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InvisibleJosex
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Josex' Poke: No Mercy for Bacteria. * 19
    #24806569 - 11/25/17 11:53 PM (2 years, 10 months ago)

K.I.S.S.


The following method is a stupid simple and extremely effective way to deal with bacteria, no matter how bad it is.
It is very 'noobish' in its simplicity and the results show quickly. One of those things you have to try or you won't believe.

We know how dirty swabs can be sometimes. Those can easily give you the most disgusting looking plates you could ever lay your eyes on.
Maybe some spores managed to germinate and there's some helpless myc in the middle of the plate surrounded by bacterial gunk with all kinds of funny colors.

Good news is, you can save a plate like that from the trash can if it means something to you, without having to resort to costly antibiotics and all the necessary equipment required to use them.

These cultures originally came from swabbed plates that were heavily bacterial and were cleaned using this method. As you can imagine, myc looked like utter garbage:


I've tried cleaning very dirty agar cultures by transferring alone and it was useless.
I've also tried the sandwich method several times and either I didn't do it properly or it just doesn't work well when there's a lot of bacteria.
Bacteria would crawl under the sandwich, make its way up and flip me the bird from on top...
:banyouverymuch:


This method revolves around two principles:
  • Making use of the good 'ol PF-tek substrate:
    BRF, verm and water. Bacteria has a hell of a time on this substrate, It isn't just hearsay or old wives' tales. It will get slowed down to a near halt, allowing myc to get ahead.
  • Taking the smallest sample possible from a contaminated culture:
    And if possible, away from the areas where there's heavier contamination.
    To achieve this, we are going to poke the culture with a thin needle to take a tiny biopsy.

This tek shares some things in common with The Biopsy Method: A different way of doing LC, so I'm just going to copy some parts with a few modifications.



The Procedure


SETTING UP THE SAB



From left to right:
  • The BRF puck.
    It's just a little glass jar with some PF-tek substrate on the bottom. More about this later.
  • The agar culture.
    As you can see, the plate is upside-down to facilitate the operation. I look through the front of my SAB, so for me it's much easier to do it this way. If you work with your head over the box, simply leave the plate upright and open the lid to take the tissue sample.
  • A 10ml syringe with some sterile water.
    More details about this later.

All has been properly sanitized and the lids of the containers have been 'pre-opened'.
Find yourself a metal rack to raise the plates above the surface of the table or towel. I use my PC rack and cover it with a paper towel soaked in alcohol, which is not really needed but me likes it for stability. :thumbup:

THE 'POKE'



Now we're going to poke that nasty bacterial culture with the needle.
  • First thing of course, is to flame the needle red hot. If you're using a torch 2 seconds will suffice, and always be careful not to melt the plastic hub at the base of the needle.
  • In the SAB, squirt some water from the syringe to cool the needle. Sometimes a drop of water may hang from the needle after doing this. Flick the syringe with your finger to get the drop out.
  • Take the agar plate, pick the spot farthest away from the more contaminated areas and poke it with the needle until you touch the bottom of the plate with the tip. Done.

To inoculate with the biopsy you have to squirt some water from the syringe into the BRF puck (see next step).

You can repeat this procedure to inoculate several BRF pucks with the same bacterial culture and using the same needle.

Now I'd like to talk a little bit about the 'poke'.
By poking I mean just that, poking a spot of the agar culture with the needle, fast and clean. It is crucial you do it this way, no fucking around needed.
There's absolutely no need to poke twice, or to do it slowly or to scrape the surface of the culture with the needle and of course, NEVER squirt water into the plate.

The biopsy you take with the poke is literally invisible to the naked eye, so don't bother looking for it, not even with the help of a magnifying glass. There's just a clean needle, no agar, no myc, just a clean pristine needle. :shrug:

It is by no means an exaggeration to say that we're inoculating with microscopic pieces of tissue, as long as you use a thin needle to do this, which you must.

This procedure works ~85% of the times on PF-tek substrate. Sometimes you will never see growth on the plate, so you should always inoculate several pucks with the same contaminated culture.

INOCULATION



The picture says it all.

Crack the lid and squirt some water from the syringe into the BRF puck.
A quick squirt will be more than enough to dislodge and expel the myc cells that got stuck inside the needle.

Screw the lid back on and call it done.

Now the waiting game. You should see the first signs of growth after 5 or 7 days.
This is slow, I know, but once the myc colonizes ~50% of the plate you can be damn sure there's a lot of clean and healthy myc there.

The bacteria that gets squirted into the pucks will get stuck hopelessly in one spot. It will usually end up on the bottom of the plate doing nothing.



We got some growth, now what?




If the agar culture was severely contaminated I recommend waiting until the mycelium colonizes at least half of the plate. Now you can take a sample with your scalpel from the leading edge and trasnfer to agar. It can be tricky to take a sample from BRF pucks. Don't try to cut a wedge as you normally would with agar. Instead, use the tip of the blade to scoop up a small piece.
Or you could use the 'poke' method again to take a biopsy from the leading edge of the culture and use it to inoculate:
  • An agar plate.
    Most likely you'll see clean growth on the agar, although myc might grow ugly on the plate with all that water. I recommend taking a look at the section 'Testing a Culture for Cleanliness' from my other thread to know what to expect.
  • Another BRF puck.
    Just in case the culture is not clean yet.
    It only took a single attempt for me to obtain clean myc every time I used this method, but in the event that it wasn't clean yet this second inoculation should take care of it.

Be careful when you poke the PF-tek substrate with the needle. Don't go all the way down to the bottom, poke it only superficially or you risk clogging the needle and when you squirt water from the syringe the jet will split in two, kinda weird.

I highly recommend keeping these BRF pucks until they pin and then transfer some pins to agar. The pins are going to be very clean and these BRF pucks pin like there's no tomorrow.


And these are the largest bastards I ever got from a BRF puck.




Prepping the Pucks


THE CONTAINERS




I use little glass jars that are awesome for this.
I recommend always using little glass jars with metal lids. Bulky plastic lids are to be avoided because they make inoculation more difficult.
Likewise, plastic containers like glad mini rounds aren't good for this for several reasons:
  • The substrate will detach from the bottom of the plastic container very easily after sterilization. This won't happen with glass.
  • They create more condensation than glass jars.
  • The bulky plastic lids of mini rounds are a bitch to open if we compare it with metal lids.
  • Mold spores can make their way under the plastic lids and into the plate (as crazy as it sounds) if you happen to have a heavy spore load in your house, and you need to keep these plates for long.

I drill a tiny hole through the lid and cover it with 3 layers of MP tape. Alternatively, you could use a nail to make the hole.

If you are going to be using little mason jars with 2 pieces metal lids I'd recommend pc'ing them with the metal lid placed upside down to make your life easier in the SAB.

PF-TEK SUBSTRATE


As easy as 2:1:1.
  • Two parts vermiculite
  • One part brown rice flour
  • One part water.

I'm not going to go into the details of how to prepare PF-tek substrate because there's lots of info about it already. Simple stupid stuff.

With 80ml of vermiculite, 40ml of BRF and ~40-45ml of water you'll have enough substrate for several pucks.

Cover the bottom of the container with some substrate and pack it down just a little, enough to make the surface level. Then take a wet paper towel and clean the inside of the jars.

I like to wrap the jars in foil entirely. No need to use paper towels to cover the MP tape.

Sterilize the jars for ~70-80 minutes at 15 psi. Probably overkill but I like overkill. :grin:
Take the jars out of the PC as soon as the dialed gauge hits zero.



Prepping the Syringe




I use a 10ml plastic syringe and a sharp 21G needle. Needles deteriorate very quickly after flaming them, so I'd recommend always using a new one per session.
Using a thin needle will ensure we're taking an extremely small sample, literally microscopic. This is crucial in order to reduce the amount of bacteria that gets transferred onto the PF puck.
Sharp thin needle for the win! :awesomenod:

For anything myco and in general, luer lock syringes are always superior than luer slip syringes (google it up).
The difference is that the luer lock syringe allows a needle to be twisted onto the tip and then locked in place. This provides a secure connection and prevents the accidental removal of the needle.

Truth is, I've only used luer slip syringes for this because I can't find luer lock syringes locally. They work just fine but I feel I need to tell you what is best.

Fill the syringe with distilled water and try to get as little air in as possible (for the pic I filled the syringe carelessly and you can see a huge air pocket).
With 10ml of water you can inoculate lots of BRF pucks.

Always use freshly sterilized syringes. Don't cut corners here and never store syringes for later use.

Cap the needle.

Now you can do 2 different things before pc'ing the syringe for 15 minutes at 15 psi.

You either:
  • Wrap the syringe in foil and introduce it in the pc always making sure the syringe is standing upright or the water will boil out during the pc cycle. To achieve this, you can prop the syringe against two jars or any thing that does the job.


Or:
  • Find a bottle tall enough, filter it and put the syringe inside.





Bonus: Using Swabs to inoculate BRF pucks


As I mentioned before, swabs are often very dirty.

Agar is an excellent media for bacteria and they'll grow quickly and happily on it as we know all too well.

On the other hand, bacteria won't propagate so readily on PF-tek substrate, so why not use it to our advantage?

Currently, I don't have any swabs with me so I'm afraid I can't post pictures to explain the following method. Words will have to do for now, so bear with me.
  • First of all, prepare a shit ton of BRF pucks. A single swab can inoculate a ridiculous amount of pucks using this method.
  • Use a little filtered container to sterilize enough water to cover the bottom half an inch.
  • In the SAB, take the swab and dip it in the sterile water to soften up the cotton fibers and to loosen up the spores. Don't over do it, 10 seconds is enough.
    This won't work with a dry swab.
  • Take your scalpel and use the tip of the blade to remove a tiny, spore-laden piece of cotton from the swab.
  • Bury the piece of cotton in the BRF puck.

You can leave the swab on a clean surface between inoculations, like a clean piece of folded foil. Never put the swab back in the container with the water.
You could also sterilize an empty container and leave the swab there between inoculations with the lid cracked.

Once you see growth you can transfer to new BRF pucks using the poke to ensure a clean culture and then finish the job on agar.


That's it! :cheers:


Edited by Josex (07/04/20 04:19 AM)


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InvisiblebodhisattaM
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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #24806682 - 11/26/17 01:31 AM (2 years, 10 months ago)

:manofapproval:
I would feel safe just stabbing one of those in vitro fruits and using that for LC


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OfflineSagescruffy
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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #24806718 - 11/26/17 02:33 AM (2 years, 10 months ago)

Dude this is tight.


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Edited by Sagescruffy (11/26/17 02:33 AM)


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Sagescruffy]
    #24806929 - 11/26/17 07:41 AM (2 years, 10 months ago)

:camping:

Its funny how i started off with brf and pf tek and over the years ive drifted away,  and now back to brf. 

Pf pucks, brf agar,  brf lc broth. Such a useful medium.

Thanks josex. Ill try this with a wild pan print i got:congrats:


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InvisibleJosex
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Re: The Biopsy Method: No Mercy for Bacteria. [Re: mushboy] * 1
    #24807117 - 11/26/17 10:30 AM (2 years, 10 months ago)

Thanks guys! :heart:

Quote:

mushboy said:
:camping:

Its funny how i started off with brf and pf tek and over the years ive drifted away,  and now back to brf.



Me too man! Such an useful tool that so often experienced growers dismiss. I started 2 years ago with no PC and doing BRF paste (no verm, just plain BRF and water) using big jelly jars.

I used the brf paste to make blenderless LI's using a bulky plastic bottle and a wire made into a ball.
Then steamed some straw bottles for 2 hours and this was the best grow I made.


I remember asking in the noob forum if it was possible to steam straw+brf bottles and Bod encouraged me to try it. Magical times.:datass:


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #24807131 - 11/26/17 10:45 AM (2 years, 10 months ago)

:takingnotes:


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http://www.shroomery.org/forums/showflat.php/Number/11917410
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OfflineWeavieWonder
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Re: The Biopsy Method: No Mercy for Bacteria. [Re: tripdawg420]
    #24807227 - 11/26/17 11:49 AM (2 years, 10 months ago)

I wasted months and hundreds of petris trying to rid a couple cultures of bacteria.  It must have been a super strain of bacteria because countless transfers, several rounds of agar sandwiches and antibiotics were of zero help.  I ran out of patience and the cultures ended up in the garbage:mad:.  If I encounter something like this again, your method will definitely be the first thing I try.  Thank you for posting this sir!:thumbup::thumbup:


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OfflineFascistbullyboy
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Re: The Biopsy Method: No Mercy for Bacteria. [Re: WeavieWonder]
    #24807253 - 11/26/17 12:07 PM (2 years, 10 months ago)

Josex I've got some spores on the side of a tub. Do you think I would be able to use some of these spores using this Tek. Or maybe I should just give it a go and see how it runs. Maybe I could use this to test an LC for trich.


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InvisibleJosex
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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Fascistbullyboy]
    #24807368 - 11/26/17 01:08 PM (2 years, 10 months ago)

Quote:

Fascistbullyboy said:
Josex I've got some spores on the side of a tub. Do you think I would be able to use some of these spores using this Tek. Or maybe I should just give it a go and see how it runs. Maybe I could use this to test an LC for trich.



It could work, crazy things like that have been done before. But those spores on the side of the tub not only have lots of bacteria, which is not the real problem, but also have tons of mold spores mixed with them. So it could be a good way to get a bunch of trich in the pucks.

Yeah you could use the pucks for testing an LC, but for testing LC agar is where is at IMO. It lets you see everything more clearly.


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #24807395 - 11/26/17 01:19 PM (2 years, 10 months ago)

Great idea and write-up.
:booooom:


One thing I've found to be useful when making BRF pucks. I use wide-mouth pint jars, and then use the bottom of a 1/4 pint jelly jar to lightly tamp down the surface.


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OfflineFascistbullyboy
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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Fungus Mountain]
    #24807437 - 11/26/17 01:42 PM (2 years, 10 months ago)

I've done some agar LI, I can't remember where the myc was from. I dunno if it's from the suspect LC or a new MS. I tested the LC I've got on agar it didn't grow anything on it for 3 weeks so I assume it's cleanish. But I have trich pop up, I've been reading in the last few days trich will hide in myc. So I thought this could be a way of testing that LC  for said contam.


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Fascistbullyboy]
    #24807819 - 11/26/17 04:32 PM (2 years, 10 months ago)

Thaaaaanks josex. Good shit as usual
:thaaannks:


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Pipefitter537]
    #24808756 - 11/27/17 01:21 AM (2 years, 10 months ago)

:takingnotes::popcorn:


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #24808791 - 11/27/17 02:03 AM (2 years, 10 months ago)

:costanza:


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: hamloaf]
    #24808854 - 11/27/17 03:39 AM (2 years, 10 months ago)

:headbang::mariopeace:
Doin' it up tomorrow, so sick of bacteria riding along when transfering from spore germination. I've always had good results using brf in any fashion so this is right up my ally. Nice adaptation of traditional cleaning techniques.
Can't believe how simple this really is.

I will try this in comparison with the sandwich to see what I get. Definitely got my faith in this as my sandwiches seem not to get all the bacteria out. I think My agar is a little thick so maybe a warm pour would work better for sandwiches.

Anyway I really like it and I definitely favor this over trying antibiotics for the first time. I'm really hoping to be able to consistently get clean cultures without constantly worrying about bacteria.

Will be trying to make some pigs fly again( bucket tek ) since my major failure wave a while back that made me rethink every process AND MINUSCULE DETAIL. OOPS especially my culture's cleanliness.

I have cracked down on every step and haven't yet to test my what I thought to be clean in the past cultures that failed with bucket tek.

I Think there must be something riding along and thinking bacteria because of the grain spawn and the way it colonizes coir. Seems bacterial but I may not be the best judge of bacteria vs mold riding along.

Sorry for the ramble but this is an easy move for all the hassle I go through to "clean up" some of these cultures to have them fail after all.

Peace and thanks for giving. You rock and have made a real impact on me. You've got me all excited for mycology again. :lookslucrative:


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: tombosley8]
    #24809357 - 11/27/17 12:10 PM (2 years, 10 months ago)

:goodday:
It'll get you a nice clean culture for sure Tom. I discovered this accidentally at the time as with almost anything I do, I just can't stop trying weird shit and some of them work. I almost feel bad about taking credit. :lol:


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #24809520 - 11/27/17 01:17 PM (2 years, 10 months ago)

So if I'm following this correctly, you're essentially making a bacterial contam'd transfer to a substrate medium where bacteria can't easily follow myc along the material, then transferring away, but writing it up for the transfer style?


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Violet]
    #24809715 - 11/27/17 03:06 PM (2 years, 10 months ago)

I don't know if I understood you correctly. You mean why I inoculate the pucks with a biopsy instead of a wedge? Less bacteria gets transferred and that speeds up the process of obtaining clean mycelium.

You can also select the less contaminated spots very easily with a needle versus cutting out an agar wedge, which is immensely huge by comparison.

As for transferring away from the pucks using the poke, it's just easier than using the scalpel. Taking a sample from a puck using the scalpel can be a bitch, but you can do it no problem.


Edited by Josex (11/27/17 03:16 PM)


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Josex]
    #24809733 - 11/27/17 03:16 PM (2 years, 10 months ago)

It may be true that less bacteria is transferred this way, but it's only true for the same reason that less mycelium is transferred as well, and if you're concerned about bacteria spreading then why propel it onto and into your substrate with water? Bacteria spreads much more readily with water's help than without...

The dynamic of bacteria spreading much less readily on such substrates is equally true for any transfer.
So I'm inclined to think the best way to "speed up the process" is the one where the mycelium run out onto that substrate the quickest, and without water potentially spreading the bacteria further than myc.  You pointed out that this process is slow, 5 to 7 days.  By that point I would hope even a contaminated wedge would already have near enough growth out into the substrate to count on a cleaner transfer.

The syringe poke is a neat and not unheard of transfer trick, but by counting on the dynamics of cross-contam and counting on small injured cells of myc to establish and start a colony from scratch you're also increasing the P value of your transfers more than most, and honestly I'm really failing to see the advantages which is why I ask


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Re: The Biopsy Method: No Mercy for Bacteria. [Re: Violet]
    #24809767 - 11/27/17 03:38 PM (2 years, 10 months ago)

Try it out Violet, noccing with a wedge I mean. I for one am all about learning and I'm not one to identify myself with a method or way of doing things unlike some. If an idea can be improved I welcome that.

Quote:

and if you're concerned about bacteria spreading then why propel it onto and into your substrate with water? Bacteria spreads much more readily with water's help than without...



This is especially true on agar, but IME the same can't be said about pf-tek substrate. The water will be absorbed in one spot quickly and I like to think bacteria would rather stay in those areas that are more wet.

Quote:

by counting on the dynamics of cross-contam



Care to elaborate that part? The tissue sample is inside a flamed needle, and the little of water you squirt is sterile. Getting cross-contams can't be applied to traditional methods?

Quote:

counting on small injured cells of myc to establish and start a colony from scratch you're also increasing the P value of your transfers more than most




Injured cells? Maybe, just maybe, you could injure some cells with the sharp point of the needle. But the tissue inside the needle got there by rubbing off from the agar, nothing is damaged. Also, I seriously doubt damaged cells could ever recover and start a colony. :shrug:
About the P value. Well, we usually seek to slim down genetics by transferring with the scalpel. If the tissue is as tiny as the biopsy you just get there doing far less transfers and much faster. I say that in the hypothetical case you wanted to do culture work by poking alone. I do culture work the traditional way.


Edited by Josex (11/27/17 10:12 PM)


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Mushrooms, Mycology and Psychedelics >> Mushroom Cultivation

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