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OfflineDinkinFlicka84
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Website Multispore to agar did not work.
    #22089552 - 08/13/15 11:09 PM (8 years, 5 months ago)

I'm fairly bummed out with the outcome of my first MS to agar. I've done agar a few times before with really great results. I'm not certain if I did it correctly, so please advise.

There were 3 strains that I used. I used  4 sterile plastic Petri dishes, which each had 3 walled-off sectinos. Therefore, I have 12 difference sections I inoculated into the agar medium. Everything looked good when I initially inoculated them. The agar was clean, I sprayed the SAB's inside with soapy water after I wiped the walls and floor with 70% isopropyl alcohol.

Flame sterilized the tip every time between inoculations. After shaking the syringe vigorously I dropped 2 drops in each sector of the  plate. One on each side, which is what I read to do.

It's now 3 days later about half of the mycelium growths have green spots.



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InvisibleGrey
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Re: Website Multispore to agar did not work. [Re: DinkinFlicka84]
    #22089603 - 08/13/15 11:31 PM (8 years, 5 months ago)

Dirty syringe most likely. Have you used it before?


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OfflineDinkinFlicka84
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Re: Website Multispore to agar did not work. [Re: Grey]
    #22089625 - 08/13/15 11:40 PM (8 years, 5 months ago)

I've used agar a number of times with great results.

So, I was going to put all 3 strains straight to agar before I inoculated any jars. Like I said, I flame sterilized each drop was put onto agar. I'm going to try again and wipe down the entire syringe with an alcohol-soaked paper towel and see if that makes a difference.

As you can see in the last picture, the Burma has one large contaminated drop and then a few small non-contaminated spots.

Would you think that it's more likely that the syringes were bad or that my technique wasn't sterile enough? I used a SAB, wiped walls with alcohol, wiped each individual plate down with an alcohol-soaked paper towel and even sprayed the walls with soapy water. Then I waited 20 minutes before I started.

I don't want to go inoculating 22 jars if there's a possibility that the strain syringes are bacteria ridden.

Any thoughts?


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Offlinegay mushroom
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Re: Website Multispore to agar did not work. [Re: DinkinFlicka84]
    #22089641 - 08/13/15 11:52 PM (8 years, 5 months ago)

Omg you need to take a chill pill budday. Just transfer when you see clean growth. The gross nasty green seems to take its time. Let the other white stuff grow out. Wait for noticeable rizos as well as any green  :joint: :lol:


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OfflineDinkinFlicka84
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Re: Website Multispore to agar did not work. [Re: gay mushroom]
    #22089668 - 08/14/15 12:03 AM (8 years, 5 months ago)

I'm going to go ahead and inoculate my jars with each of the strains regardless if there's a chance of contamination. I have a big trip that's coming up and I wanted to have these different strains ready for such a experience, which is why I'm not willing to wait on my agar. I'll still be using the agar, but I'm going to start inoculating tonight.

I had a huge issue in the past where ALL of my subs would contaminate within a week or 2 of being hit with FAE. I realized after 6 months of relentless attempts that it was my moisture content. I switched from doing LC's to G2G and that solves 99% of my problems.

So, now I'm faced with doing a MS syringe straight from a sponsor. I do dry my WBS much more than used to, which also fucked up my water content in the jars, causing bacteria. I've read a ton of posts on how much to inoculate with, but I don't want to offset my moisture in the jars, but it would be nice not having to wait 2-3 weeks for the jars to fully colonize, which happens when I would use 1cc/jar. Some people use 2cc's. Which amounts have you had the best luck with?


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Re: Website Multispore to agar did not work. [Re: gay mushroom]
    #22089678 - 08/14/15 12:07 AM (8 years, 5 months ago)

Quote:

gay mushroom said:
Omg you need to take a chill pill budday. Just transfer when you see clean growth. The gross nasty green seems to take its time. Let the other white stuff grow out. Wait for noticeable rizos as well as any green  :joint: :lol:



:canadian:


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OfflineDinkinFlicka84
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Re: Website Multispore to agar did not work. [Re: cronicr]
    #22089782 - 08/14/15 01:21 AM (8 years, 5 months ago)

Can someone explain to me what exactly causes the little green mold spt myc. What I assume is the reason is as follows. Please share some knowledge... Thanks.

1. The sponsor syringes could all 3 be "dirty" w/ bacteria wich is why the green mold spot is int he center of myc.

2. There are mold spores in the air, even in a fairly secure SAB. These spores could've come from my t-shirt or floated in from wherever and landed on the myc circle.


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Re: Website Multispore to agar did not work. [Re: DinkinFlicka84]
    #22089794 - 08/14/15 01:29 AM (8 years, 5 months ago)

ore then likely number 2....that's not the right myc on the outside that is indeed the molds mycelium


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OfflineDinkinFlicka84
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Re: Website Multispore to agar did not work. [Re: cronicr]
    #22090002 - 08/14/15 04:46 AM (8 years, 5 months ago)

Ah. Right. That makes even more sense than my half-baked answer prompt.

Thanks.


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Offlinespacechildo
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Re: Website Multispore to agar did not work. [Re: DinkinFlicka84]
    #22090014 - 08/14/15 05:05 AM (8 years, 5 months ago)

try and transfer one of the tiny pieces of white growth and see what comes of it.
or just start over if that feels more comfortable for you. probably got some good spots of myc there tho.

dont "wait for killer rhizos" thats just bull..


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OfflineNature Boy
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Re: Website Multispore to agar did not work. [Re: spacechildo]
    #22090048 - 08/14/15 05:43 AM (8 years, 5 months ago)

:whathesaid:  You'll only have lost 3 days.

N.B.


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OfflineDinkinFlicka84
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Re: Website Multispore to agar did not work. [Re: spacechildo]
    #22090077 - 08/14/15 06:01 AM (8 years, 5 months ago)

So you're saying to go ahead now and take one of those TINY little white specs and transfer to another plate or agar? I'll definitely do it, I'm just curious why I'm not waiting to see a good chunk of nice thick threaded myc, which is the whole point of agar... for me anyways.

I'm not doubting you at all, I just want to know the reason, so I can get behind it 100% and hell, I'll do it today.


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Offlinespacechildo
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Re: Website Multispore to agar did not work. [Re: DinkinFlicka84]
    #22090086 - 08/14/15 06:06 AM (8 years, 5 months ago)

the point is to transfer away from (possible) contams.


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OfflineDinkinFlicka84
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Re: Website Multispore to agar did not work. [Re: spacechildo]
    #22090280 - 08/14/15 07:58 AM (8 years, 5 months ago)

That makes sense. I was just thinking of the other reason why we do a few transfers to get a better strain. We're looking for the strongest, rope-like mycelium, which takes a few more days to show on the plate, so I would normally wait for that to happen...

BUT I see what you're saying. Take the tiny dot of colonized agar away from the green monster that's winning that colony battle in a way.

Am I far off??


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Offlinespacechildo
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Re: Website Multispore to agar did not work. [Re: DinkinFlicka84]
    #22090290 - 08/14/15 08:02 AM (8 years, 5 months ago)

well you seem focused on ropey/cottony growth while thats something you can control by removing nutes in the agar.

first and foremost I use agar to ensure clean growth. the ability to choose fast colonizers is just a bonus, I got plenty of time :tongue2:

you cant see fruiting ability on myc anyway so just aim for clean and fast.


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OfflinePinPornProducer
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Re: Website Multispore to agar did not work. [Re: DinkinFlicka84]
    #22090300 - 08/14/15 08:07 AM (8 years, 5 months ago)

Myc will sometimes start out fluffy (tomentose) until a few transfers are made and sometimes it will start roping out (rhizomorphic) on the initial drop. Luck of the draw sometimes along with the amount of nutrition in the agar. A lower nutrition agar will cause rhizo sooner than a high nutrition agar


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Re: Website Multispore to agar did not work. [Re: spacechildo]
    #22090329 - 08/14/15 08:21 AM (8 years, 5 months ago)

:whathesaid:

No syringe or prints are 100% clean either. They may be fairly clean but not totally.

Also, it is best to get a clean culture by doing agar transfers then using some wedges to inoculate grains.

While it is possible to inoculate grain jars with a MS syringe it is somewhat hit or miss. It also seems germinating spores directly on whole grains takes forever compared to using a live culture....


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OfflineDinkinFlicka84
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Re: Website Multispore to agar did not work. [Re: FreeWorldOrder]
    #22090598 - 08/14/15 10:24 AM (8 years, 5 months ago)

Yeah, I've had really bad luck with inoculating a ton of jars from a few MS syringes from respectable microscopy sites, so this time I'm going straight to agar with the MS syringes. I am curios what would happen if I poured more than a drop onto the agar for inoculating.


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Offlinespacechildo
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Re: Website Multispore to agar did not work. [Re: DinkinFlicka84]
    #22090605 - 08/14/15 10:28 AM (8 years, 5 months ago)

bacteria would happen. you dont want standing water along with spores that never are clean.

use as little as possible for best results, both in grain jars and on agar.


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OfflineDinkinFlicka84
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Re: Website Multispore to agar did not work. [Re: spacechildo]
    #22090701 - 08/14/15 11:04 AM (8 years, 5 months ago)

Right. I'll just use a single drop on either side of the agar section and transfer as soon as I see any myc growth if there happens to be contaminants surrounds good myc.

If the entire section of agar is clean with no green spots, etc., do I still need to hurry and transfer the myc again before it grows outward?
When does that portion of agar stop? When can I let the myc grow to rhyzo and then transfer the really good stuff?


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