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OfflineFishLevelMidnight
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Re: How are commercial PE syringes made? [Re: john_conner]
    #25115429 - 04/05/18 09:02 AM (5 years, 9 months ago)

Quote:

john_conner said:
for the DIY people with a 3d printer like myself i found this to be pretty interesting and might have to give it a go.

http://designawards.core77.com/Open-Design/62397/3D-Printed-Centrifuge-for-International-Health-Labs




For those of us without a 3-D printer...



How do y'all think the spores are dried? Just remove the supernatant and the non-spore bits and leave in the centrifuge tube to dry under a flowhood? Similar to an RNA pellet?

I'd like to try this with my next rounds of prints.
Any info on size and Gs needed from workman bod?

I would imagine spinning in 50 mL falcon tubes would be more efficient (It would be a PITA to add the crushed caps to 1.5 mL tubes). but I know some centrifuge inserts have a max rpm of like 4200...

Curious how easy the washing/removing the cap material is from the spore pellet. Also I wonder spore number per uL after centrifugation (or by weight of the pellet...) Sorry rambling now!


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OfflineFishLevelMidnight
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Re: How are commercial PE syringes made? [Re: john_conner]
    #25164377 - 04/25/18 11:43 AM (5 years, 8 months ago)

Maybe I should try this with APE. I'd love to get a pure spore sample and see the proportion of albino to wild type spores.

I got a bunch of dried fruits, so I would crush the caps in some water and then strain through a 22 uM filter into a centrifuge tube. Then spin that to pellet the spores? Then wash the pellet a few times.

Not sure if going to 22 microns with all of the cap material would be viable or not.

What is the wetting agent you are adding workman, is it some sort of detergent like Tween-20 or Triton X-100 to help dissociate the spores?


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Re: How are commercial PE syringes made? [Re: Asura] * 1
    #25211888 - 05/18/18 06:14 AM (5 years, 8 months ago)

Why not go with the paper and string centrifuge I posted?

Wayyyyyy easier and safer and I think it would still work for separating spores from the supernatant


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Re: How are commercial PE syringes made? [Re: john_conner]
    #25276389 - 06/18/18 11:20 AM (5 years, 7 months ago)

I’m hoping to do a trial run with some normal cube spores, then try and get spores from dried APE caps.
Summer is a little slower in the lab, so one evening I’ll bring my materials in and see what I can do. Hoping in the next month, but I will update with pics and my method if people are interested.

Good luck brother! Glad things are back on track, stay well.


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Re: How are commercial PE syringes made? [Re: john_conner]
    #25277348 - 06/18/18 07:07 PM (5 years, 7 months ago)

A nice test would be some blood. See if you can separate it.

Easy/cheap to get too :P


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Re: How are commercial PE syringes made? [Re: john_conner]
    #25278392 - 06/19/18 08:23 AM (5 years, 7 months ago)

I would use distilled or even regular tap that is PCed.

Good luck man!

How are you filtering? I plan to crush up some dried caps in water, then strain through cheesecloth, then strain through 22 uM filter into 1.5 mL tube (or 15 mL/50 mL conical depending on amount I get) for centrifugation. I will use PCed water to wash the spore pellet 2-3 X before putting to a syringe or drying on foil.


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Re: How are commercial PE syringes made? [Re: sandman420]
    #25282846 - 06/21/18 08:55 AM (5 years, 7 months ago)

Was it a fresh cap?

I want to dry with dry, not sure if you can separate from feuitbody matter once it is dry and smashes up?


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Re: How are commercial PE syringes made? [Re: john_conner]
    #25282968 - 06/21/18 10:03 AM (5 years, 7 months ago)

I doubt it, curious to find out though!


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Re: How are commercial PE syringes made? [Re: k5hd2y] * 1
    #25291160 - 06/25/18 03:05 PM (5 years, 7 months ago)

I made up my first syringes today.

Took some B+ caps from January and put into a petridish with some autoclave dh2o. Broke up with a scalpel because I forgot to break them up before adding to the water (doh)

Moved things around a bunch. Forgot anything for straining so I drew the liquid right through a 22 uM filter into a syringe, got about 30 ml with 3 normal sized caps in there.

Put the filtered liquid into 50 mL comical and centrifuge at 4200 rpm for 10 min. Remove supernatant add 20 mL water. Vortex and the centrifuge as before. I did 3 of these washes.

Added 25 mL to final pellet. Vortex to resuspend and then aspirate into 2 10 mL syringes and cap. This concentration is not super dense, but enough spores such that they were visible:


I did this all in front of a flowhood or under a flame.

Not expecting it to be clean. Hoping for clean enough and for germination (these spores were dehydrated at 160F ~24 hours before packaging, then another 24 hours after 5 months storage). Putting some spores to agar tonight and later this week if those don’t germinate.

I’ll post results here.

If I works I may make up 4-6, 3 mL syringes of B+ if others want to test it out (especially interested in those going straight to grain or pf Tek as I am testing on agar.


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Re: How are commercial PE syringes made? [Re: john_conner]
    #25292610 - 06/26/18 08:28 AM (5 years, 6 months ago)

How'd the cotton work for you?

Did you just put it in the water, then suck the water into the syringe through the cotton (eliminating some of the larger chunks)?

I might try this next time (I'm going to grind up like 5 g of B+ caps and do this a little more legit, make a bunch of small-medium syringes for giveaway) and sterilize the cotton before hand.

If things work out and I get germ on my plates, I am decapitating all my dried APE and seeing what I can get out of them. I want for crosses and to run generations/trades/giveawats and to see some albino spores, so I'll post a microscope pic later.


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Re: How are commercial PE syringes made? [Re: elasticaltiger]
    #25304112 - 07/02/18 09:00 AM (5 years, 6 months ago)

An update on my end.

Of the 2 plates I tried with the spores taken from dried caps (2X at 160F actually) one got super bacterial and the other is showing no growth at all. I think the bacteria came in from shaking the plate later on and not from the syringe, as the other plate shows nothing at all.

I'll follow it a bit further; however, it seems that spores dried at 160F are not viable. I plan to dry some batches of fruit at a lower temp to see if I can keep the integrity of the spores but also dry the fruits properly. I was thinking 100F to start, any suggestions?


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Re: How are commercial PE syringes made? [Re: john_conner]
    #25305273 - 07/02/18 08:06 PM (5 years, 6 months ago)

After collecting them the first time through the 22 uM filter I spun them down. Then I used PCed water to wash them (I dumped the supernatant, then added 20 mL, I was doing this in a 50 mL centrifuge tube) by vortexing to break up the pellet. Then I spun down and removed super and repeated 3X. Then added 25 mL and drew up into the two syringes I posted.

Anyone know the thermal tolerance of cubensis spores?


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Re: How are commercial PE syringes made? [Re: john_conner]
    #25365031 - 08/03/18 06:44 PM (5 years, 5 months ago)

Hope all is well brother. Good luck to you


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