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Offlinesonoramo
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Are syringes contamination magnets?
    #26109301 - 07/15/19 09:15 AM (4 years, 8 months ago)

I've cultivated successfully with both PF-Tek and WBS to coir. "Success" meaning "less than 50 percent of cultures getting contaminated by trich or some nasty black-spore mold." But, every time I've used a syringe for anything, the contamination rate is 100 percent. I've tried a purchased spore syringe, one-time syringes fresh from the sterile manufacturer's package (and opened inside the SAB), syringes flushed a dozen times with boiling water, syringes from the PC (many of them melted...).

All my inoculations are inside a SAB, wearing gloves. Box and gloves, jars and outside of plates carefully sanitized with 70 percent isopropyl. I've been heating the needles with alcohol flame outside the SAB before and after each inoculation, and before injecting water to a clean-looking agar jar for LI. I can hear liquid boiling for 1/4 second when the liquid goes through. I've also wiped every jar or tyvek surface that the needle penetrates before inoculating.

I use Tyvek squares from FedEx envelopes in place of canning jar lids. So the jar has a standard ring screwed down over the Tyvek, and the Tyvek poking out well past the ring thread. It looks kind of like a drum head. It makes it easy to poke an inoculation or pressure-relief hole, which I always cover with micropore tape.

When I do G2G or Agar->grain, I have to take the Tyvek "lid" off for inoculation. I expect this would make my contamination problem worse, but I get the opposite effect. I get reasonably good results when dropping the inoculant into a briefly opened jar, which I then immediately seal up, but 100 percent contamination if I inject the inoculant through the Tyvek. In both cases, I soak the Tyvek with isopropyl first.

I must be doing something that introduces contamination in the process, but what? I'm ready to try some agar experiments with sterile plates and "clean" water from syringes, just to see if I can identify how the contaminants are getting in.

Anyone care to suggest experiments that could help me identify the source of contamination?

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OfflineMorel Guy
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Re: Are syringes contamination magnets? [Re: sonoramo]
    #26109316 - 07/15/19 09:29 AM (4 years, 8 months ago)

My opinion is that sab is not great for agar.


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Offlinejbgtaa
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Re: Are syringes contamination magnets? [Re: Morel Guy] * 1
    #26153098 - 08/26/19 12:49 AM (4 years, 6 months ago)

Quote:

Morel Guy said:
My opinion is that sab is not great for agar.



...? have you tried it? SAB's have not only proved their effectiveness to myself, but to countless other people on this website. Obviously in comparison to lfh its sub-par but considering it's hundreds, if not thousands, of dollars cheaper, it's effectiveness definitely stacks up.


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Offlinesonoramo
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Re: Are syringes contamination magnets? [Re: jbgtaa]
    #26153384 - 08/26/19 08:51 AM (4 years, 6 months ago)

Quote:

jbgtaa said:
...SAB's have not only proved their effectiveness to myself, but to countless other people on this website. Obviously in comparison to lfh its sub-par but considering it's hundreds, if not thousands, of dollars cheaper, it's effectiveness definitely stacks up.




I've tried making a glove-bag by adding rubber gloves to a large "vacuum" storage bag for clothes. That was a bit better with contamination, but it's so hard to use that the SAB is faster even factoring in all the contamination losses. Also, I used the glove bag in winter, and contams are different by the season, so hard to make a head-to-head comparison.

When I was a student, I tried using a flow hood in a campus lab, but that didn't work so well either. The filter had better be clean or else it's just pumping contams directly on your work.

Original comment stands, though: I've had success by scraping spores from a surface onto agar with an exacto, success with a NiCr inoculation loop, but no success ever with a syringe. No success transferring culture cleanly with a syringe, either.

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Offlinejbgtaa
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Re: Are syringes contamination magnets? [Re: sonoramo]
    #26154296 - 08/26/19 07:58 PM (4 years, 6 months ago)

Do you not wash your hands? The fact that a flow hood didnt work for you tells me that your either not telling the truth or your sterile technique is not very good. My contamination rate is around 15% right now with SAB use ONLY. Considering that at this moment i have 30 jars colonizing, mostly receiving that were done in a SAB, and I've only thrown out 4? Thats a fucking win.

You are right though about syringes, theyre dirty. I have had minimal success inoculating a grain jar with a spore syringe. Beyond that, I've had amazing success with agar inside my SAB.

Also glove boxes are counter-productive to mycological work. The vacuum created by the "gloves" means that you create air currents with every move you make. Still air box drastically lowers air movement around your cultures.

Do you know how flow hoods work? Theyre built with a prefilter, that actively keeps the main filter, which is an inch thick, clean. So your point about "pumping contams onto your work" is just plain incorrect.


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Edited by jbgtaa (08/26/19 08:00 PM)

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Offlinesonoramo
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Re: Are syringes contamination magnets? [Re: jbgtaa]
    #26154879 - 08/27/19 08:30 AM (4 years, 6 months ago)

In my attempt with the flow hood, I think the filter was the problem. The hood was in the back of a seldom-used lab, and I could smell something musty when I turned it on. I had no idea when the filter (which was, BTW, huge) had last been replaced, and I only let the hood run a few minutes before I started work. The hood looked kind of like this.

As far as my technique,... Maybe I wasn't as methodical as I would have been at home, since I didn't want someone to come in and ask a bunch of questions that I wouldn't want to answer truthfully.

Could you clarify "success with agar in my SAB"? Do you mean success inoculating agar plates from a purchased spore syringe, from a syringe you prepared yourself, from mycelium in suspension, from submerged culture...?

Edited by sonoramo (08/27/19 08:40 AM)

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Offlinejbgtaa
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Re: Are syringes contamination magnets? [Re: sonoramo]
    #26155386 - 08/27/19 01:54 PM (4 years, 6 months ago)

I've done all of that and more in regards to agar in a still air box, successfully. ive inoculated plates (that i poured myself inside of my SAB) with spores in pretty much every way you can. I've done at least 1000 agar2agar transfer inside my sab, I've made mycelium-suspensions using water, and nutrient broth.
When i get contaminates SOLELY due to the box, its because i knocked something against the wall while working

Stop spreading false, anecdotal information. IF you have a good strerile technique you could work in even the dirtiest areas and still have success.


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Offlinesonoramo
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Re: Are syringes contamination magnets? [Re: jbgtaa]
    #26155692 - 08/27/19 04:30 PM (4 years, 6 months ago)

Quote:

jbgtaa said:
Stop spreading false, anecdotal information. IF you have a good strerile technique you could work in even the dirtiest areas and still have success.




OK, now that we have that out of the way, what do you think might be the source of contamination that seems related to using a syringe for the transfers? I'd love to clean up my technique so I can succeed with liquid transfers.

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Offlinejbgtaa
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Re: Are syringes contamination magnets? [Re: sonoramo]
    #26155916 - 08/27/19 06:38 PM (4 years, 6 months ago)

Quote:

sonoramo said:
Quote:

jbgtaa said:
Stop spreading false, anecdotal information. IF you have a good strerile technique you could work in even the dirtiest areas and still have success.




OK, now that we have that out of the way, what do you think might be the source of contamination that seems related to using a syringe for the transfers? I'd love to clean up my technique so I can succeed with liquid transfers.



What do you mean liquid transfer? Like suspending mycelium in water? If so, there are a ton of places you could catch contamination.

Outline your entire process for me including how you sterilize and maybe I can tell you where u went wrong.


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Offlinesonoramo
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Re: Are syringes contamination magnets? [Re: jbgtaa]
    #26156770 - 08/28/19 09:16 AM (4 years, 6 months ago)

Quote:

jbgtaa said:
Outline your entire process for me including how you sterilize and maybe I can tell you where u went wrong.




OK, here's a typical “failed” attempt at liquid transfer from agar to sterilized WBS.

Starting materials: 4 pint jars WBS. Tyvek tops and rings sterilized while on the WBS jars, about 1 hr at unknown pressure (gauge-less PC).  2 spice-size (5oz) jars with about 2cm-deep ME-agar on bottom, slightly broken up from an earlier transfer with exacto. Mycelium had colonized the agar in the jar, and was forming 2-3 pins in both jars. No obvious contamination on the agar. The jars were selected to make liquid transfer easier.

Equipment: SAB made by cutting two 4 inch holes in a 66-quart stackable, lockable storage bin. 5cc monoject syringes in original sterile wrapper, 1-inch blunt needles (not sure of gauge, probably 20G). Exacto with pointy blade.

Syringe cleaning: Put the needle on the syringe. Boil water in saucepan, draw it into the syringe while its boiling. Eject it back and draw a few times. By the 3rd draw, something is going on with the syringe and the plunger leaks a little. It doesn't have as much resistance. Dip Al foil in the boiling water a few seconds, pull out and give it a shake before covering the needle with it.

Wash hands, then put on rubber gloves (playtex-type).

Wipe the SAB inside surfaces with 70 percent isopropyl. Start with back, sides, floor, front and top in that order. Lock the top down.

Light the ethanol lamp.

Wash gloved hands with 70 percent, then wipe the jars and ease them into the SAB. Same with the two spice jars holding the agar and mycelium. Wipe the Tyvek tops, careful not to ooze isopropyl into the jars through the Tyvek.

Dip the exacto in the isopropyl, then flame it. Watch the pretty blue flame die out and the tippy-tip of the blade turn red. In the SAB, open an agar jar and poke a wedge from it, transfer to one of the WBS jars. Drop the exact on the SAB floor. Re-tighten the WBS jar. Noted that the Tyvek is a bit hard to handle with gloved hands, and may have touched some surface. Next time, planning to use metal lids instead with holes and micropore tape.

Wipe the syringe outside with isopropyl. By now the water is cooled to room temperature. Flame the needle till the tip turns red. Squirt a little of the water out, hear some boil in the needle.

Inside the SAB, squirt the rest of the water (about 4cc) into the agar jar, swirl it around. Try to poke it with the syringe needle to float some of the mycelium. Suck up mycelium and agar as best as possible.

inoculate the remaining jars with the syringe, in the SAB. Poking the needle through the Tyvek and sqirting at the jar edges. The loosely-fitting plunger is a problem, and one jar ends up with nearly all the water. The second one gets less than 1cc.

There's no water left to make more solution. Inoculate the last WBS jar like the first one, after flaming the exacto again. Not the original plan, but oh well.

Result: The two syringe-inoculated jars wind up with a black-spore mold in a few days. One had what looked like mycelial growth at the injection point, the other one just had the black spore mold. One wedge-inoculated jar grows a bunch of trichoderma. The final jar goes on to colonize, gets mixed with coir, produces bountiful fruits.

I like that with the syringe I don't have to open the WBS jars and juggle the lids while transfering. But the contamination is frustrating, so I gave up on it.

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Offlinejbgtaa
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Re: Are syringes contamination magnets? [Re: sonoramo]
    #26156928 - 08/28/19 11:14 AM (4 years, 6 months ago)

Get a proper pressure cooker on which you can monitor psi.

Also, if the needle is already sterile, dont boil it... there's no reason to do that? When it fills up with sterile water it will stay sterile. When the mycelium enters it it will become non-sterile. At this point you work with physics, like your SAB and soap spray, and move it as fast as possible into the jars. Also when it fills it, it doesnt "lose resistance". I've used needles for a long long time, and when you add liquid to it it becomes easier to move the plunger. What i do for large syringes is grabbing like your going to stab somebody, sharp down and thumb up. Wrap your first finger around it and use it as almost a little cushion to keep you from moving the plunger too fast, ya know?

Gotta stress if your not steriizing correctly with the pressure cooker, alot of what your doing is luck. The PC and it reaching 15PSI specifically is a major player in being able to succeed in this hobby. I would suggest trying to actually sterilize your water in the pressure cooker in a jar with your syringe full of waterin another jar. That way you have a ton of sterile water to work with if you run out again.

The fact that you dont know what pressure your cooker reaches is probably the reason everything is contaminating. You MUST be at 15psi for AT LEAST 2 hours. I personally sterilize millet (main grain in WBS) for 3 ENTIRE HOURS, with pressures ranging from 15-18psi. This step is absolutely critical, because grain is obviously the main propogator for mycelia you plan on fruiting.

Use a real scalpel. In my experience, keeping an open flame in or near my SAB draws contaminates into the SAB. Also taking your hands in and out is a bad idea. I use a regular #11 scalpel handle and i buy a 100 pack of blades and use a new blade everytime i cut something. scalpel blades cost me around 30 dollars PER YEAR when using a new blade for LITERALLY every cut. This is completely critical, as there are some TC's who use exactos with no problem

Stop wiping everything with alcohol. You cannot sterilize a SAB and sanitizing it is nearly useless. I put a towel on the table, spray it with soapy water (dawn), put the SAB on the towel, then spray the walls. Then i put all my work in it.

It sounds to me like your most major problems are:
Proper Sterilization (120min at 15psi)
Sterile technique maybe. You do a whole lot of shit and end up with contams and all i do is spray the sab and use clean scalpels. And before i use anything in the sab its goes in pressure cooker, and ive only ever ran my pressure cooker at 15psi+. Start there, also try the blenderless liquid inoculant. Follow it to a T, and invest in either a pressure gauge for your cooker, or a presto 23qt. OR if u got it like dat get an all american sterilizer. Just try following teks rather than trying to cut corners and work with what you got, because its honestly worth it. I get asked by my friends why i dont reuse alot of shit or spend so much money on supplies, but seeing a beautiful organism thrive and fruit is all worth it, straight up.


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Edited by jbgtaa (08/28/19 11:17 AM)

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Offlinesonoramo
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Re: Are syringes contamination magnets? [Re: jbgtaa]
    #26158625 - 08/29/19 08:22 AM (4 years, 6 months ago)

Quote:

jbgtaa said:
Get a proper pressure cooker on which you can monitor psi.




Done. Got a 23-quart Presto canner with gauge. It's amazing how much better it holds pressure, even though it's much bigger than my old cooker. Can't wait to see if this fixes my problems.
Quote:


Also, if the needle is already sterile, dont boil it...




The blunt needles aren't sterile. They come in bags of 10.

Pressure cooker upgrade, misting instead of wiping, avoid too much in-and-out with the SAB,... Thank you for all the help and suggestions.

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Offlinejbgtaa
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Re: Are syringes contamination magnets? [Re: sonoramo]
    #26158727 - 08/29/19 09:36 AM (4 years, 6 months ago)

No problem brother, I hope it helps. If the needles are non sterile, I’d wrap them in foil and PC them with everything else instead of boiling them. And yeah the working concept of the still airbox is that you do nothing to clean the actual air, you just spray sticky soap to catch contams and drag them to the walls/down to the floor of the sab. So as little movement anywhere around it isn’t best. I took 4mil painters plastic and sealed my ceiling and walls (working in a basement 😒) and then I covered the vent with it. Make sure your Air Conditioning is off in your home as well when you use it.


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