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OfflineAlcebiades
Stranger thanMost

Registered: 05/25/08
Posts: 39
Last seen: 11 years, 6 months
Noob Agar MSI Question HELP!
    #8469666 - 06/01/08 03:23 AM (12 years, 9 months ago)

So, a friend of a friend is trying to do their first isolation on agar.  She made up 12 plates of MEA, poured and sealed w/parafilm in a glovebox.  She waited a week before reloading the plates into the glovebox to see if any of the plates contaminated (none did, which made her happy, being her first attempt and all :laugh:) and inoculated 6 plates with tissue samples and 6 with a spore syringe.  She flame sterilized the needle, and therefore used a 10% bleach water solution to sanitize the glovebox.  She got her syringe from earths tongue, new moon B+ (she knows she was very lucky to even receive her order from them, and wont do business w/them again).  The tissue sample plates colonized like gangbusters, but the spore plates did NOTHING!  After waiting 2.5 weeks, she decided to scrap those plates, and after opening them found no evidence of contams, just dried up syringe solution...    She wonders if the bleach may have lingered in the air for the 30 minutes she let the box sterilize, and killed the spores when it settled on the surface of the agar...

Anybody have any thoughts/advice on why spores which were viable 5 weeks before, then stored in a cool dark place were unable to germinate on agar?


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No statements made in any post or message by myself should be construed to mean that I am now, or have ever been, participating in or considering participation in any activities in violation of any local, state, or federal laws. All posts are works of fiction.


Edited by Alcebiades (06/01/08 11:51 PM)


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Invisiblesandman420
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Re: Noob Agar MSI Question [Re: Alcebiades]
    #8470096 - 06/01/08 08:33 AM (12 years, 9 months ago)

does it have a greasy surface?


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OfflineNeobean
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Registered: 10/07/01
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Re: Noob Agar MSI Question [Re: Alcebiades]
    #8470289 - 06/01/08 10:59 AM (12 years, 9 months ago)

Quote:

Alcebiades said:
So, a friend of a friend is trying to do their first isolation on agar.  She made up 12 plates of MEA, poured and sealed w/parafilm in a glovebox.  She waited a week before reloading the plates into the glovebox to see if any of the plates contaminated (none did, which made her happy, being her first attempt and all :laugh:) and inoculated 6 plates with tissue samples and 6 with a spore syringe.  She flame sterilized the needle, and therefore used a 10% bleach water solution to sanitize the glovebox.  She got her syringe from earths tongue, new moon B+ (she knows she was very lucky to even receive her order from them, and wont do business w/them again).  The tissue sample plates colonized like gangbusters, but the spore plates did NOTHING!  After waiting 2.5 weeks, she decided to scrap those plates, and after opening them found no evidence of contams, just dried up syringe solution...    She wonders if the bleach may have lingered in the air for the 30 minutes she let the box sterilize, and killed the spores when it settled on the surface of the agar...

Anybody have any thoughts/advice on why spores which were viable 5 weeks before, then stored in a cool dark place were unable to germinate on agar?







From a spore syringe, it sometimes takes that long for germination to occur. How did you apply the spores onto the agar surface?


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OfflineAlcebiades
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Registered: 05/25/08
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Re: Noob Agar MSI Question [Re: Neobean]
    #8470601 - 06/01/08 12:56 PM (12 years, 9 months ago)

2 of the 6 had a greasy surface, no smell eminating from any of them though other than the agar smell. Some kind of bacterial contam?

Two drops were put onto the surface of each plate from a well mixed syringe. Those new moon syringes are really weird, big clumps of black crap come out of a well mixed syringe... :P My FoaF will stick to the sponsors from now on. She knows B+ can be slow to take off, but the rye bags she did showed colonization in 6 days... Does agar really take that long for MSI to germinate?


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No statements made in any post or message by myself should be construed to mean that I am now, or have ever been, participating in or considering participation in any activities in violation of any local, state, or federal laws. All posts are works of fiction.


Edited by Alcebiades (06/01/08 11:18 PM)


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Invisiblesandman420
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Re: Noob Agar MSI Question [Re: Alcebiades]
    #8471995 - 06/01/08 08:25 PM (12 years, 9 months ago)

im not sure exactly waht causes the greasy surface but anytime i apply spores to agar from a syringe I never have ever once had them germinate...probably some bacteria or yeast .


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OfflineAlcebiades
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Registered: 05/25/08
Posts: 39
Last seen: 11 years, 6 months
Re: Noob Agar MSI Question [Re: sandman420]
    #8472552 - 06/01/08 11:17 PM (12 years, 9 months ago)

I did a little bit of reading, and the only contam I found that fits the MO of a greasy agar surface is wetspot... My FoaF kinda freaked out when I told her, since she took a big whiff when she opened the plates. However, if it was wetspot, it should have fizzed like crazy when she poured H2O2 on it, right?


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OfflineRogerRabbitM
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Re: Noob Agar MSI Question [Re: Alcebiades]
    #8473307 - 06/02/08 02:57 AM (12 years, 9 months ago)

The upper right section of this petri dish is what bacteria contamination looks like. I don't really know how people keep finding ET, but they're the worst in the business. I think the black chunks are charcoal to make it look dark because lots of noobs have been told incorrectly that a good syringe is dark with spores.
RR


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Offlinezen_
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Registered: 06/02/08
Posts: 8
Last seen: 12 years, 8 months
Re: Noob Agar MSI Question [Re: RogerRabbit]
    #8473351 - 06/02/08 03:24 AM (12 years, 9 months ago)

<embed id="VideoPlayback" style="width:400px;height:326px" allowFullScreen="true" flashvars="fs=true" src="https://proxy.mind-media.com/proxy.php?url=http%3A%2F%2Fvideo.google.com%2Fgoo%3Cwbr%3Egleplayer.swf%3Fdocid%3D-591278924622%3Cwbr%3E0361236%26hl%3Den" type="application/x-shockwave-flash"> </embed>


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OfflineAlcebiades
Stranger thanMost

Registered: 05/25/08
Posts: 39
Last seen: 11 years, 6 months
Re: Noob Agar MSI Question [Re: RogerRabbit]
    #8473497 - 06/02/08 04:25 AM (12 years, 9 months ago)

This isn't my friends first grow... It's been a few years, and this time she's stepping things up considerably.  She had ordered gourmet spores from Ralph's before and was really happy.  She got hooked up w/ET because they also had rye bags for sale...  She didn't know about their rep until after she ordered lol... Talk about shitting a brick till her order arrived... :frown:

She decided to do some honey LC's a few days ago to test the viability of the ET syringe. It looks like there are some strands coming off of the chunks of black stuff. Then again, could just be sediments attaching to them... :tongue: She knocked the LC's up on 5-29, so we'll know for sure here soon. Anyhow, I digress...

The dishes in question didn't have any growth like the one in RR's pic (cool sectioned plates btw!). Nothing so distinct, just a greasy feel across the entirety of two of the plates. No smell and no reaction when H2O2 was introduced. I guess she must not have given the spores enough time to get going.  Then again, it's sort of a moot point since the clone plates are doing fine.

In any case, thanks to everyone who chimed in on this thread!  And to RR in particular, my friend is bidding on  a lab grade microscope at the local Goodwill (crazy what you can find there!)... If she wins it, I'll take a look at the ET syringe and let you know for sure whether or not they darken it w/charcoal. She's also looking forward to ordering your dvd next week! Your Pleurotus look so good, she can't wait to grow her own!!!


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No statements made in any post or message by myself should be construed to mean that I am now, or have ever been, participating in or considering participation in any activities in violation of any local, state, or federal laws. All posts are works of fiction.


Edited by Alcebiades (06/02/08 06:22 AM)


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