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OfflineInfinityheat
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Contamination on agar or mycelium?
    #18137672 - 04/19/13 09:20 PM (11 years, 1 month ago)

So I recent did my first time with agar.

In which I really didn't have what I needed as far as needing a flow hood

Long story short out of 7 5 seemed to be contaminated

All 4 of my lions mane were no doubt

But the other 3 which were a Thai cube strain showed no sign of contamination. Which I found pretty interesting, seeing as I did them side by side in a glove box(using a syringe which in hindsight probably wasn't the best idea

I checked last night and 2 of the Thai seem to have mycelium grown. The third looked like it might have contamination so I threw it out

So here's my question



Contamination or not?

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OfflineTmethylM
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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18137729 - 04/19/13 09:32 PM (11 years, 1 month ago)

That is healthy mycelium.


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¯\_(ツ)_/¯

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: Tmethyl]
    #18138200 - 04/19/13 11:15 PM (11 years, 1 month ago)

Alright great thanks :laugh:

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OfflineTmethylM
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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18138212 - 04/19/13 11:16 PM (11 years, 1 month ago)

:awesomenod:

Carry on with your.. devious tasks, my friend.


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Invisiblefastfred
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Re: Contamination on agar or mycelium? [Re: Tmethyl]
    #18138712 - 04/20/13 01:30 AM (11 years, 1 month ago)

Looks like a lot of yeast contamination on the left hand side though.

All you really need for sterile work is a bunsen burner and a tank of propane, along with a draft free room.

Believe it or not plenty of sterile work was done before the invention of flow hoods and HEPA filters.  In fact, the majority of sterile lab work is still not done in a flow hood, and probably never will be.


-FF


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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: fastfred]
    #18139004 - 04/20/13 05:11 AM (11 years, 1 month ago)

Hmm:/
That's what I saw on the third Thai that I threw out

Is this it?

Any chance the mycelium will over run it?

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OfflineMycoAu
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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18139023 - 04/20/13 05:24 AM (11 years, 1 month ago)

Never let your cultures run into any kind of contamination.  If you want to attempt saving the plate, always transfer away to a new dish with the cleanest material you can.  Always select tissue from as far away from the contaminate as you can.  If you already have other plates without contamination, use them and discard this plate without opening it.  You should never open a contaminated container unless you have no other way to keep that sample/species.

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: MycoAu]
    #18139055 - 04/20/13 05:46 AM (11 years, 1 month ago)

I used a glove box with doing this and sterilized everything. But I guess everything got contamination after all:/
Oh well there's always next time:)

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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18140043 - 04/20/13 12:13 PM (11 years, 1 month ago)

The important thing is to transfer any healthy sections to new dishes as soon as you can.

You should also find where that yeast is coming from.  Yeast isn't particularly airborne, and it kind of looks like when agar with yeast isn't fully sterilized.

See if the yeast is in the media or just only on the surface.  You can also do a control dish, which you should always do if you're not 100% certain of your methods and materials.

Yeast is a common contaminant.  It's sticky and present almost everywhere in the environment.  It's pretty common for prints to be contaminated.

Not sure how you did things but scraping a print with your loop or into a little water, then streaking on the plate is the best way to get clean isolates.  You can then see if the yeast is all along your streak and know the print is contaminated.

If the print is that heavily contaminated there's not much you can do with it.  Yeast is one of those annoying microbes that will slime along your whole streak and grows pretty fast.  If there's too much you won't be able to get any isolation.


-FF

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: fastfred]
    #18140612 - 04/20/13 02:59 PM (11 years, 1 month ago)

I used a spore syringe 
Each from different companies

The lions mane seemed to either have a white mold or trich

The Thai had nothing until a few days ago

Could i maybe of not sterilized the malt agar enough?
Doesn't it have yeast in it

I only have a small electric pressure cooker (just ordered large one) and it only has a "low" and "high" setting. Maybe it wasn't enough pressure to sterilize?

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Offlinemycofever
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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18140947 - 04/20/13 04:25 PM (11 years, 1 month ago)

The pc you have will work for agar It may not work for grains or sawdust blocks if you can fit one in there unless you pc for several hours. How long did you pc? I use a 921 and I pc for 30 minutes at 15 psi Then let it cool which takes about an hour. My plates come out sterile every time. Did you flame the needle before inoculation?  It could also be your technique.


--------------------
Patience will help you keep your sanity.It will insure your success if you are patient in all aspects of mushroom growing.When you rush you are prone to make mistakes and all of your efforts are wasted.

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Re: Contamination on agar or mycelium? [Re: mycofever]
    #18141162 - 04/20/13 05:27 PM (11 years, 1 month ago)

Quote:

Could i maybe of not sterilized the malt agar enough?
Doesn't it have yeast in it




I've only had yeast contam like that in not fully sterilized media with yeast in it.

Yeast contam has always been pretty common in syringes.  That's probably where it's coming from.

You need to streak to isolate.  Google "micro streak technique" and you'll find videos, lab manuals, etc..  Plenty of guides out there, so no point in explaining it here further.

The difficult part is that your spore concentration is going to be pretty low compared to the yeast.  If you have a centrifuge handy then spin down some spore solution and wash it with sterile water, vortex or shake to resuspend, then repeat a time or two.

Since I'm guessing you probably don't have a centrifuge handy... you might try storing the syringes tip down for awhile to let the spores settle to the tip.  Then put a drop in a corner of your plate and streak it out.

Otherwise just get some replacement syringes sent.  You can point them to this thread where we determined the yeast contamination makes them unsuitable for spore microscopy.  :wink:

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: fastfred]
    #18141378 - 04/20/13 06:32 PM (11 years, 1 month ago)

I pressure cooked my agar for 90 minutes

I just recently did a sawdust bag. Pressure cooked that for 2 hours. Hopefull thatll come out okay

I've never had any contamination with my BRF jars to date but I know those are a little easier

I flame sterilized each time

I was thinking about maybe innoculating some grain spawn and then putting a colonized grain in the center of the plate Could that work?

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Offlinemycofever
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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18141447 - 04/20/13 06:55 PM (11 years, 1 month ago)

90 minutes is plenty for agar even if your pc only reaches 10 psi.I Do not know what to tell ya man. The contams may have come from the vendor. You sound pretty sure of your procedures. I hope those bags turn out good. Also yes you can do a grain to agar transfer. I have done many of these they work very well. I prefer doing a solid transfer to agar rather than liquid to agar.


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Patience will help you keep your sanity.It will insure your success if you are patient in all aspects of mushroom growing.When you rush you are prone to make mistakes and all of your efforts are wasted.

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: mycofever]
    #18142433 - 04/20/13 10:50 PM (11 years, 30 days ago)

Well hopefully the grain will work better:)

I did realize after I squirted the spores that it woulda been better with a solid

It's still odd that the lions mane got a lot of mold/trich. Hopefully nothing in that syringe. Just inoculated the sawdust bag and some BRF jars with it

But it's all for experience so

:smile:

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18142682 - 04/21/13 12:10 AM (11 years, 30 days ago)

Oh I made a mistake
I sterilize my agar for 40 minutes
BRF cakes for 90

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Invisiblefastfred
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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18142811 - 04/21/13 12:50 AM (11 years, 30 days ago)

Quote:

90 minutes is plenty for agar even if your pc only reaches 10 psi.




90 minutes is badly overcooked for liquid media.  10 PSI is about 5C cooler than 15 PSI, so you should add about 25% to the sterilization time.

Here's a little info for ya...
Quote:

Although sterilization of culture media is best carried out in a steam autoclave at temperatures between 121-134°C it has to be recognised that damage is caused to the medium by the heating process.

Heat-treatment of complex culture media which contain peptides, sugars, minerals and metals results in nutrient destruction, either by direct thermal degradation or by reaction between the medium components.

Toxic products caused by chemooxidation can also be formed during heat-treatment. It is important, therefore, to optimise the heating process so that a medium is sterile after heating but minimal damage is caused to the ingredients of the medium.

A general instruction for sterilizing culture media in volumes up to one litre at 121°C for 20 minutes is given on each label.

Overheating is a common cause of pH drift, darkening, precipitation, poor gel strength and reduced bacteriological performance. These effects can also be produced if a concentrated 'pool' of ingredients at the bottom of the container is heated. All culture media should be in solution before sterilization. This will reduce the occurrence of Maillard-type reactions (non-enzymatic browning) taking place in the medium.

Overheating effects will occur if agar media are allowed to gel in bottles and are later steamed to melt the agar. They will also occur if molten media are held at 50°C for more than 3 hours before use. Agar media with pH values at or below 5.0 are very sensitive to overheating in any form because the agar is hydrolysed and the gel strength fails. It is recommended to sterilize the agar of media of a pH lower than 5.0 separately.




There's no reason to sterilize more than 1L per flask, so you should never go much more than 30 minutes on agar.  Overcooking usually isn't much of a problem, other than a little more sedimentation, caramelization, or oxidation depending on the media type.

If you don't run into problems then 30-45 minutes is fine, but even at 10 PSI you should never go over an hour.


-FF

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: fastfred]
    #18142876 - 04/21/13 01:17 AM (11 years, 30 days ago)

Well I think the high setting is suppose to be around 15psi
And I did for 40 minutes
Just made another batch
Gonna try to transfer over the good Thai, make a large plate some pink oyster culture I have and them use inoculate one with some shiitake spores i took

:smile:)

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Offlinemycofever
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Re: Contamination on agar or mycelium? [Re: fastfred]
    #18143330 - 04/21/13 05:29 AM (11 years, 30 days ago)

Quote:

Infinityheat said:
Oh I made a mistake
I sterilize my agar for 40 minutes
BRF cakes for 90



This is much closer.:lol: I was thinking that at 1 1/2 hours that agar must have been pretty dry but to say the least, sterile.  When you get colonization of those bags post a pic. If that trich was in them syringes you will know it soon.


--------------------
Patience will help you keep your sanity.It will insure your success if you are patient in all aspects of mushroom growing.When you rush you are prone to make mistakes and all of your efforts are wasted.

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: mycofever]
    #18143387 - 04/21/13 06:06 AM (11 years, 30 days ago)

Just finished 9 agar dishes :smile:



4 pink oyster( from agar dish)
3 Thai ( from agar dish)
2 shiitake( spore print)

Hopefully these will go better

And I'll post when the bag starts to colonize

If there is trich in it i won't be buying from that seller anymore
Same for if the pink oyster agar isn't pink oysters -__-

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18148464 - 04/22/13 05:53 AM (11 years, 29 days ago)

Don't want to call it too early but it looks like my lions mane jars, which probably means the sawdust bag too, has contamination. And It must be from the syringe. Never had a problem with jars. And these once even have self healing inoculation ports.

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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18149348 - 04/22/13 11:37 AM (11 years, 29 days ago)

Yeast contamination is pretty common from vendors.  It's hard to prevent and hard to spot.  You should let the vendor know so they can deal with it.


-FF

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: fastfred]
    #18150328 - 04/22/13 02:43 PM (11 years, 29 days ago)

I don't think this is yeast. But in not sure

I inoculated these Sunday at about 5am. Along with Thai jars. Which have nothing growing(to be expected)


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Re: Contamination on agar or mycelium? [Re: Infinityheat]
    #18150647 - 04/22/13 03:43 PM (11 years, 29 days ago)

Your agar plates sure look like yeast contamination.

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OfflineInfinityheat
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Re: Contamination on agar or mycelium? [Re: fastfred]
    #18150698 - 04/22/13 03:51 PM (11 years, 29 days ago)

Yes the agar is

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