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OfflineEreignis
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Registered: 07/06/06
Posts: 33
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5 failed liquid culture attempts
    #5967059 - 08/15/06 06:13 PM (17 years, 5 months ago)

First of all thanks to everyone who contributes advice & teks here.  I'm 75% through harvesting my first flush on my first batch of mushrooms and it's about 450 grams wet weight, so I'm very happy!  But, I've been trying to make a liquid culture from the fruit bodies of some of the mushrooms I've grown using the "newbie friendly guide to LC" which someone posted.  First I tried making two jars containing a solution of 1tblspoon honey and ~200ml water in a half pint jar.  I inoculated both of those with spores and got no germination after 10 days, so i threw them out to try again.  The second time, I made three jars with 5ml of a different kind of honey to 250ml of water, pressure cooked it for 15 minutes, injected a small piece of a fruiting body into two of them, and spores into one of them.  After three days, I see absolutely no signs of growth, even in the ones with mycelium in them.  It seems like I should have seen growth by now.  I have been aerating them, the hole is covered with micropore tape and I shake it to get some air exchange every day.  They've been kept at room temperature, about 75 degrees on average.  I just read in the wiki that you're supposed to keep them in a dark place, but I haven't been doing that.  Could that be my problem?  Soon I'll have to start thinking about taking a spore print if I want to have a second crack at this.  Advice appreciated :wink:


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Reason tatters
The forces tear loose from the axis
Searchlight casting
For faults in the clouds of delusion

Shall we go, you and I, while we can? Through the transitive nightfall of diamonds


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Invisiblecreamcorn
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Registered: 03/13/06
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Re: 5 failed liquid culture attempts [Re: Ereignis]
    #5967087 - 08/15/06 06:26 PM (17 years, 5 months ago)

when you say you shake them... are you literally shaking them?

you don't want the tape to get wet, ever. doing so is an invitation for bacteria to colonize through the tape and into your culture. bacteria will quickly grow in the LC. its an invisible invader sometimes, as it can be hard to see, and unfortunately, will prevent mycelium from ever growing.

that's my tek you're referring to... and maybe i didn't explain it well enough, but jars are half full or even less so so that you can swirl them around to agitate, without getting the tape wet. even then, swirling should be done gently and cautiously.

try one that is only half full, and don't disturb it at all. LC's dont need very much gas exchange in the first place, water can hold a lot of dissolved gas. the darkness doesn't matter one bit.

one other consideration, is the water you're using. i've had poor results using tap water, possibly due to chemicals its treated with. try preparing a culture with spring water or distilled water if you haven't done so yet.

and by the way, i made it really clear to measure out your honey by weight, and use no more than 4%. since it seems you're unable or unwilling to do it that way, a tip: honey weighs roughly 1.4 grams per ml. a tablespoon, as 14.7ml. so you were putting a little over 20g of honey in a 200ml solution, that's 10%. those were destined to fail from the start. your 5ml of honey to 250ml water, is within the correct range.


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OfflineEreignis
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Registered: 07/06/06
Posts: 33
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Last seen: 16 years, 10 months
Re: 5 failed liquid culture attempts [Re: creamcorn]
    #5967145 - 08/15/06 06:45 PM (17 years, 5 months ago)

Yes, it was after reading your tek that I realized I had put too much honey in the first jars.  The second batch of jars was done to your specifications, as you can see :wink:.  As for bacterial contaminants, it certainly is a possibility.  I haven't been shaking the jars so much as swirling them, but nevertheless I think at least one drop of water has gotten onto the underside of the micropore tape in each case.  I did treat the tape with lysol on both sides and let it dry before applying it to the jars, so I imagined that bacterial contamination would be less of an issue.  I've been using water run through a brita, but next time I'll use distilled water, less of it, and be more careful about contamination.  Thanks for replying.


--------------------
Reason tatters
The forces tear loose from the axis
Searchlight casting
For faults in the clouds of delusion

Shall we go, you and I, while we can? Through the transitive nightfall of diamonds


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Invisiblecreamcorn
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Registered: 03/13/06
Posts: 2,962
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Re: 5 failed liquid culture attempts [Re: Ereignis]
    #5967173 - 08/15/06 06:53 PM (17 years, 5 months ago)

that's why i swab the lid and hole with alcohol, then stick the tape on while still wet, the tape soaks it up, is "cleaned" in the process, and will stick once dry...

bacteria are small little buggers and can get through the tape.  they can get through most filter material out there, but there's a catch - they need moisture to survive, to reproduce and move around.  as long as the tape (or any type of filter material on any jar) stays dry, its not an issue.

if using a half full half pint jar doesn't seem like enough LC for your needs, use a bigger jar. :smile:  you can reuse pickle jars, spaghetti sauce jars, etc...

good luck... LCs can be a little frustrating til you work out the subtle details, but once you do they're pretty easy and convenient to work with :thumbup:


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InvisibleStinkFist13
stranger...kinda
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Registered: 05/30/06
Posts: 127
Re: 5 failed liquid culture attempts [Re: creamcorn]
    #5967470 - 08/15/06 08:20 PM (17 years, 5 months ago)

creamcorn is right, pretty much nuff said, i have had the problem creamcorn explained about the microtape bacterial growth.

i put foil or the tops of my lc's and it works fine, mark were the hole is if you try this. it doesnt filter air but it doesnt seal it air tight or leave it exposed to the open air. lc's dont need to be in dark to expand, mine are in light all day, not direct sunlight tho.

i have trouble with lc's alot too but when you get one going healthy its cool.


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2NO3- + 10e- + 12H+ → N2 + 6H2O
figure it out...i bet yall seen it once or twice


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OfflineTerillius
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Registered: 07/21/06
Posts: 1,301
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Last seen: 3 years, 11 days
Re: 5 failed liquid culture attempts [Re: creamcorn]
    #5967625 - 08/15/06 09:04 PM (17 years, 5 months ago)

I'm glad you mentioned tap water. I suddenly realize that our water tastes like shit and you can smell the chlorine in the shower every time they do their "treatement". We have decided that "treatment" is actually the half-witted maintenance man dumping good old bleach in the water main uptown.

"Durgh, how much did we say?"
"I don't 'member Earl, 5 gallons?"
"Better do 10, ya'know, to be sure."
"Kay"


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