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InvisibleBlueDruid
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Registered: 06/27/06
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Some questions on Agar
    #8093260 - 03/02/08 10:46 AM (15 years, 10 months ago)

Hi,

I've got a few questions on Agar since my last attempt didn't work out too well I wanted to clear up a few points to improve my chances next time.

1) I'll be using glass petris & I wonder what's the problem with poring the agar onto the perti rather then sterilising rather than sterilising the agar in a jar & the petris then pouring? I've seen comments against prepouring here before but no explanation as to why its a bad thing.


2) What are the chances of success (uncontaminated plates) when inoculating agar from a spore syringe rather than a print? I've got a few spore syringes (homemade) but I'm running out of prints and I'd rather not use another one as I want to use the strain/variety I've got in the syringes.

3) Why didn't culture syringes I used last time develop on nutrient agar? I was using a Shiitake cluture syringe but no mycellium developed, only left orange blobs on the plate.

4) If adding h202 to agar (when not using spores) is it necessary/worthwhile to sterilise the h202 along with the pippete? As I've got h202 in a little pippete dropper bottle and it'd be easy to sterilise it all rather than leaving the bottle out?

5) On a related issue when making master culture slants RR recommends using wooden tounge depressers in the test tubes, even when not for woodloving mushrooms. Would any type of wood do? as I don't have tounge depressers but I could easly shave a sliver of wood off some pine planks I've got, or some other type of wood.

6) When inoculating grain with mycellium on agar are any problems likely to arise if the agar contains multiple strains/sectors of mycellium? I ask as I'm on holiday soon and I want to inoculate grain jars before I go so they're ready when i get back and I won't have time to isolate individual strains.

Thanks


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Invisibledumbfounded1600
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Registered: 07/29/07
Posts: 2,624
Re: Some questions on Agar [Re: BlueDruid]
    #8093812 - 03/02/08 01:57 PM (15 years, 10 months ago)

1) Because it'll fuck up the water content and won't sterilize properly. Without a rack it can be a huge mess.
2) There hydrated and better off. Will start to germinate right away.
3) Contaminant?
4) H202 is junk. Fuck That Shit. IMHO
5) Sterilize it first.
6) Secter out the strain you want and use that. Read this

Quote:

Anno, Agar good thoughts.
I am starting my Agar experiments next week. When Isolating multi-spore cultures into single sub-strains..Explain that process and how it relates to they "aging" of myc...
Lets say you have 10 sub-strains that show up in your master dish. Simple isolation techniques give you (lets say in a perfect world) 10 sub-strain "master" dishes. From those ten, you chose two that you find to be superb, and create a few copies of each for inoculations. You discard or put into storage the remaining eight. You made the copies so you can experiment with your isolated sub-strain without damaging the original
Now, in terms of generation that was explained by agar...by the time you get to inoculation the myc is 4 generations old. Follow me:
Master (1st gen)
10 sub-strain dishes (2cnd)
Selected dish multiplied out into (3rd)
Inoculated Substrate (4th)
Now in terms of age explained by Anno, your inoculated substrate is the same generation, however it has lost some of it's youth/vigor.
Agar also explained the transfer of myc to be comparable to that of changing a baby from one womb to the next. I am in no way challenging your knowledge of the subject, however I do disagree with the analogy. We are talking about isolating and transferring the mold from one sterile (consistent) environment to the next. Scientists can't even create an artificial environment to "grow" a fetus...that says to me we are talking about apples and oranges when comparing the two...
Now. As a side note, I know I started that thread the other day talking about grains, just as this one is. I could see there being a closer comparison between grains and a womb due to the increased complexity of its nature as compared to the simplicity of agar. However, it would seem that both Agar's and Anno's answers apply to agar...hence my questions...
That meens there is a tradeoff between substrain isolation (because of the loss of vigor through age) and just innoculating from the master dish. On one hand you have selected a superior myc, but now its lost some vigor. On the other hand, you have a mix of myc, but it is all young. What gives?
-Agar




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Invisibletahoe
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Re: Some questions on Agar [Re: BlueDruid]
    #8093852 - 03/02/08 02:14 PM (15 years, 10 months ago)

1. It will work, give it a shot and tell us how messy it is.
2.if the syringe is clean the success is good. Only use a 1/4th drop of ss. You do not want liquid on your plates. It will swirl around and pick up any possible contams and spread them around
3.syringe was probably dead.
4.h202 has zero use in agar. never add anything that isnt sterilized to agar afterwards. H202 will not survive pc'ing
5.pine would, popsicle sticks will work fine
6. multispore/multistrain is best for the quick inoculation. If using the plate that has the spores on it try to use a section that is far from the point of inoculation.
I never use the #1 spore plates. I usually transfer 2 times away. It is best to be away from the possibly dirty spores. Some contams will ride on the mycelium and not show their face. This is one reason why you flip agar tranfers over when putting them to a new plate.
I usually use my third petri away from spores as my master even if it is still sectoring. I only grow outdoor stuff so I want a lot of diversity to insure a good yeild. I could get more, bigger, better looking mushrooms if I isolated but since I am allowing nature to do the work it could be years of failure before I got what I wanted.
With indoor mushrooms you cna grow out an isolate in a few weeks and be able to tell if it is a good one or not


--------------------
Stop experimenting half way through your first grow. Grow it to maturity, watch it, learn from it. Do this a few times then experiment with different ideas and figure out what works best for you.


My Legacy
https://www.shroomery.org/forums/showflat.php/Number/22140987#22140987

Teh=The
I need to proofread


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InvisibleBlueDruid
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Re: Some questions on Agar [Re: tahoe]
    #8101224 - 03/04/08 11:33 AM (15 years, 10 months ago)

Thanks guys,

I don't reckon the orange blobs on the agar are contamination as the culture syringe is orange and all plates from that syringe were affected. How long does it take for a culture syringe to die as it may well be that?


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InvisibleFooMan
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Re: Some questions on Agar [Re: BlueDruid]
    #8101344 - 03/04/08 12:12 PM (15 years, 10 months ago)

You can definitely sterilize your petri dishes with agar in them, but you need to be using a sterilizer, not a regular PC. Most PC's that use a weight to regulate pressure don't create a good enough vacuum to ensure that a contam won't get sucked into your agar as the PC cools. The sputtering of the weight also causes the agar to boil over in the plates, which creates the mess. You would need a rack to keep the dishes away from the boiling water, which can splash up into them. Petri dishes that are PC'd in a sterilizer need to be COMPLETELY cool before you move or open the sterilizer. You want the agar hardened and the vacuum from the steam gone. Once completely cooled, you can remove the plates and wrap them in parafilm, glad wrap, or micropore tape.

How can you make your PC a sterilzer? Well, if you have an All American PC, you can swap out the vent tube that the weight rests on for a toggle valve like this:



With one of these valves, you get ZERO steam noise and can PC up to 20psi fairly safely. The downside is that it's so quiet you can easily forget that your PC is going.

I just PC'd these dishes last night using the same method:



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

Quick WBS Prep


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InvisibleBlueDruid
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Posts: 807
Re: Some questions on Agar [Re: FooMan]
    #8101355 - 03/04/08 12:16 PM (15 years, 10 months ago)

That's cool man, I actually do have a steriliser, I think it's a converted pc. I've already done the agar in a jar now but I'll give it a try in petris some other time.


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OfflineSgJm
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Registered: 03/18/08
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Last seen: 15 years, 10 months
Re: Some questions on Agar [Re: BlueDruid]
    #8163680 - 03/18/08 07:28 PM (15 years, 10 months ago)

#3 My bet, the orange drops are iron rust from the needle.


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