|
Some of these posts are very old and might contain outdated information. You may wish to search for newer posts instead.
|
invitro


Registered: 05/03/13
Posts: 2,529
Last seen: 1 month, 19 days
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: bodhisatta]
#19665464 - 03/07/14 10:38 PM (9 years, 10 months ago) |
|
|
It depends on your environment, if you have a high spore count in the air, I wouldn't try it even with a sab. With a flowhood or a relatively clean work area sure.
|
bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: invitro]
#19665481 - 03/07/14 10:41 PM (9 years, 10 months ago) |
|
|
with a high spore count a SAB has to be just as effective it's all or nothing, the SAB works by not having air currents and the drag of particles encountering the still air. If you're using your SAB the right way it wont really matter. But, If you have a high spore load and use a Flow hood you're getting 99.97% filtration some stuff will make it through, that's why you work fast even in front of a flow hood(they're not sterile TEK saviors you still need excellent sterile TEK and rehearsed agility) , and it's also why the opening the petri and letting the FH blow on it isn't a good test to see if you built your FH right because even a properly built one will fail that test if you leave the dish open long enough.
Edited by Trusted cuItivator (03/07/14 10:42 PM)
|
invitro


Registered: 05/03/13
Posts: 2,529
Last seen: 1 month, 19 days
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: bodhisatta]
#19665502 - 03/07/14 10:47 PM (9 years, 10 months ago) |
|
|
What I'm saying is a SAB isn't effective enough (to get away with agar wedges in an lc) with a high spore count, because you have to put your gloves through the box, there is always some air current it's juut minimal and minimal is too much in a very high spore load situation IME.
Edited by invitro (03/07/14 10:48 PM)
|
bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: invitro]
#19665508 - 03/07/14 10:49 PM (9 years, 10 months ago) |
|
|
IME you wipe your gloves with iso inside of the SAB, it's what field mycologists use in the middle of a jungle for wild samples with success, Imagine the spore count in a humid jungle.
|
Mrcloudy
Stranger than you.



Registered: 10/01/13
Posts: 2,889
Loc: Northeast US
Last seen: 3 months, 18 days
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: bodhisatta]
#19665523 - 03/07/14 10:51 PM (9 years, 10 months ago) |
|
|
Yeah I have not had any issues inoculating LC with agar wedge In my opinion its just about the best way to go about it, treat it like a grain jar. Open it up drop the wedge in quickly then cover it back up working as quickly as you can. I admit that opening the jar is nerve racking, anything that is a potential vector of contamination makes me nervous. But no more so than doing a grain jar.
I have not yet had a contamination doing it this way. I have had some funky looking LCs though, but that was due to the variety of ways each species deals with being submerged.
--------------------
10 different Ganoderma species from across the USA AMU MrCloudys guide to North American GanodermaUpdated A rough guide to North American Ganoderma species, with an emphasis on the laccate species.
|
cronicr



Registered: 08/07/11
Posts: 61,436
Loc: Van Isle
Last seen: 2 years, 7 days
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: bodhisatta]
#19665529 - 03/07/14 10:52 PM (9 years, 10 months ago) |
|
|
all i've ever done with lc's is wedges in my sab, still air is still air, spritz water around and get to work
--------------------
  It doesn't matter what i think of you...all that matters is clean spawn I'm tired do me a favor
|
RandomFX
protege



Registered: 12/02/13
Posts: 1,015
Loc: North-East, USA
Last seen: 5 years, 1 month
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: cronicr]
#19665787 - 03/08/14 12:01 AM (9 years, 10 months ago) |
|
|
The long term storage by distilled water method, is pretty much a LC. And by pretty much I mean it is period.
I have to believe it has to be quite viable for so many major people and institutions to use it, and there is one major mycology collections group named with over 3,000 recorded specimens at the writing of the article that named them, that switched over totally to distilled water for long term storage in 1994. although I am currently doing it and testing it specifically per species I have access to, and it has been used for decades, and the specimens have been revived after decades as well. I won't say what it works with or does not specifically, I haven't tested it yet, but there's some pretty darn convincing argument for it. LC's obviously work, and work well from agar. All my species are LTS in distilled water, and not refrigerated, and only in small 13mm x 100 mm culture tubes that I get for .48 cents ea. and I am willing to bet, in the next week when I revive those cultures, which holds about 12 cc's ea. I'll easy be able to grow from less than a drop of culture, which means if the standard of 20 drops/ml is used, then a 12 ml vial of LTS mycelium can potentially revive the strain 240 times, easy. Not bad imo.
http://www.hardydiagnostics.com/articles/Saving-fungal-Cultures-Sterile-Water-By-Harris.pdf
http://www.scielo.br/scielo.php?pid=s0365-05962005000700004&script=sci_arttext&tlng=en "CONCLUSIONS
Based on the experiment, it can be concluded that:
1. Strain preservation by the distilled water method has made it possible, in a short period of time, to prove the viability and sporulation capacity of the strains submitted to this study.
2. Maintenance technique in sealed flasks containing distilled water has proven to be efficable for preserving the characteristics of sporulation of fillamentous fungi of medical interest.
3. Conservation of fungi in flasks with distilled water has easy handling, storage and transportation, besides being more economic the traditional method of successive samplings"
|
blindingleaf
blue collar underworld



Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: RandomFX]
#19666069 - 03/08/14 01:43 AM (9 years, 10 months ago) |
|
|
yea I'm with cron i only do agar to lc in an SAB. its same as agar to agar or agar to grain. the lc will start faster if u drop in a couple wedges or at least scratch up a larger wedge then drop it in, so when u shake, little parts of the myc come off the wedge and start their own colonies. i also use no RTV port or ge port, easier to shake rigorously. i only fill up the needle ONCE, when lc is two weeks in. test it on agar. if its clean, u have made a pre made sterile stringe, similar to what an edible vendor would send u. the trick is to get as much myc in ONE suck up as u can. that way u only open the lc jar twice, once to inoculate with a wedge, twice to suck up the lc with the needle. failure is still an option in any case.
-------------------- A few thoughts on cultivation MICROBIAL HUSBANDRY!!!! The whole is greater than the sum of its parts
|
tombosley8
Full on... Bossley Baggins



Registered: 10/14/13
Posts: 3,660
Last seen: 7 months, 29 days
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: blindingleaf]
#19666420 - 03/08/14 05:36 AM (9 years, 10 months ago) |
|
|
wow this is the info i've been looking for i've always feared the lc and the stigma around it but if I could make a syringe with one little wedge that'd be awesome. Can you store this at room temp or fridge for long durations?. or am i not seeing something. Yes syringe inoc seems very easy compared to agar but what are the draw backs. Just more chance of contam I guess because of the TWO times you need to open jar but seems better than exposing agar for every jar to inoc and maybe this is utilizing the agar for more quantity?
--------------------
|
blindingleaf
blue collar underworld



Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: tombosley8]
#19666440 - 03/08/14 05:47 AM (9 years, 10 months ago) |
|
|
it does have a lot of stigma. mainly b/c its an older flash in the pan style of cultivation, at least here in the forums. most ppl, when u read those old teks, are using either spore solution or clone tissue. u cannot see a contam on either of those before inoculation ur LC solution. if u start ur LC with agar, u can see a contam (most likely). i still do agar to grain 95% of the time. i ONLY use lc if I'm doing filter bags of grains, b/c i don't trust myself in an SAB unfolding the bag to dump qt of grain in. some people drink less coffee than me tho, so probably have steadier hands and g2g with filter bags are no problem for them. wish i could, g2g is faster than lc.
-------------------- A few thoughts on cultivation MICROBIAL HUSBANDRY!!!! The whole is greater than the sum of its parts
|
Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,808
Loc: Canada
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: blindingleaf]
#19666727 - 03/08/14 08:40 AM (9 years, 10 months ago) |
|
|
I gotta agree that the best way to inoculate a LC is a clean agar wedge. MS syringes are almost never clean and using one to inoculate a LC is a huge contam vector.
|
RandomFX
protege



Registered: 12/02/13
Posts: 1,015
Loc: North-East, USA
Last seen: 5 years, 1 month
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: tombosley8]
#19668180 - 03/08/14 03:39 PM (9 years, 10 months ago) |
|
|
Quote:
tombosley8 said: wow this is the info i've been looking for i've always feared the lc and the stigma around it but if I could make a syringe with one little wedge that'd be awesome. Can you store this at room temp or fridge for long durations?. or am i not seeing something. Yes syringe inoc seems very easy compared to agar but what are the draw backs. Just more chance of contam I guess because of the TWO times you need to open jar but seems better than exposing agar for every jar to inoc and maybe this is utilizing the agar for more quantity?
Read the urls I provided. it is why I provided them. There are more layers of contamination with liquid culture. first off I think when hyphae is young it is far more susceptible to contamination and has less defense to fight it off (Now it's defense may even be only something as simple as the hartig net which may hang up contamination particles for all I know and not allow them conditions to fruit or thrive. or as has been mentioned the mycelium may lay down either chemicals of it's own, or remove all the yummy nutrients and leave the area barren in a burn the fields type of defense too....I do not know, haven't followed up on it. but young hyphae is evidently more susceptible and in liquid culture, if it has nutrients that move around, plainly one could see how after being put into a spawn or other thing it could easily be susceptible also. Where as a agar wedge, is developed. and then it spreads from sort of a 'base of operations and safety', and is able to consistently reinforce itself and thus maybe fight its way through. furthermore, if you are using a nutrient rich liquid, you are going to be feeding the nasties also...they love wet and nutrient rich. agar doesn't spread the moisture love all over the place. furthermore, the concentration of the mycelium is far higher on agar pure volume of a top layer of mycelium, and it's nutrients that it is using stay with it. a small square of mycelium hartig mat can have numerous layers upon layers of cells and hyphae strands. and many more supporting the ones that are there. it is true, in a liquid culture you can have tons of cells to duplicate, but on the top of colonized agar it is far more dense with way more cells. The liquid culture will probably have more 'strands' ie more of the strings will be cut up and smaller, but that only leaves them more open to attack, not less, I would think.
|
EastBayRay

Registered: 06/06/13
Posts: 746
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: bodhisatta]
#19669417 - 03/08/14 08:44 PM (9 years, 10 months ago) |
|
|
Quote:
bodhisatta said: Silicone injection ports are always a risk. The best you can do is wipe them with alcohol which is only a sanitizer not a sterilizer so you always have a small chance of pushing contams through no matter what. Same reason why no one wipes with alcohol after flaming it's pointless. Alcohol than flame, You can't flame a silicone injection port so you're always trying your luck and every single time you use a LC you have to test it.
That's why you apply enough High Temp RTV silicone so that you can push a RED HOT needle through it and when you pull the needle out it will seal completely.
|
bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: EastBayRay]
#19669462 - 03/08/14 08:51 PM (9 years, 10 months ago) |
|
|
all silicone is high temp. RTV just means it will cure without air "room temp vulcanizing" The Red RTV us for automotive use and has some additives for corrosion resistance but it's got the same heat properties that any 100% silicone has including oven mits made from silicone etc...
besides I seriously doubt the needle has enough thermal mass and time to kill everything just from being red hot, the red hot kills anything on the needle.
Edited by Trusted cuItivator (03/08/14 08:52 PM)
|
EastBayRay

Registered: 06/06/13
Posts: 746
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: bodhisatta]
#19680400 - 03/11/14 11:10 AM (9 years, 10 months ago) |
|
|
Quote:
bodhisatta said: all silicone is high temp. RTV just means it will cure without air "room temp vulcanizing" The Red RTV us for automotive use and has some additives for corrosion resistance but it's got the same heat properties that any 100% silicone has including oven mits made from silicone etc...
besides I seriously doubt the needle has enough thermal mass and time to kill everything just from being red hot, the red hot kills anything on the needle.
Well, it's worked for me for years. Try it for yourself using proper sterile technique.
|
bodhisatta 
Smurf real estate agent



Registered: 04/30/13
Posts: 61,889
Loc: Milky way
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: EastBayRay]
#19680444 - 03/11/14 11:28 AM (9 years, 10 months ago) |
|
|
I have great sterile technique, that's why I do things the right way with agar and grain transfers rather than bullshit needle pokes. I got past that stuff after like a month of dicking around with that noob bs. 
Those techniques like SHIPs etc.. make this hobby foolproof so that anyone who can flush a toilet can have a moderate to good level of success with a low amount of failures.
Edited by Trusted cuItivator (03/11/14 11:29 AM)
|
EastBayRay

Registered: 06/06/13
Posts: 746
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: bodhisatta]
#19683135 - 03/11/14 09:30 PM (9 years, 10 months ago) |
|
|
Quote:
bodhisatta said: I have great sterile technique, that's why I do things the right way with agar and grain transfers rather than bullshit needle pokes. I got past that stuff after like a month of dicking around with that noob bs. 
Those techniques like SHIPs etc.. make this hobby foolproof so that anyone who can flush a toilet can have a moderate to good level of success with a low amount of failures.
I wasn't even insinuating you use improper technique or that you aren't experienced. Don't get all butt hurt, Jesus.
|
kotter


Registered: 01/15/11
Posts: 210
Last seen: 4 years, 7 months
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: Mrcloudy]
#19712539 - 03/18/14 10:30 AM (9 years, 10 months ago) |
|
|
Mrcloudy: Try transfering some of that culture to a thick layer of agar or some grain inside of a half pint mason jar with a filter patch and forget about it for a while in a cool location. Americanum tends to fruit small and fairly fast in that situation. It would be an easy way to tell if its right. For me, whispy branching has been common & Hericium species cultures enjoy being stored in a refrigerator -- even when forgotten for more than a year. Not too long ago I found a corolloides I'd somehow overlooked from late in 2012 and its doing great on grain and more recently on sawdust.
|
Mrcloudy
Stranger than you.



Registered: 10/01/13
Posts: 2,889
Loc: Northeast US
Last seen: 3 months, 18 days
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: kotter]
#19712946 - 03/18/14 12:13 PM (9 years, 10 months ago) |
|
|
Quote:
kotter said: Mrcloudy: Try transfering some of that culture to a thick layer of agar or some grain inside of a half pint mason jar with a filter patch and forget about it for a while in a cool location. Americanum tends to fruit small and fairly fast in that situation. It would be an easy way to tell if its right. For me, whispy branching has been common & Hericium species cultures enjoy being stored in a refrigerator -- even when forgotten for more than a year. Not too long ago I found a corolloides I'd somehow overlooked from late in 2012 and its doing great on grain and more recently on sawdust.
That is encouraging , thank you. I will definitely try it out.
--------------------
10 different Ganoderma species from across the USA AMU MrCloudys guide to North American GanodermaUpdated A rough guide to North American Ganoderma species, with an emphasis on the laccate species.
|
kotter


Registered: 01/15/11
Posts: 210
Last seen: 4 years, 7 months
|
Re: Liquid Culture De-Mystified: Ghia Style [Re: Mrcloudy]
#19714197 - 03/18/14 04:15 PM (9 years, 10 months ago) |
|
|
I wish I had a better image but this one was chopped up a bit for propagation. Despite that it will probably give a better idea than my words. All three hericium species growing here seem to be able to do this. A bit of tin foil for sake of spore collection is blocking much of the fruiting body that formed. What is growing on agar in this jar was originally transferred there from liquid culture growing from a wild fragment.



This is one I have some ID questions about I had posted earlier elsewhere here (#18933228). As soon as I have those spores I can complete that ID question appropriately. It grows up to resemble americanum more than erinaceus but if so should not be growing wild here. I thought I'd let my americanum escape but tracking back through my records showed that to be wrong.
Edited by kotter (03/18/14 04:28 PM)
|
|