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OfflineKulkulcan
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liquid culture jar setup
    #23193025 - 05/05/16 05:15 PM (8 years, 8 months ago)

In "Cybers Liquid Culture Tek" it says, before autoclaving the jars, to tighten the bands, but then loosen them by 1/4 turn to allow steam to escape.  He says if you don't do this, so much pressure may build up in the jars that they'll explode.  Is that really possible?

But even if it is, it seems to me that if air can get out of the jars, then it'll just rush back in as the jars cool.  Since Cyber doesn't appear to be using filter discs, that's gotta be risking contamination.

So here's my idea:

Use a filter disk , and drill TWO holes in the lid: an inoculation hole (a "passage way" for the inoculating needle), and a ventilation hole (to allow air into the jar so the mycelium can grow, as well as to allow steam to escape during autoclaving).

(Mycelium DOES require oxygen to grow, right?)

Set it up like this:

For the inoculation hole:  Put a filter disk on the underside of the lid.  At a point midway between the center of the lid and the rim, drill a very small hole (like maybe 1/16" in diameter) all the way through both the lid and the filter disk.  Seal the hole on both sides of the lid with silicon as Cyber describes.  Don't apply silicon to the filter disc.  Just leave the hole in the disc unsealed.  It will be protected from air going through it by the "self healing" silicon "gasket" in the lid directly above it.

For the ventilation hole:  Drill a larger hole (like maybe 1/4" in diameter) in the center of the lid.  There will be NO hole in the filter disk underneath the ventilation hole in the lid, so while air can enter the jar, contaminants can't.

Put the filter disk under the lid so that the inoculation holes in the lid and the filter disc are lined up, and then put them both on the jar full of liquid medium and screw the band on TIGHT.

Does all this sound like a good idea?

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Invisibledankington
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Re: liquid culture jar setup [Re: Kulkulcan]
    #23193044 - 05/05/16 05:21 PM (8 years, 8 months ago)

Well, if you are just starting, I wouldn't recommend LC. But this is what I'd go with:
https://www.shroomery.org/forums/showflat.php/Number/14186384#14186384
Edit: At NDStepp's suggestion: https://www.shroomery.org/forums/showflat.php/Number/19208976#19208976 the Pastyplates are where it's at.

TL has good info on using LC and making the lids for them. But you'd want a whatman filter. A regular filter may get wet when you stir the LC, which isn't good. I also don't typically condone a filter on the underside of a lid, especially with LC for the same reason.

And yes a sealed jar in the PC can definitely explode.

Edited by dankington (05/05/16 07:20 PM)

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OfflinePussyFart
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Re: liquid culture jar setup [Re: dankington]
    #23193052 - 05/05/16 05:24 PM (8 years, 8 months ago)

Quote:

dankington said:
But you'd want a whatman filter. A regular filter may get wet when you stir the LC, which isn't good. I also don't typically condone a filter on the underside of a lid, especially with LC for the same reason.

And yes a sealed jar in the PC can definitely explode.



Synthetic filter discs are water resistant and synthetic, so they can get wet and nothing will grow.

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Re: liquid culture jar setup [Re: PussyFart]
    #23193069 - 05/05/16 05:28 PM (8 years, 8 months ago)

i've always heard wet filters can wick contam through right? It's not the filter that is the problem but the nutritious water that creates a food and medium for the contam to enter or will they still be filtered when liquid passes through?

I have sfd's on my lc lids but I only fill the jar to 200 ml so I can swirl and not get near the lid or filter.


--------------------

Edited by tombosley8 (05/05/16 05:30 PM)

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Invisibledankington
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Re: liquid culture jar setup [Re: PussyFart]
    #23193079 - 05/05/16 05:32 PM (8 years, 8 months ago)

Quote:

PussyFart said:
Quote:

dankington said:
But you'd want a whatman filter. A regular filter may get wet when you stir the LC, which isn't good. I also don't typically condone a filter on the underside of a lid, especially with LC for the same reason.

And yes a sealed jar in the PC can definitely explode.



Synthetic filter discs are water resistant and synthetic, so they can get wet and nothing will grow.




Fair enough, I won't argue. Though water resistant doesn't necessarily mean water proof. You are the TC though, not I. :hatsoff:

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Re: liquid culture jar setup [Re: dankington]
    #23193087 - 05/05/16 05:35 PM (8 years, 8 months ago)

My ez felt that I use in place of sfds let's water easily pass through if say a lc is tipped onto the lid.

Sfd do allow some water to pass through just not nearly as much so definitely are water "resistant" but I've never heard of anyone using them the same as whatman's with any success


--------------------

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OfflineNDStepp84
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Re: liquid culture jar setup [Re: Kulkulcan]
    #23193379 - 05/05/16 07:09 PM (8 years, 8 months ago)

Not sure if it's your first grow, but a lot of people starting out have the misconception that it would be faster and easier to start out with LC, when in fact if done right it is one of the most advanced techniques there is, would take way more time and not worth dealing with unless you are inoculating 50 quarts or so IMO.

To be done right you would need to germinate spores on agar, transfer a few times to make sure you have a clean inoculant, use a wedge to inoculate the LC, wait for the LC to colonize, then back to agar to make sure it's clean (more waiting). Spores directly to an LC would be a big gamble and most likely be more of a waste of time than doing it right.

No spore syringe is 100% clean and you cannot look at an LC and tell if it is good or bad.
I didn't click it but I'm assuming dank linked pastys agar tek, super simple and easy. Tiger drop or TD tic tac toe to grains after you have clean plates FTW.


--------------------

"I am free, no matter what rules surround me. If I find them tolerable, I tolerate them; if I find them too obnoxious, I break them. I am free because I know that I alone am morally responsible for everything I do.
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OfflineKulkulcan
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Re: liquid culture jar setup [Re: NDStepp84]
    #23200069 - 05/07/16 07:36 PM (8 years, 8 months ago)

Quote:

dankington said:
if you are just starting, I wouldn't recommend LC.



Years ago, I had some experience with PF tek (cakes in jars), so I'm not really a noob.

I see that people are saying LC is harder than PF, but I could do a couple jars of each at the same time, so I'd learn LC while doing what I already know.

Quote:

dankington said:
you'd want a whatman filter. A regular filter may get wet when you stir the LC, which isn't good. I also don't typically condone a filter on the underside of a lid, especially with LC for the same reason.



I had never heard of Whatman filters before, but looking them up online, I see that this is a brand of paper filter.

What's the advantage that these have over synthetic or cellulose filter discs?  If they get wet, they won’t wick or get clogged?

Anyway, I was thinking that if I fill jars of LC only ¾ of the way up, I could just swirl them to mix them up, so that the LC wouldn’t touch the filter.  From what tombosley8 said, it sounds like that will work.

Quote:

tombosley8 said:
I have sfd's on my lc lids



Does "sfd" stand for "standard filter disc"?  If so, does "standard" mean "regular mouth" (70 mm)?

Quote:

dankington said:
You are the TC though



What's "TC" stand for?

Quote:

tombosley8 said:
I've never heard of anyone using (sfd) the same as whatman's with any success



To me, this sounds like Whatman filters may be used in a different way than I was talking about for regular filter discs.  Are they used in a different way?

Quote:

NDStepp84 said:
a lot of people starting out have the misconception that it would be faster and easier to start out with LC, when in fact if done right it is one of the most advanced techniques there is, would take way more time and not worth dealing with unless you are inoculating 50 quarts or so IMO.

To be done right you would need to germinate spores on agar, transfer a few times to make sure you have a clean inoculant, use a wedge to inoculate the LC, wait for the LC to colonize, then back to agar to make sure it's clean (more waiting). Spores directly to an LC would be a big gamble and most likely be more of a waste of time than doing it right.

No spore syringe is 100% clean and you cannot look at an LC and tell if it is good or bad.



OK, now I’m starting to see the difficulties.  Well, maybe later for LC, then.  Nonetheless, any answers to the questions I asked above would be appreciated.

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Invisibledankington
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Re: liquid culture jar setup [Re: Kulkulcan]
    #23200254 - 05/07/16 08:40 PM (8 years, 8 months ago)

:bongload:

Quote:

Kulkulcan said:

Years ago, I had some experience with PF tek (cakes in jars), so I'm not really a noob.

I see that people are saying LC is harder than PF, but I could do a couple jars of each at the same time, so I'd learn LC while doing what I already know.




LC is a biiiig step from PF tek. I have problems with LC and I'm fairly confident in my sterile procedure. LC is always such a crapshoot. And it's not going to save that much time over something like an LI from agar.

Quote:

Kulkulcan said:
Quote:

dankington said:
you'd want a whatman filter. A regular filter may get wet when you stir the LC, which isn't good. I also don't typically condone a filter on the underside of a lid, especially with LC for the same reason.



I had never heard of Whatman filters before, but looking them up online, I see that this is a brand of paper filter.

What's the advantage that these have over synthetic or cellulose filter discs?  If they get wet, they won’t wick or get clogged?

Anyway, I was thinking that if I fill jars of LC only ¾ of the way up, I could just swirl them to mix them up, so that the LC wouldn’t touch the filter.  From what tombosley8 said, it sounds like that will work.




They are much more likely to work for your LC jar. Just ask anyone who uses LCs. These are filters made for filtering solutions going into syringes, so their specs are fairly rigorous. Wouldn't want contaminants going into a bloodstream either, ya know?

Quote:

Kulkulcan said:
Quote:

tombosley8 said:
I have sfd's on my lc lids



Does "sfd" stand for "standard filter disc"?  If so, does "standard" mean "regular mouth" (70 mm)?




Synthetic Filter Disc.

Quote:

Kulkulcan said:
Quote:

dankington said:
You are the TC though



What's "TC" stand for?




Trusted Cultivator. Speaks for itself.

Quote:

Kulkulcan said:
Quote:

tombosley8 said:
I've never heard of anyone using (sfd) the same as whatman's with any success



To me, this sounds like Whatman filters may be used in a different way than I was talking about for regular filter discs.  Are they used in a different way?




They are usually affixed to the top of the lid via a small hole and some silicone. They would usually screw onto a luer lock syringe. They are 'repurposed' for LC work, and are the filters of choice for such.

Quote:

Kulkulcan said:
Quote:

NDStepp84 said:
a lot of people starting out have the misconception that it would be faster and easier to start out with LC, when in fact if done right it is one of the most advanced techniques there is, would take way more time and not worth dealing with unless you are inoculating 50 quarts or so IMO.

To be done right you would need to germinate spores on agar, transfer a few times to make sure you have a clean inoculant, use a wedge to inoculate the LC, wait for the LC to colonize, then back to agar to make sure it's clean (more waiting). Spores directly to an LC would be a big gamble and most likely be more of a waste of time than doing it right.

No spore syringe is 100% clean and you cannot look at an LC and tell if it is good or bad.



OK, now I’m starting to see the difficulties.  Well, maybe later for LC, then.  Nonetheless, any answers to the questions I asked above would be appreciated.




There you go! and Listen to ND. I've never been steered wrong from that man. :cheers:

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OfflineKulkulcan
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Re: liquid culture jar setup [Re: dankington]
    #23200317 - 05/07/16 09:05 PM (8 years, 8 months ago)

Many thanks, dankington, your helpful and prompt answers are much appreciated.

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Offlinecamplo
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Re: liquid culture jar setup [Re: Kulkulcan]
    #23200355 - 05/07/16 09:22 PM (8 years, 8 months ago)

Quote:

Kulkulcan said:
In "Cybers Liquid Culture Tek" it says, before autoclaving the jars, to tighten the bands, but then loosen them by 1/4 turn to allow steam to escape.  He says if you don't do this, so much pressure may build up in the jars that they'll explode.  Is that really possible?

But even if it is, it seems to me that if air can get out of the jars, then it'll just rush back in as the jars cool.  Since Cyber doesn't appear to be using filter discs, that's gotta be risking contamination.

So here's my idea:

Use a filter disk , and drill TWO holes in the lid: an inoculation hole (a "passage way" for the inoculating needle), and a ventilation hole (to allow air into the jar so the mycelium can grow, as well as to allow steam to escape during autoclaving).

(Mycelium DOES require oxygen to grow, right?)

Set it up like this:

For the inoculation hole:  Put a filter disk on the underside of the lid.  At a point midway between the center of the lid and the rim, drill a very small hole (like maybe 1/16" in diameter) all the way through both the lid and the filter disk.  Seal the hole on both sides of the lid with silicon as Cyber describes.  Don't apply silicon to the filter disc.  Just leave the hole in the disc unsealed.  It will be protected from air going through it by the "self healing" silicon "gasket" in the lid directly above it.

For the ventilation hole:  Drill a larger hole (like maybe 1/4" in diameter) in the center of the lid.  There will be NO hole in the filter disk underneath the ventilation hole in the lid, so while air can enter the jar, contaminants can't.

Put the filter disk under the lid so that the inoculation holes in the lid and the filter disc are lined up, and then put them both on the jar full of liquid medium and screw the band on TIGHT.

Does all this sound like a good idea?





I think all this is silly, I think its silly now, and I thought it was silly when I first learned about lc. What I mean is the whole rtv port deal. Its all fancy and if used right does work, and results in the same amount of success as not using them... If your sterile technique is on point, most of your contamination's are your own fault and not the method of inoculation and thus, you will be succesful.

When I do lc I pour from the jar. I am not sure why this would be a problem in a SAB as its essentially the same thing as a grain to grain. You should also use something to stir the lc. A computer fan with a hard drive magnet glued to the middle of the fan with a chopped up nail in the lc, for a stir bar, is simple and effective. Teks are on the forums. And please screw your lid on before pcing.

A simple way to approach lc as a beginner is to do small lc's in baby food jars. 1/8th hole in the lid with two pieces of micro-pore tape. It makes making syringes much more practical inside a SAB which is very limiting in movement. No injection port....unless you just are bent on it...just open the jar and suck all the lc into the syringe. If you open a lc to create a syringe, use it all up and use a little of the syringe to create 2 or 3 more baby lc's. Don't go farther than like a gen2 lc. They are done in about 3 days especially if stirred from day one. Knock up a test jar of agar or grain at the same time, and plan out a way to store and keep the lc syringe as clean as possible while you are waiting for your results.

That is basically what I did when I first started, and it worked with great success.

A practical way of testing lc is to knock up a quart of grain prepped on the dry side, with like 30ml of lc. If that jar turns out clean, use it for your grain inoculations. Then use the rest of the lc to knock up more quart masters. I recommend Liloldme's WBS Tutorial for grain used with lc though it is not the only way.


The difference between what you are supposed to do and what you are actually doing...


And omg...only inoculate the lc with a known clean source, like a tiny plug of clean ,colonized, agar, or agar li.


--------------------
:three:

Edited by camplo (05/07/16 10:17 PM)

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Offlinetump
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Re: liquid culture jar setup [Re: camplo]
    #23200876 - 05/08/16 12:59 AM (8 years, 8 months ago)

I disagree with you all about lc form spores vs flat culture off agar. True no syringe is a 100 clean. But you still use it for grain jars or pf the noobs do anyway. If you get a syringe form a trusted ventor which most people do anyway to start out then contams rate is low for spores to lc. Id say 9 out of 10 lc jars with good lids be fine. On the pther hand when i do clones or tissue samples even on agar its only 7 out of ten lc jars are fine. Mean green eats my lc every time it fails form cultures verses cobweb in spore lc. Does anyone else even see the difference of why lc is important. Any high amount of jars you need done lc is way to go.

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Offlinecamplo
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Re: liquid culture jar setup [Re: tump]
    #23200966 - 05/08/16 01:48 AM (8 years, 8 months ago)

Lc can be the way to go. It is super fast... it is the fastest that I know of, actually. I'm like the boards biggest lc advocate. I may even have some, what might appear as, edgy techniques...starting lc with spores is not good practice. Thats just my advice. As I stated above "The difference between what you are supposed to do and what you are actually doing..." If you are doing something and it works for you, great. I guess you could always make sure to test the lc afterwards. I think its a waste of spores whether its a contamination from the syringe or not taking advantage of what lc is good at, growth, and using your spores to create a master agar to create many lc's from that are clean. As long as you test the lc before knocking up 24 grain jars...or a giant bag of spawn...so be it. It's still stands that you are likely to get more contamination via spores to liquid, for sure...its the nature of the beast, regardless of vendor quality. Its not uncommon to get a hint of bacteria when spores first germinate but the mycelium beats it out and it does not carry with a transfer. You will not be forgiven in this situation in a LC. For the same reason some people  :hi:  opt to not use a master that was used to germinate spores to create a lc, They make at least one transfer and use the resulting colony.


--------------------
:three:

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InvisibleSupalemonhaze
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Re: liquid culture jar setup [Re: tump]
    #23201033 - 05/08/16 02:33 AM (8 years, 8 months ago)

Quote:

tump said:
I disagree with you all about lc form spores vs flat culture off agar. True no syringe is a 100 clean. But you still use it for grain jars or pf the noobs do anyway. If you get a syringe form a trusted ventor which most people do anyway to start out then contams rate is low for spores to lc. Id say 9 out of 10 lc jars with good lids be fine. On the pther hand when i do clones or tissue samples even on agar its only 7 out of ten lc jars are fine. Mean green eats my lc every time it fails form cultures verses cobweb in spore lc. Does anyone else even see the difference of why lc is important. Any high amount of jars you need done lc is way to go.




A pf cake and an LC are two different beasts. When you stir an LC, any contam will be chopped up to pieces and those will effectively create an inoculation point each. This will result in an LC that is littered with mold mycelium all over. When you inoculate a pf jar and the syringe is contaminated, the mushroom mycelium can overtake it enough for it to not envelop a cake until the 2nd flush. See the difference? One will go green in the jars while the other will give you a couple of flushes before going green.

It is never a good idea to make an LC from spores if you intend to use it for inoculation. Just like it's a good idea to wipe your ass after taking a dump, not really necessary but you know, it's much better. In the past almost all LCs were made by spore inoculation, one search for old LC teks and this is easily found. As well all other old and outdated info though, new more reliable methods are created, tested and finally released. It's up to you if you want to use the obsolete method but you don't really reccomend it to a newbie.

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Re: liquid culture jar setup [Re: NDStepp84]
    #23201423 - 05/08/16 08:11 AM (8 years, 8 months ago)

Quote:

NDStepp84 said:
Not sure if it's your first grow, but a lot of people starting out have the misconception that it would be faster and easier to start out with LC, when in fact if done right it is one of the most advanced techniques there is, would take way more time and not worth dealing with unless you are inoculating 50 quarts or so IMO.

To be done right you would need to germinate spores on agar, transfer a few times to make sure you have a clean inoculant, use a wedge to inoculate the LC, wait for the LC to colonize, then back to agar to make sure it's clean (more waiting). Spores directly to an LC would be a big gamble and most likely be more of a waste of time than doing it right.

No spore syringe is 100% clean and you cannot look at an LC and tell if it is good or bad.
I didn't click it but I'm assuming dank linked pastys agar tek, super simple and easy. Tiger drop or TD tic tac toe to grains after you have clean plates FTW.




:whathesaid:
I just want to add that if you plan to inoculate a LC with MS, it only takes the tiniest amount of bacteria getting into the liquid media to have a shitexplosion of them in no time and the worst thing is that you'd probably never know until it's too late and you used that LC to inoculate some projects.

Syringes are never sterile, they can be very aseptic and that's as good as it gets, meaning that it's highly probable a syringe may have bacteria. Bacteria will grow and expand at an exponential rate in the liquid media.

There are zillions of terrible and outdated teks that tell you to inoculate LC with a MS syringe.  Syringes are not clean enough for this and spores will take their sweet time to germinate and grow out, thus defeating the purpose of making this otherwise awesome inoculant, namely insane speed of colonization of the substrate you inoculate with it.

LC is a really advanced tek that should only be performed by experienced growers, and even they struggle with it sometimes. You should also test your LC on agar every time you want to use it, just to check that it's clean.

Also, in order to make a perfect LC you'd need the right equipment and set of skills:
-Agar is a must and you'd need to be proficient at it to do LC's.
-Magnetic stirrer to increase the speed of colonization of the LC.
-Watman filters and proper SHIP's.
-Maybe an Eberbach container to blend a wedge from agar.
-Malt extract instead of honey.

Edited by Josex (05/08/16 08:19 AM)

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Re: liquid culture jar setup [Re: Josex]
    #23201473 - 05/08/16 08:29 AM (8 years, 8 months ago)

I don't agree with the ports. I always ended up contaminating my LCs with them. Doesn't matter how new the port is, pieces always ended up in my jar. I prefer a hole, much safer. I don't do LCs much now anyways, g2g is just as good and more predictable.

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OfflineKulkulcan
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Re: liquid culture jar setup [Re: Supalemonhaze]
    #23203638 - 05/08/16 08:00 PM (8 years, 8 months ago)

Quote:

NDStepp84 said:
To be done right you would need to germinate spores on agar, transfer a few times to make sure you have a clean inoculant, use a wedge to inoculate the LC, wait for the LC to colonize, then

back to agar to make sure it's clean (more waiting). Spores directly to an LC would be a big gamble and most likely be more of a waste of time than doing it right.

No spore syringe is 100% clean and you cannot look at an LC and tell if it is good or bad.



Just to make sure I understand this correctly: You would have to do all this just to make sure you had no contamination, not because you were trying to obtain a single cell isolate, right?

Quote:

NDStepp84 said:
Tiger drop or TD tic tac toe to grains after you have clean plates FTW.



Sorry, "after you have clean plates" was the only part of that I understood.

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Re: liquid culture jar setup [Re: Kulkulcan]
    #23203665 - 05/08/16 08:10 PM (8 years, 8 months ago)

Yes just to make sure the inoculant is clean. That alone can take some work.

Tiger drop is when you squeeze out a glad mini round of agar into your grain. Munchauzen even made some cool videos about it. But the idea is, after having a culture you can see is clean on agar, you can make clean inoculations to your grains. If you've prepared your grains right, It's win-win.

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