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Rooster Cogburn
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Let's see what is inside of coir 1
#23542736 - 08/15/16 01:30 AM (7 years, 5 months ago) |
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This is an open experiment, I encourage anyone to try
I was having a conversation with Pasty in another thread about coir, specifically about how much life is actually in coir when we get it. The consensus seems to be that it's pretty damn clean and even heat treated before we get it. I'm skeptical about that assumption, however I do know for a FACT that coir performs well any way that it is prepared, be it just hydrated, pasteurized and treated with boiling water. It seems also the general consensus is that coir is so clean it is actually sterilized with the bucket method, and so depraved of nutrients that competitor molds and bacteria can't get a foot on it. Pasty said he wanted to do an experiment about the initial load there actually is in coir when we get it to put the subject to rest, so I was making agar tonight and thought what the hell, I will start one as well. Let's see if there is anything to germinate.
For this experiment I am using MEA

The coir being used is this, botanicare coir, I have used it for years and never had an issue with it, it's great and never has wierd debris.

This experiment will use samples from 4 different forms of coir: 1. Dry coir 2. Hydrated untreated coir 3. Pasteurized coir 4. Coir treated with boiling water
Here are the samples, the bag is pasteurized CVG


I am placing samples of each on dishes of agar and incubating them. I did this with a scalpel scooping the samples onto the dish and pushing it into the agar a bit.

Anyways, lets see how much shit is in there if you actually try to grow it and find out just why coir is the most efficient and forgiving substrate for cubensis cultivation.
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lukehighwalker710
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 I suggest adding PC sterilized coir to the list, and probably better to pastuerize some coir yourself for the experiment rather than pre-pastuerized vendor bags. Though, those could very well be another one on the list.
Either way, I'm on board to see how this turns out.
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bodhisatta 
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coir pith presses exert an enormous amount of pressure on the fiber that is mixed with a binding agent. the heat is produced from the pressing. compression = heat. it's how a diesel engine works, not to mention the friction forces from all those fibers getting pressed into such a small brick.
mycelium is able to fend for itself pretty well after the spawn step. the sterilization is only to remove all vegetative life. spores and endospores can survive if they're around but it's still called sterilization.
MEA is ok, for this test,
what you really should do is make sterile water of a known volume, and then add a coir chunk from the inside of the brick, have weights of the jar with the sterile water and then weigh it after adding the coir, then you'll know how much the coir weighed you added without weighing it and contaminating it.
then let it dissociate into the water.
take 1CC of homogeneous coir water and then do a pour plate and a spread plate.
you should use plate count agar.
you should have a control plate that you do the same techniques with just plain sterile water and carry out the exact same maneuvers.
ideally you would do the experiment twice and incubate the other half of the plates anaerobically
it's a good place to start, what you're doing. if you get any positives it would be grounds for a real investigation
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Rooster Cogburn
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That's my pasteurized coir, that is my preferred method of doing it personally. And your right I should add pc sterilized on there
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Hashed
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: bodhisatta]
#23542762 - 08/15/16 01:47 AM (7 years, 5 months ago) |
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Quote:
Trusted Cultivator said: coir pith presses exert an enormous amount of pressure on the fiber that is mixed with a binding agent. the heat is produced from the pressing. compression = heat. it's how a diesel engine works, not to mention the friction forces from all those fibers getting pressed into such a small brick.
mycelium is able to fend for itself pretty well after the spawn step. the sterilization is only to remove all vegetative life. spores and endospores can survive if they're around but it's still called sterilization.
MEA is ok, for this test,
what you really should do is make sterile water of a known volume, and then add a coir chunk from the inside of the brick, have weights of the jar with the sterile water and then weigh it after adding the coir, then you'll know how much the coir weighed you added without weighing it and contaminating it.
then let it dissociate into the water.
take 1CC of homogeneous coir water and then do a pour plate and a spread plate.
you should use plate count agar.
you should have a control plate that you do the same techniques with just plain sterile water and carry out the exact same maneuvers.
ideally you would do the experiment twice and incubate the other half of the plates anaerobically
it's a good place to start, what you're doing. if you get any positives it would be grounds for a real investigation
 Well I'm glad I posted this so we can discuss some different methods of going about this, you have some good ideas there.
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bodhisatta 
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then you can enumerate CFU/g of coir
say you use 100mL of water in a jar and sterilize it then weigh it and it weighs 300 grams we assume 100g is water. then add coir. you weigh it after adding coir and it's 310g. so we know 10g of coir in 100mL water.
we plate 1mL of coir water in a pour plate.
then if you get any colonies in the plate since you did a pour plate you can count the colonies.
then say you counted 10 colonies. you would have 10cfu per 1ml and 1ml has .1g coir since there's 10g in 100mL
so then you have a CFU count of 100 CFU/1g coir
then any one else who tests it can compare the numbers
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bodhisatta 
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Re: Let's see what is inside of coir [Re: bodhisatta]
#23542779 - 08/15/16 02:04 AM (7 years, 5 months ago) |
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these are pour plates of a badly infected beer. they're plated anaerobic since beer is obviously anaerobic. and the only spoilers of beer are hence anaerobic.
this was 1mL of finished bottled beer. there's too many colonies to count. it's easily over 500. let's just say 1000 CFU per mL
which means that 3785mL in a gal so there's at least 3.785 million bacterium in every gal. delicious. 354,800 in every 12 oz pour.
needless to say the QA plate was just for fun the beer was too gross to sip.
what you do though is pipette the 1mL sample into an empty dish then pour 47C agar over the top, just hot enough to still be liquid but not hot enough to kill any of the microorganisms. swirl the plate gently and let it harden. this way you get colonies forming in the agar as pinpoints and you can count them.
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: bodhisatta]
#23542834 - 08/15/16 02:54 AM (7 years, 5 months ago) |
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Dude, this is great info. I will definitely try this, sounds fun too. I just used the last of my MEA, what would you suggest a good recipe for this type of work if I was to go pick up ingredients tomorrow instead of ordering more premixed?
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invitro


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bodhisatta 
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Re: Let's see what is inside of coir [Re: invitro]
#23543432 - 08/15/16 10:04 AM (7 years, 5 months ago) |
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0.5% peptone 0.25% yeast extract 0.1% glucose 1.5% agar pH adjusted to neutral at 25 C.
That's plate counts recipe. Malt extract probably does plenty fine for us tho
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: bodhisatta]
#23544149 - 08/15/16 01:45 PM (7 years, 5 months ago) |
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Thanks, I have only used MEA since I started growing, I don't know why, just creature of habit and plus it's the main formula you can buy premixed. It will actually be pretty fun using a new recipe and seeing how my cultures react to it after being fed and stored on MEA for so long.
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blackout


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Worthwhile experiment 
Quote:
Rooster Cogburn said: And your right I should add pc sterilized on there
I thought the same at first, but then thought why bother, nothing will grow, this will be of no surprise or interest to anybody. Microwaving is another method people would have doubts about. I would also be interested in chemical sterilization methods like using peroxide or bleach, but this is not commonly done.
An experiment I would like to see is adding colonised grain from an isolate to coir treated in different ways and see how it performs. The problem with this is having them all equal hydration wise. I usually measure hydration levels by weighing after soaking stuff. However most people seem to find that after being heat treated that coir can take on more moisture.
I wonder if there is some way to try and get equal levels of "field capacity", rather than equal water hydration (which would be grams water per gram of dry coir). Maybe if the coir was put in a coffee french press and a weight put on top and pour off the liquid.
If actually growing with grains & coir I would be interested in seeing how the PC'd coir performs.
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bodhisatta 
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Re: Let's see what is inside of coir [Re: blackout]
#23544238 - 08/15/16 02:14 PM (7 years, 5 months ago) |
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PC sterilized acts as a control. If you get positive with that maybe your got a flaw somewhere
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Pastywhyte
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Re: Let's see what is inside of coir [Re: bodhisatta]
#23544923 - 08/15/16 06:20 PM (7 years, 5 months ago) |
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Well this saves me some trouble. Great stuff.
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: Pastywhyte]
#23544956 - 08/15/16 06:30 PM (7 years, 5 months ago) |
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I figure you have enough on your plate, ooh pun.
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Rooster Cogburn
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Ok guys, I have some action going on tonight in the bacteria department.
Let's look at these samples

First, here is the dry coir
 
And the hydrated
 
Treated with boiling water
 
And Pasteurized
 
Here are my control plates, and like you guys are saying pc sterilized coir would be a great control along with this.

Edited by Rooster Cogburn (08/15/16 09:42 PM)
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Pastywhyte
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Not bad. I wouldn't have suspected the dry to have as much as it does but it's really not even that bad really. No mold yet either.
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: Pastywhyte]
#23545515 - 08/15/16 09:58 PM (7 years, 5 months ago) |
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Yeah it's not bad at all, I expect tomorrow to be mold day if it's there or wants to germinate.
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Pastywhyte
Say hello to my little friend



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To be honest I would have expected a few more phenotypes to be present/obvious. I would love for bodhi to get that under a scope. Bet it's an endospore forming species.
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CosmoKramer
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Re: Let's see what is inside of coir [Re: Pastywhyte]
#23545673 - 08/15/16 11:10 PM (7 years, 5 months ago) |
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Cool experimental run! 24-48hrs more and they will show if present.
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bodhisatta 
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Re: Let's see what is inside of coir [Re: CosmoKramer] 1
#23545758 - 08/15/16 11:47 PM (7 years, 5 months ago) |
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I'm off third shift in a week forever. So I can actually do shit I'll try this too I think
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Supalemonhaze
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Re: Let's see what is inside of coir [Re: bodhisatta] 1
#23545980 - 08/16/16 04:26 AM (7 years, 5 months ago) |
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Yeah, definitely should have tried the PCed coir with this. As it sits, you can't be sure if it's your tools or sterile tek that's contaminating your samples. The controls you showed is simply a poured plate, not one that was used for transfers so in this case, it's only showing you that the agar you started off with was clean but nothing regarding anything that happened after.
It would be good to explain your method of obtaining the coir from the inside of the brick and your pre-cautions to make sure you do not contaminate it while getting the sample. Even with the PCed coir control, it wouldn't totally confirm that the dry sample was obtained with sterility, since you are going to be splitting your brick and taking a sample while the PCed sample is coming straight from a sterile jar.
This seems like a simple task but once you start thinking of the shit that can go wrong, you realise it's not as simple as you first thought it would be.
Quote:
Trusted Cultivator said: I'm off third shift in a week forever. So I can actually do shit I'll try this too I think
Now this I would be very interested in, I think the credibility of the user's capability is paramount in such an experiment . Can't really question someone who does this stuff for a living now, can you?
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mindbentempire



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Re: Let's see what is inside of coir [Re: Supalemonhaze]
#23546108 - 08/16/16 06:44 AM (7 years, 5 months ago) |
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Quote:
Supalemonhaze said: Yeah, definitely should have tried the PCed coir with this. As it sits, you can't be sure if it's your tools or sterile tek that's contaminating your samples. The controls you showed is simply a poured plate, not one that was used for transfers so in this case, it's only showing you that the agar you started off with was clean but nothing regarding anything that happened after.
It would be good to explain your method of obtaining the coir from the inside of the brick and your pre-cautions to make sure you do not contaminate it while getting the sample. Even with the PCed coir control, it wouldn't totally confirm that the dry sample was obtained with sterility, since you are going to be splitting your brick and taking a sample while the PCed sample is coming straight from a sterile jar.
This seems like a simple task but once you start thinking of the shit that can go wrong, you realise it's not as simple as you first thought it would be.
Quote:
Trusted Cultivator said: I'm off third shift in a week forever. So I can actually do shit I'll try this too I think
Now this I would be very interested in, I think the credibility of the user's capability is paramount in such an experiment . Can't really question someone who does this stuff for a living now, can you?
I agree, there can be lots of confounding variables that might skew the results here. But the OP is doing awesome work here. Any documented experiments will be good. Others can expand and fine tune based on the discussions this brings about.
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: Supalemonhaze]
#23546910 - 08/16/16 01:27 PM (7 years, 5 months ago) |
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Quote:
Supalemonhaze said: Yeah, definitely should have tried the PCed coir with this. As it sits, you can't be sure if it's your tools or sterile tek that's contaminating your samples. The controls you showed is simply a poured plate, not one that was used for transfers so in this case, it's only showing you that the agar you started off with was clean but nothing regarding anything that happened after.
It would be good to explain your method of obtaining the coir from the inside of the brick and your pre-cautions to make sure you do not contaminate it while getting the sample. Even with the PCed coir control, it wouldn't totally confirm that the dry sample was obtained with sterility, since you are going to be splitting your brick and taking a sample while the PCed sample is coming straight from a sterile jar.
This seems like a simple task but once you start thinking of the shit that can go wrong, you realise it's not as simple as you first thought it would be.
Quote:
Trusted Cultivator said: I'm off third shift in a week forever. So I can actually do shit I'll try this too I think
Now this I would be very interested in, I think the credibility of the user's capability is paramount in such an experiment . Can't really question someone who does this stuff for a living now, can you?
Thank you for that absolutely enlightening contribution to this discussion
The dry and hydrated samples were obtained by opening a fresh packaged 5kg brick and splitting it open all in the lab, the packaging was even cleaned well before bringing into the lab which had been scrubbed for hours ahead of time, not for this but for the agar session, and then transferred to a baggie with out touching it. Now haze, here is a trick for clean tools, you sterilize them. Now while doing work you flame sterilize the tools like your scalpel between transfers.
Haze, understand this is just the start, of course we are going to weed out the variables, and add sterilized coir. And thanks to Bohdi we are going to be taking the experiment to pour plates and start counting the CFU's. What's with the hatin' man? This is going to be both fun and informative. Never stop trying to learn Haze, that's what takes us places we never thought we would go and brings a certain sense of humbleness.
Edited by Rooster Cogburn (08/16/16 03:15 PM)
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Supalemonhaze
Spore syringe hater.



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Quote:
Rooster Cogburn said:
Now haze, here is a trick for clean tools, you sterilize them. Now while doing work you flame sterilize the tools like your scalpel between transfers.
Ah, so that's how the pros do it. 
Hating? Where is this hating?
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: Supalemonhaze]
#23548111 - 08/16/16 08:29 PM (7 years, 5 months ago) |
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Again, nice contribution to the subject
Grade A content, and as far as the hatin' goes maybe it's just the attitude, kind of unfriendly.
"Ah, so that's how the pros do it. "
What's up with that?
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Rooster Cogburn
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Alright guys I have some updates The only samples to get mold which in this case is cobweb is the dry and the hydrated, the hydrated plate filled up in less than 24 hours from not seeing anything, but I just noticed faint wisps of it's mycelium on the dry sample, I couldnt get a picture good enough to see it, but it's there and will probably be able to get a good picture of tomorrow.
Here is the hydrated sample, see the mold

And here is the dry sample

The pasteurized sample

And the bucket/boiling water treated sample

The hot water treated coir seems to have the fewest colonies out of all of them. Im going to see If I can get some better pics of the mold in few minutes
Edited by Rooster Cogburn (08/16/16 11:12 PM)
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Pastywhyte
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I doubt that is dactyllium. Probably a rhizopus sp. Still pretty interesting.
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: Pastywhyte]
#23548514 - 08/16/16 11:16 PM (7 years, 5 months ago) |
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I think your right, I just compared their pictures. Hoping to get better photos of it.
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Supalemonhaze
Spore syringe hater.



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Quote:
Rooster Cogburn said: Again, nice contribution to the subject
Grade A content, and as far as the hatin' goes maybe it's just the attitude, kind of unfriendly.
"Ah, so that's how the pros do it. "
What's up with that?
What's up with that? Really? How else do you imagine people sterilizing their tools? I mean, if you really can't recognize humour when you see it, it's no wonder I am "hating".
Hmm, nevermind, I just noticed your postcount. You still have to learn that on the shroomery, you have to leave the ego at the door.
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: Supalemonhaze]
#23548720 - 08/17/16 01:25 AM (7 years, 5 months ago) |
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Rooster Cogburn
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Where am I giving off the impression I have a big ego
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bodhisatta 
Smurf real estate agent


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lukehighwalker710
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Re: Let's see what is inside of coir [Re: bodhisatta] 1
#23548743 - 08/17/16 01:43 AM (7 years, 5 months ago) |
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--------------------
Highwalker
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Rooster Cogburn
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On it, gorilla glue , ho!
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bodhisatta 
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That's what's been floating around my part of the woods too.
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Supalemonhaze
Spore syringe hater.



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Because every reply I posted in this thread seemed to rub you the wrong way, even the one where it's clearly a joke.
Quote:
What's with the hatin' man?
Quote:
Supalemonhaze said:
Quote:
Rooster Cogburn said:
Now haze, here is a trick for clean tools, you sterilize them. Now while doing work you flame sterilize the tools like your scalpel between transfers.
Ah, so that's how the pros do it. 
Quote:
Rooster Cogburn said: Again, nice contribution to the subject
Grade A content, and as far as the hatin' goes maybe it's just the attitude, kind of unfriendly.
"Ah, so that's how the pros do it. "
What's up with that?

But OK.... I guess.
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: Supalemonhaze]
#23548806 - 08/17/16 02:32 AM (7 years, 5 months ago) |
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This was stupid shit, let's move on from it, take a with us while we are at it and tell us what you think is going on with the plates. I love this place, I'm not planning on leaving anytime soon so let's just get the fuck along
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: bodhisatta]
#23548814 - 08/17/16 02:37 AM (7 years, 5 months ago) |
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Quote:
Trusted Cultivator said: That's what's been floating around my part of the woods too.
It's almost ALL of what's been around here, I like it though.
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Mad Season
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Registered: 09/16/12
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Quote:
Rooster Cogburn said: Alright guys I have some updates The only samples to get mold which in this case is cobweb is the dry and the hydrated, the hydrated plate filled up in less than 24 hours from not seeing anything, but I just noticed faint wisps of it's mycelium on the dry sample, I couldnt get a picture good enough to see it, but it's there and will probably be able to get a good picture of tomorrow.
Here is the hydrated sample, see the mold

And here is the dry sample

The pasteurized sample

And the bucket/boiling water treated sample

The hot water treated coir seems to have the fewest colonies out of all of them. Im going to see If I can get some better pics of the mold in few minutes
pretty cool shit. It's cool that the hydrated coir is developing the most shit, and even a mold
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Kenetic
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Re: Let's see what is inside of coir [Re: bodhisatta]
#23550239 - 08/17/16 03:18 PM (7 years, 5 months ago) |
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Quote:
Trusted Cultivator said: then you can enumerate CFU/g of coir
say you use 100mL of water in a jar and sterilize it then weigh it and it weighs 300 grams we assume 100g is water. then add coir. you weigh it after adding coir and it's 310g. so we know 10g of coir in 100mL water.
we plate 1mL of coir water in a pour plate.
then if you get any colonies in the plate since you did a pour plate you can count the colonies.
then say you counted 10 colonies. you would have 10cfu per 1ml and 1ml has .1g coir since there's 10g in 100mL
CFU calulation: #of CFU/(dilution plated*dilution factor) = #of CFU/gram
CFU = colony-forming unit, and only applies to viable cells.
It has a lot of reasons to be useful in mycology, like knowing the severity of a contaminate problem before and after a period of time, whether or not your anti-contaminate countermeasures are working or getting worse, and making beer. Which is really useful in mycology imo. Cheers!
-------------------- Todo Cambia    DMT said: Everyone know's me, they just don't know it yet
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Rooster Cogburn
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Re: Let's see what is inside of coir [Re: Kenetic]
#23550532 - 08/17/16 04:51 PM (7 years, 5 months ago) |
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Thanks Mad.
Hey Pasty, the mold is rhizopus for sure, the ends of the myc turned green today as well the dry sample took off with rhizopus like crazy. I'll get some pictures later.
Edited by Rooster Cogburn (08/17/16 04:58 PM)
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ComebackKid
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Frig this makes me glad I learned about the bucket tek before just hydrating and givin'er which was my original plan...
--------------------
Substrate surface conditions / Monotub prep and care
Look around you... Everything you see exists inside the mind. Consciousness, the awareness that is experiencing this mind, is peering in from outside the universe. Our individual experiences are all part of the universe's experience of itself
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Re: Let's see what is inside of coir [Re: ComebackKid]
#23551116 - 08/17/16 08:07 PM (7 years, 5 months ago) |
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Quote:
ComebackKid said: Frig this makes me glad I learned about the bucket tek before just hydrating and givin'er which was my original plan...
Except it won't hurt to hydrate and giver. Coir is a poor food source for bacteria and mold can't germinate on it. If not for the agar media those CFU'S wouldn't stand a chance to get a foothold. Only thing that would suffer would be yield.
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ComebackKid
Multispore Enthusiast



Registered: 05/27/16
Posts: 3,951
Loc: ked in the trunk of a car
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Re: Let's see what is inside of coir [Re: Pastywhyte]
#23552329 - 08/18/16 06:05 AM (7 years, 5 months ago) |
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Science bitch! Okay that makes sense. Nutes would usually already be taken up in the spawn
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Substrate surface conditions / Monotub prep and care
Look around you... Everything you see exists inside the mind. Consciousness, the awareness that is experiencing this mind, is peering in from outside the universe. Our individual experiences are all part of the universe's experience of itself
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