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Offlinedr0384
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h2o2 syringe
    #14480949 - 05/19/11 08:50 PM (12 years, 9 months ago)

I have been having problems with bacteria in my myc syringes.
I know its not ideal, but I was going to try just have a 3% h2o2 with myc suspended in it.
What term storage would this be good for?


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InvisibleDoc_T
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Re: h2o2 syringe [Re: dr0384]
    #14480959 - 05/19/11 08:51 PM (12 years, 9 months ago)

Zero-term.

Clean up your culture on agar.


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Offlinedr0384
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Re: h2o2 syringe [Re: Doc_T]
    #14480974 - 05/19/11 08:53 PM (12 years, 9 months ago)

how do you make syringe from agar.
How I make them is make small grain jar, wait till colonize, shake, put water in, suck water out.
Bacterial contamination every time. The culture is clean to start with


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Re: h2o2 syringe [Re: dr0384]
    #14480981 - 05/19/11 08:55 PM (12 years, 9 months ago)

That should work every time. Either you have poor technique, or your cultures are not clean.

How are you sterilizing the syringes and water?
What kind of lids/filters?
How do you inject/withdraw?


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Offlinedr0384
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Re: h2o2 syringe [Re: Doc_T]
    #14481041 - 05/19/11 09:07 PM (12 years, 9 months ago)

I thought it would show up pretty clearly if my culture wasn't clean on agar.
I sterilize my jars with water in it with tyvek over the lid, no center part of the lid. Like RR does in his video. In glove box I suck up the water and spray into the jar, suck it up.
I must just be letting contaminants in when I suck it in?


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Good judgement comes from experience.
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Offline3n1gm4
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Re: h2o2 syringe [Re: dr0384]
    #14481185 - 05/19/11 09:29 PM (12 years, 9 months ago)

You should always flame sterilize your syringes b4 storing them and don't squirt solution to cool needle, use an alcohol soaked towel to cool them instead. Replace the cap, tape cap on, and store them with needles pointing up so the solution can't seep back down to the tip of the needle where contams can get in.

If you are using the same needle that has been used before for a LC chances are there is still some old myc stuck in there, take it apart,clean off the plunger and sterilize it in the PC or suck up boiling water if you don't have a PC.

GLCs will keep for about two weeks or longer at room temp, longer in the fridge but the fridge is a contam playground so I would seal them in a container b4 using the fridge.

H202 will kill myc as well as cobweb mold, myc is just more resilient therefore when sprayed with a small amount to combat cobweb the myc will survive because the H202 turns back to water in the open air b4 it can completely kill the myc, but it kills cobweb instantly.


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Re: h2o2 syringe [Re: 3n1gm4]
    #14482709 - 05/20/11 03:50 AM (12 years, 9 months ago)

:facepalm:

Don't wipe the sterile needle with anything, ever.
Flame, expel, cap.


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Offline3n1gm4
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Re: h2o2 syringe [Re: Doc_T]
    #14482852 - 05/20/11 05:21 AM (12 years, 9 months ago)

So that the needle can be full to the end for storage? A TC told me that you should flame them and store them with needles up so the liquid wouldn't seep into the needle. I have had no problems storing needles this way.


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Re: h2o2 syringe [Re: 3n1gm4]
    #14482998 - 05/20/11 06:34 AM (12 years, 9 months ago)

Quote:

I know its not ideal, but I was going to try just have a 3% h2o2 with myc suspended in it.




Immersing live mycelium in 3% H2O2 will kill it.

Quote:

3n1gm4 said:
So that the needle can be full to the end for storage? A TC told me that you should flame them and store them with needles up so the liquid wouldn't seep into the needle. I have had no problems storing needles this way.




Either way, you don't want to wipe the needle with anything.  Alcohol doesn't kill fungal or bacterial spores.  If there are spores on your cotton swab/paper towel/etc, it's just going to transfer them to the needle.

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Re: h2o2 syringe [Re: Sillicybin]
    #14483116 - 05/20/11 07:22 AM (12 years, 9 months ago)

Bacteria doesn't sporulate like mold, mold shoots the spores into the air and there are very few airborne bacteria and I am pretty sure the ones that are airborne don't affect myc. myc will eat most bacteria. I have been using the same three spore syringes for months making GLC jars with them and then flame sterilizing them before cooling them off with a fresh paper towel soaked in alcohol and then putting the cap on and the syringes are still working because I have GLC rye jars colonizing from them right now, one is getting close to a shake a week from noc the CW syringe I have has been a slow colonizer fast fruiter ever since I got it so that jar is a bit behind but it will be ready for a shake soon too.

If you oust a clean closet and the room it is in 10-15 minutes before any noc work, turn off the AC and all fans and have as little movement in the house as possible, and close the door gently when you slowly enter the closet, wear a dust mask and gloves, wipe down self healing inoc ports with vinegar towel and H2O2 towel( and myself, I do alcohol after the vinegar peroxide wipes) then there is probably a fraction of a chance for contam, but not very likely. I have only had a few things contam one was a 3 month old cake, and the others were mistakes on moisture in rye jars(heavy thumb)

So I would say I think that I could do anything that requires a flowhood or GB with at least partial success without contams. I am going to start some cultures on top of some sterile ELME/water in a half pint gar and try isolating them with this closet tek and do some GTG and see if I get any contams. I think if the room is small and clean with no moving air and you try not to sneeze on it, whatever you do in a still air area should be contam free 90-95% of the time at least.


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InvisibleSillicybin
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Re: h2o2 syringe [Re: 3n1gm4]
    #14483295 - 05/20/11 08:14 AM (12 years, 9 months ago)

:thumbup:

I'm not saying that wiping the needle with alcohol won't work, because it will, most of the time.  I did it for years before I knew better.  But success rates can improve (say, from 95% to 97%, for example) if you don't.

http://www.mushroomvideos.com/BRF-Pf-Tek

On part 2 - notice that there is no wiping the needle.  You can't get more sterile than a red-hot needle - anything that touches the needle from that point on is just another chance for contamination.

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Re: h2o2 syringe [Re: Sillicybin]
    #14483316 - 05/20/11 08:21 AM (12 years, 9 months ago)

The only reason I wipe it is for storing it without filling the needle with liquid, I get the needle so hot I am afraid it will melt the plastic threads. I don't wipe during inocs, I just flame and expel some to cool it between jars.


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Re: h2o2 syringe [Re: 3n1gm4]
    #14483321 - 05/20/11 08:24 AM (12 years, 9 months ago)

Oh. Yeah, that's valid.
Not necessary, but a valid concern.
Draw a drop of air back in if you are concerned.


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Offline3n1gm4
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Re: h2o2 syringe [Re: Doc_T]
    #14483414 - 05/20/11 09:03 AM (12 years, 9 months ago)

Drawing potentially contamed air in would be more risky than flame sterilizing and wiping with alcohol IMO.


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