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Offlinemaryxmas
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Help. Need to fight bacteria on agar
    #17794780 - 02/13/13 11:01 AM (11 years, 3 months ago)

I have some agar plates that I recently transplanted some pan cyan tissue onto.



the tissue is nice and fuzzy with fresh growth but the area around the tissue is contaminated with bacteria. I really need this clone so I don't want to pitch it into the trash.

is there a way to save the fungi or fight the bacteria so the fungi can at least out run it a little so I could get a sample to transfer to another plate.

could I maybe remove the tissue and dip it in hydrogen peroxide and transfer to another plate?

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Offlinemycofever
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Re: Help. Need to fight bacteria on agar [Re: maryxmas]
    #17794835 - 02/13/13 11:12 AM (11 years, 3 months ago)

you have to transfer. you can try scraping from the fuzzy mycelium to do this. good luck.


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InvisibleBeefy1
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Re: Help. Need to fight bacteria on agar [Re: maryxmas]
    #17798740 - 02/14/13 12:27 AM (11 years, 3 months ago)

I'm in the same situation.


I tried dipping in peroxide while transfering to a clean plate,  Killed the myc but only some of the bacteria.


Next try will be gentamycin agar

If that doesn't work I'll try the hot pour method-  basically place the tissue on a fresh agar plate and pour a layer of agar over it,  hope the myc grows up through it and the bacteria stays trapped,  at least long enough to get a clean sample.


Good luck.

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OfflineMonkeyJesusFresco
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Re: Help. Need to fight bacteria on agar [Re: Beefy1]
    #17798786 - 02/14/13 12:40 AM (11 years, 3 months ago)

out of curiosity, what advantage is there to "the hot pour" method versus laying a squar of medicated agar cut from a pre-poured dish on top of your myc. culture?


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Offlinemaryxmas
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Re: Help. Need to fight bacteria on agar [Re: MonkeyJesusFresco]
    #17799647 - 02/14/13 07:43 AM (11 years, 3 months ago)

Well I don't want to jinks it but one of my clones seems to be taking to the plate and there is no bacteria in sight on the plate..... Totally stoked if I can get one good plate to reproduce.


sadly of the three fruits I clone the plate that looks like it is going to make it was from the smallest of the three. But beggars can't be choosers and I am most def a beggar

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InvisibleBeefy1
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Re: Help. Need to fight bacteria on agar [Re: MonkeyJesusFresco]
    #17801077 - 02/14/13 01:28 PM (11 years, 3 months ago)

Quote:

MonkeyJesusFresco said:
out of curiosity, what advantage is there to "the hot pour" method versus laying a squar of medicated agar cut from a pre-poured dish on top of your myc. culture?




I think it's supposed to kinda seal it inside two layers.

If you just put a piece of premade agar on top I would guess the myc just grows between the two layers along with the bacteria instead of growing up through the agar

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Offlinemycofever
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Re: Help. Need to fight bacteria on agar [Re: MonkeyJesusFresco]
    #17803131 - 02/14/13 07:38 PM (11 years, 3 months ago)

Quote:

MonkeyJesusFresco said:
out of curiosity, what advantage is there to "the hot pour" method versus laying a squar of medicated agar cut from a pre-poured dish on top of your myc. culture?




In theory A hot pour is to set the bacteria back  and hope that a little mycelium will survive the event then grow out from the new layer as soon as it does you transfer the mycelium on top.I would not recommend this in a situation as vital as the one that maryxmas is in.It is also no good for molds this is only good for bacteria.


--------------------
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.

Edited by mycofever (02/14/13 07:57 PM)

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Offlinemaryxmas
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Re: Help. Need to fight bacteria on agar [Re: mycofever] * 1
    #17808353 - 02/15/13 04:22 PM (11 years, 3 months ago)

My one good plate has a nickel sized white fluffy patch now.



Very gorgeous

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OfflineRogerRabbitM
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Re: Help. Need to fight bacteria on agar [Re: MonkeyJesusFresco] * 1
    #17811724 - 02/16/13 04:47 AM (11 years, 3 months ago)

Quote:

MonkeyJesusFresco said:
out of curiosity, what advantage is there to "the hot pour" method versus laying a squar of medicated agar cut from a pre-poured dish on top of your myc. culture?




An enormous advantage.

The hot agar partly 'pasteurizes' the bacteria which slows it down, while leaving the mushroom mycelium unharmed so it can grow up through the new layer.  The hot agar also completely surrounds the mycelium and bacteria rather than just laying on top.

When you simply make another slab of agar and plop it on top, you don't get this advantage.
RR


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OfflineWorkmanV
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Re: Help. Need to fight bacteria on agar [Re: RogerRabbit]
    #17812638 - 02/16/13 11:11 AM (11 years, 3 months ago)

In my experience, bacteria tends to slow down naturally in a sealed plate and the mycelium may eventually overgrow the perimeter of the bacteria.  Once that happens, you should be able to snag some clean mycelium on the leading edge.  Obvious this won't work if the bacteria covers the entire plate.  But if the contamination isn't too bad, set the plates aside and see what happens.


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Edited by Workman (02/16/13 01:23 PM)

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OfflineMonkeyJesusFresco
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Re: Help. Need to fight bacteria on agar [Re: RogerRabbit]
    #17813107 - 02/16/13 01:06 PM (11 years, 3 months ago)

Cool, thank you so much. Guess I'm off to investigate more agar techniques! My agar work has been limited and dumb-luck has been a MAJOR contributing factor to any success I've had :rolleyes:

plus, just recently found reishi and (what appears to be lion's mane) on a walking trail near by, my plan is to snag tissue from them and transfer my ass off to get my techniques down and log some experience points :smile: ...also, reishi :smile:


--------------------
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OfflineRogerRabbitM
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Re: Help. Need to fight bacteria on agar [Re: MonkeyJesusFresco] * 1
    #17813757 - 02/16/13 03:26 PM (11 years, 3 months ago)

Good luck.  Lion's mane is one of the harder mushrooms to clone due to the amount of bacteria and other fungi present.

Antibiotic agar will help, even though I hate to use it.  When isolating wild cultures though, it can save the day.
RR


--------------------
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OfflineSillyputty67

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Re: Help. Need to fight bacteria on agar [Re: RogerRabbit]
    #17815292 - 02/16/13 08:07 PM (11 years, 3 months ago)

Recently ive found using inoculation loops work well for isolating from contamination. The loop allows you to grab the tiniest sample. Usually the myc grow faster than or away from the contamination. So a couple of rounds of this would be all it would require ime.


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OfflineLennybernadino
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Re: Help. Need to fight bacteria on agar [Re: Sillyputty67]
    #17819617 - 02/17/13 02:24 PM (11 years, 3 months ago)

I usually get the job done with a series of hot pours.

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OfflineChris893
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Re: Help. Need to fight bacteria on agar [Re: Lennybernadino]
    #18391399 - 06/09/13 07:11 AM (10 years, 11 months ago)

Sorry to bump an old thread but I thought it would be better than starting a new one plus I have a somewhat relevant question. I am new to mycology and microbiology in general and don't have any lab experience except for chemistry. I have recently assembled a 2x2 flow-hood and am using it to do agar work. I've had some bacterial contaminations on some of my plates along with healthy mycelial growth on the same plates. Is it safe to open these up in front of a flow hood to transfer away from? My concern is that bacterial spores will be blown all over my work space but I guess an alcohol spray and wipe following a transfer will be sufficient to kill any spores that may have contaminated my work space?

What do you all think? Recommend?

I've heard that transferring under a plastic bag is a safe route but I think that this would just give the bacterial spores the chance to contaminate my clean new agar plate which the mycelium is being transferred to. Any comments are appreciated


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OfflineTerry M
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Re: Help. Need to fight bacteria on agar [Re: Chris893]
    #18391610 - 06/09/13 09:20 AM (10 years, 11 months ago)

Not all bacteria produce spores. And the ones that can only do so when food is exhausted and they are under stress to survive. I've gotten away with transferring bacteria contaminated plates using a flow hood. But if you want to be really safe, use a glove box.


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Invisiblemicro
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Re: Help. Need to fight bacteria on agar [Re: Terry M]
    #18391641 - 06/09/13 09:35 AM (10 years, 11 months ago)

That's not how the flow hood should work... The air is flowing in a way that shouldn't allow for contamination where you are working. It might get into the filters but assuming you change the filters when you need to, and you've wiped down the area with bleach and either keep it on or let the air flow for a while before using it you should really be fine.

This is assuming the flow hood works as it is supposed to.


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OfflineChris893
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Re: Help. Need to fight bacteria on agar [Re: micro]
    #18392300 - 06/09/13 12:45 PM (10 years, 11 months ago)

I found this:

" Always transfer healthy mycelium away from the contamination, not the other way around.  In addition, never open a contaminated dish in front of a flow hood.  Always shut it off, just before opening any contaminated product, or use a glovebox.
RR "

Sounds safe enough provided I keep my dirty breath away from the plate and cause as little disturbance as possible to my surroundings while maintaining good sterile technique. I'm going to try this tonight to clean up a reishi and oyster culture.


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OfflineTerry M
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Re: Help. Need to fight bacteria on agar [Re: Chris893]
    #18392529 - 06/09/13 01:48 PM (10 years, 11 months ago)

A flow hood will keep the nasty spores away from your work area. But do you really want them scattered all over your lab room to grow? I never, ever, so much as open a plate in the lab on which there is sporulating Trichoderma or Penicillium.


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OfflineSillyputty67

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Re: Help. Need to fight bacteria on agar [Re: Terry M]
    #18392558 - 06/09/13 01:55 PM (10 years, 11 months ago)

One more reason a good Still Air Box is superior to a flow hood.


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Re: Help. Need to fight bacteria on agar [Re: Sillyputty67]
    #18392646 - 06/09/13 02:17 PM (10 years, 11 months ago)

Quote:

malicom said:
One more reason a good Still Air Box is superior to a flow hood.




I routinely use the flow hood because of the large, unobstructed area for dealing with things like 5 pound filter patch bags. But my still air box is ready to go at a moment's notice for just such situations!


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Invisible36fuckin5
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Re: Help. Need to fight bacteria on agar [Re: Sillyputty67]
    #18397177 - 06/10/13 12:46 PM (10 years, 11 months ago)

Quote:

malicom said:
One more reason a good Still Air Box is superior to a flow hood.




In this situation, sure. In general, I wouldn't do all the shit I have to do in a glovebox if you paid me.


--------------------
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A punk rock song won't ever change the world, but I can tell you about a couple that changed me.

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InvisibleOeric McKenna
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Re: Help. Need to fight bacteria on agar [Re: 36fuckin5]
    #18397220 - 06/10/13 12:55 PM (10 years, 11 months ago)

Bacteria has been hampering my latest wild clone attempts lately.
The gelatinous look of doom...


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InvisibleSatanschild
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Re: Help. Need to fight bacteria on agar [Re: Oeric McKenna]
    #18397695 - 06/10/13 02:48 PM (10 years, 11 months ago)

SAB didn't make much difference compared to open air here.
Tried to clone 14 mushrooms this week, 7 were successful and all other contaminations were exactly at the transferred tissue.
Pretty happy with the results.

Though I do use antibiotic agar, doesn't stop contamination but sure does slow it down.

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OfflineSillyputty67

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Re: Help. Need to fight bacteria on agar *DELETED* [Re: Satanschild]
    #18398380 - 06/10/13 05:05 PM (10 years, 11 months ago)

Post deleted by malicom

Reason for deletion: 1



--------------------
1) Everything I ever posted or say is a lie.

Edited by Sillyputty67 (06/10/13 05:07 PM)

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Offlinemycofever
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Re: Help. Need to fight bacteria on agar [Re: Sillyputty67]
    #18405447 - 06/11/13 09:07 PM (10 years, 11 months ago)

I need to build me a flowhood I still do all of my work in a SAB. I do agar dishes, grain jars, and sawdust blocks. They suck though. I have gotten pretty good with using the trusty SAB.


--------------------
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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InvisibleOeric McKenna
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Re: Help. Need to fight bacteria on agar [Re: mycofever]
    #18406498 - 06/12/13 01:11 AM (10 years, 11 months ago)

Sawdust blocks in a SAB?
Gotta hand it to ya man. That sounds like hell


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


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Invisible36fuckin5
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Re: Help. Need to fight bacteria on agar [Re: Sillyputty67]
    #18413015 - 06/13/13 10:52 AM (10 years, 11 months ago)

Quote:

malicom said:
Flowhood:
Need to shower,



If I've showered that day, I don't bother.

Quote:

brush teeth




I'm REALLY bad about not doing that.

Quote:

Wear surgical mask, wear tyvek sleeves




Nope and nope. I do neither.

Quote:

Have very sanitary room.




Nah, have it reasonably clean and run your hood for a while. It works.

Quote:

Major advantange: can work with lots of stuff mush quicker than a SAB. No doing 10 jars, unloading, loading, waiting 10 minutes then going to work.




Fixed.

Quote:

Major disadvantage: Fucking expensive.




Comparatively, yeah. Really, though, it's a few hundred bucks. Most people should be able to save that up in a few months or less.

Quote:

Still air box:
Sanitize with alcohol. Put on some gloves. Wait. Go to work. Unload/load. Wait. Go to work. Repeat.




Fixed.

Quote:

Major advantage: Portable. Easy to use in any environment.




Unless you don't have a room with relatively still air. I'd have to sit in a room at about 95F or more for probably a couple hours to do one run of G2G.

Quote:

I use one on a table 6 ft from a litter box. You can do anything in one of these. You will never worry about cross contamination. You can work on the most diseased culture in this without worry.




This is the one reason I still keep my SAB.

Quote:

You just make sure to sanitize it with alcohol, and tinactin antifungal spray after use.




You shouldn't use either of those. Alcohol should be avoided because there's just no damn reason to concentrate flammable fumes in your house. Tinactin because it's expensive and just not necessary.

Quote:

major disadvantage: Can be tiny. Build a big one for lots of room to move.




You ever seen a SAB that's 4 foot by 2 foot? Me neither, but that's how big my flowhood was. 2 people could very comfortable work in front of it.

Quote:

Install a glass window so you can see better when doing agar work. You dont get to feel like the rich mad scientist and buy a big toy. :wink:





Not to be a dick, but if you don't have experience with something, you shouldn't be going around saying things like they're fact.

While  the things you recommend are the best practice, you can often get away with a lot.

I don't currently use a flowhood. I use a schmuvbox, which never fails me and is much nicer to work in than a SAB. Even though the concept seems to be wrong (so much turbulent air flow) I never have contams unless I'm swiping spores, which is to be expected.


--------------------
Pat The Bunny said:
A punk rock song won't ever change the world, but I can tell you about a couple that changed me.

bodhisatta said:
i recommend common sense and figuring it out.

These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.

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OfflineSillyputty67

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Re: Help. Need to fight bacteria on agar [Re: 36fuckin5]
    #18413143 - 06/13/13 11:29 AM (10 years, 11 months ago)

I hear you. My goal was initially to get a flowhood, but after using the SAB i have no need. Im not stating facts at all. Just opinion. It was "My Weighted Comparisons".

The alcohol and tinactin work great for cleaning it before use. Im not worried about flammable vapors as i have my wife flame my tools for me from a safe distance.

Alcohol does not kill fungal spores, tinactin does. So reguardless if you feel its unnecessary, it more thoroughly sanitizes my gear. More clean is better than less, and it dont hurt my pocketbook, and i dont get contams in my sab, unless its from a print, and thats has only happened once.

Dude everyone has their own style. If it works go with it. :thumbup:


--------------------
1) Everything I ever posted or say is a lie.

Edited by Sillyputty67 (06/13/13 11:37 AM)

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Invisible36fuckin5
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Re: Help. Need to fight bacteria on agar [Re: Sillyputty67]
    #18413364 - 06/13/13 12:31 PM (10 years, 11 months ago)

Quote:

malicom said:
The alcohol and tinactin work great for cleaning it before use. Im not worried about flammable vapors as i have my wife flame my tools for me from a safe distance.




What's a safe distance? Personally, I want my red-hot metal stuff inside the SAB while it's still red so I know it's hot enough. If you let it cool too much, it can grab contams out of the air before you get it in your clean area.

Quote:

Alcohol does not kill fungal spores, tinactin does. So reguardless if you feel its unnecessary, it more thoroughly sanitizes my gear. More clean is better than less, and it dont hurt my pocketbook, and i dont get contams in my sab, unless its from a print, and thats has only happened once.




You don't need to kill the spores though. Besides, you don't get them all with anything. You just need them to not be flying around in your box. Soap and water will do this.

Quote:

Dude everyone has their own style. If it works go with it. :thumbup:




Fair enough. And yeah, if your method works, then have at it. Just trying to kick down a little experience and make things easier on you.


--------------------
Pat The Bunny said:
A punk rock song won't ever change the world, but I can tell you about a couple that changed me.

bodhisatta said:
i recommend common sense and figuring it out.

These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.

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OfflineSillyputty67

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Re: Help. Need to fight bacteria on agar [Re: 36fuckin5]
    #18413413 - 06/13/13 12:40 PM (10 years, 11 months ago)

I never get my tools red hot, and i really dont use syringes at all in my teks anymore. I just have her flame my scalpel for me, and if it sizzles when it touches the clean agar, i know its good. :smile: I appreciate the help, I have a few grows under my belt now, and am still learning.


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Offlinemannextdoor
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Re: Help. Need to fight bacteria on agar [Re: Sillyputty67]
    #18417517 - 06/14/13 05:05 AM (10 years, 11 months ago)

Just my 2 cents: I like to work with my flowhood because I think it´s much more comfortable. I hate sweating in gloves and the limited freedom of your hands in the box. But of course, for some situations a glove box isn´t that bad. And some pro´s I didn´t expect till now... Argh, now I have to build one :-)

MND

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InvisibleJuiceh
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Re: Help. Need to fight bacteria on agar [Re: mannextdoor]
    #18418713 - 06/14/13 12:38 PM (10 years, 11 months ago)

Quote:

malicom said:
Flowhood:
Need to shower, brush teeth, Wear surgical mask, wear tyvek sleeves, Have very sanitary room.




FWIW - All of the above are steps that are also recommended when using a SAB as well. I know I've seen plenty posts saying that they do these things before using their SABs.


Once you have a killer 4x2 flowhood the only thing you would possibly want a SAB for is transferring agar from contaminated dishes to fresh ones to save a culture.

I built a really nice SAB with tyvek sleeves. My first premade agar dish experiments all wound up with contams. I followed sterile procedures to the T, wore surgical face masks, gloves, hair nets and also bombed the bejesus out of the box with hot soapy water & alcohol and used tons of lysol/oust. Bottom of the SAB always had paper towels soaked with bleach solution or just soapy water. Always waited 5-10 mins after bombing the box before getting to work. I had previously had ~95% success rate with a few dozen G2G transfers in the SAB, always had soaked paper towels laying on top of jars until they were needed. I saved some of those premade agar plates to inoculate later.

Now I have a 4x2 flowhood. I inoculated the rest of those premade agar plates in front of it. 100% success. I have made a few dozen G2G transfers, 100% success. I made my own agar and poured dozens of dishes in front of the hood, inoculated them the next day. 100% success. I left a freshly poured agar plate open sitting in front of the flow hood overnight, to test the flow hood. That was about 3 weeks ago, nothing has grown on it yet and nothing probably will if it hasn't yet. Not sure you could get away with that with a SAB.

I did get one contam on a king oyster dish I poured and inoculated with tissue from a master slant once cooled. But, its because the parafilm got overstretched in one area and tore leaving an air path that aspirated every time I checked on the dish, which was clinging to the dishes below and above it. The contam is right by this tear in the parafilm. Next time I will not be cheap and I will double up the parafilm.

Now, I am going to use my SAB to transfer away from that contam. However, when I prep the SAB it will be done in front of the running flow hood. Before I even start prepping it I will just leave the top off and tip it on its side so the flow hood is blowing right into it for 10 mins. Then tip the box rightside up, line the bottom in bleach soaked paper towels and then add the items required for the work and put the top on. All still in the sterile airflow since the hood is a bit taller than the SAB. Then I'll shut off the flow hood and wait for the air in the general area to settle for a couple minutes and then get to work in the box. I think I will have much better luck with the SAB if there has been sterile air running through it for a few mins and then trapped in it when I close it up and start working in it.:grin:

While its necessary if you bomb the box with alcohol or lysol or any other flamable aerosol etc.. I don't like the idea of flaming your scalpel\needles outside the box and bringing it back in. Not only to do you run the risk of the them cooling and grabbing a contam out of the air, but you also create currents when you move your hands in and out of the box, which you're trying to do fairly quickly before your tools cool. Also, your arm & gloves you just reached out of the box with can grab contams out of the air that can be brought in and blown onto your workspace by the air currents created when your hands enter. Any time I've needed to flame my tools for work in a SAB the SAB has been washed with alcohol, then hot soapy water and the bottom lined with bleach solution paper towels, no flamable gas is allowed to accumulate in the SAB. I do not lysol\oust the inside, a misty mate with a bleach solution is used to mist up the inside of the SAB as the top is closed. I do use lysol to spray around the arm holes and around the lid gap from the outside. I then use a butane torch to flame my tools inside the SAB. Flaming outside and bringing it back in isn't much of an option when you have tyvek sleeves attached to the box and have no side door to slip hot tools through.


Bottom line, a 4x2 flowhood is glorious. But SABs can come in handy.


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Edited by Juiceh (06/14/13 12:44 PM)

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