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Invisiblecypherpunk
Stranger with candy


Registered: 10/11/09
Posts: 6
Help! Slow myc growth in PDA
    #11226512 - 10/11/09 12:56 PM (14 years, 4 months ago)

Hello to you all! I've been having some problems with my first grow recently and I was wondering if the shroomery.org inhabitants could give me some insight.

I ordered a syringe of koh samui cubensis and decided to grow it first on potato agar in order to isolate favorable rhyzomorphic mycelium for use with a rye grain substrate.
I made 6 petri dishes using sufficient sterile technique (pressure cooker, sterilizing a secluded work environment, glove box, flaming the needle, washed lab coat, sterile gloves, face mask, etc), allowed them to settle and cool for two days, injected some syringe solution (though I used a bit many CCs for the dishes; more than I probably should have. Beginner mistake ^_^;), taped them closed, covered with plastic cling wrap and set the dishes in an incubator @ 86*F total darkness to grow.

It's been about 3 weeks since starting them and the mycelium growth is sooo slow. There are faint, white, fuzzy circles (none of them larger than a dime) where the germination has occurred but certainly not a thick colonization of the agar like I've seen in pictures of other grows.
As far as I know I've given the spores an optimal environment, yet despite all my dedicated work and money they seem to not be cooperating. Checking the underside of the dishes I see little black dots in the center under some of the growth, which I believe to be spore clusters, but am not sure.

There is no visible discoloration, milky spots or sludge in the dishes to denote contamination on any of the six dishes I prepared; just slow growth. Can anyone help my poor mycelium?

Edited by cypherpunk (10/11/09 02:31 PM)

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Invisiblebadman
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Registered: 06/14/06
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Re: Help! Slow myc growth in PDA [Re: cypherpunk]
    #11226580 - 10/11/09 01:16 PM (14 years, 4 months ago)

Only transfer the white growth if it is mycelium. Cut small wedges from the old plate and move to sterile plates. Then wait and repeat when you see rhizos.

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Invisiblecypherpunk
Stranger with candy


Registered: 10/11/09
Posts: 6
Re: Help! Slow myc growth in PDA [Re: badman]
    #11226642 - 10/11/09 01:33 PM (14 years, 4 months ago)

Thank you for the reply. The myc seems to be monokaryotic still and its growth rate is very slow given the ideal incubator temperature. The areas of growth are very light compared to the thick, cottony growth and ropey strands of mature mycelium. The growth has remained light with little visible change for at least a week now.

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Invisiblebadman
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Re: Help! Slow myc growth in PDA [Re: cypherpunk]
    #11226760 - 10/11/09 01:56 PM (14 years, 4 months ago)

If the plate is wet it can slow shit down . I still suggest taking transfers because even if it is mono myc it will turn into di myc sooner of later, plus you cant tell mono and di apart with the naked eye. No change of growth for a week is never a good sign, IMO contamed agar.

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OfflineThyrax
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Registered: 01/03/08
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Loc: Montreal, Quebec
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Re: Help! Slow myc growth in PDA [Re: cypherpunk]
    #11226764 - 10/11/09 01:57 PM (14 years, 4 months ago)

Quote:

cypherpunk said:
Thank you for the reply. The myc seems to be monokaryotic still and its growth rate is very slow given the ideal incubator temperature. The areas of growth are very light compared to the thick, cottony growth and ropey strands of mature mycelium. The growth has remained light with little visible change for at least a week now.




you are using an incubator ? maybe your temp is just too high


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

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OfflineMigraine
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Registered: 09/21/09
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Loc: Alabama
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Re: Help! Slow myc growth in PDA [Re: badman]
    #11226780 - 10/11/09 02:01 PM (14 years, 4 months ago)

You don't say what your potato agar is composed of.

Three weeks seems like a long time.

Since you used spores - the white fuzz may be unmated Hyphae.

When two compatible Hyphae mate, they tend to grow faster then unmated Hyphae and usually present themself as rhyzomorphic growth.

Wait until you see sectors forming before transferring a sample to another agar dish.

As an alternative, you might let the dish grow out, thereby confirming no contams are present, and then dropping a wedge of the grown-out agar dish into different grain jars.  Grow from there as normal.

Take clones of the best mushrooms and do more agar work with them - now specializing on a specific stain that you know fruits well (isolating prior to fruiting from spore does NOT guarantee good fruits).

That is what I do - don't isolate until you know what your are getting.

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Invisiblecypherpunk
Stranger with candy


Registered: 10/11/09
Posts: 6
Re: Help! Slow myc growth in PDA [Re: Thyrax]
    #11226817 - 10/11/09 02:13 PM (14 years, 4 months ago)

My mistake, the agar is actually derived from seaweed. The agar used: http://www.nowfoods.com/Products/M004070.htm I'd be amazed if the agar in all six plates was contaminated. As I said I used very sterile techniques (everything short of having a flow hood) and a 100% contam rate for every plate is unlikely to me.

The temperature inside the incubator is at 86 Fahrenheit with only slight fluctuations (I keep the temp+humidity monitored constantly with a wireless remote sensor). In various growing guides I read that 86F is a good general incubator temperature, 10 degrees less for triggering fruiting.

Would it be simpler (and quicker) to inoculate my rye grain jar with the syringe, grow and fruit the mushrooms from the grain, take tissue samples from large, favorable mushrooms that grow and propagate those samples in agar for future grows?

Edited by cypherpunk (10/11/09 02:17 PM)

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Invisiblebadman
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Re: Help! Slow myc growth in PDA [Re: cypherpunk]
    #11226857 - 10/11/09 02:20 PM (14 years, 4 months ago)

drop the temps to 80F

You also need to add nutreints to the agar.
PDA is potato dextrose you did make PDA?

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Invisiblebadman
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Re: Help! Slow myc growth in PDA [Re: cypherpunk]
    #11226859 - 10/11/09 02:21 PM (14 years, 4 months ago)

You did add potato and dextrose to your agar yes??

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Invisiblecypherpunk
Stranger with candy


Registered: 10/11/09
Posts: 6
Re: Help! Slow myc growth in PDA [Re: badman]
    #11226903 - 10/11/09 02:28 PM (14 years, 4 months ago)

I did not. Now knowing this my guess is the mycelium growth is slow because of insufficient nutrients in the agar. I feel like the perfect ass right now -___- Please forgive my ignorance as I am new to mycology.

Edited by cypherpunk (10/11/09 02:40 PM)

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Invisiblebadman
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Re: Help! Slow myc growth in PDA [Re: cypherpunk]
    #11226975 - 10/11/09 02:38 PM (14 years, 4 months ago)

Yep theres the problem, school boy error lol

never mind just whip up some more and this will sort you right out.

http://www.shroomery.org/forums/showflat.php/Number/3077006

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Invisiblecypherpunk
Stranger with candy


Registered: 10/11/09
Posts: 6
Re: Help! Slow myc growth in PDA [Re: badman]
    #11227031 - 10/11/09 02:48 PM (14 years, 4 months ago)

Given what Migraine said I think my course of action from here on will be to inoculate the rye grain and go from there. I've already wasted a month because of my elementary mistake and it has me feeling like a failure :frown: Would I be able to take mycelium from a petri dish now and use it to colonize the grain; saving time since the germination of the spores is already complete?

If I were to order some pre-poured petri dishes could I take samples from my gimped agar plates and transfer them to the nutrient-enriched agar for culturing and isolating strains? If yes would I do better with PDA or tryptic soy agar?

Edited by cypherpunk (10/11/09 02:54 PM)

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Invisiblebadman
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Re: Help! Slow myc growth in PDA [Re: cypherpunk]
    #11227088 - 10/11/09 02:55 PM (14 years, 4 months ago)

youll be fine with PDA

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OfflineMigraine
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Registered: 09/21/09
Posts: 158
Loc: Alabama
Last seen: 3 months, 22 days
Re: Help! Slow myc growth in PDA [Re: cypherpunk]
    #11227137 - 10/11/09 03:00 PM (14 years, 4 months ago)

In a sterile environment, cut around the mycelium you have and drop in into your grain jar/bag.

Make sure you have some sort of gas exchange filter present.

Once you drop it in, shake the grain real well as this will tend to tear off some of the mycelium from the agar and help the growth process.

If the slowness is due to your agar mix, then getting it exposed to rye grain should kick it off.

Wait until jar is 30% colonized (white) and then shake hard again - this will redistribute the mycelium throughout the grain. If grain is in a jar - hit the jar against a tire - not your hand to shake and break up the grain!!!

Then wait until its 100% before moving to your next step.

I just dropped some agar wedges in several rye grain jars two days ago and I have spots of mycelium growth already.

Good Luck.

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Invisiblecypherpunk
Stranger with candy


Registered: 10/11/09
Posts: 6
Re: Help! Slow myc growth in PDA [Re: Migraine]
    #11227375 - 10/11/09 03:36 PM (14 years, 4 months ago)

Thanks to everyone for the help. I just cut a healthy section from a dish and transferred it over to the rye jar. Hopefully now the growth will speed up drastically with proper nutrition in addition to a sterile, warm environment :rolleyes:

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