'm going to take your advice and now start with agar. I just got agar the easy way (pre-made,add water and pour). This should help a lot.  I'll get mini-jars (i do have glass petri). I'll use the petri/mason jars for agar culture and see which I like best.

I've also had the best success with agar.Which I've been thinking about.  Now I got a hood/clean box things should be easier with agar.

To note, my last few agars that were home made are still in the hood drying. No contam growth. Petris are not sealed so full vent flow over them. This is my other test as I know my "seales" for ventaliztion are good under the hood. This is me confirming unsealed agar/petri should be fine. If this proves true, and my hood is good then I'm pretty sure the cultures were fucked in the syringe.

Yes this is a full hood/clean box.  Yes it's dyi, yes i'm not fucking dumb.  I've had 5 jars that I inoculated hot.  Meaning the spores didn't take,too high temp. Those jars sat in the hood for over a week with no contams. So is it my technique?

I really think the syringes were contam.

Edited by mushroomboy (06/01/22 08:13 PM)

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InvisibleMysticMycologist
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Re: ~ [Re: mushroomboy] * 1
    #27802001 - 06/01/22 09:02 PM (2 years, 7 months ago)

The syringes are dirty, like I said. All spores are dirty, by their nature. Post a picture of your hood? No one thinks you’re an idiot, but we see a lot of dyi hoods and “clean boxes” that lack laminar flow and are thus useless. We are just trying to help you, not deride you.


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Offlinemushroomboy
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Re: ~ [Re: MysticMycologist]
    #27810233 - 06/07/22 09:28 PM (2 years, 7 months ago)

I'll get a pic but it's pretty much a SAB with a fan/filter (edit: basic tote box upside down, holes drilled with gloves attached, nothing special).  I've recently just started using it as a SAB and heavily wetting the walls.  I let things settle and then go to work.

The problem is, even if I don't have contam syringes.  Syringes that get growth typically pull in/ball all the fucking spores. So I get a few chances at that to even put to agar or something else to better technique.

To me, this is fucking awful.  One vendor has nice syringes, where things aren't clumped or other bullshit. I get consistent results with them, the other I get fucking shit results. How can I learn with inconsistency.

The other major problem is I can only do credit/paypal rn.  And my other vendor rn doesn't do credit.  So I'm stuck with the shit vendor.

How can I do any work when 90% of my spores are in a fucking ball?

So this isn't just a contam, though every syringe I've had from this vendor does this:

It colonizes well, for a bit.  Then contams set and it colonizes fast but with grey mold. 

I've sprayed the box, I've wiped everything with alcohol.  I even have brewyers iodine (edit: yeast to iodine) to wipe shit down, same shit. Yet I can get LQs from other vendors (edibles cause it's cheap) and have had 100% success.  It fucking blows.  I've taken live to home made agar and "juggeled" things till healthy cultures.

So what the fuck am I doing wrong?  Standard SAB as a box/holes/taped gloves type shit (has a hole w/fan and filter but last experiments had the fan off). I right now have an almost full bag of kings going on rye.  Just iodine wipe/rubbing alcohol to wipe/pull the iodine out.  Inject with LQ, boom done. 

I've got myc saved from contaminated jars on agar.  I did this by juggling contams as well.  2 perfect samples finally and 2 wild samples saved. The samples saved were PE6, Golden Oyster, Blue Oyster.  Which I dealt with bacteria and outside mold.  RN there in small agar jars with no air holes. I'm putting them upside down with a loose lid for gas exchange. If they stall further I'll re-transfer to another healthy agar with filters.

However all that success is one thing, why the fuck can't I inoculate grain with any syringe of this vendor.  And why should I deal with half the spores clumped in a fucking ball.  It's god damn stupid.

Edited by mushroomboy (06/07/22 09:38 PM)

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Offlinemtshrooms
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Re: ~ [Re: mushroomboy]
    #27810366 - 06/08/22 12:40 AM (2 years, 7 months ago)

So, one could assume ALL syringes are dirty, and it shouldn't affect much, as you should clean it in agar first. Stop trying to blame your troubles on the supplier, this is helping nothing. I say this because you bring it up on all of your posts, let it go, it's gonna be ok.

If your using your box as a SAB, I feel like you would gain from reading a bit about how to use it and why they work. I say this because you said you are heavily wetting the walls before using it? It has/had a fan? It's too complicated!

Clear box, 2 holes.

Wipe it down with 70% iso if you feel like it, then set it up. Then place a wet paper towel on the bottom under a wire rack to give you a raised surface. Give the inside a couple mists of soapy water and wait for things to settle.

When using a scalpel or syringe, heat the needle/scalpel blade till it is red hot outside of the SAB with a Torch before using it in the SAB. I do this several times while working with agar.

Also, a SAB is NOT the same as a glove box, and if your using a glove box, that's also a source of your problem.

Shake the syringes like he'll before you use them perhaps, idk, but spores are microscopic, so they are more then likely all over in that liquid, we just can't see them.

Why can't you inoculate grain with your syringes? You shouldn't be able too. Syringes are DIRTY.

Stop blaming the vendors, we are all trying to help, please keep it chill, let's figure this out!

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InvisibleMysticMycologist
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Re: ~ [Re: mtshrooms]
    #27810422 - 06/08/22 03:38 AM (2 years, 7 months ago)

Ditch your turbulent air glove box, that is not a flow hood.  Build a $10 SAB with big 7” holes, and that’s it. Watch your success skyrocket.

Read this post by P9. It was written for you my friend.


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OfflineThe Tao
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Re: Syringe contam? [Re: PBJ710]
    #27810459 - 06/08/22 04:44 AM (2 years, 7 months ago)

Quote:

PBJ710 said:
:whathesaid:

You need to clean your culture before you expand it otherwise the latent contams will expand faster than the mycelium can.

By your usage of terms, I'm guessing you have some kind of turbulent air box and not an actual flow hood which is likely causing alot of your issues.  Use a properly made SAB with decent sterile technique and your contam rates should drop significantly.

If the LC in your gallery is what you're referring to, it's in really bad shape.  Looks really bacterial from the cloudiness and the growth in it does not look healthy.  IME cultures that float to the top or stick to the glass are usually molds.




The picture OP has in his gallery is from a honey LC they made 10 years and 5 months ago.
OP, post some current pictures of the inoculation box, syringes, current LC and agar.
Is the inoculation box set up the original from 2011?

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OfflineRotnpins
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Re: Syringe contam? [Re: mushroomboy]
    #27810544 - 06/08/22 06:44 AM (2 years, 7 months ago)

Check out this write up.. it seems like you may find it relevant,  it explains why spore syringes should always be considered  dirty.. I hope you find the information helpful

:cheers:

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