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Celestialexplorer1



Registered: 03/25/20
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Re: vacutainers? [Re: D3_Myc]
#27010744 - 10/29/20 04:18 PM (3 years, 2 months ago) |
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Would having a specific official thread for things like liquid culture be worth it or best to just leave it in general cultivation discussion
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 To spend just one moment in eternity
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Asura
Cyantist


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mushboy's thread was going strong for awhile but it kind of died off.
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mushhead
Potato Devourer



Registered: 08/22/14
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Re: Offical LC Thread [Re: Asura] 1
#27012316 - 10/30/20 03:15 PM (3 years, 2 months ago) |
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Right now I've done 2 quarts of straight grain water LC sterilized at 15psi for 45min Dropped some clean APE agar culture into it. I have no choice at the moment but to use regular jar lids. Each lid is punctured with a small hole and covered with a double layer of micropore tape. I don't have the ability to spin it However I have been stirring it by gently mixing daily. This will be my third attempt at LC since I'm in love with A2G, both previous attempts with LC were successful.
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PitcherCrab
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Re: Offical LC Thread [Re: mushhead]
#27014915 - 11/01/20 12:07 AM (3 years, 2 months ago) |
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Thanks for the write up Blue Helix! I've been following your grows for over a decade and have had some amazing Pan canopies following your LC techniques. I'm stoked to see you're back!
I've got a couple questions for you. First I'm wondering if you recommend inoculating your LCs with mycelium vs spore. I remember in some of your old Pan grows you went to LC straight from spore. Everything I know tells me I should start on agar first then go to LC to make sure my culture is clean, but I'm curious what your process is these days.
Second, is there a way to reuse vacutainers? I have a grip of old ones I got back in 2010 or so but they no longer have their vacuum anymore. After reading this post you've inspired me to try LCs again and it would be awesome if I don't need to buy more new stuff.
Finally, does the pore size on the syringe filters matter? I went to go buy the 32mm 0.22um ones you linked to in your LC lid thread, but it looks like in 32mm all that's currently available is the 0.45um.
Edit: I just ended up ordering some of these syringe filters. I expect they will do the trick! Link: https://www.ebay.com/itm/100PCS-PTFE-Syringe-Filter-0-22%CE%BCm-25mm-Non-Sterile-Hydrophobic-Filtration-Fit-US/254729572774?ssPageName=STRK%3AMEBIDX%3AIT&var=554515200001&_trksid=p2057872.m2749.l2649
-------------------- PC's LAGM 2021 (TOC & TWC)P. natalensis Growlog 2021Pans for PC Fall 2021 Growlog “I must not fear. Fear is the mind-killer. Fear is the little death that brings total obliteration. I will face my fear. I will permit it to pass over me and through me. And when it has gone past I will turn the inner eye to see its path. Where the fear has gone there will be nothing. Only I will remain.” - Bene Gesserit Litany Against Fear
Edited by PitcherCrab (11/01/20 01:16 AM)
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Nurkurzda23
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Registered: 07/13/20
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hi
I don't have enough time to take pictures of all my glasses at the moment. if they look like the one in this photo, that's a good sign, isn't it?
if so, how long should they be in the incubator before I can use them? Is it possible to incubate for too long?
And is it safe without a glovebox to make syringes of I work as sterile as I can?
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Mateja


Registered: 07/14/16
Posts: 7,948
Loc: Here
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How did you inoculate and how long ago? Are you stirring the LC? The broth looks clear (no turbidity) indicating there's no bacterial colony in the broth. And there is no wrong from wrong when you're growing out your LC, you decide when to use the LC if you think you have grown it enough
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Nurkurzda23
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Re: Some notes about LCs [Re: Mateja]
#27015334 - 11/01/20 08:50 AM (3 years, 2 months ago) |
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I inculated on 24.10.
I tried 3 different recipes to look Wich work best This is with less one and it works great 0;7g to 500ml
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Blue Helix
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Registered: 02/02/03
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Quote:
PitcherCrab said: Thanks for the write up Blue Helix! I've been following your grows for over a decade and have had some amazing Pan canopies following your LC techniques. I'm stoked to see you're back!
I've got a couple questions for you. First I'm wondering if you recommend inoculating your LCs with mycelium vs spore. I remember in some of your old Pan grows you went to LC straight from spore. Everything I know tells me I should start on agar first then go to LC to make sure my culture is clean, but I'm curious what your process is these days.
Hello! Here's my take on this: LCs can be inoculated with spores and I've done that many times, but you open yourself up to some big problems if the print isn't just perfect. And as for spore syringes, they often have too much bacteria for the LC to take off. Because of these problems that I've had and that I've read other have, I would say if you can do an agar plate first, you probably will save yourself from a lot of trouble. If you really don't like dicking with agar--and I sure don't--you can take your chances at inoculating a LC with spores, but always with spores, remember, less is more. You'd be better of having a tiny bit of spore print or syringe in there versus a lot. For example, when I use a spore print directly, I simply open it in a SAB above the LC and take the print between my index finger and thumb and rub the foil against itself. Even if you don't see any spores falling, I promise you that enough went into the LC. But, just to repeat myself, most of the time--and particularly if the printer didn't use sterile techniques to make the print--you are better off spores to agar and taking a bit of good growth (be it T1 or T2) to the LC.
Quote:
Second, is there a way to reuse vacutainers? I have a grip of old ones I got back in 2010 or so but they no longer have their vacuum anymore. After reading this post you've inspired me to try LCs again and it would be awesome if I don't need to buy more new stuff.
Sure you can use vacutainers over - but because they are unsterile more than they've lost their vacuum. Old vacutainers might lose their vacuum through gas diffusion through the septum, but that doesn't means mold/bacteria/etc. enter it. They definitely will not. If you do want to reuse a dirty one, it's as simple as rinsing it out, drying it with a piece of toilet paper or whatever, putting the septum back on, drawing out as much air as you can with a syringe, and then pressure cook it for 15 to 30 minutes. The reason you need to do all that is because otherwise the septum will pop out in the pressure changes of the cooker.
Quote:
Finally, does the pore size on the syringe filters matter? I went to go buy the 32mm 0.22um ones you linked to in your LC lid thread, but it looks like in 32mm all that's currently available is the 0.45um.
Edit: I just ended up ordering some of these syringe filters. I expect they will do the trick! Link: https://www.ebay.com/itm/100PCS-PTFE-Syringe-Filter-0-22%CE%BCm-25mm-Non-Sterile-Hydrophobic-Filtration-Fit-US/254729572774?ssPageName=STRK%3AMEBIDX%3AIT&var=554515200001&_trksid=p2057872.m2749.l2649
Well, I have not tried the 0.45um ones myself, but I was listening to a guy who runs a mushroom farm that uses LCs on YouTube, and he said either was fine and that they used both in their commercial production (and I verified that in his video actually). Also, spawn bags come in various filter types, and 0.5 um isn't even the largest pore size for a spawn bag. Some spawn bags have 5.0 um even (although I don't recommend that type for this purpose). The point is that 0.45um is still very small and will probably be just fine for this purpose. Also, being realistic, it's unlikely that the seal between your filter disc and the lid is actually tighter than 0.45 um unless you are using some kind of glue (I never do), so you are fooling yourself if you think you need smaller than that. Lastly, keep in mind that even IF a tiny bit of bacteria or a spore somehow made it in there, the LC remains very hot during the cooldown so would kill it. And by the time you are drawing the stuff out for use (and air comes into the LC jar to fill the displaced LC volume), the LC is so well established that you probably aren't going to get enough contamination in it to take off.
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Blue Helix
bold hand


Registered: 02/02/03
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Quote:
Nurkurzda23 said: I inculated on 24.10.
I tried 3 different recipes to look Wich work best This is with less one and it works great 0;7g to 500ml
I've read folks who say 1.5% sugar works just as well, if not better, than my 4% solution (i.e. 20g per 500ml). And if that is true, you might be better off with less sugar since you'll have less chances at bacterial issues getting the upper hand in there. I just haven't tried it, but I bet it works great.
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Blue Helix
bold hand


Registered: 02/02/03
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Last seen: 6 months, 18 days
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Quote:
Nurkurzda23 said: hi
I don't have enough time to take pictures of all my glasses at the moment. if they look like the one in this photo, that's a good sign, isn't it?
if so, how long should they be in the incubator before I can use them? Is it possible to incubate for too long?
And is it safe without a glovebox to make syringes of I work as sterile as I can?

The one in the pictures looks normal for a LC that is not being spun. For a spun LC, that would not be expected, though.
How long in the incubator? Well, I've had them in there for 48 hours when it was plenty well colonized to use and I've left them going before for 3 or 4 weeks even (longer in cold storage). LCs should not go bad if you go an extra few weeks. They can get so thick that it's harder to draw them out, though, so I usually just refrigerate it if it looks well enough colonized.
Yes, it is ABSOLUTELY safe to make syringes without a glovebox as long as your are injecting through a rubber septum. For me, half of the point of LCs is to not have to mess around with still air boxes, flow hoods, or excessively sterile techniques. As long as what you are doing is injection-based, you don't need those things - ever. In fact I inject the huge spawn bags with an 140 ml of LC right out in the open, with probably molding crap around me since I'm in the kitchen. I've never had a problem either. Just make sure if you do anything besides injections, you use a SAB or flow hood. And when you do inject your spawn bags, try to avoid having the spawn bag draw much outside air in the injection hole, and always immediately seal the hole with a nice big blob of hot glue over the tape reinforcement as I say in my technique.
 
PS - in one of those two pictures the tape reinforcement was far too low. It should actually be across from the filter patch and well above the substrate.
Edited by Blue Helix (11/01/20 02:52 PM)
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Nurkurzda23
Stranger

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Re: Some notes about LCs [Re: Blue Helix]
#27016740 - 11/02/20 04:51 AM (3 years, 2 months ago) |
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The lids with syringe filters:
is it safe to wash the lids with syringe filters in the dishwasher or is it bad for the syringe filters?
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Nurkurzda23
Stranger

Registered: 07/13/20
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Last seen: 2 years, 1 month
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how much LC do you use for a 2,5kg / 5.5 pound sack of rye for inoculating? I use 10-20ml multispore syringes. how much do i use with LC syringes? 10ml or less?
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mushhead
Potato Devourer



Registered: 08/22/14
Posts: 2,215
Loc: Dimension X-124
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Quote:
Nurkurzda23 said: The lids with syringe filters:
is it safe to wash the lids with syringe filters in the dishwasher or is it bad for the syringe filters?
Look out for rust m8. rust is the reason why I hate regular jar lids. If you're using plastic lids just wash them by hand because you'd have to do so with a dishwasher anyway, depending on the type of washer it could leave a variety of thing behind.
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sonoramo
Contaminant



Registered: 02/27/19
Posts: 851
Loc: California, baby!
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Re: Some notes about LCs [Re: Mateja]
#27017914 - 11/02/20 05:33 PM (3 years, 2 months ago) |
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Quote:
Mateah said:
... I made 5 liquid cultures in varying strengths of ELME at 0.1%, 0.2%, 0.4%, 0.8%, and 1.5% strength. I inoculated all LC´s with the same clone using The Biopsy Method by Josex.
Very, very nice work! Can you elaborate, ELME is powdered "Extra Light Malt Extract"? Is it different from "LME" or what Amazon calls "Organic Light Dried Malt Extract DME - Maltoferm 10001"? Are the percentages relative weight of powder and water, or something else?
Thanks!
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trippleblack
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i used as little as 15 ml for a 5 pound bag, can take over 2+ weeks, but i much rather use 50ml for a 5 pound spawn bag, you'll be nearly at full colonization in ten days..
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sonoramo
Contaminant



Registered: 02/27/19
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Loc: California, baby!
Last seen: 8 hours, 18 minutes
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Re: Some notes about LCs [Re: mushhead]
#27017940 - 11/02/20 05:41 PM (3 years, 2 months ago) |
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Quote:
mushhead said: Look out for rust m8. rust is the reason why I hate regular jar lids. If you're using plastic lids just wash them by hand because you'd have to do so with a dishwasher anyway, depending on the type of washer it could leave a variety of thing behind.
Just curious,... why care about rust? So long as it doesn't rust all the way through the lid, it just puts extra iron in the broth, no? I've even used home-made stir-bars made from 1/4 inch scrap steel rod that I magnetized. They look nasty, but the rust doesn't seem to hurt anything.
Regarding the dishwasher,... two-layer Tyvek circles epoxied onto the lids seem to go through the dishwasher with no problem.
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mushhead
Potato Devourer



Registered: 08/22/14
Posts: 2,215
Loc: Dimension X-124
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Re: Some notes about LCs [Re: sonoramo]
#27017960 - 11/02/20 05:46 PM (3 years, 2 months ago) |
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Quote:
sonoramo said:
Quote:
mushhead said: Look out for rust m8. rust is the reason why I hate regular jar lids. If you're using plastic lids just wash them by hand because you'd have to do so with a dishwasher anyway, depending on the type of washer it could leave a variety of thing behind.
Just curious,... why care about rust? So long as it doesn't rust all the way through the lid, it just puts extra iron in the broth, no? I've even used home-made stir-bars made from 1/4 inch scrap steel rod that I magnetized. They look nasty, but the rust doesn't seem to hurt anything.
Regarding the dishwasher,... two-layer Tyvek circles epoxied onto the lids seem to go through the dishwasher with no problem.
Rust can easily harbor contams ime. Tyvek is easily sterilized and is at best disposable from any standpoint. Plastics like the white plastic lids can pick up residue leading to contams. I'm a germaphobe, I think of these things.
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Blue Helix
bold hand


Registered: 02/02/03
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Last seen: 6 months, 18 days
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Quote:
Nurkurzda23 said: The lids with syringe filters:
is it safe to wash the lids with syringe filters in the dishwasher or is it bad for the syringe filters?
I do not recommend washing them. I rinse the lids a little, but I would never throw something like that in the dishwasher. How do you know that the crap they put in the soap to prevent films on the dishes won't clog the pores of the filter? I just won't do it unless you remove the syringe filter first.
Edited by Blue Helix (11/02/20 08:09 PM)
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Blue Helix
bold hand


Registered: 02/02/03
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Quote:
Nurkurzda23 said: how much LC do you use for a 2,5kg / 5.5 pound sack of rye for inoculating? I use 10-20ml multispore syringes. how much do i use with LC syringes? 10ml or less?
I use 120 to 140ml per SUBSTRATE spawn bag since my large syringe is 140ml. I do not do spawning anymore, and I was never very impressed with the results I got with spawning substrates to be honest. The yields were shitter overall. As far as how much to use, you can use FAR FAR less than 140ml, but I like to have enough liquid going in there that I can spray it down the sides. That way when I knead the bag, I don't have to mix it quite as much. Whatever you are using in a spore solution, will work fine with an LC. Let us know if 10ml works for a 5-6 pound bag. I'm curious since I never have used that little.
Here's is what I put in my bags again:
Quote:
Horse-Manure-based Substrate
- Dehydrated Manure 44 oz
- WBS or other grain 10 oz
- Vermiculite 1.5 L (for the brand I use this is about 6.6 oz)
- Water ~100 oz
Makes: ~10 pounds total
The above makes 2 x 5-pound spawn bag. I often use 7.5-pound bags for cubes, though, since they need more substrate. I sterilize for about 4 to 5 hours. Anything over 2 x 7.5-pound bags starts to take too long to sterilize in my 21-quart pressure cooker (like over 5 hours) because my cooker is too packed to allow for spacing between the bags, which makes heat penetration difficult.
Edited by Blue Helix (11/02/20 08:10 PM)
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Blue Helix
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Re: Some notes about LCs [Re: sonoramo] 1
#27018225 - 11/02/20 08:05 PM (3 years, 2 months ago) |
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Quote:
sonoramo said:
Quote:
mushhead said: Look out for rust m8. rust is the reason why I hate regular jar lids. If you're using plastic lids just wash them by hand because you'd have to do so with a dishwasher anyway, depending on the type of washer it could leave a variety of thing behind.
Just curious,... why care about rust? So long as it doesn't rust all the way through the lid, it just puts extra iron in the broth, no? I've even used home-made stir-bars made from 1/4 inch scrap steel rod that I magnetized. They look nasty, but the rust doesn't seem to hurt anything.
Regarding the dishwasher,... two-layer Tyvek circles epoxied onto the lids seem to go through the dishwasher with no problem.
I do not think rust can harbor contamination, but I do think it could end up making things not seal that well. Eventually the rust WILL go through, so keep an eye out for it. I used to use metal lids in a different style of LC lid than my current recommendation:
  
One thing I did that helped them rust a lot slower is to make sure you cover the metal lid with at least one layer of Tyvek before you put the band over it. The metal-on-metal initiates faster rusting by scratching the protective coating they put on the lids, so that Tyvek helped slow rusting issues a lot by helping protect the anti-rust coating. Also, Tyvek is good as extra filtration anyway.
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