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InvisibleJvF
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Registered: 02/13/14
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Agar plauged by bacteria
    #21354718 - 03/03/15 01:15 AM (9 years, 10 months ago)

So for the past couple months ive been working with agar. Ive been germinating spores and have beem minorly successful.  Ive made about 40 dishes. At this point im at about a 50% contamination rate. Ive had a few dishes with trich and one with cobweb, but the rest have all shown signs of bacteria about 2 days afyer inoculation. Not ao much after the germination of the spores, id say moreso after transfers are made. I havent madr transfers from dishes showing bacteria, but even at the third transfer from the initial spore germination im still facing difficulty with bacteria.

My agar recipe is currently 3.5 Gs agar agar to 3.5 Gs potato flakes and one cup of water for 10 dishes. Ive also been adding a bit of karo for the past couple goes and have noticed no significant change. 

I would consider my sterile procedure to be quick and efficient. I work in a glove box and go through all the motions of flaming my scalpel and completely running my arms and gloves down with alcohol. I work quickly and dont mess up or fumble when maling transfers.

At this point im just at a loss as to why my attempts are failing so badly. What can I do to prevent bacteria? It all seems to be the same type.

Ive read about dipping the wedge about to be transferred into peroxide. Ive also read about antibiotic agar. Are these viable options worth tryong?


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Invisiblehummingbird

Registered: 06/29/14
Posts: 2,282
Re: Agar plauged by bacteria [Re: JvF]
    #21354727 - 03/03/15 01:21 AM (9 years, 10 months ago)

Where is the bacteria starting from...more on the edges or more towards the center of the plates?

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InvisibleVal
Were just Marbles in a Cave...

Registered: 01/31/15
Posts: 120
Re: Agar plauged by bacteria [Re: JvF]
    #21354728 - 03/03/15 01:21 AM (9 years, 10 months ago)

Media not sterilized long enough, or Dirty spores....maybe?

Are you leaving some blanks?

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InvisibleJvF
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Re: Agar plauged by bacteria [Re: Val]
    #21354734 - 03/03/15 01:23 AM (9 years, 10 months ago)

The bacteria starts mostly where the transferred wedge is about 3/4 of thr time. The rest of the time starts randomly nowhere near the inoculation point.


And no, I havent left any blanks. Thats a good idea to get an idea if whats happening


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Invisiblehummingbird

Registered: 06/29/14
Posts: 2,282
Re: Agar plauged by bacteria [Re: JvF]
    #21354743 - 03/03/15 01:28 AM (9 years, 10 months ago)

You could try adding peroxide to your agar for transfers. I'm not sure about antibiotic agar, I haven't used it yet myself. I have been curious about it though, seems like it could help with dirty/wild prints and cloning.

If you don't seem contams as much on the germination plates, its probably either a flaw somewhere in your sterile technique or under cooked agar.




Edited by hummingbird (03/03/15 01:44 AM)

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InvisibleJvF
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Re: Agar plauged by bacteria [Re: hummingbird]
    #21354752 - 03/03/15 01:34 AM (9 years, 10 months ago)

I originally germinated 10 dishes with abou 6 different prints. Of those, only 1 got bacteria. Come to think of it, after I started adding karo to the mix the bacteria seems to have increased.  It could be that, its something I didnt consider.

ill make some more plates without it, could definitely be that


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Invisiblehummingbird

Registered: 06/29/14
Posts: 2,282
Re: Agar plauged by bacteria [Re: JvF]
    #21354769 - 03/03/15 01:47 AM (9 years, 10 months ago)

I've only used malt or dextrose for agar. I have used karo for LC with success though.

How long do you cook/sterilize your agar for?

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InvisibleJvF
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Re: Agar plauged by bacteria [Re: hummingbird]
    #21354777 - 03/03/15 01:55 AM (9 years, 10 months ago)

I PC for 30 min at 15psi. Using the recipe for 10 dishes I stated above


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Offlinesaintburger
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Registered: 09/18/14
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Re: Agar plauged by bacteria [Re: JvF]
    #21354799 - 03/03/15 02:15 AM (9 years, 10 months ago)

Quote:

jfischer218 said:
I originally germinated 10 dishes with abou 6 different prints. Of those, only 1 got bacteria. Come to think of it, after I started adding karo to the mix the bacteria seems to have increased.  It could be that, its something I didnt consider.

ill make some more plates without it, could definitely be that




I could be wrong but I don't think Karo is too common in the mix. I used some grain soak water in my agar mix last time and some cultures are tearing it up!


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Set goals for yourself, or wander around aimlessly.

"One day Alice came to a fork in the road
and saw a Cheshsire cat in a tree.
'Which road do I take?' she asked.
'Where do you want to go?' was his response
'I don't know', Alice answered.
'Then', said the cat, 'it doesn't matter.'"

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OfflineKizzle
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Re: Agar plauged by bacteria [Re: saintburger]
    #21357798 - 03/03/15 07:00 PM (9 years, 10 months ago)

Using a low nutrient agar can help a lot if you don't have antibiotic agar. I always use it for the initial spore germination or clone plates to make it easier to isolate some clean mycelium.


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InvisibleJvF
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Re: Agar plauged by bacteria [Re: Kizzle]
    #21357862 - 03/03/15 07:12 PM (9 years, 10 months ago)

What would you classify as a low nutrient agar? Right now im using the 1:1 agar agar to potato flakea. Would a 2:1 ratio be better?


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OfflineDirdyD
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Re: Agar plauged by bacteria [Re: JvF]
    #21357908 - 03/03/15 07:22 PM (9 years, 10 months ago)

:takingnotes:

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InvisibleVal
Were just Marbles in a Cave...

Registered: 01/31/15
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Re: Agar plauged by bacteria [Re: DirdyD]
    #21358031 - 03/03/15 07:50 PM (9 years, 10 months ago)

I would guess that its less of of the potato flakes your using, or malt if your doing MEA....and the agar stays the same since its what solidifies.

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Offlinetourrat
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Re: Agar plauged by bacteria [Re: Val]
    #21358082 - 03/03/15 08:02 PM (9 years, 10 months ago)

Not to say its your only problem but that's WAY more potato flakes than I would use...and nix the karo also. I personally have been having good results with a very weak nutrient mix... the myc stretches out fast looking for food. The bacteria that's coming from the transfer could very well be hitchhiking... what transfer are you on?


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OfflineKizzle
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Re: Agar plauged by bacteria [Re: DirdyD]
    #21358144 - 03/03/15 08:19 PM (9 years, 10 months ago)

If you're literally adding the flakes you should only be adding water the flakes were boiled in and not the flakes themselves.


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InvisibleJvF
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Re: Agar plauged by bacteria [Re: Kizzle]
    #21358621 - 03/03/15 10:18 PM (9 years, 10 months ago)

Yea ive been adding the flakes directly to the mix.

So basically I would boil potato flakes into water and use that boiled water, the same way as if I were using fresh potatoes?

And tourrat, thats an excellent point about thr myc stretching out looking for nutrients. I hadnt thougjt of it in that way, I was assuming more nutrients would be better and promote stronger growth


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Invisiblefilthyknees
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Re: Agar plauged by bacteria [Re: JvF]
    #21358630 - 03/03/15 10:19 PM (9 years, 10 months ago)

I just dump the agar, potato flakes and water in a jar and pc it, never had any issues :shrug:

I've done like 100+ plates this way


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OfflineKizzle
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Re: Agar plauged by bacteria [Re: filthyknees]
    #21358783 - 03/03/15 10:49 PM (9 years, 10 months ago)

It's not that adding the flakes causes problems in itself it's just that the additional nutrients support rapid bacterial growth which makes it harder to isolate clean mycelium when bacteria is also present.

Quote:

So basically I would boil potato flakes into water and use that boiled water, the same way as if I were using fresh potatoes?



Pretty much. Just pour it through a coffee filter or something to remove the solids.


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Invisiblefilthyknees
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Re: Agar plauged by bacteria [Re: Kizzle]
    #21358814 - 03/03/15 10:56 PM (9 years, 10 months ago)

:takingnotes:


--------------------
But if you're in a hurry, and really got to go
If you're in a hurry, might have to find out slow
That it's one thing to try and another to fly
You get there quicker just a step at a time
It's one thing to bark, another to bite
The show ain't over till you pack up at night

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InvisibleNumeroEno
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Re: Agar plauged by bacteria [Re: filthyknees]
    #21358933 - 03/03/15 11:39 PM (9 years, 10 months ago)

I recently cleaned up all my bacterial agar. I hade plates with bacillus, pseudomonas, and both. I made a low-ish nutrient PDA (7 grams potato starch, 7 grams dextrose, 10 grams agar, 500ml water) with 25 milligrams of gentamicin sulfate. Every transfer was from the cleanest looking parts of the bacterial plates, and of all 20 new plates, not a single one has bacteria.

I've never messed with peroxidated agar, but a medium to low nutrient antibiotic agar will work miracles.


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InvisibleJvF
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Re: Agar plauged by bacteria [Re: NumeroEno]
    #21361825 - 03/04/15 03:34 PM (9 years, 10 months ago)

Sounds very good, thanks for all the input everyone. This was the third transfer from spores and still seeing bacteria. Ill whip up a new batch today and hopefully ill be able to use the next transfer to inoculate some grains


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OfflineKizzle
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Re: Agar plauged by bacteria [Re: JvF]
    #21361860 - 03/04/15 03:41 PM (9 years, 10 months ago)

I had a bacteria colony on one of those plates (agar+potato broth) and the colony didn't even grow. So I did an experiment and took my unsterile blade and touched it to the agar. A bacteria colony formed where I had touched it but once again the colony remained localized, it didn't even colonize the plate at least not visibly.


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InvisibleJvF
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Re: Agar plauged by bacteria [Re: Kizzle]
    #21361896 - 03/04/15 03:49 PM (9 years, 10 months ago)



Im seeing alot of what youre describing with regard to the localized colonies. In one case, the myc even ate the colony completely. That was the transkei strain and is some of the most aggressive myc ive ever seen. The fluffy parts were all where the bacteria was and as you can see, once it 'ate' the bacteria, its grown incredibly healthy


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InvisibleVal
Were just Marbles in a Cave...

Registered: 01/31/15
Posts: 120
Re: Agar plauged by bacteria [Re: JvF]
    #21362171 - 03/04/15 04:37 PM (9 years, 10 months ago)

Cut out that ropey part and transfer to a few plates.
Only need a small chunk 1-2mm x 1-2mm @3,5,6 oclock

Also just a question is there any condensation on your plate lids?

Edited by Val (03/04/15 04:44 PM)

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InvisibleJvF
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Re: Agar plauged by bacteria [Re: Val]
    #21362238 - 03/04/15 04:55 PM (9 years, 10 months ago)

Yea im planning on tranafering in a few hours :laugh:

and yea, usually for thr top 2 or so of the stadk theres condensation but it disappears by the next day or so


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