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Needle Biopsy for LC's


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#1 phlegmbae

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Posted 10 June 2021 - 05:47 PM

Tonight I'm going to put some sterile Cubensis flesh to agar plates. I would like to try some LC's too. Would it aid sterility by using a syringe that has 3% hydrogen peroxide in it to push a biopsy into a sterile LC? Just enough to get the myc into the LC.

 

I'm thinking about PCing a syringe full of h2o2, and using an unopened 14ga needle for each LC biopsy. How long should I PC a syringe of h2o2 @ 15lbs?

 

By biopsy I don't mean just sticking the needle into the stalk. I will split the FB to expose the sterile myc in the center, and extract that. Hmmmm..... Now I wonder. Could I use this tek to inoculate agar plates too, instead of using a scalpel?



#2 coorsmikey

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Posted 10 June 2021 - 09:18 PM

Peroxide weakens the desire mycelium/mold that you are trying to clone, but on the positive side it will give you a false hope that you sterile tech is that much better than it really is. If your tek is sterile like you think then why would you need to add peroxide? What do you think the peroxide does to mycelium if it kills contaminants? If it don't kill your mycelium the how well do you think it will kill the very contaminants that you are trying to prevent? 

 

Enough of that dead horse! People are alway gonna get lucky and post peroxide as their savior. What you really need to do is a few practice runs of your needle biopsy. Before you get your sterile goods out and loose everything cuz you think its easy to do a needle biopsy. Get a few practice runs in first to build that muscle memory before to try it with sterile media and IMO that will be far more effective that knocking the mycelium back and hoping a stressed contaminant doest win the race.

 

Seriously, how bout you go stab a few stems with a needle and report back? Let us know how that goes so we can advise more from that point. Its a pita especially when working in SAB, done correctly no peroxide is needed and it works good enough to do in open air as long as you are fast and efficient.


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#3 Oldpunk

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Posted 10 June 2021 - 09:22 PM

Think so. I wanna try this route too. A while back Rockyfungus sent me a link to a very detailed post at shroomery. I don't know about the h2o2.

But I also would like to know the answer. I tried plucking a couple strands with a sterilized tweezer a few days ago. I'd like to hear and see how it works out for you
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#4 pastyoureyes

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Posted 10 June 2021 - 11:46 PM

Is 14 gauge too small for biopsy? I remember having difficulty getting a biopsy when I attempted it a few years ago due to a small gauge needle, pretty sure it was a 14.
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#5 phlegmbae

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Posted 11 June 2021 - 12:24 AM

I don't know about that. I think they use 14's for donating plasma. They're not the biggest guage needles I've ever seen, but the biggest needle I've ever been stuck with. I think it's the biggest guage any of the shroom suppliers commonly sell. I bet they do a helluva job on the self-healing injection ports! Needle gauges go up the smaller they are. Diabetics use 31's usually.



#6 pastyoureyes

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Posted 11 June 2021 - 02:37 AM

Maybe they were 18? Cant remember which is bigger at the moment. I may have had it backward.

#7 Cuboid

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Posted 11 June 2021 - 03:32 AM

I've not done much work with needles and the couple that I have I don't even have an idea what gauge they are so this thread piqued my interest as it is an area I ought to know more about as they are clearly very useful tools in our hobby.

 

I found this on the following US needle manufacturer's/supplier's website, which I hope is useful.

 

https://www.hamilton...dle-gauge-chart

 

Text version here for convenience, pretty version on their website, I have an aversion to reproducing this kind of data as a 'picture' as you then can't copy 'n' paste the data when trying to share info which bugs me. Couldn't figure out how to insert basic html version of the table here either so good old fashioned text it is !

Gauge Number	OD(mm)	ID(mm)	WT(mm)	Dead Volume(µL/25.4 mm)
34		0.159 	0.051 	0.051 	0.052 µL/25.4 mm
33		0.21 	0.108 	0.051 	0.233 µL/25.4 mm
32		0.235 	0.108 	0.064 	0.233 µL/25.4 mm
31		0.261 	0.133 	0.064 	0.353 µL/25.4 mm
30		0.312 	0.159 	0.076 	0.504 µL/25.4 mm
29		0.337 	0.184 	0.076 	0.675 µL/25.4mm
28		0.362 	0.184 	0.089 	0.675 µL/25.4 mm
27		0.413 	0.21 	0.102 	0.876 µL/25.4 mm
26s		0.474 	0.127 	0.178 	0.322 µL/25.4 mm
26		0.464 	0.26 	0.102 	1.349 µL/25.4 mm
25s		0.515 	0.153 	0.178 	0.464 µL/25.4 mm
25		0.515 	0.26 	0.127 	1.349 µL/25.4 mm
24		0.566 	0.311 	0.127 	1.93 µL/25.4 mm
23s		0.642 	0.116 	0.267 	0.268 µL/25.4 mm
23		0.642 	0.337 	0.152 	2.266 µL/25.4 mm
22s		0.718 	0.168 	0.279 	0.563 µL/25.4 mm
22		0.718 	0.413 	0.152 	3.403 µL/25.4 mm
21		0.819 	0.514 	0.152 	5.271 µL/25.4 mm
20		0.908 	0.603 	0.152 	7.255 µL/25.4 mm
19		1.067 	0.686 	0.191 	9.389 µL/25.4 mm
18		1.27 	0.838 	0.216 	14.011 µL/25.4 mm
17		1.473 	1.067 	0.203 	22.715 µL/25.4 mm
16		1.651 	1.194 	0.229 	28.444 µL/25.4 mm
15		1.829 	1.372 	0.229 	37.529 µL/25.4 mm
14		2.109 	1.6 	0.254 	51.076 µL/25.4 mm
13		2.413 	1.804 	0.305 	64.895 µL/25.4 mm
12		2.769 	2.159 	0.305 	93 µL/25.4 mm
11		3.048 	2.388 	0.33 	113.728 µL/25.4 mm
10		3.404 	2.693 	0.356 	144.641 µL/25.4 mm

OD == Outer Diameter of needle

ID == Inner diameter of needle

WT == Wall thickness of needle

'Dead volume' == internal volume of needle, probably not too relevant in this particular context but maybe useful info non the less.

 

 


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#8 phlegmbae

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Posted 11 June 2021 - 01:54 PM

I got the agar work done this morning. I think coorsmikey is correct in recommending a dry run first. FWIW I used a scalpel this morning. This was my first work with agar plates. It was not as easy as it looks. Of course I cut myself. Note to self.... be very gentle with a fresh scalpel blade. Now if I can figure out how to take the used disposable blade off.

 

I've got some over grown white enoki, and some agaricus brunescens in my fridge that I'm going to practice the needle biopsy with. Then I'll try it on the shroom stalks for LC's with fresh unopened 14ga needles. I'll try to take some pics, and post. I've never had any luck with posting pics here yet. I'm an old poop. I'm not very tech savvy.

 

Oooh! I got a fresh script of ketamine in the mail today! Whee! At least daddy's gonna be happy this weekend! Happy Father's day folks!


Edited by phlegmbae, 11 June 2021 - 01:59 PM.

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#9 Oldpunk

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Posted 11 June 2021 - 03:45 PM

It is a little tricky with a scalpel. That's why I tried a tweezer this time. Which worked well. My strands are sprouting mycelium to the agar nicely.

I've got a bunch of mushrooms maturing this week so I think I will try a couple needle pokes. I need to pc my syringes tonight. Think they're 18ga. Whatever they sent me with spores.
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#10 phlegmbae

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Posted 11 June 2021 - 07:21 PM

Well get on it OP. Those syringes won't PC themselves. I see no reason why 18ga needles wouldn't work. I got my info to do this from a youtube channel video put out by Mossy Creek Mushrooms. He does the biopsy with oyster mushrooms. Check it out!

 

 

He's got a lot of pertinent information on his channel.

 

 

Philly Golden Teacher makes it look so easy that he builds your confidence enough to just do it. I've noticed a lot of folk just get scared of failure when you mention spawning to bulk, and you best be prepared for a lot of failure in this hobby. But failure builds character, and if you can learn from your mistakes, you've got it beat. I used PGT's CVG recipe, and the sub came out at a perfect field capacity. Think I'll stick to just coco from now on though. For some reason PGT sorta reminds me of Uncle Fester.

Much of the other information I snarfed for this grow was from here.

 

https://www.youtube....7cZ1h46Nn9NiSCQ

 

I just love doing 6 qt shoe boxes. They're so convenient. Easy to move into your clean area. I like that each one uses a quart of spawn.

 

 

 

 

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#11 pastyoureyes

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Posted 12 June 2021 - 02:51 AM

I remember having trouble doing a biopsy with a smaller gauge needle I bought at the store. Just something to be aware of if you buy them yourself instead of vendor supplied needles.
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#12 phlegmbae

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Posted 12 June 2021 - 12:51 PM

After a half hour of practice on my overgrown enoki's last night, I've decided it's much easier just to use a scalpel. Oyster's have a different consistency in their flesh. Notice he chooses the toughest stem also. Enoki's have a stringy texture very similar to the inner mycelium of Cubensis.

 

I will have to mark this experiment down as a failure. Practice makes perfect, and coorsmikey..... I most definitely took your advice into consideration in this instance. Even practicing with a scalpel on the Enoki's got me so I'm not just tearing out strings that may stick up out of the agar too far, and lead to contamination. it was worth a try.

 

Golden Teacher's really do drop a heavy spore load. Prolly make 3 prints from one cap. How long should one dry their spore prints before closing them up?


Edited by phlegmbae, 12 June 2021 - 12:59 PM.





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