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Offlinespatulaxx
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How common are contaminated syringes
    #20736495 - 10/22/14 12:51 AM (9 years, 5 months ago)

Ok, so I'm still fairly new to this but I've inoculated 11 total containers on 3 separate occasions with 2 syringes from the same vendor (a reputable one). Every one so far has contaminated. The first time I open air inoculated two presterilized bags of rye berries. The second I inoculated 4 1 pint jars with a still air box and the third 4 more pint jars in a glove box. If they were all different contaminants I'd be like "hey I think I suck at this" but every single container contaminated with pink bread mold. My apt is not exactly a clean environment so the fact that it's the same contaminant and not any of the other several types of mold I bunk with makes me suspicious. I'm pretty sure that the syringes are contaminated but like I said, I'm new at this. I've already got mda agar with gentimicin on the way so I'm already taking steps to develop my technique. I'm basically just wondering if anyone can confirm my suspicions and just answer whether or not this is normal seeing as they were sold for use in microscopy... And I know enough about sterile technique to be confident that I'm minimizing my risk of contamination as much as I can with what materials are available.

thanks in advance for the informative responses

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Offlineuglyman
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Re: How common are contaminated syringes [Re: spatulaxx]
    #20736509 - 10/22/14 12:56 AM (9 years, 5 months ago)

I know you said you know enough about being sterile but still, do you flame sterilize the syringes before and after each inoculation and before putting the cap back on?

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Re: How common are contaminated syringes [Re: uglyman]
    #20736518 - 10/22/14 12:59 AM (9 years, 5 months ago)

And are you sure to never wipe the needle with alcohol?


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Offlinespatulaxx
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Re: How common are contaminated syringes [Re: elasticaltiger]
    #20736600 - 10/22/14 01:38 AM (9 years, 5 months ago)

I do flame sterilize the needle between inoculation points and do not use alcohol wipes. Unfortunately so far I have been using a butane bic lighter which is not ideal, I know, being that it leaves soot on the needle. I have ordered an alcohol lamp for my next go. I flame sterilize for about 8 seconds and cool the needle by ejecting a small amount before inserting it into the container. However I do not flame sterilize before I put the cap back on which extremely good advice that I will keep in mind. The first syring was used in it's entirety on the two bags and the second syringe I have used twice on the 2 batches of jars and still have about 3 mL. I read a post about aspirating a sterile liquid growth medium into the syringe to check for contaminants but have yet to actually try it.

Edited by spatulaxx (10/22/14 01:45 AM)

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OfflineToolhead
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Re: How common are contaminated syringes *DELETED* [Re: spatulaxx]
    #20736643 - 10/22/14 02:05 AM (9 years, 5 months ago)

Post deleted by Toolhead

Reason for deletion: x


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Offlinespatulaxx
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Re: How common are contaminated syringes [Re: Toolhead]
    #20736659 - 10/22/14 02:18 AM (9 years, 5 months ago)

Yes, I do have a pressure cooker. The second time I prepared jars I inadvertently made the holes on one of the lids too small and couldn't inoculate it, it basically ended up being a control jar and I left it while the others colonized. After 2 or 3 weeks when it was obvious the other jars were contaminated it showed no signs of colonization by any organism.

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OfflineLhouse
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Re: How common are contaminated syringes [Re: spatulaxx]
    #20736661 - 10/22/14 02:20 AM (9 years, 5 months ago)

I had all kinds of problems. Then I started buying new spore syringes from *********** and Substrate jars from **************. I take the syringe from its box, jar from its box and inject. Zero problems that way.  Newbie way to do it but it works.

Read where someone said, 'Contamination is like being pregnant, you are or you are not... no in between'.

Edited by PussyFart
Reason: It's against the forum rules to name vendors.

3.  Spores are sold for microscopy use only so mycologists can ID mushrooms. If you're going to misuse them to grow a controlled substance, keep it to yourself.  Any post which mentions 'such and such' vendor, will be deleted and you'll receive an official warning.  Do not plug non sponsors.

Rules Thread-READ BEFORE POSTING

Edited by PussyFart (11/05/14 04:28 PM)

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Offlineuglyman
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Re: How common are contaminated syringes [Re: Lhouse]
    #20736690 - 10/22/14 02:44 AM (9 years, 5 months ago)

Well, that definitely sounds weird. I had a similar instance. The only thing was, I didn't spray the air down with disinfectant after a nasty contamination clean up. I was rushing.8  of flaming seconds sounds plenty. As a matter of fact, it sounds a little bit excessive and you should be careful not to break the plastic but if it works then don't change it. Maybe it was the syringe but if you had to put the needle together and it was in an unfit environment, that could have contaminated it. From what you're saying I think it's that or the syringe was contaminated if you're as sterile as you say you are. I guess the only thing you could do is walk us through your exact process from start to finish. Maybe somewhere along the line you did something small. Hopefully because it would suck to get a contaminated syringe and twice in a row is unlikely if the vendor is reputable.

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OfflineToolhead
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Re: How common are contaminated syringes *DELETED* *DELETED* [Re: Lhouse]
    #20736722 - 10/22/14 03:12 AM (9 years, 5 months ago)

Post deleted by Toolhead

Reason for deletion: x


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Edited by Toolhead (10/22/14 03:21 AM)

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Offlinespatulaxx
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Re: How common are contaminated syringes [Re: Toolhead]
    #20737375 - 10/22/14 10:19 AM (9 years, 5 months ago)

OK so here's my process so far. The first time when I inoculated grow bags I cleaned my area with Clorox surface/air disinfectant 3 times over the course of an hour, fogging the room each time I left. After thirty minutes I came back with rubber gloves and a face mask. Each one got a third of the syringe (excessive I know). I did not flame sterilize but used only 70% isopropyl alcohol wipes to wipe the needle applying friction before and between bags. These bags contaminated but in the end it was my fault because I had the temp at like 83F. The myc was sweating and a about 2 mL of fluid pooled at the bottom. They took forever to colonize and ended up contaminating.

The second time I followed PF tek to prepare jars. I used whole brown rice ground finely in a coffee mill. I used a still air box. I placed all my items into the still air box after wiping them and the box with 70% isopropyl. I fog the air box with aerosol Clorox let it sit for 15 minutes came back with rubber gloves and a mask to inoculate sterilizing with a bic lighter between each jar. The jars all contaminated within 2 weeks and were stored at 72F.

The third time I followed PF tek except this time i punched one hole in the middle of the jar instead of four to minimize inoculation points. Same process as the second time except using a glove box. Wiped everything with 70% isopropyl put it in the box fogged it with Clorox. I then tied a trash back around the lid and the bin (its made from a storage bin) and duct taped it to the bin to ensure minimal or no airflow. Flame sterilized with a bic lighter in the box before and between inoculation points. Instead of using a full mL for these I used about 0.3 mL each jar. They all began colonizing nicely and then about 2-3 weeks in, contamination. However these ones are showing significantly less than the second batch where I used about 0.4 mL x 4 per jar. I'm not entirely sure if this is because I used less of the syringe that I suspect is contaminated but I think its a possibility.

I place masking tape over the holes in my jars before I put them into the PC and do not peel it back until they are in the still/glove box and I immediately inoculate. My PC only read about 12-13 PSI but I leave them in there for an hour. and I'm 98% sure that not having high enough pressure is not an issue. ( Control jar did not contaminate. )

I also want to mention that when I attach the needle to the syringe I do it in the bag they came in. Nothing leaves the bag until the needle is set up completely. I do not remove the needle only recap it and place it into the bag.

If you would like to ask any specific questions feel free. I'm an open book dude man guys.

Edited by spatulaxx (10/22/14 10:29 AM)

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Offlineuglyman
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Re: How common are contaminated syringes [Re: spatulaxx]
    #20737672 - 10/22/14 11:49 AM (9 years, 5 months ago)

just a note: Don't wipe your items with alcohol before putting them in a still air box. We had an incident where the air box caught fire because alcohol is flammable.

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Offlineuglyman
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Re: How common are contaminated syringes [Re: uglyman]
    #20737678 - 10/22/14 11:51 AM (9 years, 5 months ago)

I always clean my gloves too, with some kind of disinfectant and dry them.

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Invisibleazur
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Re: How common are contaminated syringes [Re: uglyman]
    #20737680 - 10/22/14 11:52 AM (9 years, 5 months ago)

Agar is your friend


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Offlineuglyman
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Re: How common are contaminated syringes [Re: azur]
    #20737716 - 10/22/14 12:03 PM (9 years, 5 months ago)

can you explain that to me? I never did understand why people like agar so much. It seems like it would lead to contamination to me since you have to take it out and openly inoculate but everyone seems to love it.

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OfflineFreeWorldOrder
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Re: How common are contaminated syringes [Re: uglyman]
    #20737750 - 10/22/14 12:13 PM (9 years, 5 months ago)

lol... just the opposite. When using agar wedges you select clean mycelium to inoculate with. Plus isolate strains for size and potency.
I started with spore syringes as well. But really they are a shot in the dark as far as what genetics you end up with.


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Offlinespatulaxx
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Re: How common are contaminated syringes [Re: FreeWorldOrder]
    #20738063 - 10/22/14 01:38 PM (9 years, 5 months ago)

Hey now! I think the posts are beginning to stray from the original topic.

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Offlineuglyman
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Re: How common are contaminated syringes [Re: spatulaxx]
    #20738073 - 10/22/14 01:40 PM (9 years, 5 months ago)

you're right my apologies for making that agar post. I was just curious.

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InvisibleSpitballJedi
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Re: How common are contaminated syringes [Re: spatulaxx]
    #20738257 - 10/22/14 02:22 PM (9 years, 5 months ago)

Quote:

spatulaxx said:
OK so here's my process so far. The first time when I inoculated grow bags I cleaned my area with Clorox surface/air disinfectant 3 times over the course of an hour, fogging the room each time I left. After thirty minutes I came back with rubber gloves and a face mask. Each one got a third of the syringe (excessive I know). I did not flame sterilize but used only 70% isopropyl alcohol wipes to wipe the needle applying friction before and between bags. These bags contaminated but in the end it was my fault because I had the temp at like 83F. The myc was sweating and a about 2 mL of fluid pooled at the bottom. They took forever to colonize and ended up contaminating.




All that extra work with bleach and stuff trying to clean the air is overkill in my opinion. You only need still air as in a SAB. I only ever wipe stuff down with 70% IPA and spray soap water in my SAB. Occasionaly, I may spray the air with an air disinfectant.

The pooling water is likely because of over wet grains and/or bacteria. The fact that it was 83F had little, if anything, to do with your contamination. If your grains are already borderline wet, a third of a syringe could be the tipping point too.

Spore syringes are rarely 100% contam free and the more you squirt in, the higher chance of contamination. Also, you didn't flame sterilize your needle.

To this day, I still prefer using jars instead of bags.

Quote:

spatulaxx said:The second time I followed PF tek to prepare jars. I used whole brown rice ground finely in a coffee mill. I used a still air box. I placed all my items into the still air box after wiping them and the box with 70% isopropyl. I fog the air box with aerosol Clorox let it sit for 15 minutes came back with rubber gloves and a mask to inoculate sterilizing with a bic lighter between each jar. The jars all contaminated within 2 weeks and were stored at 72F.




Shit happens even when you do everything right. Did you have a dry vermiculite layer as you contam barrier? Didi you move the jars around much after inoculating? Did you sterilize your cakes properly? Again, spore syringes are rarely 100% contam free and some can be more contaminated than others. Less is more.

Quote:

spatulaxx said:The third time I followed PF tek except this time i punched one hole in the middle of the jar instead of four to minimize inoculation points. Same process as the second time except using a glove box. Wiped everything with 70% isopropyl put it in the box fogged it with Clorox. I then tied a trash back around the lid and the bin (its made from a storage bin) and duct taped it to the bin to ensure minimal or no airflow. Flame sterilized with a bic lighter in the box before and between inoculation points. Instead of using a full mL for these I used about 0.3 mL each jar. They all began colonizing nicely and then about 2-3 weeks in, contamination. However these ones are showing significantly less than the second batch where I used about 0.4 mL x 4 per jar. I'm not entirely sure if this is because I used less of the syringe that I suspect is contaminated but I think its a possibility.




Using less is better. You only need about 1/4cc per hole.

Don't flame inside the box. The heat creates air currents and the air is no longer still. Also, you said glovebox. Don't attach the gloves to the box; wear the gloves instead. This works better for this hobby.
Here's my SAB


Quote:

spatulaxx said:I place masking tape over the holes in my jars before I put them into the PC and do not peel it back until they are in the still/glove box and I immediately inoculate. My PC only read about 12-13 PSI but I leave them in there for an hour. and I'm 98% sure that not having high enough pressure is not an issue. ( Control jar did not contaminate. )




Good call. If you didn't inoculate your control jar or never opened it and all the other jars did contaminate, then that shows it's most likely either your technique or the syringe or both. It looks like you need to work on your technique a little, but it also looks like you have a very contaminated syringe as well.

Being as your PC only gets to 12-13 psi, I highly recommend PCing for 90 mins to be safe.

Quote:

spatulaxx said:I also want to mention that when I attach the needle to the syringe I do it in the bag they came in. Nothing leaves the bag until the needle is set up completely. I do not remove the needle only recap it and place it into the bag.




I open the needle and do the swap in my SAB. I do one last flame before putting the cap over the needle when I'm done. Make sure your needle is glowing red when flame sterilizing and do it outside the box


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Offlinespatulaxx
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Re: How common are contaminated syringes [Re: SpitballJedi]
    #20739217 - 10/22/14 05:35 PM (9 years, 5 months ago)

I did use a dry vermiculite layer and with the last batch I tamped the substrate down a little so I could fit a thicker layer in the jar. I've read that pink bread mold is a slippery little bastard and wanted to see if it would have an effect.... I'm getting off topic though.

Thank you very much for the info I can honestly say I learners a couple things

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OfflineToolhead
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Re: How common are contaminated syringes *DELETED* [Re: spatulaxx]
    #20739771 - 10/22/14 07:09 PM (9 years, 5 months ago)

Post deleted by Toolhead

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