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Invisiblecheesenoonions
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Antibiotic concentrations in Agar
    #1160865 - 12/21/02 07:35 PM (14 years, 7 months ago)

Can someone give me some concentrations for Streptamycin, gentamycin and others.  Please put them in mg/mL.  i am using MEA and am having some trouble with a wild strain.  I get off white colonies that resemble E. coli in a day or so.  :frown: 


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OfflineAnnoA
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Re: Antibiotic concentrations in Agar [Re: cheesenoonions]
    #1161177 - 12/21/02 11:49 PM (14 years, 7 months ago)

Streptomycin brakes down when pressure cooking.

Gentamycin: 100mg of 60-80% clean gentamycin sulfate per liter.


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Invisiblecheesenoonions
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Re: Antibiotic concentrations in Agar [Re: Anno]
    #1161380 - 12/22/02 05:08 AM (14 years, 7 months ago)

I know streptamycin breaks down, I can add after sterilization. I have gentamycin, i don't know if it is gentamycin sulfate, the bottle just reads gentamycin. So any words of advice about taming a wild strain, Anno?


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Re: Antibiotic concentrations in Agar [Re: cheesenoonions]
    #1161547 - 12/22/02 07:08 AM (14 years, 7 months ago)

Ampicillin is cheap; I use 50 ug/mL. Tetracycline, I don't know what is optimal, but 30 mg/mL works. [both post-sterilization, of course].

I've regularly come across environmental bacteria that are resistant to both of these antibiotics, particularly Pseudomonas spp. and Flavomonas spp. They are both very motile bacteria and thus can hitch rides easily on expanding mycelia. One trick is to let the transfer 'fluff out' a little bit, so that you can pick a tiny aerial strand for a new transfer. You need only the tiniest of pieces.

Another possibility is to prepare a plate with a little peroxide; That may do the trick.

What species are you cloning?


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Invisiblecheesenoonions
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Re: Antibiotic concentrations in Agar [Re: Suntzu]
    #1161573 - 12/22/02 07:22 AM (14 years, 7 months ago)

I am trying to tame a local strain of cubensis. I looked long and hard all over for a local specimen. I'm thinking of doing some grad work on them.


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Offlineyoungmycologist
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Re: Antibiotic concentrations in Agar [Re: cheesenoonions]
    #1162642 - 12/22/02 04:31 PM (14 years, 7 months ago)

That sounds like alot of fun Cheese.I've always wanted to find and domesticate a wild strain.Good luck with your work.Keep us posted.


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OfflineH2O2shrooms
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Re: Antibiotic concentrations in Agar [Re: youngmycologist]
    #1163186 - 12/22/02 07:49 PM (14 years, 7 months ago)

innoculating the bottom af the agar may also help. Mycelium, will grow through agar while most contaminants grow on the surface.

Putting some mycelium on the bottom of the agar and then harvesting on the top portion will eliminate the bacteria that cannot grow through agar.



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Mycology is a lot like quantum mechanics in that we don?t have causal relationships like in Newtonian physics, only probabilities of various outcomes.<=== Misapropriated from Mycofile


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Invisiblecheesenoonions
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Re: Antibiotic concentrations in Agar [Re: H2O2shrooms]
    #1163834 - 12/23/02 05:49 AM (14 years, 7 months ago)

ok here are some pics

and again

the dark spots on the agar are clumps of spores. how do you inooc the bottom of the agar? Do you just put your loop all the way to the bottom? What about pH as a control, can I pH the agar to an optimal range for cubensis, but not for bacteria or is the range of bacteria too broad?


Edited by cheesenoonions (12/23/02 06:42 AM)


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Re: Antibiotic concentrations in Agar [Re: cheesenoonions]
    #1176676 - 12/29/02 07:45 AM (14 years, 7 months ago)

Looks like a healthy culture of Bacillus. Unfortunately, those plates are history. . .even with antibiotics the bacteria have likely killed the spores already. The pH question is an interesting one, I would guess that the pH zone for spore germination is more restrictive than the pH for bacterial proliferation.
I'd say your best bet is to formulate some antibiotic plates. Bacillus responds very well to just about all of them, penicillin, ampicillin, tetracycline. The other character you could be dealing with there is an environmental Pseudomonas, Flavomonas [gram negatives that can look snotty as well] in which case gentamicin would be your preferred antibiotic, though ampicillin may work as well.

In addition to being heat-tolerant, gentamicin is better at catching more gram positive and negative bacteria. . .two reasons it seems to be favored in this hobby.


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InvisibleZen Peddler
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Re: Antibiotic concentrations in Agar [Re: Suntzu]
    #1178486 - 12/30/02 01:40 AM (14 years, 7 months ago)

Domesticating wild varities of mushrooms like Ps.Cubensis is quite easy. Dont bother with gentamycin and the like as most bacterial contaminations will be resistant to it. Bacteria can be easily isolated.
I suggest streaking your spores across six normal mea plates and attempting to isolate a clean innoculation from that. It may sound like hit and miss, but from six plates you should get atleast one point on one of the plates that is clean mycelia.
Tranfers this point straight away to a couple of peroxidated agar plates and do the same again when sectoring occurs. This should clean them up so you can go about isolating mycelia upon rhizomorphic sectors or primordia formation performance (this can be tested easiky with cubies as a good isolate will develop them as soon as it fully colonises the plate.)
have fun.


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