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OfflineDomno
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What am I doing wrong?
    #26490657 - 02/17/20 06:44 PM (3 years, 11 months ago)

So I just tossed out my second set of agar plates.  The first one was very contaminated and looked like snot was on the plates. And now the same, with four different syringes.  They all have the same looking contamination and non of them seem to have any mycelium growing. It looks like white or sometimes slightly off-white snot. It smells like an old dirty sponge or wash rag.   

I use a SAB in my kitchen table.  I have racks that keep everything about 2 inches up and I have wet paper towels on the surface of the table.  I clean and wipe down the table top, racks and box with alcohol then put the it down on the paper towels. I put in my agar cups on the left and syringes on the right, a small bowl with alcohol and cotton balls, and a spray bottle. 

I spray the walls and towel completely down and cover the holes and let it sit for 10+ minutes. Then I go inside, wipe the edges of the jar with alcohol, wipe the syringe down.  Then I uncap the syringe, pull it out, flame it, bring it back in, lift the agar and put 1-2 drops (trying for 1), then snap the lid. 


I made the green containers about a week prior to the red ones, which were also a few days old at the time f inoculation. All the plates were clean and new looking when I inoculated them.

All of the containers had a lot of condensation, but I had had them top down since they came out of the pressure cooker.  Treds had a bit less.

I am really trying to figure out where I am going wrong.  I have had the same results with all 5 syringes.  I would understand if most of them were bad, but I am pretty sure I haven't seen any mycelium growing which would only make since if I was being extremely careless. 

If anyone can tell what kind of contam this is and where I may be going wrong, I would much appreciate it. 







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Invisiblec10h12n2o
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Re: What am I doing wrong? [Re: Domno]
    #26490679 - 02/17/20 07:11 PM (3 years, 11 months ago)

That's bacterial contamination

Did you have any plates you didnt put spores in? If so, did it contam too?

That could let you know if the issue is your agar prep or if it's your spore solution. Or at least figure out what point in the process it is going wrong


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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OfflineFrankRhizo
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Re: What am I doing wrong? [Re: Domno]
    #26490681 - 02/17/20 07:12 PM (3 years, 11 months ago)

Bacteria. Do you have any control plates?


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OfflineBilliswow
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Re: What am I doing wrong? [Re: Domno]
    #26490695 - 02/17/20 07:24 PM (3 years, 11 months ago)

Broooooo! Same here!

Did we buy syringes from the same place?? :lol:

I'm mainly commenting to see what other people recommend here because I'm having the exact same problem buuut... heres my 2 cents. If you have contaminated syringes (very possible) it might help to do a streak rather than a drop on your plates. It might be hard because your tubs are a little deeper than a petri dish but worth a try. You can squirt some of the solution into a sterilised shot glass and then streak a tiny amount of it on the agar with an inoculation loop or sterilised swab. It dilutes the solution so if you have tons of bacteria and a few spores, you have less of each and hopefully some distance between them on the plate. Also reduces the amount of liquid swimming about on the surface.


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OfflineDomno
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Re: What am I doing wrong? [Re: FrankRhizo]
    #26490703 - 02/17/20 07:28 PM (3 years, 11 months ago)

Thanks much for the replies. 

I did not take a control plate. That would have been a great idea though and I will do that from now on.  For a control plate, I just open it and close it in the SAB? or is there more to it? Also, I used the other half of the green plates a week prior, so the ones that I used this round had been at room temp with no contams while the last four were contaminated during the first inoculation. I am pretty sure the contams came at least after opening the container in the SAB. 

I have 8 more plates that I did later, new agar, new containers that I can actually see through.  These ones I made an inoculation loop so I could keep the dishes facing down.


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OfflineDomno
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Re: What am I doing wrong? [Re: Billiswow]
    #26490709 - 02/17/20 07:34 PM (3 years, 11 months ago)

Quote:

Billiswow said:
Broooooo! Same here!

Did we buy syringes from the same place?? :lol:

I'm mainly commenting to see what other people recommend here because I'm having the exact same problem buuut... heres my 2 cents. If you have contaminated syringes (very possible) it might help to do a streak rather than a drop on your plates. It might be hard because your tubs are a little deeper than a petri dish but worth a try. You can squirt some of the solution into a sterilised shot glass and then streak a tiny amount of it on the agar with an inoculation loop or sterilised swab. It dilutes the solution so if you have tons of bacteria and a few spores, you have less of each and hopefully some distance between them on the plate. Also reduces the amount of liquid swimming about on the surface.




Dude,

I got these syringes from mushroomprints.com. (Yes I know they are not a sponsor, no I did not know that resources was available when I purchased the syringe. If if my last plates are failures in the next week, I'm going to purchase a print from a sponsor.) Anyway, I bought the one syringe, then won a contest and they sent me 4 more. So I am pretty stoked.  But I am having a hell of a time.  I tried a loop this last time, so we will see.


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OfflineBilliswow
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Re: What am I doing wrong? [Re: Domno]
    #26490731 - 02/17/20 07:48 PM (3 years, 11 months ago)

I know, proper irritating innit. I haven't even bothered buying tubs for fruiting yet...

Lets hope using the lööps put an end to our woes.

Best of luck :heart:


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OfflineSockadin
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Re: What am I doing wrong? [Re: Billiswow]
    #26490792 - 02/17/20 08:36 PM (3 years, 11 months ago)

I'm gonna venture and say that your Sterilization need some work. The food coloring isn't helping either.


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InvisibleSmartattack
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Re: What am I doing wrong? [Re: Sockadin]
    #26490799 - 02/17/20 08:40 PM (3 years, 11 months ago)

It's the syringes. I'm putting my 1000 shroomery dollars on it here and now.


Well. Hmm. The bacteria appears to be all over the damn place in your dishes. So maybe not, unless you've been inverting your cups after inoculating?


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Edited by Smartattack (02/17/20 08:42 PM)


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InvisiblePiptheGreAtest
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Re: What am I doing wrong? [Re: Sockadin]
    #26490803 - 02/17/20 08:41 PM (3 years, 11 months ago)

I was having this same problem last week had nothing do with my sterilization but agar prep. What I would do to test they theory is buy 10 premade plates on amazon that are sterilized use the same procedures. If you get good growth on those you know somewhere in your agar prep you are screwing up or bad syringe(p.s. never had bad syringe from SporeWorks) Here picture of my most recent ones from amazon.


Here before premade plates


--------------------


Edited by PiptheGreAtest (02/17/20 08:43 PM)


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Offlinenarmaduke
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Re: What am I doing wrong? [Re: Sockadin]
    #26490807 - 02/17/20 08:43 PM (3 years, 11 months ago)

Looks like my plates from a recent B+ syringe. It could easily be the syringe (it was for me) and I had to grow out at least 10 plates till finally there was about 1 cm of mycelium on one plate that was not touching any of the bacteria. Took some surgeon like precision to make that transfer to another plate but after about three transfers I had some clean plates haha


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OfflineJohnRainyII
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Re: What am I doing wrong? [Re: Domno]
    #26490827 - 02/17/20 09:01 PM (3 years, 11 months ago)

Hard to say what's wrong. 


The way they are covered with bac does seem a bit like it's not the syringe.  If you  just put a drop on the agar, the bac would grow from the drop, you've got it everywhere, unless you waited for it to spread before taking those pictures...

So, it could be that the lids aren't sealing the way they are supposed to.

When this shit happens to me, I just begin changing things.  You get the old brain a stormin', and you make changes to your procedure.  I just keep thinking and changing until it works again.  Because the germs can't be seen, it's all blind.  Sometimes I think up some pretty stupid things.  No matter, just keep trying until you've got something that works for you.

If I was going through what you are going through right now, I'd get some proper petris.  That's the technology that's actually used for microbiology. 

At least make some of those agar containers again and let them sit to see if they contaminate so you can rule that out.

The other thing is to use prints.  I don't understand what's so great about syringes.  I thought the only appropriate use for them is inoculating PF cakes, because you don't shake them, so, you use liquid to help spread the spores around. 

Other than that, syringes are worse, because they have a higher contamination rate, and they come in a box which gets more scrutiny if going through customs, or your mom's mailbox, or whatever.  Prints come in an envelope that looks like any other mail.

Syringes are a value added product that vendors can make dozens of with one mushroom, and charge more than a whole print too.  It's a convenient racket, IMHO.


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InvisiblemushboyMDiscord
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Re: What am I doing wrong? [Re: JohnRainyII] * 1
    #26490830 - 02/17/20 09:06 PM (3 years, 11 months ago)

It's the syringes use the sponsors link or hit up the marketplace.

Here was my last non vendor syringe


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InvisibleJessicaRabbit88
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Re: What am I doing wrong? [Re: mushboy]
    #26490904 - 02/17/20 10:45 PM (3 years, 11 months ago)

I have read that the kitchen is the most bacteria infected room in your home. Second is the bathroom. So I stopped doing my sterile work in my kitchen and instead emptied my office to just have a standing desk and a small bookcase. Run my hepa air filter for an hour, wipe it all down with 10% bleach/water/a few drops of dawn. Then wipe everything going into my SAB with 70% alcohol. I wear gloves and wash them with iso, flame sterilize my scalpel and continuously wipe my items in my SAB with ISO. I never rush through anything but work quickly and methodically, making sure not to put my gloved hands over any part of exposed grains or agar.

This has definitely helped reduce contaminated agar plates.


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Invisiblec10h12n2o
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Re: What am I doing wrong? [Re: JessicaRabbit88] * 1
    #26490936 - 02/17/20 11:20 PM (3 years, 11 months ago)

I wouldnt be using premade agar as someone else recommended. Just dial in your technique

It's probably the syringes

I second the streaking, especially if you use the technique in my sig agar guide it will make it much easier to isolate any healthy myc. Just use one drop on the loop. You will be most likely to see clean growth in the later zones. Its basically a serial dilution on the plate


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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OfflineDomno
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Re: What am I doing wrong? [Re: c10h12n2o]
    #26491299 - 02/18/20 07:39 AM (3 years, 11 months ago)

Thank you so much for the feedback everyone. I have gotten some small deli dishes and they are much better for seeing what is going on. The the contams only showed up after inoculation.  Prior to that they were sitting out, then the contams start showing 4 days after inoculation. On the other hand, my first plates were very wet and I think the water definitely smeared some of that stuff around. I did let them grow a while because when they first showed up they were hard to see through the lids. Then it looked really bad and I just popped them open, took pics and tossed them out.

So now I have 40 more cups, 8 with inoculation using a loop and a z shape. I'll let the uninoculated plates sit for a bit to make sure they dont contaminate as well, but it's been 4 days of them sitting in a 70F room, which is usually when the plates have been showing the first signs of growth. If those 8 fail I will probably look into getting a print and/or try c10's streaking technique.

New dishes fresh out of the pc (4 days ago). I flipped them right after the agar set.


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Invisiblec10h12n2o
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Re: What am I doing wrong? [Re: Domno]
    #26491849 - 02/18/20 02:13 PM (3 years, 11 months ago)

Nice :smile:

Just to be clear, with that streaking technique you just use one drop of spore solution if you are using a syringe


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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